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Rhinovirus infection of the human airway epithelium : in vitro characterization of viral replication,… Machala, Anna 2007

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RHINO VIRUS INFECTION OF THE HUMAN AIRWAY EPITHELIUM: IN VITRO CHARACTERIZATION OF VIRAL REPLICATION, INFLAMMATORY RESPONSE, AND IMMUNEMODULATING EFFECTS OF ECHINACEA by Anna Machala BSc, University of British Columbia, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR T H E DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA March 2007 © Anna Machala, 2007  ABSTRACT R h i n o v i r u s e s ( R V s ) are the l e a d i n g cause o f upper-respiratory tract i n f e c t i o n s i n h u m a n s . T o date r e l a t i v e l y little is k n o w n about the m e c h a n i s m o f R V i n f e c t i o n and no cure or p r e v e n t i o n exists. M o u n t i n g evidence shows that R V replicates v e r y little i n its host a i r w a y e p i t h e l i a l cells l e a d i n g researchers to h y p o t h e s i z e that the illness associated w i t h R V i n f e c t i o n is the result o f the host's i m m u n e response, but not necessarily R V r e p l i c a t i o n . T h i s study characterized R V i n f e c t i o n in vitro in terms o f v i r a l r e p l i c a t i o n , v i r a l R N A , and p r o - i n f l a m m a t o r y c y t o k i n e / c h e m o k i n e secretion over the course o f a t y p i c a l i n f e c t i o n u s i n g t w o distinct a i r w a y e p i t h e l i a l c e l l lines ( B E A S - 2 B and A 5 4 9 ) and t w o different receptor-utilizing R V serotypes ( R V 1 4 and R V 1 A ) . C e l l s were i n f e c t e d w i t h k n o w n amounts o f R V , s a m p l e d over 1 w e e k , and assayed f o r infectious v i r u s , R V 1 4 R N A , and i n t e r l e u k i n (IL)-6 and/or IL-8 secretion. F o r B E A S - 2 B and A 5 4 9 cells v i r a l r e p l i c a t i o n p e a k e d between day 1 ( D l ) and D 2 post-infection f o r both R V 1 4 and R V 1 A , and no s i g n i f i c a n t v i r a l r e p l i c a t i o n was observed after D 3 . S t i m u l a t i o n o f LL-6 and IL-8 was t y p i c a l l y not observed before D 2 and r e m a i n e d elevated up to D 7 . O v e r a l l , B E A S - 2 B cells w e r e m o r e susceptible to R V i n f e c t i o n than A 5 4 9 , and s i m i l a r trends were observed f o r R V 1 4 and R V 1 A , except R V 1 4 f a i l e d to replicate i n the A 5 4 9 c e l l s . F u r t h e r m o r e , U V i n a c t i v a t i o n o f b o t h R V serotypes c o m p l e t e l y i n h i b i t e d v i r a l r e p l i c a t i o n and IL-6 secretion in the B E A S - 2 B m o d e l , suggesting the necessity o f g e n e t i c a l l y intact v i r u s to stimulate the IL-6 response. F i n a l l y , the effects o f two c h e m i c a l l y distinct E c h i n a c e a extracts o n v i r a l r e p l i c a t i o n and IL-6 secretion were investigated i n the B E A S - 2 B m o d e l . N e i t h e r o f the E c h i n a c e a extracts had any effect o n R V r e p l i c a t i o n , nor d i d they stimulate IL6 secretion i n u n i n f e c t e d cells. H o w e v e r , E c h i n a c e a treatment o f R V i n f e c t e d cells s i g n i f i c a n t l y affected IL-6 secretion, but a different trend was o b s e r v e d between  RV  serotypes, and f o r the t w o herb preparations. O v e r a l l , R V i n f e c t i o n o f a i r w a y e p i t h e l i a l cells results i n r e l a t i v e l y l o w levels o f R V r e p l i c a t i o n but a p r o n o u n c e d p r o i n f l a m m a t o r y c y t o k i n e / c h e m o k i n e response w h i c h is the l i k e l y cause o f c o l d s y m p t o m s and a potential target for therapeutics.  TABLE OF CONTENTS ii  Abstract Table of Contents  iii v  List of Figures List of Abbreviations  vii  Acknowledgements  viii  Co-Authorship Statement Chapter 1: Introduction and O b j e c t i v e s References  ix 1 28  Chapter 2 : In Vitro C h a r a c t e r i z a t i o n o f R h i n o v i r u s Infection i n A i r w a y Epithelial Cells (Growth Curves) Figures References  43 59 68  Chapter 3: T h e E f f e c t s o f U l t r a v i o l e t Inactivated R h i n o v i r u s o n A i r w a y Epithelial Cells  Figures References  Chapter 4: T h e E f f e c t s o f E c h i n a c e a E x t r a c t s on R h i n o v i r u s Infected and Uninfected A i r w a y Epithelial Cells Figures  73  82 87  88 97  References  99  Chapter 5: G e n e r a l D i s c u s s i o n and C o n c l u s i o n s References  103 113  Appendices: Appendix A : B i o h a z a r d C e r t i f i c a t e  116  Appendix B : G r o w t h C u r v e E x p e r i m e n t a l D e s i g n  117  Appendix C : E f f e c t o f P u r i f i e d and U n p u r i f i e d R h i n o v i r u s on V i r a l R e p l i c a t i o n and C y t o k i n e / C h e m o k i n e Secretion f r o m B E A S - 2 B and A 5 4 9 C e l l s  118  Appendix D: C e l l C o u n t s for R V Infected and U n i n f e c t e d B E A S - 2 B and A 5 4 9 C e l l s  Appendix E : H I , B E A S - 2 B and A 5 4 9 C e l l C o u n t s at C o n f l u e n c e  121  123  Appendix F : R h i n o v i r u s S t a b i l i t y  124  Appendix G : I n t e r l e u k i n - 6 and Interleukin-8 S t a b i l i t y  126  in  A p p e n d i x H: Image o f P l a q u e A s s a y  ....129  iv  LIST OF FIGURES  Figure 2.1: Effect of a) R V 1 4 and b) R V 1 A on viral replication in H I cells over time  59  Figure 2.2: Effect of R V 1 4 on: a) viral replication and b) and c) IL-6 secretion in B E A S - 2 B cells over time  60  Figure 2.3: Effect of R V 1 A on: a) viral replication and b) and c) IL-6 secretion in B E A S - 2 B cells over time  61  Figure 2.4: Effect of R V 1 4 on: a) viral replication and b) and c) IL-8 secretion in A 5 4 9 cells over time  62  Figure 2.5: Effect of R V 1 A on: a) viral replication and b) and c) IL-8 secretion in A 5 4 9 cells over time  63  Figure 2.6: R V 1 4 R N A for H I , B E A S - 2 B and A 5 4 9 cells over time  64  Figure 2.7: Effect of R V 1 4 on: a) viral replication and b) IL-8 secretion and c) IL-6 secretion in simultaneously cultured (tandem) B E A S - 2 B and A549 cells over time  65  Figure 2.8: Effect of R V 1 A on: a) viral replication and b) IL-8 secretion and c) IL-6 secretion in simultaneously cultured (tandem) B E A S - 2 B and A 5 4 9 cells over time  66  Figure 2.9: R V 1 4 R N A levels in tandem cultures of B E A S - 2 B and A 5 4 9 cells over time  67  Figure 3.1: Trial 1. Effect of U V treated and untreated R V 1 4 on a) viral replication and b) IL-6 secretion in B E A S - 2 B cells after 48 hours  82  Figure 3.2: Trial 2. Effect of U V treated and untreated R V 1 4 on a) viral replication and b) IL-6 secretion in B E A S - 2 B cells after 48 hours  83  Figure 3.3: Trial 1. Effect of U V treated and untreated R V 1 A on a) viral replication and b) TL-6 secretion in B E A S - 2 B cells after 48 hours  84  Figure 3.4: Trial 2. Effect of U V treated and untreated R V 1 A on a) viral replication and b) IL-6 secretion in B E A S - 2 B cells after 48 hours  85  Figure 3.5: Effect of U V treated and untreated R V 1 4 inocula on R V 1 4 R N A levels in B E A S - 2 B cells after 48 hours  86  v  F i g u r e 4.1: Effects of Echinacea extracts ( E l , E2) and ethanol on a) R V 1 4 replication and b) IL-6 secretion in R V 1 4 infected and uninfected B E A S - 2 B cells  97  F i g u r e 4.2: Effects of Echinacea extracts ( E l , E2) and ethanol on a) R V 1 A replication and b) IL-6 secretion in R V 1 A infected and uninfected B E A S - 2 B cells  98  vi  LIST OF ABBREVIATIONS RV  rhinovirus  COPD  c h r o n i c obstructive p u l m o n a r y d i s o r d e r  UV  ultraviolet  ICAM-1  intercellular adhesion molecule-1  LDLR  l o w density l i p o p r o t e i n receptor  VLDLR  v e r y l o w density l i p o p r o t e i n receptor  HID TCID™ CPE  5 0 % h u m a n i n f e c t i o u s dose 5 0 % tissue culture i n f e c t i o u s dose  TNF  t u m o r necrosis factor  IL  interleukin  5 0  c y t o p a t h i c effects  NFKB  nuclear factor kappa-B  ATCC  American Type Culture Collection  qRT-PCR  quantitative real-time p o l y m e r a s e c h a i n reaction  ELISA  e n z y m e - l i n k e d i m m u n o s o r b e n t assay  ANOVA MOI D(0-7)  analysis o f variance multiplicity of infection  days after R V i n f e c t i o n / i n o c u l a t i o n  pfu  plaque f o r m i n g units  FBS  fetal b o v i n e serum  DMEM  Dulbecco's Modified Eagle's M e d i u m  MEM  Modified Eagle's M e d i u m  ACKNOWLEDGEMENTS F i r s t and f o r e m o s t , I w o u l d l i k e to thank m y co-supervisors D r . James H u d s o n and D r . C o l i n B r a u n e r f o r their support and m e n t o r s h i p . D r H u d s o n , thank y o u f o r s p a r k i n g m y interest i n the w o r l d o f viruses and f o r y o u r c o n t i n u e d encouragement d u r i n g this project. D r . B r a u n e r , thank y o u f o r t a k i n g m e into y o u r lab and a l l o w i n g m e to get m y hands a b i t wet w i t h the C h i n o o k . I w o u l d also l i k e to thank D r . R o b e r t H a r r i s w i t h o u t w h o m this thesis w o u l d not be p o s s i b l e . I w o u l d l i k e to a c k n o w l e d g e D r . M a n j u S h a r m a and D r . S e l v e r a n i V i m a l a n a t h a n f o r their h e l p d u r i n g m y project. F i n a l l y , I w o u l d l i k e to thank the B r a u n e r and N a n d a n L a b s f o r a l l their support o v e r the past f e w years.  viii  CO-AUTHORSHIP STATEMENT  A l l of the work required for this thesis was completed by me independently; however, my supervisors (Dr. James Hudson, Dr. Robert Harris, and Dr. C o l i n Brauner) provided me with mentorship and supervision, and w i l l be listed as co-authors on the publications arising from this project.  ix  Chapter 1: Literature Review and Thesis Objectives  INTRODUCTION T h e most prevalent i n f e c t i o n i n humans is the acute upper-respiratory tract i n f e c t i o n , also k n o w n as the c o m m o n c o l d ( M o n t o , 2 0 0 2 ) . O v e r 100 R h i n o v i r u s e s ( R V s ) persist i n o u r w o r l d today and are the l e a d i n g cause o f such respiratory tract infections ( A r r u d a et a l . , 1 9 9 7 ; Johnston et a l . , 1 9 9 5 ; v a n G a g e l d o n k - L a f e b e r et a l . , 2 0 0 5 ) . In healthy i n d i v i d u a l s R V infections are t y p i c a l l y short-lived and s e l f - l i m i t i n g ; h o w e v e r , f o r susceptible groups such as infants, the e l d e r l y , and the i m m u n o - c o m p r o m i z e d R V i n f e c t i o n c a n be lifethreatening. F u r t h e r m o r e , f o r people already affected b y diseases such as asthma or c h r o n i c obstructive p u l m o n a r y disorder ( C O P D ) , R V i n f e c t i o n is k n o w n to cause serious exacerbations o f those c o n d i t i o n s ( B a r d i n , 1 9 9 2 ; F r a e n k e l et a l . , 1 9 9 5 ; G e r n , 2 0 0 2 ; G e r n & B u s s e , 1 9 9 9 ; G r e e n b e r g , 2 0 0 2 ; G r u n b e r g & Sterk, 1 9 9 9 ; M a l l i a et a l . , 2 0 0 6 ; M e s s a g e & J o h n s t o n , 2 0 0 4 ; S e e m u n g a l , 2 0 0 1 ; S e e m u n g a l , 2 0 0 0 ; T e r a n et a l . , 1997). In the case o f asthma, recent studies suggest that early c h i l d h o o d R V i n f e c t i o n m a y even p l a y a causative role i n the d e v e l o p m e n t o f the disease ( G e r n , 2 0 0 4 ; S i n g h et a l . , 2 0 0 7 ) . T o date, re l ati v e l y little is k n o w n about the m e c h a n i s m o f R V i n f e c t i o n and no cure o r p r e v e n t i o n exists. M o u n t i n g evidence shows that R V replicates v e r y little i n its host a i r w a y e p i t h e l i a l tissues ( L o p e z - S o u z a et a l . , 2 0 0 4 ) , l e a d i n g researchers to h y p o t h e s i z e that the illness associated w i t h R V i n f e c t i o n is the result o f the host's i m m u n e response to some v i r a l trigger, but not necessarily to the l e v e l o f v i r a l r e p l i c a t i o n itself. B e a r i n g this i n m i n d , new therapies w h i c h modulate the i m m u n e system and mitigate R V associated s y m p t o m s are b e c o m i n g i n c r e a s i n g l y interesting to scientists. O n e such candidate therapy is the natural herb extract E c h i n a c e a . E c h i n a c e a use has recently g a i n e d w i d e s p r e a d p o p u l a r i t y i n W e s t e r n culture, and c o m m e r c i a l products are w i d e l y available to c o n s u m e r s . A l t h o u g h the q u a l i t y o f m a n y c o m m e r c i a l f o r m u l a t i o n s is questionable, there is g r o w i n g e v i d e n c e that E c h i n a c e a has diverse effects o n the i m m u n e system (Brousseau & M i l l e r , 2 0 0 5 ; B r u s h et al., 2 0 0 6 ; C u r r i e r & M i l l e r , 2 0 0 0 ; G o e l , 2 0 0 5 ; S h a r m a et a l . , 2 0 0 6 ) . In terms o f m i t i g a t i n g R V - r e l a t e d i l l n e s s , E c h i n a c e a m a y down-regulate the i n f l a m m a t o r y response p r o v o k e d b y v i r a l i n f e c t i o n ; h o w e v e r , its m e c h a n i s m o f action is l a r g e l y u n k n o w n ( S h a r m a et a l . , 2 0 0 6 ) .  1  In my experiments, I aimed to characterize R V infection in immortalized human airway epithelial cells and to evaluate the effects of Echinacea extracts on R V infected and uninfected cells. This first chapter provides background material relevant to this thesis and concludes with general objectives and hypotheses. In Chapter 2 {In Vitro Characterization of Rhinovirus Infection in Airway Epithelial Cells) I characterized R V infection in two distinctive airway epithelial cell models using two different receptor-utilizing R V serotypes. I measured levels of viral replication, viral R N A , and cell secretion of proinflammatory cytokines and chemokines over the course of a typical infection and compared those parameters over time, between cell models, and between R V serotypes. In Chapter 3 (The Effects Ultraviolet Inactivated Rhinovirus on A i r w a y Epithelial Cells) I investigated the potential viral trigger for cell inflammatory mediator secretion by using ultraviolet ( U V ) inactivated R V to assess its ability to infect cells and/or elicit an inflammatory response. In Chapter 4 (The Effects of Echinacea Extracts on Rhinovirus Infected and Uninfected A i r w a y Epithelial Cells) I examined the effects of two chemically distinct Echinacea extracts on infected and uninfected bronchial epithelial cells in terms of viral replication, viral R N A , and cell pro-inflammatory cytokine secretion. Finally, Chapter 5 presents a general discussion of my findings, overall significance and conclusions, and future directions for investigation.  The Host: The A i r w a y E p i t h e l i u m The human airway epithelium is a pseudostratified cell layer which provides a physical barrier between the internal and external environments of the air passages while playing a vital role in processes such as the maintenance of lung fluid balance, mediation of smooth muscles, clearance and metabolism of irritants and pathogens, and activation of the inflammatory response. The human airway epithelium consists of at least 8 morphologically different cell types which can be functionally subdivided into: columnar ciliated epithelial cells, mucous cells, and basal cells (Jeffery & Reid, 1975; Spina, 1998). The ciliated cells are in contact with the external environment and are the predominant airway epithelial cell type (Halama et al., 1990). The cilia of these cells beat in a rhythmic fashion to move any trapped debris out of the respiratory tract to be coughed out or swallowed. The mucous cells (e.g. goblet and Clara cells) secrete acidic-mucin granules  2  into the airway lumen which mix with water forming an outer mucus lining (Stinson & Loosli, 1978). Finally, the basal cells are attached to the underlying basement membrane and anchor the other cells of the epithelium. They are also the primary stem cells to the other cell types (Knight & Holgate, 2003). The cells themselves function as a barrier to the outside environment, and furthermore paracellular diffusion is restricted by the formation of tight junctions between the apices of adjacent cells (Qu, 2005). The cells of the airway epithelium also secrete many important molecules such as: lipid mediators, growth factors, broncho-constricting peptides, arachidonic acid metabolites, cytokines, and chemokines, which play diverse roles in ensuring proper respiratory functioning (Knight & Holgate, 2003). The human respiratory tract is susceptible to infection by many bacteria, fungi, and viruses. The most common viruses to infect the airway epithelial cells are the R V s often resulting in acute upper-respiratory tract infections known as the common cold.  M o d e l s of the A i r w a y E p i t h e l i u m In order to facilitate laboratory research, many in vitro models of the human airway epithelium have been developed by scientists. The most commonly used models are cultured cell lines derived from native airway epithelial cells. These cell lines have typically been transformed by viruses, or derived from cancerous growths, rendering them immortalized and amenable to repeated culture. Examples of immortalized airway epithelial cell lines include: S V 4 0 adenovirus transformed bronchial epithelial ( B E A S - 2 B ) cells (Ke et al., 1988), and human adenocarcinoma derived type II alveolar-like (A549) cells (Lieber et al., 1976). The above cell lines retain much of their native cell functions including the ability to form monolayers and the capability to secrete various biological molecules (Ke et al., 1988; Lieber et al., 1976; Reddel et al., 1988; Shapiro et al., 1978; Smith, 1977). When grown submersed in culture medium immortalized cells undergo limited differentiation and thus resemble basal epithelial cells most closely (Albright et al., 1990). They also lack distinct apical and basolateral membranes; however, junctional complexes can be observed (Albright et al., 1990). Overall, immortalized cell lines are an approachable and widely accepted model for many scientific experiments. It is possible to induce further differentiation in the aforementioned cell lines by manipulating their growth environment. For example, culturing B E A S - 2 B or A549 cells on  3  permeable supports or s p e c i a l i z e d membranes w i t h an air-liquid interface produces m o n o l a y e r s w i t h a p i c a l and basolateral sides and increases the e x p r e s s i o n o f tight j u n c t i o n proteins ( B l a n k et a l . , 2 0 0 6 ) . F i n a l l y , w i t h access to l i v e a i r w a y e p i t h e l i a l tissues scientists can establish p r i m a r y cultures b y p r o c e s s i n g and p l a t i n g cells u s i n g s p e c i a l i z e d procedures. P r i m a r y cultures are t y p i c a l l y created f r o m nasal, tracheal or b r o n c h i a l tissues d e r i v e d f r o m s u r g i c a l procedures such as p o l y p e c t o m i e s ( D o n n i n g e r et a l . , 2 0 0 3 ; L o p e z - S o u z a et a l . , 2 0 0 4 ; S u z u k i et a l . , 2002). O b v i o u s l y , such cultures are the truest in vitro m o d e l s o f the a i r w a y e p i t h e l i u m retaining native e p i t h e l i u m characteristics most c l o s e l y . H o w e v e r , l i v e tissues are often d i f f i c u l t to obtain (especially i n s u f f i c i e n t amounts), culture techniques are more sensitive and c o m p l i c a t e d , and cells o n l y a l l o w f o r 1-2 passages before u n d e r g o i n g t e r m i n a l differentiation. F u r t h e r m o r e , p r i m a r y tissues d e r i v e d f r o m surgeries m a y be affected b y the presence o f pre-operative drugs such as anesthetics. A l l o f the a b o v e m o d e l s are susceptible to R V i n f e c t i o n at least to s o m e degree. L o p e z S o u z a et al. (2004) f o u n d that the least differentiated cells ( i m m o r t a l i z e d c e l l cultures) were the most v u l n e r a b l e to R V i n f e c t i o n and the most differentiated cells ( p r i m a r y cultures) w e r e m u c h m o r e resistant. U n d i f f e r e n t i a t e d cultured c e l l s p r o d u c e d v i r a l titers 30 to 130 times that o f their differentiated p r i m a r y culture counterparts, w h i l e i m m o r t a l i z e d cells g r o w n o n permeable supports p r o d u c e d intermediate v i r a l concentrations. A s i m i l a r trend was observed f o r R V i n d u c e d i n f l a m m a t o r y m e d i a t o r secretion and suggests that undifferentiated a i r w a y c e l l m o d e l s m a y represent a somewhat exaggerated response to R V infection.  Rhinovirus (RV) R V s b e l o n g to the Picornaviridae f a m i l y and are i c o s a h e d r a l w i t h a protein c a p s i d s u r r o u n d i n g genetic material e n c o d e d i n single stranded R N A ( B e l l a & R o s s m a n , 1999). T h e i r genetic i n f o r m a t i o n carries a p o s i t i v e p o l a r i t y and is translated into a p o l y p r o t e i n u p o n entry into the c y t o p l a s m . T h i s p o l y p r o t e i n is then a u t o m a t i c a l l y c l e a v e d b y the c e l l and further processed b y v i r a l proteases, eventually f o r m i n g the v i r a l c a p s i d proteins and non-structural proteins i n v o l v e d i n the r e p l i c a t i o n process ( R e i t h m a y e r et a l . , 2 0 0 2 ) . T o date, over 100 R V serotypes have been i d e n t i f i e d based on the presence o f u n i q u e and  4  specific antigens (Bella & Rossman, 1999). The R V capsid is about 30nm in diameter and consists of 60 copies of four distinct viral proteins called: V P 1 , V P 2 , V P 3 , and V P 4 . The first three viral proteins form the outer protein shell while V P 4 exists interiorly in contact with the single stranded R N A . The R V structure is characterized by protuberances on a fivefold vertex with depressions called "canyons" between the vertices (Bella & Rossmann, 1999; K i m & K i m , 1989; Oliveira et al., 1993; Rossmann, 1989; Zhao et al., 1996).  R h i n o v i r u s Receptors The R V viral binding sites have been determined by antibody binding studies (Bella & Rossmann, 1999). Binding sites were located by analyzing mutant viruses that were unaffected by antibodies. Four specific antigenic areas were located and mapped onto the known structure of R V 1 4 . These antigenic sites correspond to the outer edges of the canyons and are the exposed parts of the virus (Bella & Rossmann, 1999; Rossmann, 1989). The viral binding site for receptors is largely found within the canyons although it has been shown that this binding does extend over the rims of the canyons (Bella & Rossmann, 1999; Smith et al., 1996). The R V s are often classified by the type of cell receptor that they bind as identified by monoclonal antibody studies where antibodies recognized a 95kd glycoprotein on both human cells and mouse transfectants (Greve et al., 1989). Most R V s (more than 90) belong to the "major"group, sharing a common receptor called the intercellular adhesion molecule1 ( I C A M - 1 ) (Bella & Rossmann, 1999; Greve et al., 1989; Staunton et al., 1989). The "minor"group consists of 10 serotypes which utilize receptors in the low density lipoprotein receptor ( L D L R ) family (Bella & Rossmann, 1999; Hofer et al., 1994). Finally, human R V 8 7 does not bind to either of the above receptors and has recently been reclassified as an enterovirus based on genome sequences, although receptor-binding studies are lacking (Oberste et al., 2004; Uncapher et al., 1991). It has been proposed that once bound to their receptors some viruses enter the cytoplasm by cellular endosomes (Bayer et al., 1998; Zeichhardt et al., 1985). Such a mechanism has been demonstrated for R V 2 (a minor R V ) and also for the foot-and-mouth disease virus (Baxt, 1987). This mechanism is characterized by the low p H internal to  5  endosomes which causes the release of capsid-bound R N A into the cellular environment (Baxt 1987; Bayer et al., 1998; Prchla et al., 1994; Zeichhardt et al., 1985). Other studies have demonstrated that the I C A M - 1 receptor (major R V s ) is capable of uncoating both R V s and polioviruses at normal physiological p H in soluble form, suggesting the passage of the virus without and endocytic step (Perez & Carrasco, 1993).  Intercellular Adhesion Molecule-1 (ICAM-1) Receptor The I C A M - 1 receptors are transmembrane glycoprotein cell adhesion molecules (Bella & Rossmann, 1999). Exteriorly they consist of a row of immunoglobulin domains. It is these domains that are the basic building blocks of antibodies (Bella & Rossmann, 1999). The I C A M - 1 ligand is a pair of integrin receptors that is largely found on leukocyte cells. In the leukocyte the I C A M - 1 receptors' main functions are to promote cell adhesion to the extracellular matrix and also to lymphocytes (Bella & Rossmann, 1999). This role is important in the inflammatory response when the expression of I C A M - 1 receptors is elevated in endothelial cells, thus causing these cells to adhere to leukocytes passing in the blood and drawing leukocytes to locations of injury or infection (Bella & Rossmann, 1999). Although there is evidence that RV-stimulated cytokine secretion increases the permeability of lung endothelial cells (Sedgwick et al., 2002), it is unknown whether R V can or does directly infect the airway endothelium. The major R V s have exploited the I C A M - 1 receptors and utilized them as viral binding sites binding I C A M - 1 receptors at the deepest canyon site on their surface (Olson et al., 1993). The binding of receptor arid virus is the first step of infection which is followed by viral genetic uncoating and entry into the cell by crossing the plasma membrane. It has been demonstrated that major group R V infection causes a rapid up-regulation in membrane bound I C A M - 1 expression in airway epithelial cells. (Grunberg, 2000; Papi & Johnston, 1999; Papi, 2002; Winther et al., 2002). For example, Papi, (2002) found that upon infection with R V , I C A M - 1 expression in cell lines and primary cultures increased 4.2 to 6 times respectively, when compared to controls. This R V induced increase in I C A M - 1 expression seems to occur mostly in basal cells and to a lesser extent in ciliated cells (Grunberg, 2000). I C A M - 1 expression peaks 8-24 hours post-infection and gradually declines back to control levels usually by day 5 (Papi & Johnston, 1999; Winther et al.,  6  2002). The up-regulation in I C A M - 1 expression is correlated to the airway inflammatory response which can be triggered by irritants, pathogens, and as a complication from diseases such as asthma and C O P D where this response is unnaturally exaggerated. Researchers have found a direct link between viral infection and the increase of inflammatory mediators (e.g. cytokines), leading to the up-regulation of I C A M - 1 and other airway inflammatory response genes (Grunberg, 2000; Higashimoto et al., 1999; Papi, 2002; Winther et al., 2002 ).  Low Density Lipoprotein Receptors (LDLRs) The L D L R receptors are comprised of: L D L R proper, very low density lipoprotein receptor ( V L D L R ) , LDLR-related protein, megalin, and other membrane proteins (Hofer et al., 1994; Reithmayer et al., 2002). The ligands for this family of receptors are binding domains consisting of cysteine residue repeats, although particular ligands are quite remarkably divergent (Reithmayer et al., 2002). A s such, the mechanism of receptor recognition is still largely unknown. The L D L R type R V 2 and R V 1 A have been successfully replicated in mouse cell lines which also carry L D L R - t y p e receptors (Lomax & Y i n , 1989; Reithmayer et al., 2002). The L D L R receptors are strongly correlated with the endocytic mechanism (Brabec et al., 2006). It is thought that the low p H of the endosomes triggers the opening of virus induced pores in the plasma membrane thus allowing the viral R N A to enter the cell (Bayer et al., 1998; Prchla et al., 1995). The L D L R receptors have been definitively shown to bind some minor R V serotypes (Hofer et al., 1994). Interestingly, cells lacking or with suppressed L D L R receptor expression do allow limited viral entry of some serotypes suggesting the possibility of additional points of entry (Hofer et al., 1994).  The Common Cold: Infection & Illness R V s are the cause of over 50% of upper respiratory tract infections in humans (Arruda et al., 1997; lohnston et al., 1995). Other viruses that can cause the common cold include the respiratory syncytial virus, adenoviruses, and coronaviruses (Arruda et al., 1997). Most adults experience an average of 2-4 colds a year, while children may experience 6-8 colds per year (Monto & Sullivan, 1993). Infections are more common in temperate climates  7  during the colder months of the year (Couch, 1996; Monto, 2002). Individuals usually become infected by contact with contaminated surfaces or inhalation of large particle aerosols like those arising from coughing (D'Alessio et al., 1976; Dick et al., 1987; Gwaltney et al., 1978; Gwaltney & Hendley, 1982). While infection is usually initiated in the nasopharyngeal area, most of the airway tissues are susceptible to infection at least to some degree (Winther et al., 1986). Unlike enteroviruses, R V s have not been found to replicate in the gastrointestinal tract and are rapidly inactivated in the stomach (cited in Couch, 1996). The 50% human infectious dose (HID50) for R V s is generally regarded as low; ranging from 0.032 to 0.4 of the 50% tissue culture infectious dose (TCID o) in human fibroblasts. S  However, this infectious dose is variable, for example; R V 1 4 HID50 was found to be 5.7 times the fibroblast T C I D  5 0  (cited in Couch, 1996). Individuals with existing R V antibodies  are resistant to infection and the viral dose required to cause illness is higher than for individuals lacking antibodies (Alper et al., 1996). The incubation period of R V leading to first viral shedding in vivo is 10-12 hours while the viral replication cycle duration is 6-8 hours (Harris & Gwaltney, 1996). Onset of illness typically begins with a "scratchy" throat, followed by symptoms including: nasal discharge, nasal obstruction, sneezing, sore throat, cough, headache, myalgia, malaise, and rarely fever (Arruda et al., 1997). Overall, the systemic symptoms are much milder than those caused by viruses such as the influenza virus. Computer tomographic scans of individuals infected with colds show: occlusion and abnormalities of the sinus cavities, thickening of nasal passage walls, and engorged turbinates (Gwaltney et al., 1994). The mean duration of the cold is 7-11 days (Arruda et al., 1997) with peak symptoms occurring between the second and third day. In healthy patients R V can be recovered for up to 3 weeks (Jartti et al., 2004). K l i n g et al. (2005) found that R V R N A persisted in 44% of asthmatic children 6 weeks after infection. Additionally chronic R V infections, persisting for more than 12 months, have been described in some lung transplant recipients (Kaiser et al., 2006).  R h i n o v i r u s Infection of the A i r w a y s The underlying histology of R V infection of the airways is not totally understood; however, there is mounting evidence the R V infection does not manifest itself as a  8  w i d e s p r e a d m u c o s a l i n f e c t i o n but as l o c a l i z e d f o c i f r o m where i n f l a m m a t o r y responses are generated. In vivo, this has been demonstrated w i t h nasal b i o p s i e s w h i c h c l e a r l y p o i n t to s m a l l c e l l u l a r areas o f l o c a l i z e d i n f e c t i o n , w h i l e s h o w i n g f e w other h i s t o l o g i c a l abnormalities ( D o u g l a s et a l . , 1968; H a m o r y et a l . , 1 9 7 7 ; W i n t h e r et a l . , 1984). M o r e recent i m m u n o h i s t o c h e m i c a l e v i d e n c e also supports the f i n d i n g that R V i n f e c t i o n o f the a i r w a y s emerges i n a patch-like f a s h i o n ( M o s s e r et a l . , 2 0 0 5 ) . M o s s e r et al. (2002) demonstrated that o n l y 5 - 1 0 % o f p r i m a r y a i r w a y e p i t h e l i a l cells were susceptible to R V regardless o f the i n f e c t i o n dose used. A r r u d a et al. (1995) also f o u n d that o n l y a v e r y s m a l l p r o p o r t i o n o f nasal e p i t h e l i a l cells w e r e infected w i t h R V i n e x p e r i m e n t a l l y i n o c u l a t e d volunteers w h o had i n d e e d d e v e l o p e d c o l d s y m p t o m s . F u r t h e r m o r e , i n tissue and c e l l cultures l i k e : B E A S - 2 B , A 5 4 9 , or p r i m a r y tracheal cells there are no cytopathic effects ( C P E ) such as: c e l l r o u n d i n g , w r i n k l i n g , rupture, or death observed u p o n i n f e c t i o n w i t h R V (Johnston et a l . , 1 9 9 8 ; S u z u k i et a l . , 2 0 0 1 ) , although v i r a l r e p l i c a t i o n can e a s i l y be detected. T h e level o f R V r e p l i c a t i o n is also c o n s i d e r e d r e l a t i v e l y l o w w i t h i n c r e a s i n g l y differentiated cells p r o d u c i n g decreased titers o f v i r u s ( L o p e z - S o u z a et a l . , 2 0 0 4 ) . C o n s e q u e n t l y , there is a g r o w i n g b e l i e f that it is the p r o - i n f l a m m a t o r y c y t o k i n e s and c h e m o k i n e s stimulated b y R V i n f e c t i o n that cause the s y m p t o m s and pathogenicity o f infection.  Immune Response to Infection In order f o r R V to infect the a i r w a y e p i t h e l i u m several defensive barriers must first be penetrated. F i r s t l y , the v i r u s must breach the m u c o s a l layer o f the e p i t h e l i u m , and i f s u c c e s s f u l , the v i r u s m a y adhere to the e p i t h e l i a l layer, but o n l y i f it can out-compete the natural f l o r a o f the a i r w a y s , and evade phagocytes w h i c h are e s p e c i a l l y r i c h i n the l u n g tissues. O n c e attached its host receptor, v i r a l entry and r e p l i c a t i o n m a y o c c u r , and l o c a l i n f e c t i o n m a y ensue. O n c e i n s i d e the c e l l , the v i r u s is not e x p o s e d to most elements o f the i m m u n e system but as v i r a l p r o g e n y are released f r o m infected c e l l s , these v i r i o n s are c o n f r o n t e d b y the anticipatory i m m u n e response. T h e tissues defend themselves against potential threats such as p h y s i c a l i n j u r y , irritants and pathogens b y t w o types o f i m m u n i t y c a l l e d : innate and adaptive. T h e innate response is fast acting, triggered w i t h i n 0-4 hours and acts over several days. It initiates the same  9  cascade of events regardless of threat (Janeway, 2005). The adaptive response is slower acting, usually activated after 96 hours of exposure, and results in the production of specific antibodies which resolve infections while ensuring long-term or life-time immunity against specific antigens (Janeway, 2005).  Innate Immunity The innate immune response is the airway cells' first line of defense once a pathogen has penetrated the epithelial layer. In vivo, pathogens are first met by phagocytic macrophages residing in the airway tissues, which both engulf threats and secrete inflammatory mediators to initiate an inflammatory response. These mediators include lipids such as prostaglandins, leukotrienes, and platelet-activating factor, and protein mediators called cytokines and chemokines (Janeway, 2005). The airway epithelial cells themselves are also capable of secreting cytokines and chemokines, and thus this portion of the innate cellular response can be retained in vitro ( K i m et al., 2000; Sharma et al., 2006; Zhu et al., 1996). A multitude of different cytokines and chemokines are secreted from various cells interacting to coordinate the initial inflammatory response and prime the impending adaptive response. Inflammation functions to increase microcirculation to the site of infection allowing for increased entry of white blood cells and also increases lymph circulation for initiation of the adaptive response by antibody formation (Janeway, 2005). During inflammation the principal cells recruited to the injured site are neutrophils, which engulf and destroy the invading pathogens. Neutrophil levels are typically elevated during the first day post-infection but quickly return to normal levels (Couch, 1996). The characteristic symptoms associated with this response: swelling, redness, heat and pain are thought to be caused by the inflammatory mediator induced effects on local blood vessels, such as increased dilation and permeability (Janeway, 2005). The inflammatory response is critical in controlling infections while initiating the slower acting adaptive response.  Cytokines & Chemokines Cytokines are soluble intercellular signaling glycoproteins which are secreted by cells and affect the behaviour of other cells bearing receptors for them (Janeway, 2005). To date,  10  over 100 members of the cytokine family have been identified (Haddad, 2002). In general, cytokines are secreted in response to various injuries and stresses (including viral infection), and act in a paracrine fashion to initiate responses from their neighbours (Janeway, 2005). However, more recently cytokines have been ascribed diverse immunological roles in antigen presentation, adhesion molecule expression, and bone marrow differentiation, thus cytokine roles are much more complex than initially thought (Borish & Steinke, 2003). A t the site of injury, which type of immune response initiated (humoural, allergic, cell mediated, or cytotoxic) is dictated by the types and combinations of cytokines secreted (Borish & Steinke, 2003). Generally, cytokines can be divided into two functional groups: pro-inflammatory and anti-inflammatory, where pro-inflammatory cytokines initiate and/or amplify inflammation while anti-inflammatory cytokines negate such effects (Calixto et al., 2004). Cytokines bear specialized receptors which recognize specific patterns present on pathogens thus initiating an immune response (Borish & Steinke, 2003). These receptors activate tyrosine kinase signaling pathways, which include the janus kinases and signal transducer and activators of transcription acting in a hormonal fashion to affect transcription of specific genes (Darnell et al., 1994; Jhle et al., 1994). Chemokines are small (8-12kD) secreted proteins which attract other chemokine receptor bearing cells (e.g. neutrophils and monocytes) from the bloodstream to the site of injury. The hallmark function of chemokines is the induction of chemotaxis in the various immune cells. Chemokine activity is controlled by G-protein coupled receptors, of which 18 different types have been identified. So far 47 different chemokines have been discovered, thus there is likely some redundancy in receptor binding (Borish & Steinke, 2003). A s a group, chemokines are 20-50% homologous, and their differences are largely due to variable positions of cysteine residues. Most chemokines are considered proinflammatory, although again more complex roles (e.g. in adaptive immunity and lymphocyte development) are constantly being discovered. Different tissues bear diverse numbers of chemokine receptors from 3000 per cell to 50,000 per cell in some white blood cells (Borish & Steinke, 2003). Both cytokines and chemokines are released by macrophages, which phagocytoze pathogens by surface receptor recognition, and are also released by the host airway epithelial cells themselves (Borish & Steinke, 2003). These mediators initiate the process of inflammation. Cytokine and chemokine mediators are  11  often grouped according to their functions. Distinct categories of cytokines include: lymphokines, tumour necrosis factors (TNFs), and interferons, while chemokines are often divided into: C C chemokines and C X C chemokines, based on their tertiary protein structure (Janeway, 2005). Those mediators thought to directly act on leukocytes (chemokines or cytokines) are termed interleukins (ILs), of which 33 different proteins have been identified (Janeway, 2005). It is important to note however, that the groupings and nomenclatures of the various cytokines and chemokines are not necessarily accurate due to the pleiotropic nature of these proteins. IL-6 is a cytokine which has many different functions. In general, IL-6 is released from cells in response to stress and injury (Janeway et al, 2005). Excreted IL-6 stimulates neighbouring cells to release more IL-6 themselves helping to activate white blood cells such as T lymphocytes, and Ig production of B lymphocytes (Borish & Steinke, 2003). The primary source of IL-6 is from mononuclear phagocytic cells; however, IL-6 is also secreted from: epithelial cells, endothelial cells, B and T lymphocytes, fibroblasts, keratinocytes, hepatinocytes, and bone marrow cells (Akira et al., 1993; Borish & Steinke, 2003). Most of the effects of IL-6 are considered pro-inflammatory, although in some tissues IL-6 also takes on anti-inflammatory roles by decreasing the secretion of other proinflammatory cytokines such as IL-1 and T N F (Borish & Steinke, 2003). Scientists have shown that IL-6 is an important mediator of inflammation in the respiratory tract and plays a key role in IgA antibody production (Fraenkel et al., 1995; Gwaltney & Ruckert, 1997; Gwaltney et al., 1966). IL-8 (systemic name C X C L 8 ) is a chemokine which is also secreted from various cells (mononuclear phagocytes, endothelial and epithelial cells) in response to stress and injury (Janeway, 2005). It has a multitude of functions, the most notable being the chemotactic attraction of neutrophils and adherence to endothelial cells. T w o receptors for IL-8 have been identified: C X C R 1 and C X C R 2 (Borish & Steinke, 2003). Dysregulations in IL-8 levels have been implicated in various airway diseases such as asthma and cystic fibrosis (Damme, 1994). Its secretion is usually triggered by the presence of other cytokines such as IL-1 and T N F , and also by viruses. R V infection can directly stimulate the release of many cytokines and chemokines, including IL-6 and IL-8, from various pulmonary cells both in vitro and in vivo ( K i m et al.,  12  2000; Papadopoulos et al., 2001; Terajima et al., 1997; Zhu et al., 1996 and 1997). Zhu et al. (1996) showed that IL-6 and IL-8 were released from A549 cells upon stimulation with R V 1 4 and R V 1 A . I L levels began to increases within 4-8 hours post-infection and peaked at 24 hrs for both R V serotypes. K i m et al. (2000) used B E A S - 2 B cells infected with R V 1 6 and found that maximum IL-6 and IL-8 protein release occurred at 4-6 hours post-infection and that the maximal m R N A signal for both ILs was detected within 1 hour of infection. Lopez-Souza et al. (2004) infected fully differentiated primary nasal cultures with R V 1 6 and found increases in both IL-6 and IL-8 over 50 hours. However, other studies report conflicting findings in the pattern of I L release from infected cells. For example, Johnston et al. (1998) infected A549 cell lines with R V 9 and found that IL-8 secretion continued to increase over time for the entire 120 hours in which experimental samples were taken. They also found that m R N A for the ILs increased within and hour, but peaked at 3-4 hours post-infection and had disappeared by 96 hours. The cause of these discrepancies is not clear but may be the result of: method used, R V serotype, cell type, cell age, or other factors. For example, the levels of cytokines excreted by cells generally decreases with passage number (Sanders et al., 1998). A l s o , R V serotypes have similar but not identical properties, and furthermore varying amounts of viral titers and different infection protocols were used in the various experiments. The mechanism of R V induced cytokine and chemokine secretion is still largely unknown. Both major and minor type R V s have been shown to induce cytokine and chemokine responses despite their differential use of receptors. There is evidence of virus induced inflammatory mediator secretion through nuclear factor kappa-B ( N F K B ) activation (Zhu et al., 1996; Zhu et al., 1997). The N F K B transcription factor family [ N F K B I (pl05/p50), N F K B 2 ( 100/p52), R e l A (p65), R e l B , and c-Rel] is known to mediate P  responses to stress and injury (Caamano & Hunter, 2002). Under normal conditions these transcription factors are bound to inhibitory IkB proteins in the cytoplasm. In response to various stressor stimuli they dissociate from their inhibitors, enter the nucleus and regulate transcription of a variety of genes. Zhu et al. (1996) found evidence of increased transcription of the IL-6 gene upon R V infection which was blocked 90-95% by mutation of the N F K B binding site in the IL-6 promoter region. Furthermore, they demonstrated that R V infection selectively induced N F K B binding in lung cells mediated by p65 and p50.  13  Similar evidence was also found for IL-8 (Zhu et al., 1997). Other studies have found that the IL-8 gene contains binding sites for several transcription factors including: activating protein-1, activating protein-2, hepatic nuclear factor-1, interferon regulatory factor-1, glucocorticoid response element, N F - I L - 6 and N F K B (Oliveira et al., 1994). Clearly cytokine and chemokine transcription is more complicated than simple N F K B binding as inflammatory mediator transcription is affected by various other promoter regions and associated transcription factors are known to interact in complex ways. For example, Sharma et al. (2006) showed increased nuclear content of more than 30 transcription factors, including N F K B , upon R V infection of airway epithelial cells.  Ultraviolet (UV) Inactivation of Rhinovirus The specific R V trigger for inciting the host inflammatory response is unknown. Traditionally it was thought that virus replication levels were responsible for illness since such a pathophysiology is characteristic of many other viral infections (e.g. human immunodeficiency virus) where systemic viral titers are proportional to disease severity (Clark & Shaw, 1993). However, considering the low levels of R V replication in the airway tissues it is reasonable to suggest that this is not the case for R V . Furthermore, it is unknown whether viral entry or replication is even necessary to induce R V symptoms and illness, or i f a mere interaction of R V with its cellular receptor or some other host recognition site may be sufficient to provoke an inflammatory cascade. One method of investigating this potential trigger is by using U V inactivated R V for infection of tissues or cells. M a n y D N A and R N A viruses, including R V , can be inactivated by U V irradiation at a wavelength of 260nm ( U V C ) (Hughes et al., 1979). The first site of R V inactivation is the viral nucleic acid. Hughes et al. (1979) found that this site may be destroyed in less than 10 seconds in dilute R V 1 7 and R V 4 0 preparations, and up to 90 seconds for more concentrated stocks. However, R V antigenic specificities were retained for much longer; 7 minutes of U V C exposure resulted in a less than ten-fold decrease in the ability to evoke neutralizing antibodies in guinea pigs, and R V s exposed to U V C for over 13 minutes (the maximum time experimentally considered) were still capable of inducing antibodies. Thus theoretically, i f R V is exposed to U V C under optimal conditions it should be possible to create genetically damaged but intact capsids. If this inactivated virus is capable of  14  inducing an inflammatory response in cells, then the existence of a non-replicative trigger for R V illness can be substantiated. Some evidence of such a phenomenon exists; Johnston et al. (1998) found that 30 minute U V irradiation of R V 9 completely halted viral replication in A 5 4 9 cells, but only reduced IL-8 secretion and m R N A content by about half. However, Griego et al. (2000) observed that B E A S - 2 B cells challenged with U V inactivated R V 3 9 (20 minute irradiation) only produced slightly elevated levels of IL-6 and IL-8 relative to control. Hence, identifying and defining the trigger for RV-associated illness is a crucial component of understanding R V infection and paramount in the development of appropriate therapeutics.  Upper Versus Lower Respiratory Tract Infections R V infections have traditionally been thought of as upper respiratory tract infections. However, the discovery that R V s may be the cause of exacerbations of pulmonary diseases such as asthma and C O P D , which largely involve the lower airways, has lead researchers to the likelihood of lower airway R V infection. Although lower airway cells have been shown to possess R V receptors, such as I C A M 1, it is not clear whether their receptors are as vulnerable to R V infection, or whether R V virions would routinely reach the receptors residing deep in the airways (Papi, 2002). The greatest debate in terms of lower airway R V infection has been on the subject of temperature. It has been widely accepted that R V replicates and infects optimally at upper airway temperatures, which are typically from 33°C to 35°C (Hayden, 2004). A s such, it has been assumed that R V infections were typically confined to these upper regions and not the lower airways which experienced temperatures closer to the core body temperature of 37°C. However, evidence has suggested that R V viral replication can and does occur in the lower respiratory tracts, and that perhaps temperature is not as limiting as once thought (Papadopolous et al., 1999 and 2000). A s previously mentioned, the upper respiratory temperature has traditionally been experimentally set at 33-35°C, while the lower airway temperature is experimentally set at 37°C. Airstream monitoring of airway temperatures by thermistors from the trachea to sub-segmental bronchi showed inspiratory quiet breathing temperatures of 33.2°C in the upper airways to 35.5°C in the lower passages, while  15  expiratory temperatures were 32.9°C and 36.3°C respectively (McFadden et al., 1985). Furthermore, when test subjects breathed very cold air (-18.6°C) temperatures in the airways declined by 3-4°C, and during increased ventilation at normal ambient temperature lower airway temperatures were very similar to those of the upper airways (McFadden et al., 1985). Thus, due to the constant fluctuation in airway temperature in response to ventilation and ambient temperature, the designation of an upper and lower airway temperature is perhaps quite arbitrary. Furthermore, it has been demonstrated that R V can replicate efficiently at both 33°C and 37°C. Papadopoulos et al. (1999) demonstrated in H e L a cells that of 8 wild-type R V s , 4 replicated just as well at 37°C, and in fact one serotype replicated more efficiently at 37°C. Recently evidence, both in vivo and in vitro, has emerged showing the replication of R V in the lower airways. Mosser et al. (2004) experimentally infected volunteers with R V and obtained samples from the upper airways (nasal lavage), sputum, and from bronchoalveolar lavage. They detected R V in all of the upper respiratory tract samples, all sputum samples, and 5 of 19 bronchoalveolar samples 4 days post-infection. Immunohistochemistry of the lower airway samples showed patches of infected cells, which were similar to the infection pattern seen in the upper respiratory tract. It is possible that the lower incidence of R V infection in the bronchoalveolar patches was more the result of R V inocula not reaching the lower airways than the vulnerability of the cells themselves. In vitro, alveolar A549 cells inoculated with R V at 37°C and incubated at 35°C produced significant increases in inflammatory cytokines compared to control, suggesting the successful viral infection of lower airway cells (Zhu et al., 1996). The inflammatory response, and subsequent release of cytokines, is temperature sensitive. For example, increases in cytokines can be triggered in response to both hypothermia and hyperthermia. Fairchild et al. (2004) showed that monocytic T H P - 1 cells increase secretion of IL-1 in response to moderate hypothermia (32°C). Bouchama et al. (2005) demonstrated increased plasma IL-6 concentrations after moderate heatstroke temperatures of 42.5°C. Clearly inflammatory cytokine and chemokine secretion from R V infected airway epithelial cells plays a dominant role in the presentation of cold symptoms. Thus  16  comprehending the patterns of inflammatory mediator secretion and the mechanisms by which they are secreted are crucial in understanding the pathology of the common cold.  Rhinovirus Drugs The effective development of preventatives and treatments for R V infection has proved itself a formidable task. Vaccines are generally regarded as impractical because over 100 serotypes of the virus persist with limited cross-neutralization capabilities amongst them. M a n y different approaches to R V therapy have been investigated, all with limited benefits. From a preventative perspective, a soluble competitive I C A M - 1 receptor spray, Tremacamra, has been formulated (Turner et al., 1999), and shows some promise in warding off R V infection, but only when administered just prior to or during inoculation with a major group R V serotype. Specific picornavirus anti-viral agents, which typically disrupt viral replication or attachment to cell membranes, have recently entered clinical trials. Pleconaril is a capsid-binding agent which may interfere with viral entry across the cell membrane. It has been shown to delay viral shedding in enterovirus infection, and to inhibit cell attachment during R V infection in cultured cells (Zhang et al., 2004). The United States Institutes of Health are currently recruiting volunteers for phase II clinical trials evaluating Pleconaril as a treatment for natural colds and asthma exacerbations (United States Food and Drug Administration, [http://www.clinicaltrials.gov/ct/gui/show/NCT00394914]), and Pleconaril is the first antiR V drug to be evaluated by the United States Food and Drug Administration. Another antiR V drug, Ruprintrivir (formerly AG7088), is a 3C protease enzyme inhibitor which prevents the cleavage of a newly synthesized R V polyprotein into its functional subunits. Its antiviral effects have been demonstrated in vitro by Zalman et al. (2000). In clinical trials with experimental R V infection, Ruprintrivir intranasal prophylaxis decreased: proportion of subjects with positive cultures, viral titers, and severity of illness, but had no effect on frequency of colds (Hayden et al., 2003). Bearing in mind the mounting evidence that R V associated illness has little to do with viral replication and is largely associated to host secretion of inflammatory mediators, the development of anti-virals may be futile. More recently, researchers have become interested in immune-modulating compounds which may be able to mitigate RV-related symptoms through the host response.  17  Investigation into the use of anti-histamines and corticosteroids has resulted in inconclusive results so far (Doull et al., 1997; Gaffey et al., 1988; Muether & Gwaltney, 2001). These compounds have shown the ability to mitigate secretion of inflammatory mediators (e.g. IL-8) in some cases, but far more research must be conducted. A limited number of chemokine receptor antagonists have been developed but they have not yet been evaluated in an R V infection context (Akahori et al., 2006; Purandare et al., 2006; Tsutsumi et al., 2006). Two natural treatments associated with R V infection include the use of zinc and the natural herb Echinacea. Zinc has been shown to have an anti-viral effect by inhibiting protease 3C in vitro, but clinical trials have produced inconclusive results (Eby et al., 1984; Macknin et al., 1998; Mossad et al., 1996; Turner & Cetnarowski, 2000). Echinacea is a popular natural herb extract (discussed in detail below) with demonstrated immunemodulating activities which may potentially mitigate RV-related illness by directly affecting host inflammatory mediator secretion.  Echinacea Recently in Western culture there has been marked increase in interest in natural and alternative medicines. This interest may in some cases be due to the perceived shortcomings of traditional Western medicine, or perhaps as explorations into complementary therapies. In either case, it seems that these various therapies are becoming more deeply embedded into our culture. For example, most American medical schools, including Harvard and Johns Hopkins, now offer courses in alternative medicine (Harvard Medical School, [http://www.hms.harvard.edu/news/releases/0700compmed.html];  Johns Hopkins  School of Medicine, [http://www.hopkinsmedicine.org/CAM/]). According to a survey published in the Journal of the American Medical Association there was a 47% increase per household of visits to alternative practitioners from 1990-1997 (Eisenberg et al., 1998). This new interest has resulted in a boom in the alternative medicine industry which was estimated at 21.2 billion dollars in 1997 (Eisenberg et al., 1998). One facet of this boom has been a surge in the use of natural medicines such as herbs and dietary supplements. A n herbal extract that has gained widespread popularity for the potential treatment and/or prevention of upper respiratory tract infections is Echinacea.  18  Echinacea sales in the United States are estimated at 300 million dollars annually (Barrett, 2003; Brevoort, 1998). Echinacea is a natural extract derived from one or a combination from 3 species from the genus Echinacea: Echinacea purpurea (common name: purple coneflower), Echinacea angustifolia, and Echinacea pallida. Echinacea plants are perennial prairie wildflowers native to North America and were first used by Native Americans to treat a variety of infections and illnesses (Barrett, 2003). Extracts can be made from various combinations of species and plant parts (including roots, leaves, petals and seeds). Extractions are performed with diverse solvents, most commonly ethanol or water. Echinacea supplements are sold in various forms such as: capsules, pills, tinctures, lozenges, and teas which are widely available in supermarkets and pharmacies. Recently, there has been much controversy surrounding Echinacea and its potential health benefits, with numerous contradictory studies being published (Goel et al., 2004; Sperber et al., 2004; Turner et al., 2005; Turner et al., 2000). The challenge that Echinacea faces seems to be two-fold. Firstly, the lack of regulation for this industry puts into question the quality of the extracts available on the market. For example, when Gilroy et al. (2003) examined 59 commercial Echinacea preparations they found huge variations in extract quality and labeling. The daily recommended therapeutic dose by the German Commission E , where Echinacea is approved for the treatment of upper-respiratory tract infections, is 900mg/day. Commercial extracts ranged in recommended doses from 451600mg between brands. The quality of the Echinacea extracts itself was also hugely variable. 10% of the extracts had no detectable levels of Echinacea at all, 52% were consistent with their label, while 39% either contained more or less Echinacea than indicated. A l s o 20% of the products did not have any expiration dates, and there is evidence that many of the active compounds undergo enzymatic degradation, especially in alcoholic extracts (Wolkart, 2004). Hence, improper quality control, dosage, and labeling are key problems in the elucidation of Echinacea's health benefits. The second challenge that Echinacea faces is the demonstration of its health benefits in vitro and especially in vivo using properly standardized and characterized extracts. A s mentioned previously, Echinacea extracts are used as preventions and/or treatments for upper-respiratory tract infections. Most cold cases are caused by infection by the R V s and  19  so the Echinacea-RV model is the most commonly studied (Goel et al., 2005; Koenig & Roehr, 2006; Sharma et al., 2006; Turner et al., 2000). The mechanism of Echinacea's effects on R V infected cells is largely unknown. It is thought that the extract may mediate the immune response in such a way that prevents or mitigates RV-associated symptoms. Confusing claims are often made about Echinacea, referring to it as "immune-stimulatory", "immune-supportive", or "immune-modulatory". None o f these claims are necessarily untrue; however, one must keep in mind that the immune response is a complex network with no clear "up" or "down". Echinacea is also sometimes called an "antiviral"; this claim is misleading because although some virucidal effects have been demonstrated in viruses such as herpes simplex virus-1 (Binns et al., 2002), there is no evidence that Echinacea affects viral attachment or replication in any way. Regardless, any potential anti-viral activity may be completely irrelevant, considering the low levels o f R V replication during airway infection. M a n y biologically active compounds have been identified in Echinacea extracts. The major compounds are grouped as: polysaccharides, alkamides and caffeic acid derivatives. Such compounds are used as markers for standardization which typically include: cichoric acid, 6-O-caffeoylechinacoside, echinacoside, verbascoside, cynarine and chlorogenic acid and 6 defined alkylamides (Sloley et al., 2001). Preparations from different plant species and plant parts often have different profiles of these constituents. For example, Echinacea purpurea root extracts are typically rich in alkamides while Echinacea pallida aerial parts are richest in cichoric acid (Binns et al., 2002). Further research is needed to investigate the specific effects of the above compounds both individually and in combination (as found in natural crude extracts), as evidence exists that constituents may act in a synergistic fashion (Dalby-Brown et al., 2005). The actions of these compounds have been demonstrated in vitro. Absent o f virus, Echinacea extracts tend to stimulate and activate cells such as macrophages (Burger et al., 1997), monocytes, natural killer cells (Gan et al., 2003), and to cause immune and airway epithelial cells to secrete a variety of cytokines and chemokines like IL-6, IL-8, and T N F alpha (Hwang et al., 2004). In combination with R V infection these effects may be more complex. For example, Sharma et al. (2006) showed increased cytokine and chemokine levels with Echinacea  20  stimulation, but decreased levels of mediator release in R V infected samples treated with Echinacea than those infected with R V alone. This pattern held true for over 20 of the cytokines and chemokines tested (including IL-6 and IL-8). Although the specific mechanism of Echinacea is still unknown, it has been suggested that Echinacea extracts may affect the cell secretion of cytokines and chemokines through a modulation of the N F K B pathway (Sharma et al., 2006). However, considering the diverse chemical profiles of Echinacea extracts and the unique trends observed when Echinacea interacts with R V infected cells it seems unlikely that one simple mechanism exists. Sharma et al. (2006) showed that Echinacea alone increased the nuclear content of over 30 transcription factors (including  NFKB)  in airway epithelial cells and in R V infected cells treated with Echinacea  those transcription factors were significantly down-regulated nearing control levels. Other research has found that the alkylamides in Echinacea bind the cannabinoid type 2 receptor, and that a subsequent up-regulation TNF-alpha is mediated through cyclic adenosine monophosphate, p38/mitogen activated protein kinase and J N K signaling, as well as  NFKB  and activating transcription factor-2/cAMP responsive element binding protein-1 activation (Gertsch et al., 2004; Raduner et al., 2006; Woelkart et al., 2005). H o w the effects of Echinacea may translate into improved health remains unclear. It has been postulated that the stimulatory effects may enhance a depressed immune system bringing it back to balance (cited in Barrett, 2003). But it seems more likely that an association between R V and Echinacea down-regulates the inflammatory mediators resulting in fewer symptoms of infection. In vivo, Echinacea extracts have been found to increase white blood cells in mice, and Echinacea treatment resulted in faster recovery of normal cell counts following radiation therapy (Mishima et al., 2004). Furthermore another study found that 74% of mice fed Echinacea from birth survived to 13 months of age, while only 46% of control mice remained alive at 13 months (Brousseau & Miller, 2005). Echinacea administration to aging mice has resulted in the synthesis of natural killer cells de novo in the bone marrow (Currier & M i l l e r , 2000). In humans, administration of Echinacea has also been shown to: elevate white blood cell counts, activate immune cells, increase heat-shock protein expression, and prevent free radical damage of red blood cells (Agnew et al., 2005; Brush et al., 2006).  21  Clinical studies investigating the potential benefits of Echinacea extracts in treating and preventing R V infections have shown indeterminate results. Some clinical trials have found no health benefits in Echinacea treatment of R V infection (Koenig & Roehr, 2006; Turner et al., 2000), while others have found significant positive effects (Goel et al., 2005). The contradictory findings may be due to many of the issues discussed above (including extract quality and dosage), and also affected by timing of extract administration. It is unclear whether Echinacea is most effective when administered prior to, during, or after R V challenge, and time of administration varies from study to study. A l s o , i f Echinacea does not act on the virus itself, then measuring viral titers as a means of assessing effectiveness, as i f often done, may be completely inappropriate. Finally, the use of patient symptom scoring which is entirely subjective may lead to inconclusive results, especially in smaller studies. In vivo, researchers must also consider whether the biologically active compounds in Echinacea reach their target tissues. Very few studies have been conducted to assess the bioavailability of consumed Echinacea extracts. Matthias et al. (2004) found evidence that alkylamides cross gut cell monolayers quite readily; however, the caffeic acid derivatives in Echinacea diffused poorly across cultured gut cells. Other studies found evidence of bioactive alkylamides in the blood of test subjects after both consuming Echinacea tablets and after the oral administration of 60% ethanolic tinctures (Dietz et al., 2001; Woelkart et al., 2005). However, Matthias et al. (2005) found no evidence of caffeic acid conjugates in any blood samples following Echinacea tablet ingestion. There is also evidence that alkylamides are oxidized by cytochrome P450 enzymes in the liver generating novel metabolites which may have divergent effects (Cech, 2006). On the other hand, the blood bioavailability of Echinacea may not be relevant considering this extract may be most beneficial by direct application to affected airway epithelial tissues, such as in the case of tinctures, sprays, and teas. Consequently, further studies should investigate Echinacea's bioavailability, as well as research the most effective mode of administration for this extract.  22  RATIONALE AND OBJECTIVES FOR THESIS Although R V s have been studied for many years, relatively little is known about their mechanism of infection and the central role of the host immune response is still a relatively novel discovery. Furthermore, R V treatments and preventions remain in their primitive stages and no specific R V chemotherapy exists. The mounting evidence suggesting low levels of viral replication and induction of the host immune response requires further support and investigation, as many questions have not yet been fully explained. For example, the patterns of viral replication and cell inflammatory mediator secretion over the course of infection have not been adequately documented. Furthermore, such studies have not compared the effects of R V infection in different airway epithelial cell models, or potential differences in infection between R V serotypes. It is also unknown what the specific viral trigger for R V illness may be. Considering the low levels of viral replication it is feasible that some other viral trigger (other than R V replication) may be responsible for stimulating some or all of the host inflammatory response. For example, i f the inflammatory response can be stimulated by a mere virusreceptor or virus-cell interaction, then it is possible that viral entry and replication are not even necessary to provoke illness. The use of U V irradiated non-replicative R V has generated limited data to support this idea; however, more studies are necessary (Johnston et al., 1998). Finally, the lack of therapy to treat R V infection has lead scientists to explore many medicinal avenues. A growing enthusiasm for natural therapies has sparked interest in the potential benefits of using Echinacea to prevent and/or treat R V infections. Immunemodulating Echinacea components have been identified; however, the effects of such extracts directly on the host airway epithelial cells have only been investigated by our laboratory. The combined effects of Echinacea in R V infected airway epithelial cells published by our research group (Sharma et al., 2006) suggest a more complex interaction between Echinacea and R V infection which must be further investigated.  23  G e n e r a l Objectives:  C h a p t e r 2 (In Vitro C h a r a c t e r i z a t i o n o f R h i n o v i r u s Infection i n A i r w a y E p i t h e l i a l C e l l s G r o w t h C u r v e s ) : T o characterize R V i n f e c t i o n in vitro i n terms o f v i r a l r e p l i c a t i o n , v i r a l R N A , and c e l l secretion o f p r o - i n f l a m m a t o r y c y t o k i n e s / c h e m o k i n e s o v e r the t y p i c a l course o f i n f e c t i o n u s i n g t w o different r e c e p t o r - u t i l i z i n g R V serotypes i n t w o distinct c u l t u r e d human airway epithelial cell models.  A l t h o u g h in vitro m o d e l s f o r R V i n f e c t i o n m a y not m i m i c in vivo results, they are c r i t i c a l to o u r understanding o f R V i n f e c t i o n because they offer h i g h l y c o n t r o l l e d and standardized c o n d i t i o n s under w h i c h R V i n f e c t i o n m a y be investigated, w i t h f e w e r variables than i n in vivo m o d e l s . A c c o r d i n g l y , m o r e subtle a n d m e c h a n i s t i c differences m a y be discernable. F r o m a l o g i s t i c p o i n t o f v i e w , in vitro experiments are m o r e approachable, less dangerous, and less c o s t l y than their in vivo counterparts, and thus p l a y a c r i t i c a l role i n v i r a l research. I chose to study R V i n f e c t i o n i n t w o different i m m o r t a l i z e d h u m a n a i r w a y e p i t h e l i a l c e l l m o d e l s : a b r o n c h i a l e p i t h e l i a l c e l l m o d e l ( B E A S - 2 B ) , and a type II a l v e o l a r c e l l m o d e l ( A 5 4 9 ) . I selected these m o d e l s because they are well-characterized a n d w i d e l y used i n s i m i l a r R V experiments. A l t h o u g h both c e l l lines are d e r i v e d f r o m the m i d to l o w e r r e g i o n o f the a i r w a y s the B E A S - 2 B cells represent cells higher i n the respiratory tract, w h i l e the alveolar cells are the l o w e s t cells o f the respiratory e p i t h e l i u m . In the a b o v e c e l l m o d e l s I investigated the effects o f t w o different receptor-utilizing R V serotypes: R V 1 4 (major group) and R V 1 A ( m i n o r group) i n order to observe the extent o f differences p o s s i b l e between R V serotypes. In order to address m y objectives and characterize R V i n f e c t i o n , I chose to measure v i r a l r e p l i c a t i o n , v i r a l R N A levels (for R V 1 4 ) , and c e l l IL-6 a n d IL-8 secretion at d a i l y t i m e intervals o v e r the course o f a t y p i c a l i n f e c t i o n (about 7 days), a n d termed these experiments " G r o w t h C u r v e s " . V i r a l r e p l i c a t i o n w a s assessed b y plaque assay, w h i c h detects the amount o f infectious ( f u l l y f o r m e d r e p l i c a t i n g units) virus present i n a g i v e n s a m p l e , and thus increases i n values f r o m controls indicate v i r a l r e p l i c a t i o n . T o assess whether a i r w a y e p i t h e l i a l cells a l l o w e d o n l y l o w levels o f R V r e p l i c a t i o n , v i r a l titers were c o m p a r e d to those o b t a i n e d i n i d e n t i c a l  24  experiments i n the p e r m i s s i v e H I e p i t h e l i a l c e l l l i n e . H I cells are c o n s i d e r e d p e r m i s s i v e because they a l l o w for the highest R V titers o f any k n o w n cells f o l l o w i n g R V i n f e c t i o n ( A r r u d a et a l . , 1996). V i r a l R N A levels were assessed i n b o t h c e l l m o d e l s f o r R V 1 4 o n l y , as the entire R V 1 A g e n o m e sequence is not f u l l y sequenced and p r i m e r design is p r o b l e m a t i c . V i r a l R N A levels are i n d i c a t i v e o f the n u m b e r o f v i r a l genomes present i n the s a m p l e , regardless o f whether those genomes are present i n f u l l y i n f e c t i o u s and r e p l i c a t i n g units. C o n s e q u e n t l y , increases i n v i r a l R N A m a y not m i r r o r plaque assay results as they represent e f f i c i e n c y i n p r o d u c t i o n o f v i r a l R N A ; f u n c t i o n a l o r not. R N A was detected u s i n g quantitative real-time p o l y m e r a s e c h a i n reaction ( q R T - P C R ) w h i c h has been s h o w n to be 10 times m o r e sensitive to detect R V than c o n v e n t i o n a l P C R ( D a g h e r et a l . , 2004). F i n a l l y i n f l a m m a t o r y m e d i a t o r secretion w a s measured u s i n g e n z y m e - l i n k e d i m m u n o s o r b e n t assays ( E L I S A s ) f o r s p e c i f i c c y t o k i n e s and/or c h e m o k i n e s . In the B E A S 2 B m o d e l , IL-6 secretion w a s d e t e r m i n e d , w h i l e i n A 5 4 9 cells IL-8 secretion w a s measured based o n p r e v i o u s laboratory data (unpublished) w h i c h i n d i c a t e d p r o n o u n c e d secretion o f those s p e c i f i c proteins i n the respective c e l l m o d e l s . A n increase i n i n f l a m m a t o r y m e d i a t o r secretion w a s used as an i n d i c a t o r for the s t i m u l a t i o n o f the host i n f l a m m a t o r y response as is routine practice f o r such studies.  Hypotheses:  1)  R V i n f e c t i o n o f a i r w a y e p i t h e l i a l cells w i l l result i n R V r e p l i c a t i o n and s t i m u l a t i o n o f  the host i n f l a m m a t o r y response as represented b y p r o - i n f l a m m a t o r y c y t o k i n e / c h e m o k i n e secretion f r o m c e l l s . 2)  R V infected a i r w a y e p i t h e l i a l cells w i l l not e x h i b i t C P E because R V i n f e c t i o n results i n  little o r n o c e l l death o r c y t o t o x i c i t y . 3)  R V r e p l i c a t i o n and R N A levels i n a i r w a y e p i t h e l i a l cells w i l l be r e l a t i v e l y l o w as  c o m p a r e d to p e r m i s s i v e H I cells (by at least one order o f magnitude). 4)  V i r a l r e p l i c a t i o n , v i r a l R N A , and c e l l p r o - i n f l a m m a t o r y IL-6/IL-8 secretions w i l l  increase post R V i n f e c t i o n and g r a d u a l l y d e c l i n e to c o n t r o l levels b y d a y 7 as the i n f e c t i o n is r e s o l v e d .  25  5)  M a x i m u m R V r e p l i c a t i o n and R N A levels w i l l o c c u r earlier d u r i n g the time-course o f  i n f e c t i o n than peak c y t o k i n e / c h e m o k i n e secretion i n f l a m m a t o r y m e d i a t o r secretion, and c y t o k i n e / c h e m o k i n e l e v e l s w i l l r e m a i n elevated l o n g e r than R V r e p l i c a t i o n and R V R N A . 6)  R V 1 4 and R V 1 A serotypes w i l l p r o d u c e s i m i l a r i n f e c t i o n patterns because they b e l o n g  to the same v i r u s f a m i l y . 7)  A l v e o l a r A 5 4 9 c e l l s w i l l be less susceptible to R V i n f e c t i o n than b r o n c h i a l B E A S - 2 B  c e l l s because they are d e r i v e d f r o m l o w e r regions o f the a i r w a y s .  Chapter 3 (The E f f e c t s U l t r a v i o l e t Inactivated R h i n o v i r u s on A i r w a y E p i t h e l i a l C e l l s ) : T o investigate the potential for a non-replicative v i r a l trigger for i n i t i a t i n g the c e l l u l a r i n f l a m m a t o r y response b y e x p o s i n g b r o n c h i a l e p i t h e l i a l ( B E A S - 2 B ) c e l l s to p a r t i a l l y and f u l l y U V inactivated v i r u s and q u a n t i f y i n g subsequent v i r a l r e p l i c a t i o n , v i r a l R N A and i n f l a m m a t o r y mediator secretion.  These experiments were c o n d u c t e d i n B E A S - 2 B c e l l s f o r both R V 1 4 and R V 1 A . R V stocks were U V - i r r a d i a t e d f o r different lengths o f t i m e and those samples used d u r i n g i n f e c t i o n procedures i n order to observe the effects o f U V treated R V o n r e p l i c a t i o n , v i r a l R N A ( R V 1 4 o n l y ) , and IL-6 secretion i n order to assess w h e t h e r R V w i t h d a m a g e d genetic material c o u l d e l i c i t an i n f l a m m a t o r y response.  Hypotheses:  1)  T h e v i r a l trigger f o r the observed host i n f l a m m a t o r y response is not related to v i r a l  r e p l i c a t i o n , therefore R V s irradiated w i t h U V C s u f f i c i e n t l y to damage g e n o m i c R N A material but not protein structure, w i l l e l i c i t an IL-6 response w h e n used to infect B E A S 2 B cells. 2)  A s i m i l a r IL-6 response f r o m B E A S - 2 B c e l l s w i l l o c c u r f o r both R V 1 4 and R V 1 A  because they b e l o n g to the same virus f a m i l y .  Chapter 4 (The E f f e c t s o f E c h i n a c e a E x t r a c t s o n R h i n o v i r u s Infected and U n i n f e c t e d A i r w a y E p i t h e l i a l C e l l s ) : T o assess the effects o f 2 c h e m i c a l l y distinct E c h i n a c e a extracts  26  o n R V infected and u n i n f e c t e d B E A S - 2 B c e l l s , i n terms o f v i r a l r e p l i c a t i o n and p r o i n f l a m m a t o r y IL-6 secretion, f o r R V 1 4 and R V 1 A .  E x p e r i m e n t s were c o n d u c t e d i n the B E A S - 2 B c e l l m o d e l w i t h both R V serotypes. C e l l s were infected w i t h either R V 1 4 o r R V 1 A and then treated w i t h one o f t w o c h e m i c a l l y distinct E c h i n a c e a extracts ( E l o r E 2 ) i m m e d i a t e l y post-infection. E l w a s an aqueous E c h i n a c e a extract and E 2 an a l c o h o l i c tincture. B o t h extracts' c h e m i c a l p r o f i l e s were p r e v i o u s l y d e t e r m i n e d b y h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y ( B i n n s et a l . , 2 0 0 2 ) . V i r a l r e p l i c a t i o n w a s assessed at s p e c i f i c time intervals for R V infected s a m p l e s , and IL-6 secretion w a s measured i n R V infected and u n i n f e c t e d c e l l s treated w i t h the E c h i n a c e a extracts.  Hypotheses: 1)  Treatment o f R V - i n f e c t e d B E A S - 2 B c e l l s w i t h E c h i n a c e a w i l l not affect v i r a l  r e p l i c a t i o n because E c h i n a c e a has no effect o n the R V r e p l i c a t i o n c y c l e . 2)  Treatment o f u n i n f e c t e d B E A S - 2 B c e l l s w i t h E c h i n a c e a w i l l i n v o k e an i n f l a m m a t o r y  response and result i n increased secretion o f IL-6. 3)  Treatment o f R V - i n f e c t e d B E A S - 2 B cells w i t h E c h i n a c e a w i l l i n h i b i t R V i n d u c e d IL-6  secretion because o f c o m p l e x interactions between E c h i n a c e a and the v i r u s infected c e l l s . 4)  E l and E 2 w i l l affect IL-6 secretion d i f f e r e n t l y because they are d e r i v e d f r o m distinct  extract preparations and have different active constituent p r o f i l e s . 5)  E c h i n a c e a extracts w i l l have the same effects o n R V 1 4 and R V 1 A because the viruses  b e l o n g to the same f a m i l y .  M y research offers c o n t r o l l e d and standardized e x p e r i m e n t s w h i c h w i l l help to elucidate the effects o f R V i n f e c t i o n i n h u m a n a i r w a y e p i t h e l i a l c e l l s and to investigate the i m m u n e - m o d u l a t o r y effects o f E c h i n a c e a extracts. U n d e r s t a n d i n g the steps a n d m e c h a n i s m s o f R V and the roles that natural m e d i c i n e s c o u l d p l a y i n p r e v e n t i n g and/or a l l e v i a t i n g infections and s y m p t o m s m a y help to d i m i n i s h their impact o n both our health and the e c o n o m y .  27  REFERENCES Agnew, L . , S. Guffogg, M . A , R. Lehmann, K . Bone, and K . Watson 2005. Echinacea intake induces an immune response through altered expression of leukocyte hsp70, increased white cell counts and improved erythrocyte antioxidant defenses. Journal of Clinical Pharmacy and Therapeutics. 30:363-369. Akahori, T., H . Kashizuka, T. Nomi, H . Kanehiro, and Y . 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T h e J o u r n a l o f C l i n i c a l Investigation. 9 7 : 4 2 1 - 4 3 0 .  42  Chapter 2: In Vitro Characterization of Rhinovirus Infection in Airway Epithelial Cells (Growth Curves) 1  BACKGROUND R h i n o v i r u s e s ( R V s ) are the l e a d i n g cause o f upper respiratory tract i n f e c t i o n s i n h u m a n s ( A r r u d a et a l . , 1997). F o r people already affected b y other respiratory diseases, s u c h as asthma o r c h r o n i c obstructive p u l m o n a r y disorder ( C O P D ) , R V i n f e c t i o n can lead to dangerous exacerbations o f those c o n d i t i o n s ( B a r d i n , 1 9 9 2 ; F r a e n k e l et a l . , 1 9 9 5 ; G e r n & B u s s e , 1 9 9 9 ; G r u n b e r g & Sterk, 1 9 9 9 ; G r e e n b e r g , 2 0 0 2 ; H a l p e r i n , 1 9 8 5 ; J o h n s t o n , 1 9 9 3 ; Johnston et a l . , 1 9 9 5 ; J o h n s t o n , 2 0 0 5 ; M e s s a g e , 2 0 0 1 ; S e e m u n g a l , 2 0 0 0 ; S e e m u n g a l , 2001). F u r t h e r m o r e , i n the case o f asthma R V i n f e c t i o n s have recently been attributed a causative role i n the p a t h o l o g y o f the disease ( S i n g h et a l . , 2 0 0 6 ) . A s o f yet, n o prevention or cure f o r R V i n f e c t i o n exists and m e d i c a t i o n s o n l y act to alleviate s y m p t o m s . T h e s p e c i f i c m e c h a n i s m o f R V i n f e c t i o n is still l a r g e l y u n k n o w n . R V s generally infect the upper a i r w a y e p i t h e l i a b y direct contact w i t h infected i n d i v i d u a l s , c o n t a m i n a t e d surfaces, o r large particle aerosols (Turner, 2 0 0 1 ) . H o w e v e r , there is g r o w i n g e v i d e n c e that the l o w e r a i r w a y s are also susceptible to R V i n f e c t i o n ( F r a e n k e l et a l . , 1 9 9 5 ; G e r n et a l . , 1997; G e r n et a l . , 2 0 0 0 ; H a y d e n , 2 0 0 4 ; M o s s e r et a l . , 2 0 0 2 ; M o s s e r et a l . , 2 0 0 5 ; N i c h o l s o n et a l . , 1996; P a p a d o p o u l o s et a l . , 2 0 0 0 ; S c h r o t h et a l . , 1999). T r a d i t i o n a l l y , it was also thought that the s y m p t o m s caused b y R V i n f e c t i o n : r u n n y nose, c o u g h , s n e e z i n g , were d i r e c t l y correlated to the levels o f v i r a l r e p l i c a t i o n i n the a i r w a y tissues. H o w e v e r , recent e v i d e n c e shows that R V r e p l i c a t i o n occurs at r e l a t i v e l y l o w levels and m a y not be attributable to the extent o f these s y m p t o m s ( L o p e z - S o u z a et a l . , 2004). F o r e x a m p l e , M o s s e r et al. (2002) s h o w e d that o n l y 5 - 1 0 % o f p r i m a r y a i r w a y e p i t h e l i a l c e l l s w e r e susceptible to R V n o matter what the infectious dose. T h u s , it has been p r o p o s e d that it i s the host i m m u n e response to R V that causes the wide-spread s y m p t o m s o f illness. F o r e x a m p l e , m o u n t i n g e v i d e n c e suggests that R V i n f e c t i o n does not manifest i t s e l f as a w i d e s p r e a d m u c o s a l i n f e c t i o n but as s m a l l l o c a l i z e d f o c i o f infected c e l l s f r o m w h i c h w i d e s p r e a d i n f l a m m a t o r y responses are generated ( A r r u d a et a l . , 1 9 9 5 ; M o s s e r et a l . ,  ' A version of this chapter will be submitted for publication. Machala, A . M . , Harris, R . A . , Brauner, C.J., Hudson, J.B.  43  2002 and 2005). It is this inflammatory response that then causes the cascade of cold symptoms. A t the cellular level, the inflammatory response can be measured by cytokine and chemokine secretion. Cytokines are proteins that are secreted by cells which in turn cause their neighbours to secrete cytokines, thus modulating and attracting various immune cells or mediators (Janeway, 2005). Chemokines are secreted proteins which attract other cells, such as neutrophils and monocytes, from the bloodstream to the site of infection (Janeway, 2005). R V infection of humans can trigger pro-inflammatory cytokine and chemokine secretion from a variety of cells (Gern et al., 2000; Grunberg et al., 1997; Johnston, 1997; Message & Johnston, 2004; K i m et al., 2000; Zhu et al., 1997; Zhu et al., 1996; Subauste et al., 1995; Teran et al., 1997; Turner, 1998). More specifically, airway epithelial cells have demonstrated the ability to secrete various cytokines and chemokines in response to viral infection (Arnold, 1994; Griego et al., 2000; Johnston et al., 1998; Konno et al., 2002; Lopez-Souza et al., 2004; Zhu et al., 1996). This study aimed to elucidate the biology of R V infection in vitro in cultured human respiratory epithelial cells by relating: cytopathic effects (CPE), viral replication, secretion of pro-inflammatory cytokines/chemokines, and viral R N A over the course of infection for two separate R V serotypes, R V 1 4 (major group) and R V 1 A (minor group), using two distinct airway epithelial cell lines. These experiments have been termed "Growth Curves" as they follow the effects of R V infection for the duration of a typical infection and address the hypothesis that R V infection is characterized by low levels of viral replication and a pronounced inflammatory response.  MATERIALS AND METHODS A l l viral, cell culture and molecular work was conducted under sterile conditions in a type II biosafety cabinet. A l l protocols were pre-approved by the U B C biosafety committee in certificate H04-0061 (Appendix A ) .  G r o w t h C u r v e E x p e r i m e n t s (see Appendix B for experimental design): Growth Curves were conducted for the permissive epithelial H I cell lines, and for the airway epithelial models using bronchial B E A S - 2 B , and alveolar A549 cell lines (described below), and two  44  different receptor-utilizing R V serotypes: R V 1 4 a major group i n t e r c e l l u l a r adhesion molecule-1 ( I C A M - 1 ) u t i l i z i n g R V , and R V 1 A a m i n o r group l o w density l i p o p r o t e i n receptor ( L D L R ) u t i l i z i n g R V . C e l l s were infected w i t h R V 1 4 or R V 1 A at a m u l t i p l i c i t y o f i n f e c t i o n ( M O I ) o f 1, c o r r e s p o n d i n g to 1 infectious v i r a l particle per c e l l , and parameters such as C P E , v i r a l r e p l i c a t i o n , i n t e r l e u k i n (IL)-6/IL-8 secretion, and R V 1 4 R N A were measured o v e r the course o f i n f e c t i o n , t y p i c a l l y between days 0-7 ( D 0 - D 7 ) after R V i n o c u l a t i o n . T w o separate G r o w t h C u r v e trials were c o n d u c t e d f o r each c e l l type and each R V serotype, and a d d i t i o n a l l y a tandem A 5 4 9 / B E A S - 2 B G r o w t h C u r v e was carried out b y s i m u l t a n e o u s l y c u l t u r i n g , i n f e c t i n g , and s a m p l i n g those c e l l l i n e s , i n order to a l l o w f o r a better direct c o m p a r i s o n . H I c e l l G r o w t h C u r v e s ( F i g u r e 2.1) were c o n d u c t e d to serve as p o s i t i v e controls for the a i r w a y e p i t h e l i a l m o d e l s . H I cells are e p i t h e l i a l cells k n o w n to be p e r m i s s i v e to R V i n f e c t i o n a l l o w i n g for h i g h levels o f v i r a l r e p l i c a t i o n . B e c a u s e R V i n f e c t i o n o f H I  cells  causes c e l l death and l y s i s (and therefore secreted c y t o k i n e / c h e m o k i n e cannot be d i s t i n g u i s h e d f r o m l y s e d c e l l mediator release) c y t o k i n e / c h e m o k i n e levels were not assayed f o r H I c e l l s . G r o w t h C u r v e samples were c o l l e c t e d o n l y up to D 4 because c o m p l e t e c e l l death had o c c u r r e d b y this time. F o r B E A S - 2 B cells (Figures 2.2 and 2.3): C P E , R V 1 4 v i r a l r e p l i c a t i o n , v i r a l R N A , and IL-6 secretion w e r e assessed f o r i n f e c t i o n s w i t h both R V serotypes. IL-6 was assayed as an i n d i c a t o r y i n f l a m m a t o r y mediator i n the B E A S - 2 B m o d e l because p r e v i o u s laboratory data (not s h o w n ) indicated a g o o d response f o r IL-6 secretion f r o m R V infected B E A S - 2 B c e l l s . F o r the A 5 4 9 c e l l m o d e l (Figures 2.4 and 2.5): C P E , v i r a l r e p l i c a t i o n , v i r a l R N A , and IL-8 secretion were assessed for both R V serotypes. H e r e , IL-8 was chosen as an i n f l a m m a t o r y i n d i c a t o r based on previous data (not shown) w h i c h suggested a p r o n o u n c e d IL-8 response i n A 5 4 9 c e l l s c o m p a r e d to a w e a k IL-6 response. F i n a l l y , f o r the tandem A 5 4 9 / B E A S - 2 B (Figures 2.7-2.9) the same parameters w e r e assessed, i n c l u d i n g both IL-6 and IL-8 secretion and R V 1 4 R N A . Cell Culture: T h e S V 4 0 adenovirus transformed h u m a n b r o n c h i a l e p i t h e l i a l c e l l l i n e ( B E A S - 2 B ) was obtained f r o m the A m e r i c a n T y p e C u l t u r e C o l l e c t i o n ( A T C C ) ( R o c k v i l l e , M D ) and c u l t u r e d i n 7 5 m m flasks i n 5 0 : 5 0 D u l b e c c o ' s M o d i f i e d E a g l e ' s M e d i u m 2  ( D M E M ) and H a m ' s F 1 2 w i t h 1 0 % e n d o t o x i n free fetal b o v i n e serum ( F B S ) . C u l t u r e  45  reagents w e r e obtained f r o m Invitrogen ( V a n c o u v e r , C a n a d a ) . C e l l s were passaged w e e k l y and incubated at 35-37°C w i t h 5 % c a r b o n d i o x i d e i n 9 5 % air. A 5 4 9 c e l l s , a transformed a l v e o l a r e p i t h e l i a l c e l l l i n e , w e r e also c u l t u r e d under s i m i l a r c o n d i t i o n s but i n D M E M  and  5 % F B S . F i n a l l y , R V - s e n s i t i v e H I cells ( f r o m A T C C ) were c u l t u r e d under the same c o n d i t i o n s w i t h D M E M and 5 % F B S . V i r u s e s : B o t h R V 1 4 and R V 1 A were obtained f r o m the A T C C . R V s were propagated b y i n f e c t i n g H I cells g r o w n to c o n f l u e n c e i n 7 5 m m flasks c o n t a i n i n g D M E M , a l l o w i n g f o r 2  f u l l C P E . Cell-free culture f l u i d was harvested w h e n C P E were at a m a x i m u m b y centrifugation at 10,000 x g f o r 2 0 minutes at 4 ° C . T h i s centrifugation c l a r i f i e d the virus suspension f r o m bacteria and c e l l debris. T h e stock v i r u s suspension was a l i q u o t e d into c r y o v i a l s and stored at -80°C f o r e x p e r i m e n t a l use. T i t e r o f v i r a l stock was determined b y v i r a l plaque assay (see b e l o w ) o f s e r i a l l y d i l u t e d stock, and expressed i n plaque f o r m i n g units (pfu) per m L . P f u represent the n u m b e r o f i n f e c t i o u s R V particles present i n a k n o w n v o l u m e o f sample. F o r e x p e r i m e n t s , aliquoted stock v i r u s was r a p i d l y thawed at 37°C and v o r t e x e d p r i o r to use. B e c a u s e such c l a r i f i e d v i r a l stocks contain H I c e l l remnants (e.g. s o l u b l e proteins, organelles) c o n t r o l experiments were c o n d u c t e d to c o n f i r m that any observed changes i n IL-6 and IL-8 secretion were due to v i r u s , and not some other c e l l u l a r c o m p o n e n t present i n the i n o c u l a ( A p p e n d i x C ) .  V i r a l Infections a n d G r o w t h C u r v e s : H I , B E A S - 2 B , o r A 5 4 9 cells were g r o w n until f r e s h l y confluent under standardized c o n d i t i o n s i n sterile 6-well plates. O n c e cells reached c o n f l u e n c e it was assumed that c e l l n u m b e r d i d not change s i g n i f i c a n t l y o v e r the course o f e x p e r i m e n t s f o r either c o n t r o l o f R V i n f e c t e d cultures. T h i s a s s u m p t i o n w a s c o n f i r m e d e x p e r i m e n t a l l y ( A p p e n d i x D ) . C e l l n u m b e r per w e l l (6-well plate) at c o n f l u e n c e was p r e determined for c e l l lines ( A p p e n d i x E ) and used to calculate v i r a l dose. D u r i n g i n f e c t i o n , cells w e r e inoculated w i t h R V 1 4 or R V 1 A at an M O I = l , o r m o c k infected w i t h m e d i u m , and incubated at 35°C for 1 hour. A f t e r i n f e c t i o n , c e l l s were w a s h e d 3 times w i t h l m L o f m e d i u m t o r e m o v e any e x o g e n o u s v i r u s , and i n c u b a t e d at 35°C i n 3 m L o f fresh m e d i u m and 1 % F B S . W a s h e s were assayed to c o n f i r m v i r u s r e m o v a l (not s h o w n ) . S a m p l e s were c o l l e c t e d at the same time d a i l y between D O ( i m m e d i a t e l y post-infection and washes) a n d  46  D 7 . S a m p l i n g c o n s i s t e d o f first r e m o v i n g l m L o f supernatant f r o m appropriate w e l l s , c e n t r i f u g i n g at 1000 x g to r e m o v e c e l l u l a r debris, and f r e e z i n g samples at -20°C f o r future c y t o k i n e / c h e m o k i n e assays. C e l l s f r o m R V infected samples were scraped into the r e m a i n d e r o f the m e d i u m ( 2 m L ) , pipetted into c r y o v i a l s , and stored at -80°C f o r plaque assays. F i n a l l y , f o r quantitative real-time p o l y m e r a s e c h a i n reaction ( q R T - P C R ) designated samples, m e d i u m was r e m o v e d , c e l l m o n o l a y e r s w a s h e d t w i c e w i t h sterile phosphate b u f f e r e d saline, and l m L o f T R I Z O L reagent was added to each w e l l , a l l o w i n g f o r c e l l l y s i s to occur. T h i s suspension was stored i n E p p e n d o r f tubes at -80°C f o r R N A e x t r a c t i o n . C o n t r o l e x p e r i m e n t s were c o n d u c t e d i n order to assess the stability o f the R V s and IL-6/IL8 under e x p e r i m e n t a l c o n d i t i o n s ( A p p e n d i c e s F and G ) .  P l a q u e Assays (See A p p e n d i x H f o r i m a g e ) : V i r a l i n f e c t i v i t y and r e p l i c a t i o n were measured b y plaque assays i n p e r m i s s i v e H I c e l l s . P r e v i o u s l y f r o z e n R V infected c e l l samples f r o m G r o w t h C u r v e s were r a p i d l y f r o z e n and thawed t w i c e at 37°C to rupture cells and release v i r u s . These samples were then s e r i a l l y d i l u t e d and used to infect p e r m i s s i v e H I cells i n duplicate w h e r e 0 . 7 5 m L o f sample was added onto freshly c o n f l u e n t H I c e l l s g r o w n i n 6-well trays, and a l l o w e d incubated f o r 1 hour at 35°C. A f t e r i n f e c t i o n , the i n o c u l a were aspirated and replaced w i t h a 5 0 : 5 0 l i q u i d m i x t u r e o f 2x M E M ( w i t h 5 % F B S ) and 1 % sterile agarose i n d H 2 0 . T h e agarose was a l l o w e d to s o l i d i f y at r o o m temperature, and plates were incubated at 35°C f o r 4 days. A s the v i r u s replicated i n infected c e l l s , l y s i s o c c u r r e d i n the H I c e l l s f o r m i n g r o u n d areas o f c e l l death c a l l e d " p l a q u e s " . O n c e i n c u b a t i o n was c o m p l e t e plates were f i x e d w i t h 3 % f o r m a l d e h y d e i n phosphate b u f f e r e d saline, agarose was r e m o v e d , and H I c e l l s were stained w i t h 1 % crystal violet i n d H 2 0 to reveal the clear unstained plaques. Plaques were counted and reported as p f u / m L (infectious v i r i o n s / m L ) . A n increase i n infectious virus o v e r time (relative to DO) indicated viral replication.  C y t o k i n e s a n d C h e m o k i n e s : IL-6 and IL-8 were assayed i n duplicate f r o m thawed G r o w t h C u r v e supernatant samples a c c o r d i n g to standard p r o t o c o l s p r o v i d e d b y e n z y m e l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) c o m m e r c i a l kits f r o m I m m u n o t o o l s (Friesoythe, G e r m a n y ) . A b s o r b e n c i e s were d e t e r m i n e d b y an E L I S A plate reader (Pasteur D i a g n o s t i c s  47  L P 4 0 0 ) at a 4 5 0 n m w a v e l e n g t h . H i g h l y concentrated samples were d i l u t e d w i t h m e d i u m and re-assayed to f a l l w i t h i n the standard assay range w h i c h was f r o m 0-450 p g / m L f o r b o t h IL-6 and IL-8. S e n s i t i v i t y o f the E L I S A s was 4 p g / m L .  RNA E x t r a c t i o n : R N A was extracted f o l l o w i n g standard T R I Z O L reagent p r o t o c o l and reconstituted i n 5 0 p L o f R N A s e / D N A s e free water and stored at -80°C f o r q R T - P C R . R N A f o r R V 1 4 standard curves was extracted f r o m stock R V 1 4 suspensions u s i n g the Q I A G E N R N e a s y kit (Mississauga, O N ) .  qRT-PCR:  q R T - P C R was c o n d u c t e d f o r R V 1 4 samples. P r i m e r s w e r e designed u s i n g the  k n o w n R V 1 4 genetic sequence f r o m the N a t i o n a l C e n t e r f o r B i o t e c h n o l o g y I n f o r m a t i o n Database and p u r c h a s e d f r o m O p e r o n ( H u n t s v i l l e , A L ) . P r i m e r s w e r e d e s i g n e d w i t h the f o l l o w i n g sequences: f o r w a r d 5' G A C A T G G T G T G A A G A C T C G C 3' and reverse 5' T C T G T G T A G A A A C C T G A G C G C 3 ' creating a 2 3 8 base p a i r product. P r i m e r s were tested b y c o n v e n t i o n a l two-step P C R o f k n o w n R V infected samples, and P C R products c o n f i r m e d b y gel electrophoresis (not s h o w n ) . F o r R V 1 4 R N A samples a one-step q R T - P C R k i t f r o m Q I A G E N ( M i s s i s s a u g a , O N ) was used. T h e P C R m i x t u r e c o n t a i n e d : 2 5 u L M a s t e r M i x ( H o t S t a r T a q D N A P o l y m e r a s e , Q u a n t i T e c t S Y B R G r e e n B u f f e r , S Y B R G r e e n I d y e , 1 . 5 m M M g C 1 2 , 2 0 0 u M each d N T P ) , 2 . 5 u L o f each p r i m e r , 0.5 u L R T m i x ( O m n i s c r i p t and S e n s i s c r i p t R e v e r s e Transcriptases), and l O u L o f the R N A template to a f i n a l v o l u m e o f 5 0 u L . Standard c u r v e R V concentrations were d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y and s e r i a l l y d i l u t e d at k n o w n concentrations. D u p l i c a t e reactions were c a r r i e d out i n an M J R e s e a r c h D N A E n g i n e O p t i c o n C o n t i n u o u s F l u o r e s c e n c e Detector ( O P 0 0 0 5 3 7 ) p r o g r a m m e d to: incubate at 4 2 ° C f o r 5 5 m i n , 94°C f o r 5 m i n , 94°C f o r 30 sec, 55°C f o r 3 0 sec, 72°C f o r l m i n , repeat f o r 3 5 c y c l e s , 72°C f o r l O m i n , m e l t i n g c u r v e f r o m 55°C to 95°C read e v e r y 0.5°C, incubate 72°C f o r l O m i n , and 4 ° C forever. D a t a were a n a l y z e d u s i n g O p t i c o n M o n i t o r analysis software v e r s i o n 1.07.  Statistics: G r o w t h C u r v e infectious virus (replication) data (n=4 f o r each day per trial) were subjected to K r u s k a l - W a l l i s ranks analysis o f variance ( A N O V A ) w i t h day as a  48  factor, f o l l o w e d b y post hoc m u l t i p l e c o m p a r i s o n s D u n n e t ' s tests i n order to c o m p a r e d a i l y data relative to D O c o n t r o l s . Non-parametric testing was e m p l o y e d because data d i d not satisfy parametric assumptions (normality and/or equal variances) and c o u l d not be transformed. A s i g n i f i c a n t increase i n i n f e c t i o u s v i r u s o v e r t i m e (relative to DO) i n d i c a t e d v i r a l r e p l i c a t i o n . E L I S A c y t o k i n e / c h e m o k i n e data (n=3 per d a y f o r each treatment) were a n a l y z e d u s i n g t w o - w a y A N O V A s w i t h day and treatment (control u n i n f e c t e d and R V infected cells) as factors and post-hoc m u l t i p l e c o m p a r i s o n s D u n n e t ' s tests w i t h d a i l y • c o n t r o l treatments as c o n t r o l s . In the case o f B E A S - 2 B / A 5 4 9 t a n d e m G r o w t h C u r v e s , a 2w a y A N O V A was p e r f o r m e d f o r i n f e c t i o u s v i r u s data u s i n g d a y and treatment as factors f o l l o w e d b y a post-hoc D u n n e t ' s test u s i n g D O as a c o n t r o l . F o r IL-6 and IL-8 data a 3-way A N O V A was p e r f o r m e d w i t h : day, treatment, and c e l l type as factors and a post-hoc D u n n e t ' s test w i t h d a i l y c o n t r o l treatments as c o n t r o l s . R N A data (n=2 per c e l l type f o r each day) w e r e a n a l y z e d b y 2-way A N O V A w i t h day and c e l l type as factors and post-hoc T u k e y tests. A l l data w e r e a n a l y z e d u s i n g S i g m a Stat 3.0 software and statistical s i g n i f i c a n c e was set at a = 0 . 0 5 . S i g n i f i c a n t differences c o r r e s p o n d to p<0.05, and h i g h l y s i g n i f i c a n t differences c o r r e s p o n d to p<0.001. R e s u l t s are presented as m e a n ± standard error o f the mean.  RESULTS F o r all G r o w t h C u r v e experiments trials were s i g n i f i c a n t l y different f r o m each other (data c o u l d not be p o o l e d ) .  H I Cells ( F i g u r e 2.1): C P E : F o r H I cells infected w i t h R V 1 4 C P E began to o c c u r on D l , and manifested themselves as r o u n d e d c e l l s w h i c h e v e n t u a l l y b e c a m e detached f r o m the substratum. F u l l C P E and c e l l death was observed f o r a l l samples and u s u a l l y c o m p l e t e b y D 4 . H I cells infected w i t h R V 1 A also s h o w e d C P E resulting i n c o m p l e t e c e l l death b y the D 3 or D 4 . A n e c d o t a l l y , the occurrence o f C P E seemed to o c c u r faster w i t h R V 1 A than R V 1 4 . RV14  R e p l i c a t i o n ( F i g u r e 2.1a): F o r both trials, s i g n i f i c a n t v i r a l r e p l i c a t i o n (relative to  DO) o c c u r r e d o n D l and D 2 . F o r trial 1, m a x i m u m infectious v i r u s was o b s e r v e d on D l (at  49  1.8 x 10 ±2.8 x 1 0 p f u / m L ) , and for trial 2 i n f e c t i o u s virus also peaked s i g n i f i c a n t l y o n 7  6  D l at 4.4 x 1 0 ± 1.1 x 1 0 p f u / m L . 6  RV1A  s  Replication ( F i g u r e 2.1b): A g a i n , f o r both trials, s i g n i f i c a n t R V r e p l i c a t i o n (relative  to DO) was o b s e r v e d on D l and D 2 . P e a k v i r a l titers were measured on D l (3.7 x 1 0 ± 2.4 7  x 1 0 p f u / m L and 1.9 x 10 ± 1.2 x 1 0 p f u / m L respectively). 6  7  6  BEAS-2B Cells (Figures 2.2 and 2.3): U p o n i n f e c t i o n o f B E A S - 2 B c e l l s w i t h R V 1 4 or R V 1 A , no C P E w e r e o b s e r v e d at  CPE:  any point d u r i n g G r o w t h C u r v e s . Replication ( F i g u r e 2.2a): F o r trial 1, no s i g n i f i c a n t r e p l i c a t i o n was observed  RV14  relative to DO. P e a k v i r a l titers o c c u r r e d o n D l (4.4 x 10' ± 1.3 x 10 p f u / m L ) , d e c l i n i n g to 1.7 x 1 0 ± 2.9 x 1 0 p f u / m L b y D 7 . 2  1  In trial 2, s i g n i f i c a n t v i r a l r e p l i c a t i o n (relative to DO) o c c u r r e d on D l at 2.2 x 1 0  4  ±1.2  x 10 pfu/mL. 3  RV14  and IL-6 Secretion (Figures 2.2b and c ) : F o r trial 1 ( F i g u r e 2.2b), no s i g n i f i c a n t  differences were o b s e r v e d between u n i n f e c t e d (control) and R V 1 4 infected c e l l s at any t i m e post-infection. F o r trial 2 ( F i g u r e 2.2c), IL-6 secretion i n R V 1 4 infected c e l l s was h i g h l y s i g n i f i c a n t w h e n c o m p a r e d to u n i n f e c t e d controls o n D 3 , D 5 , and D 7 post-infection. Peak IL-6 concentration was o b s e r v e d o n D 7 at 3 5 1 7 . 9 ± 9 9 3 . 8 , and o n D 3 IL-6 concentration in R V infected c e l l s was n e a r l y 1375 times its c o n t r o l . RV1A  Replication ( F i g u r e 2.3a): F o r trial 1, v i r a l r e p l i c a t i o n was s i g n i f i c a n t l y different  f r o m D O on D 2 and D 3 . Peak v i r a l titers o f 7.3 x 1 0 ± 2.6 x 1 0 p f u / m L w e r e observed on 4  3  D2. In trial 2, s i g n i f i c a n t v i r a l r e p l i c a t i o n o c c u r r e d on D l relative to DO, w h i c h represented the peak R V titer at 2.9 x 10 ± 1.4 x 1 0 p f u / m L . 4  RV1A  3  and IL-6 secretion (Figures 2.3b and c): In t r i a l 1, IL-6 concentration ( F i g u r e  2.3b) was s i g n i f i c a n t l y different between treatments oh D 3 , D 5 , and D 7 . P e a k IL-6 concentration o c c u r r e d o n D 7 at 2 4 3 4 . 3 ± 215.3 p g / m L w h i c h represented an 8-fold increase o v e r its c o n t r o l .  50  F o r trial 2 ( F i g u r e 2.3c), there was a s i g n i f i c a n t d i f f e r e n c e between u n i n f e c t e d and R V 1 A infected c e l l s o n D 3 , D 5 , a n d D 7 . Peak IL-6 concentrations w e r e measured o n D 7 at 4 2 2 4 . 3 ± 5 3 0 . 0 p g / m L , w h i c h was 5 7 0 times c o n t r o l levels.  A549 Cells (Figures 2.4 and 2.5): CPE:  N o C P E or c e l l death was observed at any point, D 0 - D 7 , for A 5 4 9 c e l l s infected  with R V 1 4 o r R V 1 A . RV14  Replication ( F i g u r e 2.4a): F o r both trials, n o s i g n i f i c a n t R V 1 4 r e p l i c a t i o n o c c u r r e d  relative to DO. RV14  and IL-8 Secretion (Figures 2.4b and c ) : In the first trial (Figure 2.4b), no  s i g n i f i c a n t differences were observed i n IL-8 secretion between treatments. F o r trial 2 ( F i g u r e 2.4c), there were statistically s i g n i f i c a n t differences between treatments o n D l , D 2 , D 3 , and D 5 . Peak IL-8 concentration was o b s e r v e d o n D 5 at 302.3 ± 39.5 p g / m L , w h i l e o n D 2 the largest increase relative to c o n t r o l was measured (5-fold). RV1A  Replication ( F i g u r e 2.5a): F o r the first trial, a s i g n i f i c a n t d i f f e r e n c e i n infectious  v i r u s w a s o b s e r v e d o n D l and D 2 relative to DO. Peak v i r a l r e p l i c a t i o n w a s measured o n D l w i t h 3.7 x 1 0 ± 6.9 x 1 0 p f u / m L . s  3  In trial 2, a s i g n i f i c a n t d i f f e r e n c e i n v i r a l r e p l i c a t i o n was also observed on D l relative to DO. Peak R V 1 A titers were measured o n this day at 5.6 x 10 ± 1.3 x 1 0 4  RV1A  4  pfu/mL.  and IL-8 Secretion (Figures 2.5b and c ) : F o r trial 1 (Figure 2.5b), IL-8  concentrations were h i g h l y s i g n i f i c a n t between treatments on D l , D 2 , D 3 , and D 6 . Peak IL-8 concentration was observed o n D 6 at 7 6 9 . 0 ± 57.4 p g / m L ; h o w e v e r , the largest increase i n IL-8 relative to c o n t r o l was measured o n D l (3.5-fold). In trial 2 (Figure 2.5c), there was a h i g h l y s i g n i f i c a n t increase in IL-8 secretion between treatments for D 2 , D 3 , and D 6 . Peak IL-8 concentration was 1864.9 ± 25.3 p g / m L on D 6 , and the largest increase i n IL-8 secretion relative to its c o n t r o l was observed o n D 2 (5.2 times).  Growth Curve RV14 R N A Levels ( F i g u r e 2.6): T h e r e is a statistically s i g n i f i c a n t d i f f e r e n c e i n R N A levels between H I c e l l s and b o t h B E A S - 2 B and A 5 4 9 c e l l s (all days c o m b i n e d ) . H I c e l l s p r o d u c e d i n c r e a s i n g R N A levels to  51  a peak o f 2.8 x 1 0 ±1.4xl0 pg/well at D 3 , and a l l time-points after D O w e r e i n the 1 0 " p g 5  4  3  5  range. F o r B E A S - 2 B c e l l s peak R V 1 4 R N A levels o c c u r r e d o n D 3 m e a s u r i n g 17.7 ± 7.2 pg/well and then decreasing to 0.005 ± 0.001 pg/well b y D 7 . F o r A 5 4 9 c e l l s , peak R V 1 4 R N A was measured o n D O at 1.7 x 10" ± 6.7 x 10" p g / w e l l , and then d e c l i n e d to 2.3 x 1 0 " 6  ± 7.8 x 1 0 "  11  7  10  pg/well o n D l , after w h i c h R V 1 4 R N A was undetectable f o r D 2 , D 3 and D 6 .  W i t h i n the H I c e l l s , R V 1 4 R N A is s i g n i f i c a n t l y h i g h e r o n D 2 relative to D O . F o r B E A S - 2 B and A 5 4 9 c e l l s there w e r e no s i g n i f i c a n t differences observed w i t h i n the respective c e l l types w h e n c o m p a r e d to DO. BEAS-2B/A549 Tandem (Figures 2.7-2.9): C P E : N o C P E or c e l l death was o b s e r v e d i n any o f the samples infected w i t h either R V 1 4 or R V 1 A f o r B E A S - 2 B or A 5 4 9 c e l l s . RV14 Replication ( F i g u r e 2.7a): R e p l i c a t i o n levels f o r B E A S - 2 B c e l l s were s i g n i f i c a n t l y different f r o m A 5 4 9 c e l l s o n D l , D 2 and D 3 . W i t h i n B E A S - 2 B c e l l s there was a s i g n i f i c a n t difference observed o n D l and D 2 relative to DO. Peak v i r a l titer was measured on D l at 1.6 x 10 ± 9.6 x 1 0 p f u / m L . W i t h i n A 5 4 9 c e l l s there were no statistically s i g n i f i c a n t 4  2  differences i n R V 1 4 r e p l i c a t i o n . IL-8 Secretion ( F i g u r e 2.7b): 11-8 concentrations were h i g h l y s i g n i f i c a n t between B E A S 2 B and A 5 4 9 c e l l s (all days c o m b i n e d ) ; h o w e v e r , no s i g n i f i c a n t differences between infected and u n i n f e c t e d c e l l s were observed i n either c e l l l i n e f o r IL-8 secretion. T h e m a x i m u m LL-6 concentration measured was 327.1 ±42.3 p g / m L i n the B E A S - 2 B c e l l s o n D7. IL-6 Secretion ( F i g u r e 2.7c): A g a i n mediator secretion was h i g h l y s i g n i f i c a n t between B E A S - 2 B and A 5 4 9 c e l l s (all days c o m b i n e d ) , but there were no differences observed between treatments. M a x i m u m IL-6 concentration i n B E A S - 2 B c e l l s was measured o n D l at 27.4 ± 2.8 p g / m L . RV1A  Viral Replication (Figure 2.8a): R e p l i c a t i o n levels i n B E A S - 2 B c e l l s were h i g h l y  s i g n i f i c a n t c o m p a r e d to A 5 4 9 c e l l s as observed o n D l and D 2 . W i t h i n B E A S - 2 B c e l l s h i g h l y s i g n i f i c a n t increases in v i r a l r e p l i c a t i o n w e r e measured o n D l and D 2 . Peak v i r a l r e p l i c a t i o n (relative to DO) o c c u r r e d o n D l at 3.4 x 10 ± 3.6 x 1 0 p f u / m L . W i t h i n A 5 4 9 4  3  cells n o s i g n i f i c a n t differences i n R V 1 A r e p l i c a t i o n were o b s e r v e d , a l t h o u g h a t y p i c a l  52  G r o w t h C u r v e trend w a s observed w i t h peak v i r a l titer o f 4.5 x 1 0 ± 1.4 x 1 0 p f u / m L o n 3  2  Dl. IL-8 Secretion ( F i g u r e 2.8b): IL-8 concentrations were s i g n i f i c a n t l y different between B E A S - 2 B a n d A 5 4 9 c e l l s (all days c o m b i n e d ) . W i t h i n the B E A S - 2 B c e l l s those infected w i t h R V were h i g h l y s i g n i f i c a n t f r o m their respective controls f o r D 2 , D 3 , D 5 , and D 7 . P e a k IL-8 concentration w a s measured o n D 5 at 2 2 3 6 . 7 ± 60.4 p g / m L . T h e same trend w a s observed f o r A 5 4 9 c e l l s where there w a s a h i g h l y s i g n i f i c a n t difference between treatments f o r D 2 , D 3 , D 5 , and D 7 a n d m a x i m u m LL-8 concentration w a s measured o n D 3 (1966.7 ± 83.3 p g / m L ) . IL-6 Secretion ( F i g u r e 2.8c): D i f f e r e n c e s between B E A S - 2 B a n d A 5 4 9 c e l l s were h i g h l y s i g n i f i c a n t ( a l l days c o m b i n e d ) . W i t h i n B E A S - 2 B c e l l s , treatments were s i g n i f i c a n t l y different o n D 3 and h i g h l y s i g n i f i c a n t o n D 7 . P e a k LL-6 concentration f o r B E A S - 2 B w a s 2 6 1 5 ± 124.1 p g / m L w h i c h w a s 53 times its c o n t r o l . In A 5 4 9 c e l l s both D 3 and D 7 were h i g h l y s i g n i f i c a n t between treatments. P e a k IL-6 concentration f o r A 5 4 9 c e l l s w a s measured o n D 7 at 1114.7 ± 105.5 p g / m L w h i c h represented the m a x i m u m increase relative to c o n t r o l at 124 times.  B E A S - 2 B / A 5 4 9 T a n d e m R V 1 4 R N A ( F i g u r e 2.9): There w a s a statistically s i g n i f i c a n t difference i n R V 1 4 R N A levels between B E A S - 2 B a n d A 5 4 9 c e l l s as observed o n D l a n d D 2 . F o r B E A S - 2 B peak R N A levels were measured o n D 2 at 11.6 ± 5.8 p g / w e l l , w h i l e peak R V 1 4 R N A i n A 5 4 9 c e l l s was observed o n D O at 4.0 x 10~ ± 9.4 x 10~ p g / w e l l . 2  3  H o w e v e r , w i t h i n c e l l lines n o s i g n i f i c a n t l y differences were observed relative to D O .  DISCUSSION R V i n f e c t i o n o f the a i r w a y e p i t h e l i a l c e l l s ( B E A S - 2 B and A 5 4 9 ) resulted i n s i g n i f i c a n t v i r a l r e p l i c a t i o n a n d s t i m u l a t i o n o f c y t o k i n e / c h e m o k i n e secretion i n the o v e r w h e l m i n g majority o f e x p e r i m e n t s , certainly s u p p o r t i n g the g r o w i n g consensus that R V i n f e c t i o n does p r o v o k e an i n f l a m m a t o r y response i n its host cells. C P E : P r e v i o u s studies o f in vitro c e l l l i n e e x p e r i m e n t s , c u l t u r e d p r i m a r y c e l l s , and b i o p s y d e r i v e d a i r w a y e p i t h e l i a l tissues i n d i c a t e d that n o C P E or c e l l death w a s o b s e r v e d d u r i n g  53  R V i n f e c t i o n ( M o s s e r et a l . , 2 0 0 2 ; G r i e g o et a l 2 0 0 0 , L o p e z - S o u z a et a l , 2 0 0 4 ) . F o r e x a m p l e , J a n g et al. (2005) f o u n d that nasal turbinate m u c o s a infected w i t h R V 1 6 d i d not cause any observable damage to the pseudostratified c o l u m n a r e p i t h e l i u m , basement m e m b r a n e , or c i l i a . M y results also support these f i n d i n g s since no C P E or c e l l death w e r e observed f o r either the B E A S - 2 B or A 5 4 9 c e l l lines at any p o i n t d u r i n g G r o w t h C u r v e e x p e r i m e n t s f o r either R V serotype. In contrast, a l l p e r m i s s i v e H I c e l l s infected w i t h R V e x h i b i t e d C P E b y D l and c o m p l e t e c e l l death b y D 4 (a t y p i c a l l y t i c c y c l e ) . These observations suggest that R V has some u n k n o w n m e c h a n i s m o f c r o s s i n g out the p l a s m a m e m b r a n e w i t h o u t r u p t u r i n g the c e l l , or alternatively, a m i n u t e fraction o f c e l l s l y s e , and these l o w l o c a l i z e d levels o f R V are s u f f i c i e n t to i n d u c e a p r o n o u n c e d i m m u n e response. It m a y be that both the a b o v e m e c h a n i s m s do o c c u r , and act s y n e r g i s t i c a l l y to e l i c i t the observed i n f l a m m a t o r y response. L o w levels of R e p l i c a t i o n C o m p a r e d to H I Cells: There is g r o w i n g consensus that R V replicates at v e r y l o w l e v e l s i n the a i r w a y e p i t h e l i a l tissues. W h e n c o m p a r i n g m y G r o w t h C u r v e data between H I c e l l s and the a i r w a y c e l l m o d e l s this hypothesis c e r t a i n l y seems to be supported. H I c e l l s p r o d u c e d peak R V titers at 3-4 orders o f m a g n i t u d e higher than B E A S - 2 B c e l l s , and this trend was generally m o r e exaggerated f o r the A 5 4 9 c e l l m o d e l . E v e n t a k i n g into account the 1.4-fold greater H I c e l l n u m b e r at c o n f l u e n c e (see A p p e n d i x E ) the levels o f R V r e p l i c a t i o n r e m a i n i m p r e s s i v e . W h a t m a k e s the a i r w a y c e l l s less susceptible to R V i n f e c t i o n than the p e r m i s s i v e c e l l s remains u n k n o w n . It is feasible that the a i r w a y c e l l s a l l o w less R V - r e c e p t o r b i n d i n g and entry than H I c e l l s because o f f e w e r surface I C A M - 1 or L D L R receptors. S i n c e R V receptor e x p r e s s i o n is a h i g h l y d y n a m i c process this hypothesis is d i f f i c u l t to test. There is evidence that R V i n f e c t i o n causes a r a p i d up-regulation i n the surface e x p r e s s i o n o f I C A M - 1 ( G r u n b e r g , 2 0 0 0 ; P a p i & J o h n s t o n , 1999; W h i t e m a n et a l . , 2 0 0 3 ; W i n t h e r et a l . , 2 0 0 2 ) , but perhaps this process is not as e f f i c i e n t i n the a i r w a y c e l l s w h e n c o m p a r e d to p e r m i s s i v e c e l l s . T a k i n g the G r o w t h C u r v e data into c o n s i d e r a t i o n , it seems that H I c e l l s m a y a l l o w f o r increased v i r a l passage across the c e l l m e m b r a n e . D O levels i n H I c e l l s are o n the order o f 1 0 , w h i l e the a i r w a y 4  c e l l s D O values are l o w e r , even b e a r i n g i n m i n d i n i t i a l c e l l numbers and v i r a l dose. F u r t h e r m o r e , regardless o f i n f e c t i o n s u s c e p t i b i l i t y , a i r w a y e p i t h e l i a l c e l l s m a y o n l y a l l o w  54  f o r l i m i t e d R V r e p l i c a t i o n . T h i s c o u l d potentially o c c u r b y an active m e c h a n i s m , o r p a s s i v e l y due to s l o w e r and/or less efficient r e p l i c a t i o n m a c h i n e r y . G r o w t h C u r v e R e p l i c a t i o n T r e n d s : In the vast majority o f the G r o w t h C u r v e r e p l i c a t i o n data a s i g n i f i c a n t l e v e l o f v i r a l r e p l i c a t i o n was observed between D l and D 2 (relative to DO), w i t h the majority o f peak titers b e i n g measured o n D l . F o l l o w i n g the increases i n R V r e p l i c a t i o n titers tended to g r a d u a l l y decrease to starting levels and were not s i g n i f i c a n t l y different f r o m D O after D 2 . H o w these cultured cells manage to resolve R V i n f e c t i o n is s t i l l l a r g e l y a m y s t e r y . In vivo, i n f l a m m a t o r y mediators recruit and activate w h i t e b l o o d cells to e n g u l f pathogens, and a n t i b o d y f o r m a t i o n i s thought to u l t i m a t e l y r i d the b o d y o f i n f e c t i o n (Janeway, 2 0 0 5 ) . H o w e v e r the a i r w a y c e l l m o d e l s also seem to be capable o f m i t i g a t i n g R V i n f e c t i o n w i t h o u t the a i d o f i m m u n e c e l l s . It is p o s s i b l e that the e p i t h e l i a l c e l l is able to r e c o g n i z e v i r a l r e p l i c a t i o n b y some intra-cellular i m m u n e process and down-regulate its g e n o m i c m a c h i n e r y i n response to R V . T h e r e is e m e r g i n g e v i d e n c e o f intra-cellular i m m u n i t y against retroviruses, and furthermore, s o m e eukaryotic cells are capable o f b l o c k i n g p o l y c i s t r o n i c m R N A translation (Fire, 2 0 0 5 ; Z h e n g et a l . , 2 0 0 5 ) . R V i n d u c e d c y t o k i n e / c h e m o k i n e secretion f r o m affected c e l l s m a y also alert n e i g h b o u r i n g c e l l s to decrease s u s c e p t i b i l i t y to R V i n f e c t i o n . T h i s c o u l d potentially be a c c o m p l i s h e d b y affecting the transcriptional cascades needed b y R V . T h e question remains whether R V infects m a n y cells i n the m o n o l a y e r a l l p r o d u c i n g l o w R V titers, or i f o n l y a f e w cells b e c o m e infected each r e p l i c a t i n g R V at h i g h levels. M o s s e r et al. (2005) f o u n d that i n b i o p s y derived tissues o n l y 5 - 1 0 % o f the cells were infected regardless o f v i r a l dose, and h i s t o l o g i c a l l y R V i n f e c t i o n m a n i f e s t e d itself as s m a l l l o c a l i z e d clusters o f infected c e l l s . H o w e v e r , it remains unclear i f v i r a l entry is e v e n necessary to induce an i n f l a m m a t o r y response, arguably a mere R V - r e c e p t o r or c e l l interaction is sufficient to trigger c y t o k i n e / c h e m o k i n e secretion. F o r e x a m p l e , one study f o u n d evidence o f c y t o k i n e / c h e m o k i n e s t i m u l a t i o n b y ultraviolet ( U V ) inactivated R V (Johnston et a l . , 1998). In such a case, R V r e p l i c a t i o n levels and the n u m b e r o f infected cells m a y be c o m p l e t e l y irrelevant c o n s i d e r i n g that R V c o u l d stimulate e p i t h e l i a l cells to secrete i n f l a m m a t o r y c y t o k i n e s / c h e m o k i n e s irrespective o f R V c r o s s i n g the p l a s m a m e m b r a n e . A t the same t i m e , it is clear that R V does infect at least s o m e cells and s i g n i f i c a n t l y replicate i n the a i r w a y e p i t h e l i u m . F o r a m a j o r i t y o f patients presenting w i t h  55  self-diagnosed c o l d s R V can be consistently isolated f r o m a i r w a y lavages and tissues ( A r r u d a et a l . , 1 9 9 7 ; Johnston et a l . , 1 9 9 5 ; v a n G a g e l d o n k - L a f e b e r et a l . , 2 0 0 5 ) . Perhaps R V r e p l i c a t i o n is not the cause o f c o l d s y m p t o m s but p l a y s a c r u c i a l role i n the propagation o f the i n f e c t i o n b y c a u s i n g the release o f n e w v i r a l p r o g e n y onto yet unaffected c e l l s and generating an additive i n f l a m m a t o r y response. Immune Response Hypothesis: B e c a u s e o f the observed l o w levels o f R V r e p l i c a t i o n , there is a g r o w i n g hypothesis that the i l l n e s s associated w i t h R V i n f e c t i o n is initiated b y the v i r u s , but propagated and a m p l i f i e d b y the host i m m u n e system through the secretion o f p r o - i n f l a m m a t o r y c y t o k i n e s and c h e m o k i n e s l i k e IL-6 and IL-8. S u c h a m e c h a n i s m is v e r y different f r o m m a n y viruses where c e l l death caused b y v i r a l r e p l i c a t i o n is the source o f virus p a t h o l o g y . Johnston et al. (1998) f o u n d a p r o l o n g e d release o f IL-8 up to 120 hours i n the p u l m o n a r y e p i t h e l i u m to l o w doses o f R V 9 even though r e p l i c a t i o n p e a k e d at 24 hours. G e r n et al. (2000) also f o u n d increased IL-8 secretion i n response to R V 1 6 in vivo w i t h n o c o r r e l a t i o n to quantities o f v i r a l s h e d d i n g . In e x p e r i m e n t a l c o l d s r h i n o r r h e a s y m p t o m s u s u a l l y peaked o n D 2 w h i l e i n natural c o l d s the m a x i m u m o c c u r r e d o n D 3 , but throat and c o u g h s y m p t o m s p e a k e d closer to D 4 , i n d i c a t i n g i n c r e a s i n g s y m p t o m s w e l l b e y o n d m a x i m u m v i r a l r e p l i c a t i o n ( G w a l t n e y et a l . , 2 0 0 3 ) . In an in vitro context, m y c y t o k i n e / c h e m o k i n e data also support these f i n d i n g s . A s p r e v i o u s l y d e s c r i b e d , peak v i r a l titers were c o n s i s t e n t l y measured between D l and D 2 f o r a l l c e l l m o d e l s w i t h no s i g n i f i c a n t v i r a l r e p l i c a t i o n f r o m D 3 o n w a r d . H o w e v e r , elevated c y t o k i n e / c h e m o k i n e secretions and m a x i m u m increases w h e n c o m p a r e d to respective controls were t y p i c a l l y o b s e r v e d later i n i n f e c t i o n (often D 2 - 7 ) . A l s o c y t o k i n e and c h e m o k i n e secretion r e m a i n e d elevated w e l l b e y o n d detectable R V r e p l i c a t i o n . T h e m e c h a n i s m l e a d i n g f r o m R V interaction/infection w i t h its host c e l l to the secretion o f p r o - i n f l a m m a t o r y c y t o k i n e s / c h e m o k i n e s (such as IL-6 and IL-8) is not w e l l d e s c r i b e d . IL-6 and IL-8 transcription is c o n t r o l l e d b y various transcription factors i n c l u d i n g nuclear factor k a p p a - B ( N F K B ) ( C a a m a n o & H u n t e r , 2 0 0 2 ; O l i v e i r a et a l . , 1994). N F K B is a c y t o s o l i c p r o t e i n , n o r m a l l y b o u n d b y i n h i b i t o r s , w h i c h b e c o m e s u n b o u n d i n response to various stressors and b i n d s its p r o m o t e r sites w i t h i n the nucleus r e s u l t i n g i n the increased transcription o f various c y t o k i n e s / c h e m o k i n e s ( C a a m a n o & H u n t e r , 2 0 0 2 ) . T h e r e is some e v i d e n c e that R V i n f e c t i o n is m e d i a t e d through an N F K B m e c h a n i s m r e s u l t i n g i n the up-  56  regulation o f i n f l a m m a t o r y m e d i a t o r secretion ( S p u r r e l l et a l . , 2 0 0 5 ; Z h u et a l . , 1997). H o w e v e r , S h a r m a et al. (2006) s h o w e d elevated e x p r e s s i o n o f o v e r 3 0 transcription factors ( i n c l u d i n g N F K B ) u p o n R V i n f e c t i o n , thus c l e a r l y the R V m e c h a n i s m remains p o o r l y understood. Cell Model and R V Serotype Comparisons: A direct c o m p a r i s o n o f the t w o a i r w a y e p i t h e l i a l m o d e l s s h o w e d s i g n i f i c a n t l y m o r e R V r e p l i c a t i o n , R N A , and c y t o k i n e / c h e m o k i n e secretion i n B E A S - 2 B c e l l s than observed i n A 5 4 9 c e l l s regardless o f R V serotype. A cause f o r this increased s u s c e p t i b i l i t y o f B E A S - 2 B c e l l s to i n f e c t i o n is not k n o w n but m a y be attributable to increased e x p r e s s i o n o f R V receptors, and/or a m o r e e f f i c i e n t or faster r e p l i c a t i o n c y c l e . W h e t h e r this increased B E A S - 2 B s u s c e p t i b i l i t y is representative o f c e l l s o c c u r r i n g higher i n the a i r w a y s is d i f f i c u l t to say c o n s i d e r i n g that c u l t u r e d c e l l s m a y not represent the native a i r w a y e p i t h e l i u m adequately, and o v e r a l l b o t h c e l l lines are d e r i v e d f r o m the m i d to l o w e r respiratory tract. F u r t h e r m o r e , A 5 4 9 c e l l s represent surfactant secreting l u n g cells w h i c h are i n t r i n s i c a l l y different f r o m b r o n c h i a l e p i t h e l i a l tissues. H o w e v e r , i t seems that at least in vitro both b r o n c h i a l and alveolard e r i v e d c e l l s can be i n f e c t e d b y R V and initiate i n f l a m m a t o r y responses. T h i s evidence further supports g r o w i n g evidence that l o w e r a i r w a y c e l l s are v u l n e r a b l e to R V i n f e c t i o n ( G e r n et a l . , 1 9 9 7 ; H a y d e n , 2 0 0 4 ) , and is important i n the p a t h o l o g y o f diseases such as asthma and C O P D w h i c h are c o n s i d e r e d l o w e r a i r w a y diseases. R V 1 A p r o d u c e d m o r e p r o n o u n c e d effects than R V 1 4 i n terms o f v i r a l r e p l i c a t i o n and IL-6/IL-8 secretion f o r b o t h B E A S - 2 B and A 5 4 9 c e l l s , and a d d i t i o n a l l y R V 1 4 f a i l e d to replicate i n the A 5 4 9 c e l l s . T h i s m a y be due to d i f f e r e n t i a l e x p r e s s i o n o f L D L R receptor versus I C A M - 1 receptors but c o u l d also be caused b y i n t r i n s i c differences between the t w o serotypes. T h e R V 1 4 g e n o m e has been f u l l y sequenced ( S t a n w a y et a l . , 1984); h o w e v e r , o n l y certain portions o f the R V 1 A genome are k n o w n thus sequence h o m o l o g y between the t w o is u n d e t e r m i n e d . R V studies m a y u t i l i z e any o f 100+ serotypes and although serotypes are d o c u m e n t e d the potential differences amongst them are l a r g e l y i g n o r e d . M y data suggest that serotype selection m a y be an important factor f o r R V research.  RV14  experiments were also m u c h m o r e variable i n their IL-6 and IL-8 responses than R V 1 A . F o r some G r o w t h C u r v e s R V 1 4 f a i l e d to stimulate a p r o - i n f l a m m a t o r y response ( F i g u r e 2.2b), w h i l e i n other e x p e r i m e n t a l l y identical e x p e r i m e n t s IL-6/IL-8 s t i m u l a t i o n was h i g h l y  57  s i g n i f i c a n t ( F i g u r e 2.2c). T h e source o f this v a r i a b i l i t y is u n k n o w n , but perhaps the R V 1 4 v i r a l dose was near s o m e threshold l e v e l f o r t r i g g e r i n g a c y t o k i n e / c h e m o k i n e response i n a i r w a y e p i t h e l i a l c e l l s . O v e r a l l , the B E A S - 2 B / R V 1 A is the most consistent m o d e l f o r R V i n f e c t i o n o f the a i r w a y s . R V i n f e c t i o n o f cultured a i r w a y e p i t h e l i a l cells results i n r e l a t i v e l y l o w levels o f v i r a l r e p l i c a t i o n a c c o m p a n i e d b y a p r o l o n g e d secretion o f c y t o k i n e s and c h e m o k i n e s such as IL-6 and IL-8. Further research s h o u l d focus on the s p e c i f i c l i n k s between R V i n f e c t i o n and the e l i c i t e d a i r w a y e p i t h e l i a l i n f l a m m a t o r y response i n order to further define the m e c h a n i s m s i n v o l v e d .  58  FIGURES a)  Figure 2.1: Effect of a) RV14 and b) R V 1 A on viral replication over time in HI cells. Each trial represents n=4. A n asterisk (*) indicates a significant difference (ct=0.05) in infectious virus relative to DO within a given trial.  59  a)  0  1  3  2  4  5  6  7  Time After RV Infection (Days)  b) 6000 5000 Q_  c 4000 i o 2  3000  CD 2000  1000  H  0  1  2  3  5  7  Time After RV Infection (Days)  c)  6000 5000  F = l  Control RV14  Trial 2: BEAS-2B/RV14  O)  -2= 4000 *  S 3000 cz CD O  2000 H  1000 •{  1 0  1  2  3  5  7  Time After RV Infection (Days)  Figure 2.2: Effect of RV14 on: a) viral replication and b) and c) IL-6 secretion over time in BEAS-2B cells. In a) two trials (n=4 each) are depicted, and an asterisk (*) indicates a significant difference (a=0.05) in infectious virus relative to DO within a given trial. In b) IL-6 secretions correspond to trial 1. A l l measurements (control and R V for all days) are plotted but some are not visible because they are so close to 0. In c) IL-6 secretion corresponds to trial 2. An asterisk (*) indicates a significant difference in IL-6 concentration between R V infected and uninfected controls (n=3) at a given time (see b for further details).  60  a)  6000  Trial 1: BEAS-2B/RV1A  Control RV1A  -5 5000  a. §  4000  •£ 3000 a> o o 2000 CD - 1000  J L 0  1  2  3  I  i 5  7  Time After RV Infection (Days)  c)  0  1  2  3  5  7  Time After RV Infection (Days)  Figure 2.3: Effect of R V 1 A on: a) viral replication and b) and c) IL-6 secretion over time in BEAS-2B cells. See Fig. 2.2 legend for further details.  61  a)  0  1  2  3  5  7  Time After RV Infection (Days) Figure 2.4: Effect of RV14 on: a) viral replication and b) and c) IL-8 secretion over time in A549 cells. See Fig. 2.2 legend for further details.  62  a) A549/RV1A  Trial 1  E  Trial 2  £ 6 a o o> 2 5 3  g o  0  1  2  3  4  5  6  Time After RV Infection (Days)  b) 2500  Trial 1: A549/RV1A  Control  E  2000 -j  RV1A  a. o 1500 g 1000 c o O 500  I 0  1  2  3  i  6  Time After RV Infection (Days) c) 2500  0  1  2  3  6  Time After RV Infection (Days)  Figure 2.5: Effect of R V 1 A on: a) viral replication and b) and c) IL-8 secretion over time in A549 cells. See Fig. 2.2 legend for further details.  63  0  1  2  3  4  5  6  7  Time After RV Infection (Days) Figure 2.6: RV14 R N A for H I , BEAS-2B, and A549 cells over time. Daily RV14 R N A levels were measured for each cell line (n=2 each). An asterisk (*) indicates a significant difference (ct=0.05) in RV14 R N A within a given cell type relative to its DO. There is a significant difference in R N A levels (all days combined) between HI cells and both BEAS-2B and A549 cells. RV14 R N A in A549 cells was undetectable after D l .  64  a)  0  1  2  3  4  5  6  7  Time After R V Infection (Days)  b) 500  IL-8/RV14  Control RV14  E  400 ] BEAS-2B  300  g  200  A549  -I  c o O °?  100  *F W' 1  -F 0  2  3  5  7  0  2  3  5  7  Time After R V Infection (Days)  C) 500  IL-6/RV14  Control RV14  E  400 300  c o O CD  200 A549  BEAS-2B 100  L0_ 1  3  7  JLEL 1  3  7  Time After R V Infection (Days)  Figure 2.7: Effect of RV14 on: a) viral replication and b) IL-8 secretion and c) IL-6 secretion in simultaneously cultured (tandem) BEAS-2B and A549 cells over time. In a) an asterisk (*) indicates a significant difference (a=0.05) in infectious virus relative to DO within a cell type (n=3). A plus (+) denotes a significant difference in infectious virus between cell types on a given day. In b) IL-8 secretion from B E A S 2B and A549 cells corresponds to R V infections shown in a. A significant difference (a=0.05) was found in secretion patterns between cell types (all days combined). A l l measurements (control and R V for all days) are plotted but some are not visible because they are so close to 0. In c) IL-6 secretion for BEAS-2B and A549 cells is shown (see b for further information).  65  4000  Control  IL-8/RV1A  RV1A  E  "Bi 3000 o. c g  BEAS-2B  A549  2 2000 c CD o c o ° 1000 co  ll  J, 0  2  3  5  7  0  2  3  5  7  Time After RV Infection (Days)  c)  4000  Control  IL-6/RV1A  RV1A  E  "Q, 3000 g 2  2000  A549  BEAS-2B  <D  o c o O  CD  1000 -\  JL 1  3  7  1  3  7  Time After RV Infection (Days)  Figure 2.8: Effect of R V 1 A on: a) viral replication b) IL-8 secretion and c) IL-6 secretion in simultaneously cultured (tandem) BEAS-2B and A549 cells over time. In b) and c) an asterisk (*) indicates a significant difference in IL-6 or IL-8 concentration between R V infected and uninfected controls (n=3 each) at a given time. See Fig. 2.7 legend for further details.  66  Figure 2.9: RV14 R N A levels over time in tandem cultures of BEAS-2B and A549 cells. No significant differences (a=0.05) were found in RV14 R N A levels within the same cell type over time (n=2 each). A pi (+) indicates a significant difference in RV14 R N A levels between cell types on a given day.  67  REFERENCES Arnold, R. 1994. Interleukin 9, i n t e r l e u k i n 6, and soluble t u m o u r necrosis factor  receptor type 1 release f r o m a h u m a n p u l m o n a r y e p i t h e l i a l c e l l line ( A 5 4 9 ) e x p o s e d to respiratory s y n c y t i a l v i r u s . I m m u n o l o g y . 82:126.  Arruda, E . , A . Pitkaranta, T . Witek, Jr, C . Doyle, and F . Hayden 1997. 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R e s p i r a t o r y e p i t h e l i a l c e l l e x p r e s s i o n o f vascular  c e l l adhesion molecule-1 and its up-regulation b y r h i n o v i r u s i n f e c t i o n v i a N F - k a p p a B and G A T A transcription factors. J o u r n a l o f B i o l o g i c a l C h e m i s t r y . 274:9707-9720.  Schroth, M . K . , E . Grimm, P. Frindt, D. M . Galagan, S.-I. Konno, R. Love, and J . E . Gern 1999. R h i n o v i r u s r e p l i c a t i o n causes R A N T E S p r o d u c t i o n i n p r i m a r y b r o n c h i a l epithelial c e l l s . A m e r i c a n J o u r n a l o f R e s p i r a t o r y C e l l s and M o l e c u l a r B i o l o g y . 20:12201228.  70  Seemungal, T . 2 0 0 0 . D e t e c t i o n o f r h i n o v i r u s i n i n d u c e d s p u t u m and exacerbation o f  c h r o n i c obstructive p u l m o n a r y disease. E u r o p e a n R e s p i r a t o r y J o u r n a l . 16:677-83.  Seemungal, T . 2 0 0 1 . R e s p i r a t o r y viruses, s y m p t o m s and i n f l a m m a t o r y markers i n  acute exacerbations and stable c h r o n i c obstructive p u l m o n a r y disease. A m e r i c a n Journal o f  R e s p i r a t i o n and C r i t i c a l C a r e M e d i c i n e . 164:168-1623.  Sharma, M . , J . Arnason, and J . Hudson 2 0 0 6 . E c h i n a c e a extracts modulate the  p r o d u c t i o n o f m u l t i p l e transcription factors i n uninfected cells and rhinovirus-infected cells. P h y t o t h e r a p y R e s e a r c h . 20:1074-1079.  Spurrell, J . C . L . , S. Wiehler, R. S. Zaheer, S. P. Sanders, and D. Proud 2 0 0 5 .  H u m a n a i r w a y e p i t h e l i a l cells produce IP-10 ( C X C L 1 0 ) in vitro and in vivo u p o n  r h i n o v i r u s i n f e c t i o n . A m e r i c a n J o u r n a l o f P h y s i o l o g y L u n g C e l l u l a r and M o l e c u l a r P h y s i o l o g y . 289:L85-95.  Stanway, G . , P. J . Hughes, R. C . Mountford, P. D. Minor, and J . W . Almond 1984. T h e c o m p l e t e n u c l e o t i d e sequence o f a c o m m o n c o l d v i r u s : H u m a n r h i n o v i r u s 14. N u c l e i c A c i d s R e s e a r c h . 12:7859-7875. Subauste, M . C , and D. Proud 2 0 0 1 . E f f e c t s o f t u m o r necrosis factor-a, e p i d e r m a l g r o w t h factor and t r a n s f o r m i n g g r o w t h factor-a o n interleukin-8 p r o d u c t i o n b y , and h u m a n r h i n o v i r u s r e p l i c a t i o n i n , b r o n c h i a l epithelial c e l l s . International I m m u n o p h a r m a c o l o g y . 1:1229-1234. Teran, L . , S. Johnston, J . Schroder, M . Church, and S. Holgate 1997. R o l e o f nasal  interleukin-8-in neutrophil recruitment and activation i n c h i l d r e n w i t h virus-induced  asthma. A m e r i c a n J o u r n a l o f R e s p i r a t i o n and C r i t i c a l C a r e M e d i c i n e . 155:1362-1366.  Turner, R. B . 1998. A s s o c i a t i o n between interleukin-8 concentration i n nasal secretions and severity o f s y m p t o m s o f e x p e r i m e n t a l r h i n o v i r u s c o l d s . C l i n i c a l Infectious Diseases. 26: 840-846. Turner, R. B . 2 0 0 1 . R e v i e w : T h e treatment o f r h i n o v i r u s i n f e c t i o n s : Progress and  potential. A n t i v i r a l R e s e a r c h . 49:1-14.  van Gageldonk-Lafeber, A . , M . Heijnen, A . Bareids, M . Peters, S. van der Plas, and B . Wilbrink 2 0 0 5 . A case-control study o f acute respiratory tract i n f e c t i o n i n general practice patients i n the N e t h e r l a n d s . C l i n i c a l Infectious Diseases. 41:490-497.  Whiteman, S. C , A . Bianco, R. A . Knight, and M . A . Spiteri 2 0 0 3 . H u m a n  r h i n o v i r u s selectively modulates m e m b r a n o u s and soluble f o r m s o f its intercellular  adhesion molecule-1 ( I C A M - 1 ) receptor to p r o m o t e epithelial c e l l i n f e c t i v i t y . J o u r n a l o f  B i o l o g i c a l C h e m i s t r y . 278:11954-11961.  71  Winther, B . , E . Arruda, T . J . Witek, S. D . Marlin, M . M . Tsianco, D . J . Innes, and F. G . Hayden 2 0 0 2 . E x p r e s s i o n o f I C A M - 1 i n nasal e p i t h e l i u m and levels o f s o l u b l e  I C A M - 1 i n nasal l a v a g e f l u i d d u r i n g h u m a n e x p e r i m e n t a l r h i n o v i r u s i n f e c t i o n . A r c h i v e s o f O t o l a r y n g o l o l g y - H e a d & N e c k Surgery. 128:131-136.  Zheng, Y . H . , and M . Peterlin 2 0 0 5 . Intracellular i m m u n i t y to H I V - 1 : N e w l y d e f i n e d  retroviral battles inside infected c e l l s . R e t r o v i r o l o g y . 2:25-38.  Zhu, Z . , W . Tang, J . M . Gwaltney, Jr., Y . W u , and J . A . Elias 1997. R h i n o v i r u s s t i m u l a t i o n o f interleukin-8 in vivo and in vitro: R o l e o f N F - k a p p a B. A m e r i c a n J o u r n a l P h y s i o l o g y L u n g C e l l u l a r and M o l e c u l a r P h y s i o l o g y . 273:L814-824. Zhu, Z . , W . Tang, A . Ray, Y . W u , O . Einarsson, M . L . Landry, and J . M . Gwaltney, J r . 1996. R h i n o v i r u s s t i m u l a t i o n o f interleukin-6 in vivo and in vitro. T h e J o u r n a l o f C l i n i c a l Investigation. 97:421-430.  72  CHAPTER 3- The Effects Ultraviolet Inactivated Rhinovirus on Airway Epithelial Cells BACKGROUND D e s p i t e the fact that r h i n o v i r u s ( R V ) c o l d s are the most prevalent i l l n e s s i n h u m a n s , v e r y little is k n o w n about R V pathogenesis. S i n c e e v i d e n c e suggests that R V s cause little or n o damage to their host tissues a n d replicate at r e l a t i v e l y l o w levels w i t h i n the host, it has been postulated that the s y m p t o m s o f c o m m o n c o l d s are the result o f a host i n f l a m m a t o r y response characterized b y the secretion o f p r o - i n f l a m m a t o r y c y t o k i n e s (and c h e m o k i n e s ) such as i n t e r l e u k i n (IL)-6, a n d not R V r e p l i c a t i o n per se ( L o p e z - S o u z a et a l . , 2 0 0 4 ; M o s s e r et a l , 2 0 0 5 ; M o s s e r et a l . , 2 0 0 2 ) . F u r t h e r m o r e , a l i n k has been demonstrated between the severity o f c o l d s y m p t o m s (e.g. r h i n o r r h e a , sore throat, c o u g h i n g , malaise) and increases i n i n f l a m m a t o r y mediator secretion ( G w a l t n e y et a l . , 2 0 0 3 ) . If r e p l i c a t i o n is not the cause o f R V p a t h o l o g y then it is c o n c e i v a b l e that R V triggers an i n f l a m m a t o r y response i n its host a i r w a y e p i t h e l i a l cells b y s o m e virus-cell interaction w i t h o u t the necessity o f R V r e p l i c a t i o n o r passage across the p l a s m a m e m b r a n e . O n e m e t h o d f o r i n v e s t i g a t i n g this hypothesis is b y u s i n g ultraviolet ( U V ) inactivated R V f o r e x p e r i m e n t a l i n f e c t i o n s . There is e v i d e n c e that U V C ( 2 6 0 n m ) i rradi ati on o f R V first affects the v i r a l n u c l e i c a c i d site before i m p a c t i n g protein structures. C o n s e q u e n t l y U V exposure causes i n f e c t i v i t y to be lost before observed changes i n antigen s p e c i f i c i t y ( H u g h e s et a l . , 1979). U V p r i m a r i l y causes damage i n the f o r m o f c y c l o b u t y l p y r i m i d i n e d i m e r s a n d photoproducts w h i c h i n h i b i t the r e p l i c a t i o n o f genetic material ( M y a t t et a l . , 2003). If n o n i n f e c t i o u s R V is capable o f s t i m u l a t i n g an i n f l a m m a t o r y response i n host c e l l s , then this supports the i d e a o f a non-replicative trigger f o r R V p a t h o l o g y . V e r y f e w studies have investigated such a h y p o t h e s i s ; h o w e v e r , there is l i m i t e d evidence that inactivated R V m a y retain some o f its c y t o k i n e s t i m u l a t i n g abilities. F o r e x a m p l e , Johnston et al. (1998) f o u n d that 3 0 m i n u t e U V C i rradi ati on o f R V 9 c o m p l e t e l y i n h i b i t e d v i r a l r e p l i c a t i o n i n A 5 4 9 c e l l s , but o n l y reduced IL-8 secretion b y about one half. O n the other  A version of this chapter will be submitted for publication. Machala, A . M . , Harris, R.A., Brauner, C.J., Hudson, J.B.  2  73  h a n d , G r i e g o et al. (2000) f o u n d that U V treated R V d i d not i n d u c e m u c h IL-6 or IL-8 secretion i n B E A S - 2 B c e l l s b e y o n d c o n t r o l values. It is u n c l e a r whether n o n i n f e c t i o u s R V is capable o f e l i c i t i n g an i m m u n e response i n a i r w a y e p i t h e l i a l c e l l s . T h i s study investigated the a b i l i t y o f U V - t r e a t e d R V to stimulate IL-6 secretion i n the b r o n c h i a l B E A S - 2 B c e l l line.  MATERIALS AND METHODS A l l v i r a l , c e l l culture and m o l e c u l a r w o r k was c o n d u c t e d under sterile c o n d i t i o n s i n a type II b i o s a f e t y cabinet. A l l p r o t o c o l s were pre-approved b y the U B C b i o s a f e t y c o m m i t t e e i n certificate H 0 4 - 0 0 6 1 ( A p p e n d i x A ) .  E x p e r i m e n t s : C u l t u r e d B E A S - 2 B c e l l s were i n o c u l a t e d w i t h either R V 1 4 or R V 1 A p r e v i o u s l y treated w i t h U V C for exposure times o f 2, 5, 10, 15 and 3 0 minutes ( U V 2 , U V 5 , U V 1 0 , U V 1 5 , and U V 3 0 respectively), or w i t h untreated R V ( N O U V 0 , N O U V 3 0 ) . Infectious v i r u s , IL-6 secretion, and v i r a l R N A w e r e measured at time=0 (Ohrs) i m m e d i a t e l y post-infection, and 4 8 hours later (48hrs). A l t h o u g h the n u c l e i c a c i d site i s thought to be affected b y U V first, the exact t i m i n g o f n u c l e i c a c i d and protein d i s r u p t i o n i s l a r g e l y u n k n o w n thus R V s were e x p o s e d to U V f o r different lengths o f t i m e to p r o d u c e n o n i n f e c t i o u s virus w h i c h retained as m a n y o f its other characteristics (e.g. antigenic activity) as p o s s i b l e . T w o separate trials were c o n d u c t e d f o r each R V serotype (n=3 each).  C e l l C u l t u r e : T h e S V 4 0 adenovirus transformed h u m a n b r o n c h i a l e p i t h e l i a l c e l l l i n e ( B E A S - 2 B ) was obtained f r o m the A m e r i c a n T y p e C u l t u r e C o l l e c t i o n ( A T C C ) ( R o c k v i l l e , M D ) and c u l t u r e d i n 7 5 m m flasks i n 5 0 : 5 0 D u l b e c c o ' s M o d i f i e d E a g l e ' s M e d i u m 2  ( D M E M ) and H a m ' s F 1 2 w i t h 1 0 % e n d o t o x i n free fetal b o v i n e serum ( F B S ) . C u l t u r e reagents were obtained f r o m Invitrogen ( V a n c o u v e r , C a n a d a ) . C e l l s w e r e t r y p s i n i z e d and passaged w e e k l y and incubated at 35-37°C w i t h 5 % c a r b o n d i o x i d e i n 9 5 % air. R V sensitive H I c e l l s ( f r o m A T C C ) were c u l t u r e d under the same c o n d i t i o n s w i t h D M E M and 5%  FBS.  74  Viruses: B o t h R V 1 4 and R V 1 A were obtained f r o m the A T C C . R V s were propagated b y i n f e c t i n g H I c e l l s g r o w n to c o n f l u e n c e i n 7 5 m m f l a s k s c o n t a i n i n g D M E M , a l l o w i n g f o r 2  f u l l cytopathic effects ( C P E ) . Cell-free culture f l u i d was harvested w h e n C P E were at a m a x i m u m b y centrifugation at 10,000 x g f o r 2 0 minutes at 4 ° C . T h e stock v i r u s suspension was a l i q u o t e d into c r y o v i a l s and stored at -80°C f o r e x p e r i m e n t a l use. T i t e r o f v i r a l stock was determined b y v i r a l plaque assay (see b e l o w ) o f s e r i a l l y d i l u t e d stock, and expressed i n plaque f o r m i n g units (pfu) per m L . P f u represent the n u m b e r o f i n f e c t i o u s R V particles present i n a k n o w n v o l u m e o f sample. F o r e x p e r i m e n t s , a l i q u o t e d stock v i r u s was r a p i d l y thawed at 37°C and v o r t e x e d p r i o r to use. B e c a u s e such c l a r i f i e d v i r a l stocks c o n t a i n H I c e l l remnants (e.g. s o l u b l e proteins, organelles) c o n t r o l experiments were c o n d u c t e d to c o n f i r m that any o b s e r v e d changes i n IL-6 and IL-8 secretion were due to v i r u s and not some other c o m p o n e n t present i n the i n o c u l a ( A p p e n d i x C ) .  Preparation of U V inactivated virus: S t o c k suspensions o f either R V 1 4 or R V 1 A w i t h k n o w n v i r a l titers ( 1 0 p f u / m L ) and equal v o l u m e s were p l a c e d i n s i n g l e - w e l l culture plates 8  (lids r e m o v e d ) and irradiated b y a 2 6 0 n m U V C light source p l a c e d 10 c m a w a y in a dark r o o m . Plates were gently agitated throughout U V C exposure. A t s p e c i f i c durations o f U V treatment: 2, 5, 10, 15 and 3 0 minutes ( U V 2 , U V 5 , U V 1 0 , U V 1 5 , and U V 3 0 respectively), equal v o l u m e s o f R V stocks were r e m o v e d , p l a c e d i n c r y o v i a l s , and i m m e d i a t e l y stored at -80°C. S u f f i c i e n t stock R V v o l u m e was used to a v o i d d r y i n g o f samples d u r i n g U V exposure. C o n t r o l R V stocks were prepared s i m u l t a n e o u s l y under the same c o n d i t i o n s except s h i e l d e d f r o m l i g h t exposure i n order to c o n t r o l f o r R V stability o v e r the 3 0 m i n u t e s a m p l i n g p e r i o d . T h e c o n t r o l samples were c o l l e c t e d j u s t p r i o r to U V treatment ( N O U V 0 ) , and at the 30 m i n u t e endpoints ( N O U V 3 0 ) .  Infections with U V inactivated R V :  B E A S - 2 B c e l l s were plated into 6-well trays and  g r o w n u n t i l f r e s h l y c o n f l u e n t . O n c e c e l l s reached c o n f l u e n c e it was assumed that c e l l n u m b e r d i d not change s i g n i f i c a n t l y o v e r the course o f experiments f o r either c o n t r o l or R V infected cultures. T h i s a s s u m p t i o n was c o n f i r m e d e x p e r i m e n t a l l y ( A p p e n d i x D ) . C e l l n u m b e r per w e l l (6-well plate) at c o n f l u e n c e was pre-determined f o r c e l l lines ( A p p e n d i x E ) and used to calculate v i r a l dose. R V i n o c u l a were prepared in culture m e d i u m (no  75  FBS)  f r o m the above d e s c r i b e d U V treated stocks ( N O U V O , N O U V 3 0 , U V 2 , U V 5 , U V 1 0 , U V 1 5 , U V 3 0 ) w i t h equivalent amounts o f v i r u s , based o n a p r e - U V treatment v i r a l dose o f M O I = l . A t i n f e c t i o n , m e d i u m was aspirated f r o m c e l l s and replaced w i t h l m L o f the appropriate U V - t r e a t e d R V i n o c u l a , or w i t h m e d i u m alone (control), and c e l l s were incubated for 1 h o u r at 35°C. A f t e r i n f e c t i o n , i n o c u l a were aspirated and c e l l s washed 3 times w i t h l m L culture m e d i u m to r e m o v e exogenous virus (virus r e m o v a l e x p e r i m e n t a l l y c o n f i r m e d , not s h o w n ) . F o l l o w i n g this, 3 m L o f fresh m e d i u m ( 1 % F B S ) was added to the c e l l s and one subset o f the plates was s a m p l e d at Ohrs w h i l e the r e m a i n d e r were i n c u b a t e d at 35°C and s a m p l e d at 48hrs. S a m p l i n g consisted o f first r e m o v i n g l m L o f supernatant f r o m appropriate w e l l s , c e n t r i f u g i n g at 1000 x g to r e m o v e c e l l u l a r debris, and f r e e z i n g samples at -20°C f o r future IL-6 assays. C e l l s f r o m R V infected samples were scraped into the remainder o f the m e d i u m ( 2 m L ) , pipetted into c r y o v i a l s , and stored at -80°C f o r plaque assays. F i n a l l y , f o r quantitative real-time p o l y m e r a s e c h a i n reaction ( q R T - P C R ) designated samples, m e d i u m was r e m o v e d , c e l l m o n o l a y e r s w a s h e d t w i c e w i t h sterile phosphate b u f f e r e d saline, and l m L o f T R I Z O L reagent was added to each w e l l a l l o w i n g f o r c e l l l y s i s to o c c u r . T h i s suspension was stored i n E p p e n d o r f tubes at -80°C for subsequent R N A extraction.  P l a q u e Assays (See A p p e n d i x H f o r i m a g e ) : V i r a l i n f e c t i v i t y and r e p l i c a t i o n were measured b y plaque assay i n p e r m i s s i v e H I cells. O n c e a l l the c e l l s c r a p i n g samples were c o l l e c t e d , they were r a p i d l y f r o z e n and thawed at 37°C t w i c e , to rupture c e l l s and release v i r u s . These samples w e r e used at serial d i l u t i o n s to infect the p e r m i s s i v e H I cells i n duplicate. 0 . 7 5 m L o f s a m p l e was added onto f r e s h l y c o n f l u e n t H I c e l l s g r o w n i n 6-well trays and a l l o w e d to infect f o r 1 hour at 35°C. A f t e r i n f e c t i o n , the i n o c u l a were aspirated and replaced w i t h a 5 0 : 5 0 l i q u i d m i x t u r e o f 2x M E M ( w i t h 5 % F B S ) and 1 % agarose g e l . T h e agarose gel was a l l o w e d to s o l i d i f y at r o o m temperature, and then the plates w e r e incubated at 35°C f o r 4 days. A s the virus replicated i n infected c e l l s , l y s i s o c c u r r e d i n the H I c e l l s f o r m i n g r o u n d areas o f c e l l death c a l l e d " p l a q u e s " . O n c e i n c u b a t i o n was c o m p l e t e , plates were f i x e d w i t h 3 % f o r m a l d e h y d e , agarose was r e m o v e d , and c e l l s stained w i t h crystal v i o l e t to reveal the clear unstained plaques. These plaques w e r e then counted and  76  reported as p f u / m L (infectious v i r i o n s ) present i n the sample. A n increase i n infectious v i r u s o v e r time (relative to DO) i n d i c a t e d v i r a l r e p l i c a t i o n .  C y t o k i n e s : IL-6 was assayed i n duplicate f r o m supernatant samples u s i n g standard p r o t o c o l p r o v i d e d b y c o m m e r c i a l l y available e n z y m e - l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) kits f r o m I m m u n o t o o l s (Friesoythe, G e r m a n y ) . A b s o r b e n c i e s w e r e read o n an E L I S A plate reader (Pasteur D i a g n o s t i c s L P 4 0 0 ) at a 4 5 0 n m w a v e l e n g t h . H i g h l y concentrated samples w e r e d i l u t e d w i t h m e d i u m and re-assayed to f a l l w i t h i n the standard assay range w h i c h was f r o m 0-450 p g / m L . S e n s i t i v i t y o f the assay was 4 p g / m L .  R N A E x t r a c t i o n : R N A was extracted f o l l o w i n g standard T R I Z O L reagent p r o t o c o l and reconstituted i n 5 0 u L o f R N A s e / D N A s e free water and stored at -80°C for q R T - P C R . R N A f o r R V 1 4 standard curves was extracted f r o m stock R V 1 4 suspensions u s i n g the Q I A G E N R N e a s y kit (Mississauga, O N ) .  q R T - P C R : q R T - P C R was c o n d u c t e d for R V 1 4 samples. P r i m e r s w e r e designed u s i n g the k n o w n R V 1 4 genetic sequence f r o m the N a t i o n a l C e n t e r for B i o t e c h n o l o g y I n f o r m a t i o n database and purchased f r o m O p e r o n ( H u n t s v i l l e , A L ) . P r i m e r s w e r e d e s i g n e d w i t h the f o l l o w i n g sequences: f o r w a r d 5' G A C A T G G T G T G A A G A C T C G C 3 ' and reverse 5' T C T G T G T A G A A A C C T G A G C G C 3 ' creating a 2 3 8 base p a i r product. P r i m e r s were tested b y c o n v e n t i o n a l two-step P C R o f k n o w n R V infected s a m p l e s , and P C R products c o n f i r m e d b y gel electrophoresis (not s h o w n ) . F o r R V 1 4 R N A samples a one-step q R T - P C R k i t f r o m Q I A G E N ( M i s s i s s a u g a , O N ) was used. T h e P C R m i x t u r e c o n t a i n e d : 2 5 u L M a s t e r M i x ( H o t S t a r T a q D N A P o l y m e r a s e , Q u a n t i T e c t S Y B R G r e e n B u f f e r , S Y B R G r e e n I d y e , 1 . 5 m M M g C 1 2 , 2 0 0 u M each d N T P ) , 2 . 5 u L o f each p r i m e r , 0.5 u L R T m i x ( O m n i s c r i p t and S e n s i s c r i p t R e v e r s e Transcriptases), and l O u L o f the R N A template to a f i n a l v o l u m e o f 5 0 u L . Standard c u r v e R V concentrations were d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y and s e r i a l l y d i l u t e d at k n o w n concentrations. F o r standards and samples, duplicate reactions were c a r r i e d out i n a M J Research D N A Engine Opticon Continuous Fluorescence Detector (OP000537) p r o g r a m m e d to: incubate at 4 2 ° C for 5 5 m i n , 94°C for 5 m i n , 94°C f o r 3 0 sec, 55°C f o r 3 0  77  sec, 72°C f o r l m i n , repeat for 3 5 c y c l e s , 72°C f o r l O m i n , m e l t i n g curve f r o m 55°C to 95°C read e v e r y 0.5°C, incubate 72°C for l O m i n , and 4 ° C forever. D a t a were a n a l y z e d u s i n g O p t i c o n M o n i t o r analysis software v e r s i o n 1.07.  Statistics: Infectious v i r u s (replication) was a n a l y z e d b y t w o - w a y analysis o f variance ( A N O V A ) w i t h time and treatment as factors, and post-hoc D u n n e t ' s test w i t h Ohrs as a c o n t r o l . IL-6 data f o r each serotype were subjected to a 1-way A N O V A w i t h treatment as a factor and post-hoc D u n n e t ' s test to c o m p a r e treatments w i t h c o n t r o l ( C ) . R N A data were a n a l y z e d b y one-way A N O V A w i t h treatment as a factor and post-hoc D u n n e t ' s test w i t h untreated R V ( N O U V O ) as a c o n t r o l . A l l data were a n a l y z e d u s i n g S i g m a Stat 3.0 software and statistical s i g n i f i c a n c e was set at a = 0 . 0 5 . R e s u l t s are presented as m e a n ± standard error o f the m e a n . A l l statistical differences w e r e c o n s i d e r e d s i g n i f i c a n t w h e n p < 0 . 0 5 ; h o w e v e r , i n most cases, w h e n differences were f o u n d they were h i g h l y s i g n i f i c a n t (p<0.001).  RESULTS CPE:  N o C P E were o b s e r v e d i n the B E A S - 2 B c e l l s f o r any o f the c o n t r o l , R V i n f e c t e d , or  U V - t r e a t e d R V infected samples. RV14  Replication ( F i g u r e s 3.1a and 3.2a): T h e r e was a h i g h l y s i g n i f i c a n t difference  between trial 1 and 2; h o w e v e r , the trends observed were the same. F o r b o t h e x p e r i m e n t s , h i g h l y s i g n i f i c a n t v i r a l r e p l i c a t i o n was measured f r o m Ohrs to 48hrs f o r b o t h the N O U V O and N O U V 3 0 samples. H o w e v e r , no s i g n i f i c a n t differences w e r e f o u n d between N O U V O and N O U V 3 0 samples f o r either sample time (i.e. Ohrs or 4 8 h r s ) , i n d i c a t i n g that no measurable ( n o n - U V ) R V 1 4 degradation had o c c u r r e d d u r i n g preparation o f U V irradiated virus. F u r t h e r m o r e , no s i g n i f i c a n t R V r e p l i c a t i o n was o b s e r v e d i n B E A S - 2 B c e l l s infected w i t h any o f the U V treated ( U V 2 - U V 3 0 ) samples demonstrating a c o m p l e t e i n h i b i t i o n o f R V 1 4 replication by U V . RV14  and IL-6 Secretion (Figures 3.1b and 3.2b): T h e differences betw een trial 1 and 2  were h i g h l y s i g n i f i c a n t , and trends observed were not s i m i l a r . In the first t r i a l , R V 1 4 f a i l e d to stimulate an IL-6 response f r o m B E A S - 2 B c e l l s w h e n c o m p a r e d to c o n t r o l ( C ) . A l t h o u g h there was s o m e statistical differences f o u n d ( N O U V O and U V 5 ) , c o n s i d e r i n g the  78  v e r y l o w concentrations o f IL-6 ( m a x i m u m 4 0 . 0 ± 8.0 p g / m L ) , it is h i g h l y d o u b t f u l that this was o f any b i o l o g i c a l relevance. H o w e v e r , in trial 2, R V 1 4 i n d u c e d h i g h l y s i g n i f i c a n t IL-6 secretion i n the c e l l s for N O U V O and N O U V 3 0 treatments, but none o f the U V irradiated R V 1 4 treatments stimulated IL-6 secretion i n B E A S - 2 B c e l l s . RV1A  Replication (Figures 3.3a and 3.4a): T h e r e was also a h i g h l y s i g n i f i c a n t statistical  difference between trials 1 and 2; h o w e v e r , the trends observed were again the same between e x p e r i m e n t s and the same as seen f o r R V 1 A r e p l i c a t i o n . T h e r e w a s a h i g h l y s i g n i f i c a n t difference f r o m Ohrs to 48hrs f o r N O U V O and N O U V 3 0 , i n d i c a t i n g R V r e p l i c a t i o n . H o w e v e r , n o significant differences were f o u n d between N O U V O and N O U V 3 0 samples at either Ohrs or 4 8 h r s , i n d i c a t i n g that no measurable ( n o n - U V ) R V 1 A degradation had o c c u r r e d d u r i n g preparation o f U V irradiated virus. F u r t h e r m o r e , no s i g n i f i c a n t R V r e p l i c a t i o n was o b s e r v e d i n B E A S - 2 B c e l l s infected w i t h any o f the U V treated ( U V 2 - U V 3 0 ) s a m p l e s , demonstrating a c o m p l e t e i n h i b i t i o n o f R V 1 A r e p l i c a t i o n b y UV. RV1A  and IL-6 Secretion (Figures 3.3b and 3.4b): A h i g h l y s i g n i f i c a n t d i f f e r e n c e was  f o u n d between trials 1 and 2, but w i t h the same trends as p r e v i o u s l y d e s c r i b e d . A g a i n the N O U V O and N O U V 3 0 treatments p r o d u c e d h i g h l y s i g n i f i c a n t differences w h e n c o m p a r e d to c o n t r o l ( C ) . N o n e o f the U V - i r r a d i a t e d R V 1 A treatments stimulated s i g n i f i c a n t IL-6 secretion f r o m c e l l s w h e n c o m p a r e d to c o n t r o l s . RV14  R N A ( F i g u r e 3.5): There was a h i g h l y s i g n i f i c a n t difference between the U V  untreated ( N O U V O ) R V 1 4 R N A treatment, w h i c h contained 14 pg/well o f v i r a l R N A , and all other treatments. T h i s i n d i c a t e d damage to the v i r a l genetic material f o r a l l o f the U V treated samples.  DISCUSSION T h e postulate that R V s m a y p r o v o k e the secretion o f p r o - i n f l a m m a t o r y c y t o k i n e s such as IL-6 w i t h o u t actually i n f e c t i n g a i r w a y e p i t h e l i a l c e l l s is certainly p r o v o c a t i v e . A l t h o u g h c y t o k i n e secretion has been l i n k e d more c l o s e l y to c o l d s y m p t o m s than R V r e p l i c a t i o n ( G w a l t n e y et a l . , 2 0 0 3 ) , there is no e v i d e n c e that n o n i n f e c t i o u s v i r u s is actually capable o f c a u s i n g i l l n e s s . V e r y f e w studies have investigated the effects o f U V inactivated R V o n a i r w a y e p i t h e l i a l c e l l s , and those p u b l i s h e d have y i e l d e d i n c o n c l u s i v e results. F o r e x a m p l e ,  79  Johnston et al. (1998) f o u n d that 3 0 m i n u t e U V i n a c t i v a t i o n o f R V 9 i n h i b i t e d v i r a l r e p l i c a t i o n c o m p l e t e l y but o n l y reduced IL-8 secretion b y h a l f i n A 5 4 9 c e l l s . H o w e v e r , b o t h G r i e g o et al. (2000) and P a p a d o p o l o u s et al. (2001) demonstrated that U V i n a c t i v a t i o n o f R V l b and R V 3 9 halted b o t h R V r e p l i c a t i o n and IL-8, IL-6, and R A N T E S secretion f r o m B E A S - 2 B c e l l s . M y results support the latter t w o studies, as no increased IL-6 secretion was o b s e r v e d f o r U V inactivated R V 1 4 or R V 1 A f o r any U V exposure time. E v e n 2 minutes o f U V C exposure was sufficient to arrest b o t h v i r a l r e p l i c a t i o n and IL-6 secretion f r o m the B E A S - 2 B c e l l s . H u g h e s et al. (1979) s h o w e d that U V C treatment o f R V 1 7 and R V 4 0 inactivated the v i r a l n u c l e i c a c i d i n less than 10 seconds f o r dilute virus preparations and up to 9 0 seconds f o r m o r e concentrated samples. H o w e v e r , the same study demonstrated that antigenic a c t i v i t y ( p r o v o c a t i o n o f a n t i b o d y f o r m a t i o n ) o f R V c o u l d be retained w i t h at least 13 minutes o f U V exposure. It is p o s s i b l e that the c a p a c i t y to i n d u c e antibody f o r m a t i o n is retained l o n g e r than the a b i l i t y to stimulate an i n f l a m m a t o r y response since w h i t e b l o o d c e l l s m a y r e c o g n i z e R V more e f f e c t i v e l y than the a i r w a y e p i t h e l i a l c e l l s . M y e x p e r i m e n t s also s h o w e d a s i g n i f i c a n t d i f f e r e n c e in R V 1 4 R N A f o r U V treated samples w h e n c o m p a r e d to untreated samples. T h i s result is interesting because there have been c o n f l i c t i n g reports suggesting that U V treatment o f p o l i o v i r u s and R V m a y or m a y not affect P C R assays ( M a et a l . , 1 9 9 4 ; M y a t t et a l . , 2 0 0 3 ) . T h e reason f o r the d i s c r e p a n c y between the Johnston et al. (1998) study and the other studies ( i n c l u d i n g this one) is not clear. T h e c h o i c e o f c e l l l i n e c o u l d be the source o f the difference since o n l y Johnston et al. (1998) used A 5 4 9 c e l l s (versus B E A S - 2 B ) . M o r e o v e r , R V serotype differences c o u l d also contribute to the observed differences, f o r e x a m p l e the R V 9 c a p s i d m a y be m o r e resistant to U V damage than other R V s . S i n c e U V i n a c t i v a t i o n o f R V is also affected b y i n i t i a l v i r u s concentration and U V intensity, it is p o s s i b l e that even the U V 2 v i r u s treatment had both non-functional genetic material and protein structure w h i c h c o u l d not p r o v o k e an IL-6 response. F i n a l l y , the c h o i c e o f the c y t o k i n e or c h e m o k i n e measured m a y affect results, b e a r i n g i n m i n d that numerous interacting i n f l a m m a t o r y mediators are released f r o m a i r w a y c e l l s i n response to R V i n f e c t i o n . Interestingly, f o r one o f the R V 1 4 experiments no IL-6 response was o b s e r v e d f o r untreated R V e v e n t h o u g h s i g n i f i c a n t v i r a l r e p l i c a t i o n was apparent. T h i s same p h e n o m e n o n was observed i n p r e v i o u s e x p e r i m e n t s (see C h a p t e r 2 ) ; h o w e v e r , the  80  e x p l a n a t i o n remains unclear. Perhaps the R V 1 4 dosage is near s o m e threshold v a l u e f o r e l i c i t i n g a c y t o k i n e response f r o m the c e l l s . In c o n c l u s i o n , these e x p e r i m e n t s demonstrated that U V inactivated R V was not capable o f e l i c i t i n g IL-6 secretion f r o m B E A S - 2 B c e l l s f o r either R V 1 4 or R V 1 A . T h i s suggests that R V i n f e c t i o n and/or r e p l i c a t i o n m a y be necessary to the p a t h o l o g y o f the c o m m o n cold.  81  FIGURES a)  Trial 1: RV14  E 3  mam  Ohrs  (aajsi 48hrs  4  Q.  O  O CO  3  CO  O  1  0)  a  i  NO UVO N O U V 3 0  a.y  a,y  UV2  UV5  a,y  a,y  UV10  UV15  UV30  Treatment  b) 600  N O UVO  NOUV30  UV2  UV5  UV10  UV15  UV30  Treatment  Figure 3.1: Trial 1. Effect of U V treated and untreated RV14 on a) viral replication and b) IL-6 secretion in BEAS-2B cells after 48 hours. RV14 was pre-treated with U V C (260nm) for exposure times of 2, 5, 10, 15 or 30 minutes (UV2, U V 5 , UV10, UV15 and UV30 respectively). Simultaneously, control RV14 samples, which were shielded from light, were prepared at time=0 (NO UVO) and after 30 minutes (NO UV30). In a) an asterisk (*) indicates a significant (a=0.05) difference in infectious virus from 0 to 48hrs within a treatment (n=3). Symbols that differ (a for Ohrs; x,y for 48hrs) indicate significant differences in infectious virus between treatments at a given time (i.e. 0 or 48hrs). A l l measurements are plotted but some are not visible because they are so close to 0. In b) an asterisk (*) indicates a significant difference in IL-6 concentration between control (C) cells and other treatments (n=3).  82  a)  Trial 2: RV14 E "3 4  Ohrs [-""I 48hrs  H—  Q. O O) O  co  3  3  CO 3 O  €  2  CD  NO UVO N O U V 3 0  UV2  a,y  a.y  UV5  UV10  a,y UV15  UV30  Treatment  b) 600  C  NOUV0 NOUV30  UV2  UV5  UV10  UV15  UV30  Treatment  Figure 3.2: Trial 2. Effect of U V treated and untreated RV14 on a) viral replication and b) IL-6 secretion in BEAS-2B cells after 48 hours. See legend Fig. 3.1 for further details.  83  a)  Trial 1: RV1A  wmm  Ohrs  ZEEB  48hrs  3  5 o o  4  co 3  a i  1  co 3  •  O OD C j;  l| ii  1  NO UVO NO UV30  a.y  UV2  a.y  ii  UV5  a.y  a.y  a.y  I  IB I  UV10  UV15  I  UV30  Treatment b)  -i———i C  1—'—'—i—'—'—i—'—'—i—'—•—\—*—'—i— NO UVO NOUV30 UV2 UV5 UV10 UV15 UV30  Treatment  Figure 3.3: Trial 1. Effect of U V treated and untreated R V 1 A on a) viral replication and b) IL-6 secretion in BEAS-2B cells after 48 hours. See legend Fig. 3.1 for further details.  84  a)  NO UVO NO UV30  UV2  UV5  UV10  UV15  UV30  Treatment b) 600  C  NO UVO NO UV30  UV2  UV5  UV10  UV15  UV30  Treatment  Figure 3.4: Trial 2. Effect of U V treated and untreated R V 1 A on a) viral replication and b) IL-6 secretion in BEAS-2B cells after 48 hours. See legend Fig. 3.1 for further details.  85  2 RV14/48hrs  °  0  •1  H  NO UVO  UV2  UV5  UV15  UV30  Treatment  Figure 3.5: Effect of U V treated and untreated RV14 inocula on RV14 R N A levels in BEAS-2B cells after 48 hours. Cells were inoculated with untreated RV14 (NO U V ) , and RV14 pre-treated with U V C for 2, 5, 15 or 30 minutes (UV2, U V 5 , UV15, and UV30 respectively). An asterisk (*) indicates a significant difference (a=0.05) in RV14 R N A levels relative to untreated RV14 (NO UVO) control (n=2).  86  REFERENCES Griego, S. D., C . B . Weston, J . L . Adams, R. Tal-Singer, and S. B. Dillon 2 0 0 0 .  R o l e o f p 3 8 mitogen-activated protein kinase i n r h i n o v i r u s - i n d u c e d c y t o k i n e p r o d u c t i o n b y b r o n c h i a l epithelial c e l l s . T h e J o u r n a l o f I m m u n o l o g y . 165:5211-5220.  Gwaltney, J . M . , J . O . Hendley, and J . T. Patrie 2 0 0 3 . S y m p t o m severity patterns i n  e x p e r i m e n t a l c o m m o n c o l d s and their usefulness i n t i m i n g onset o f illness i n natural c o l d s . C l i n i c a l Infectious Diseases. 36:714-23.  Hughes, J . H . , M . Mitchell, and V . Hamparian 1979. R h i n o v i r u s e s : K i n e t i c s o f  ultraviolet i n a c t i v a t i o n and effects o f U V and heat on i m m u n o g e n i c i t y . A r c h i v e s o f V i r o l o g y . 61:313-319.  Johnston, S. L . , A . Papi, P. J . Bates, J . G . Mastronarde, M . M . Monick, and G . W . Hunninghake 1998. L o w grade r h i n o v i r u s i n f e c t i o n induces a p r o l o n g e d release o f IL-8 i n p u l m o n a r y e p i t h e l i u m . T h e J o u r n a l o f I m m u n o l o g y . 160:6172-6181. Lopez-Souza, N . , G . Dolganov, R. Dubin, L . A . Sachs, L . Sassina, H . Sporer, S. Yagi, D. Schnurr, H . A . Boushey, and J . H . Widdicombe 2 0 0 4 . Resistance o f differentiated h u m a n a i r w a y e p i t h e l i u m to i n f e c t i o n b y r h i n o v i r u s . A m e r i c a n J o u r n a l o f P h y s i o l o g y L u n g C e l l u l a r and M o l e c u l a r P h y s i o l o g y . 286:L373-381. Ma, J.F. S., T . Pepper, and C . Gerba 1994. C e l l culture and P C R determination o f p o l i o v i r u s i n a c t i v a t i o n b y disinfectants. A p p l i e d and E n v i r o n m e n t a l M i c r o b i o l o g y . 60:4203-4206. Mosser, A . G . , R. A . Brockman-Schneider, S. Amireva, L . Burchell, J . B. Sedgwick, W . W . Busse, and J . E . Gern 2 0 0 2 . S i m i l a r frequency o f r h i n o v i r u s - i n f e c t i b l e  cells i n upper and l o w e r a i r w a y e p i t h e l i u m . J o u r n a l o f Infectious Diseases. 185:734-743. Mosser, A . G . , R. Vrtis, L . Burchell, W . M .  Lee, C . R. Dick, E . Weisshaar, D. Bock,  C. A . Swenson, R. D. Cornwell, K . C . Meyer, N . N . Jarjour, W . W . Busse, and J . E . Gern 2 0 0 5 . Quantitative and qualitative analysis o f r h i n o v i r u s i n f e c t i o n i n b r o n c h i a l  tissues. T h e A m e r i c a n J o u r n a l o f R e s p i r a t i o n and C r i t i c a l C a r e M e d i c i n e . 171:645-651. Myatt, T . A . , S. L . Johston, S. Rudnick, and D. K . Milton 2 0 0 3 . A i r b o r n e r h i n o v i r u s  detection and effect o f ultraviolet i r r a d i a t i o n on detection b y a semi-nested R T - P C R assay. B i o M e d C e n t r a l P u b l i c H e a l t h . 3:5-12.  Papadopoulos, N . G . , A . Papi, J . Meyer, L . A . Stanciu, S. Salvi, S. T . Holgate, and S. L . Johnston 2 0 0 1 . R h i n o v i r u s i n f e c t i o n up-regulates e o t a x i n and eotaxin-2 e x p r e s s i o n i n b r o n c h i a l epithelial c e l l s . C l i n i c a l and E x p e r i m e n t a l A l l e r g y . 31:1060-1066.  87  CHAPTER 4: The Effects of Echinacea Extracts on RV Infected and Uninfected Airway Epithelial Cells 3  BACKGROUND R h i n o v i r u s ( R V ) infections o f a i r w a y e p i t h e l i a l c e l l s are the most important cause o f the c o m m o n c o l d ( M o n t o , 2 0 0 2 ) . F o r people already affected b y respiratory diseases such as asthma a n d c h r o n i c obstructive p u l m o n a r y disorder ( C O P D ) , R V i n f e c t i o n m a y cause dangerous exacerbations o f those c o n d i t i o n s ( B a r d i n , 1 9 9 2 ; G e r n & B u s s e , 1 9 9 9 ; G e r n , 2 0 0 2 ; G r u n b e r g et a l . , 1 9 9 7 ; G r u n b e r g & Sterk, 1 9 9 9 ; H a l p e r i n , 1 9 8 5 ; Johnston et a l . , 1995; S i n g h et a l . , 2 0 0 6 ) . S u r p r i s i n g l y , R V s replicate at r e l a t i v e l y l o w levels i n the a i r w a y e p i t h e l i a l tissues, and m o u n t i n g e v i d e n c e suggests that c o l d s y m p t o m s are the result o f a p r o n o u n c e d host i n f l a m m a t o r y response to i n f e c t i o n characterized b y the secretion o f p r o i n f l a m m a t o r y c y t o k i n e s s u c h as i n t e r l e u k i n (IL)-6, a n d not R V r e p l i c a t i o n ( G w a l t n e y , 2 0 0 2 ; G r u n b e r g et a l . , 1 9 9 7 ; H e n d l e y & G w a l t n e y , 2 0 0 4 ; L o p e z - S o u z a et a l . , 2 0 0 4 ) . F u r t h e r m o r e , in vivo studies have l i n k e d increases i n p r o - i n f l a m m a t o r y c y t o k i n e secretion to increases i n severity o f c o l d s y m p t o m s ( G w a l t n e y et a l . , 2 0 0 3 ) . A i r w a y e p i t h e l i a l c e l l s , both in vivo and in vitro, have demonstrated the a b i l i t y to secrete various c y t o k i n e s ( i n c l u d i n g IL-6) i n response to e n v i r o n m e n t a l stresses i n c l u d i n g R V i n f e c t i o n ( A r n o l d , 1994; B e r g et a l . , 2 0 0 4 ; Johnston et a l . , 1 9 9 8 ; L o p e z - S o u z a et a l . , 2 0 0 4 ; S h a r m a et a l . , 2 0 0 6 ; S p a n n h a k e et a l . , 2 0 0 2 ; T a k i z a w a et a l . , 2 0 0 0 ; et a l . , 1 9 9 8 ; Z h u et a l . , 1 9 9 7 ; Z h u et al., 1996). N o cure o r p r e v e n t i o n f o r R V i n f e c t i o n exists a n d most drugs o n l y act to alleviate s y m p t o m s . V a c c i n e s have been d i f f i c u l t to d e v e l o p because o v e r 100 p o o r l y crossn e u t r a l i z i n g serotypes o f R V persist. Scientists have d e v e l o p e d v a r i o u s R V anti-viral c o m p o u n d s , such as P l e c o n a r i l a n d R u p r i n t r i v i r , w h i c h prevent R V r e p l i c a t i o n o r entry across the p l a s m a m e m b r a n e ( H a y d e n et a l . , 2 0 0 3 ; Z h a n g et a l . , 2 0 0 4 ) . H o w e v e r , c o n s i d e r i n g the p o s s i b i l i t y that R V m a y stimulate c y t o k i n e secretion f r o m c e l l s w i t h o u t the necessity o f R V r e p l i c a t i o n o r c e l l entry, anti-viral d e v e l o p m e n t m a y b e f u t i l e .  A version of this chapter will be submitted for publication. Machala, A . M . , Harris, R.A., Brauner, C.J., Hudson, J.B.  3  88  A g r o w i n g n u m b e r o f researchers are i n v e s t i g a t i n g the a b i l i t y o f i m m u n e - m o d u l a t i n g c o m p o u n d s to mitigate R V - a s s o c i a t e d s y m p t o m s . F o r e x a m p l e , c o m p o u n d s capable o f down-regulating p r o - i n f l a m m a t o r y c y t o k i n e secretion c o u l d c o n c e i v a b l y reduce c o l d s y m p t o m s . E c h i n a c e a is a p o p u l a r natural herb extract w h i c h is thought to have i m m u n e m o d u l a t i n g effects. T h e r e is evidence that E c h i n a c e a has s t i m u l a t o r y effects on macrophages, m o n o c y t e s , T l y m p h o c y t e s , natural k i l l e r c e l l s , and e p i t h e l i a l c e l l s , resulting i n increased p r o - i n f l a m m a t o r y c y t o k i n e release (Brousseau & M i l l e r , 2 0 0 5 ; B r u s h et a l . , 2 0 0 6 ; C u r r i e r & M i l l e r , 2 0 0 0 ; G o e l , 2 0 0 5 ; M o z z a r o n i et a l . , 2 0 0 5 ; S a s a g a w a et a l . , 2 0 0 6 ; S h a r m a et a l , 2 0 0 6 ) . H o w e v e r , S h a r m a et al. (2006) s h o w e d that w h e n E c h i n a c e a was administered to R V infected a i r w a y e p i t h e l i a l c e l l s , R V - i n d u c e d c y t o k i n e secretion was i n h i b i t e d , suggesting a m o r e c o m p l e x interaction between the herb extract and virus infected c e l l s than that observed i n u n i n f e c t e d c e l l s . C l i n i c a l trials i n v e s t i g a t i n g E c h i n a c e a have y i e l d e d i n c o n c l u s i v e results ( G o e l et a l . , 2 0 0 4 ; Sperber et a l . , 2 0 0 4 ; T u r n e r et a l . , 2 0 0 5 ; T u r n e r et a l . , 2 0 0 0 ) , and the q u a l i t y o f m a n y c o m m e r c i a l f o r m u l a t i o n s is questionable ( G i l r o y et a l . , 2 0 0 3 ; K r o c h m a l et a l . , 2 0 0 4 ) . S t a n d a r d i z e d in vitro studies are needed to further elucidate the effects o f E c h i n a c e a i n R V infected a i r w a y e p i t h e l i a l c e l l s . T h i s study a i m e d to assess the effect o f t w o c h e m i c a l l y distinct E c h i n a c e a extracts on cultured b r o n c h i a l e p i t h e l i a l cells ( B E A S - 2 B ) infected w i t h R V 1 4 or R V 1 A . V i r a l r e p l i c a t i o n and IL-6 secretion were measured i n order to address the hypothesis that E c h i n a c e a is i m m u n e - m o d u l a t o r y and stimulates IL-6 i n u n i n f e c t e d c e l l s , but i n h i b i t s R V i n d u c e d c y t o k i n e secretion i n c u l t u r e d a i r w a y e p i t h e l i a l c e l l s .  MATERIALS AND METHODS A l l v i r a l , c e l l culture and m o l e c u l a r w o r k was c o n d u c t e d under sterile c o n d i t i o n s i n a type II b i o s a f e t y cabinet. A l l p r o t o c o l s were pre-approved b y the U B C b i o s a f e t y c o m m i t t e e i n certificate H 0 4 - 0 0 6 1 ( A p p e n d i x A ) .  C e l l C u l t u r e : T h e S V 4 0 adenovirus transformed h u m a n b r o n c h i a l e p i t h e l i a l c e l l l i n e ( B E A S - 2 B ) was obtained f r o m the A m e r i c a n T y p e C u l t u r e C o l l e c t i o n ( A T C C , R o c k v i l l e , M D ) and c u l t u r e d i n 7 5 m m flasks i n 5 0 : 5 0 D u l b e c c o ' s M o d i f i e d E a g l e ' s M e d i u m 2  89  ( D M E M ) and H a m ' s F 1 2 w i t h 1 0 % e n d o t o x i n free fetal b o v i n e s e r u m ( F B S ) . C u l t u r e reagents were obtained f r o m Invitrogen ( V a n c o u v e r , C a n a d a ) . C e l l s were passaged w e e k l y and incubated at 35-37°C w i t h 5 % c a r b o n d i o x i d e i n 9 5 % air. R V - s e n s i t i v e H I c e l l s ( f r o m A T C C ) w e r e cultured under the same c o n d i t i o n s w i t h D M E M and 5 % F B S .  Echinacea Extracts: T w o c o m m e r c i a l preparations ( E l and E 2 ) were a n a l y z e d f o r their major constituents. E l was a spray d r i e d expressed j u i c e extract o f the aerial parts o f E. purpurea (accession n u m b e r U O 1 9 1 8 0 ) . E l was r i c h i n water extractable p o l y s a c c h a r i d e s , w i t h a total extractable p o l y s a c c h a r i d e content o f 2 3 . 7 % w/w ( S h a r m a et a l . , 2 0 0 6 ) . E 2 was a 5 5 % ethanolic tincture f r o m E. purpurea roots (1:9 w/v). H i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y analysis o f the E 2 tincture s h o w e d the presence o f a l k a m i d e s and c a f f e i c a c i d derivatives (caftaric a c i d 5 9 . 5 u g / m L , c h l o r o g e n i c a c i d 1 9 . 3 p g / m L , c a f f e i c a c i d 2 . 4 p g / m L , c y n a r i n e O p g / m L , e c h i n a c o s i d e O p g / m L , c i c h o r i c a c i d 3 7 u g / m L and tetraene a l k y l a m i d e s 8 0 . 5 u g / m L ) . B o t h o f these extracts were a n a l y z e d p r e v i o u s l y b y B i n n s et al. (2002) and p r o v i d e d to our laboratory. Extracts w e r e filtered at 0 . 2 p m (precharacterization), d i l u t e d i n culture m e d i u m , and stored at -20°C. Viruses: B o t h R V 1 4 and R V 1 A were obtained f r o m the A T C C . R V s were propagated b y i n f e c t i n g H I c e l l s g r o w n to c o n f l u e n c e i n 7 5 m m flasks c o n t a i n i n g D M E M , a l l o w i n g f o r 2  f u l l cytopathic effects ( C P E ) . Cell-free culture f l u i d was harvested w h e n C P E were at a m a x i m u m b y centrifugation at 10,000 x g for 2 0 minutes at 4 ° C . T h e stock v i r u s suspension was a l i q u o t e d into c r y o v i a l s and stored at -80°C f o r e x p e r i m e n t a l use. T i t e r o f v i r a l stock was determined b y v i r a l plaque assay (see b e l o w ) o f s e r i a l l y d i l u t e d stock, and expressed i n plaque f o r m i n g units (pfu) per m L . P f u represent the n u m b e r o f infectious R V particles present i n a k n o w n v o l u m e o f sample. F o r e x p e r i m e n t s , a l i q u o t e d stock virus was r a p i d l y thawed at 37°C and v o r t e x e d p r i o r to use. B e c a u s e such c l a r i f i e d v i r a l stocks c o n t a i n H I c e l l remnants (e.g. s o l u b l e proteins, organelles) c o n t r o l e x p e r i m e n t s were c o n d u c t e d to c o n f i r m that any o b s e r v e d changes i n IL-6 and IL-8 secretion w e r e due to v i r u s and not some other c e l l u l a r c o m p o n e n t present i n the i n o c u l a ( A p p e n d i x C ) .  90  Viral Infections: B E A S - 2 B  c e l l s w e r e c u l t u r e d i n 6-well plates u n t i l freshly c o n f l u e n t i n  5 0 : 5 0 D M E M : H a m ' s F 1 2 m i x w i t h 1 0 % F B S . O n c e c e l l s reached c o n f l u e n c e it was assumed that c e l l n u m b e r d i d not change s i g n i f i c a n t l y o v e r the course o f experiments f o r either c o n t r o l o f R V infected cultures. T h i s a s s u m p t i o n was c o n f i r m e d e x p e r i m e n t a l l y ( A p p e n d i x D ) . C e l l n u m b e r per w e l l (6-well plate) at c o n f l u e n c e was pre-determined f o r c e l l lines ( A p p e n d i x E ) and used to calculate v i r a l dose. P r i o r to infections m e d i a was aspirated and replaced w i t h 0 . 7 5 m L o f R V 1 A or R V 1 4 i n o c u l a at a m u l t i p l i c i t y o f i n f e c t i o n ( M O I ) = l , o r m o c k i n f e c t e d w i t h m e d i u m . C e l l s were incubated at 35°C f o r 1 hour. F o l l o w i n g i n f e c t i o n i n o c u l a were aspirated and c e l l s were w a s h e d 3 times w i t h l m L o f m e d i u m i n order to r e m o v e exogenous v i r u s (virus r e m o v a l e x p e r i m e n t a l l y c o n f i r m e d , not s h o w n ) . F o l l o w i n g this, fresh culture m e d i u m c o n t a i n i n g either: 5 0 p g / m L o f E l , 1:50 E 2 / m e d i u m d i l u t i o n , 0 . 9 % ethanol ( E 2 v e h i c l e c o n t r o l ) , or m e d i u m alone (control) w a s added to w e l l s , and c e l l s were incubated at 35°C i n a 5 % c a r b o n d i o x i d e incubator. S a m p l e s were c o l l e c t e d at the same t i m e i m m e d i a t e l y post-infection and washes (Ohrs), and 4 8 hours (48hrs), and 96 hours (96hrs) later. S a m p l i n g consisted o f first r e m o v i n g l m L o f supernatant f r o m appropriate w e l l s , c e n t r i f u g i n g at 1000 x g to r e m o v e c e l l u l a r debris, and f r e e z i n g samples at -20°C f o r future c y t o k i n e / c h e m o k i n e assays. C e l l s f r o m R V infected samples were scraped into the r e m a i n d e r o f the m e d i u m ( 2 m L ) , pipetted into c r y o v i a l s , and stored at -80°C f o r plaque assays.  Plaque Assays:  V i r a l i n f e c t i v i t y and r e p l i c a t i o n were measured b y plaque assays i n  p e r m i s s i v e H I c e l l s . P r e v i o u s l y f r o z e n R V infected c e l l samples f r o m G r o w t h C u r v e s were r a p i d l y f r o z e n and t h a w e d t w i c e at 37°C to rupture c e l l s and release v i r u s . These samples were then s e r i a l l y d i l u t e d and used to infect p e r m i s s i v e H I c e l l s i n duplicate where 0 . 7 5 m L o f s a m p l e was added onto freshly c o n f l u e n t H I c e l l s g r o w n i n 6-well trays and a l l o w e d incubated f o r 1 h o u r at 35°C. A f t e r i n f e c t i o n the i n o c u l a w e r e aspirated and replaced w i t h a 5 0 : 5 0 l i q u i d m i x t u r e o f 2x M E M ( w i t h 5 % F B S ) and 1 % sterile agarose i n d H 2 0 . T h e agarose was a l l o w e d to s o l i d i f y at r o o m temperature, and plates were i n c u b a t e d at 35°C for 4 days. A s the v i r u s replicated i n infected c e l l s , l y s i s o c c u r r e d i n the H I c e l l s f o r m i n g r o u n d areas o f c e l l death c a l l e d " p l a q u e s " . O n c e i n c u b a t i o n was c o m p l e t e , plates were f i x e d w i t h 3 % f o r m a l d e h y d e i n phosphate b u f f e r e d saline, agarose was r e m o v e d , and H I  91  c e l l s were stained w i t h 1 % c r y s t a l v i o l e t i n d H 2 0 to reveal the clear unstained plaques. Plaques were counted and reported as p f u / m L . A n increase i n infectious v i r u s over time (relative to DO) i n d i c a t e d v i r a l r e p l i c a t i o n .  Cytokines IL-6 w a s assayed f r o m supernatant samples u s i n g standard p r o t o c o l p r o v i d e d b y c o m m e r c i a l l y a v a i l a b l e e n z y m e - l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) kits f r o m I m m u n o t o o l s (Friesoythe, G e r m a n y ) . A b s o r b e n c i e s were read on an E L I S A plate reader (Pasteur D i a g n o s t i c s L P 4 0 0 ) at a 4 5 0 n m w a v e l e n g t h . H i g h l y concentrated samples were d i l u t e d w i t h m e d i u m and re-assayed to f a l l w i t h i n the standard assay range w h i c h was f r o m 0-450 p g / m L . S e n s i t i v i t y o f the assay was 4 p g / m L .  S t a t i s t i c s : Infectious virus (replication) data (n=3 per treatment f o r each s a m p l i n g time) were a n a l y z e d b y two-way analysis o f variance ( A N O V A ) w i t h day and treatment as factors, f o l l o w e d b y post-hoc T u k e y tests. F o r IL-6 secretion (n=3 for each treatment) a one-way A N O V A w i t h treatment as a factor was p e r f o r m e d w i t h post-hoc T u k e y tests in order to c o m p a r e a l l treatments. A l l data w e r e a n a l y z e d u s i n g S i g m a Stat 3.0 software and statistical s i g n i f i c a n c e was set at a = 0 . 0 5 . S i g n i f i c a n t statistical differences c o r r e s p o n d to p<0.05, and h i g h l y s i g n i f i c a n t statistical differences c o r r e s p o n d to p<0.001. R e s u l t s are presented as m e a n ± standard error o f the m e a n .  RESULTS C P E : N o C P E were observed f o r any o f the samples i n c l u d i n g all R V infected c e l l s , E l and E 2 treated c e l l s , and R V + E c h i n a c e a treated c o m b i n a t i o n s . R V 1 4 R e p l i c a t i o n ( F i g u r e 4.1a): T h e r e was a h i g h l y s i g n i f i c a n t difference i n infectious v i r u s between Ohrs and 48hrs (all treatments c o m b i n e d ) i n d i c a t i n g v i r a l r e p l i c a t i o n , but no v i r a l r e p l i c a t i o n was detected at 96hrs (relative to Ohrs). A v e r a g e o f Ohrs treatment p f u / m L values (all treatments c o m b i n e d ) was 3.6 x 1 0 ± 9.3 x 1 0 p f u / m L , i n c r e a s i n g to 3.5 x 1 0 ± 3  1  4  7.9 x 1 0 p f u / m L at 48hrs. In c o m p a r i n g treatments, o n l y R V and R V + E t o h were 2  s i g n i f i c a n t l y different, and this difference was o n l y o b s e r v e d at 4 8 h r s , but was h i g h l y significant.  92  RV14  and IL-6 Secretion ( F i g u r e 4.1b): T h e r e is a h i g h l y s i g n i f i c a n t difference between  treatment groups. N o n e o f the u n i n f e c t e d c e l l treatments ( C , E l , E 2 , E t o h ) were s i g n i f i c a n t l y different f r o m each other. A l l R V infected treatments: R V , R V + E 1 , R V + E 2 , and R V + E t o h were f o u n d to be s i g n i f i c a n t l y different f r o m u n i n f e c t e d c e l l s . W i t h i n R V infected c e l l s there were no s i g n i f i c a n t differences between R V , R V + E 1 or R V + E t o h ; h o w e v e r , there was a s i g n i f i c a n t difference between R V + E 2 and a l l other treatments. R V + E 2 represented peak IL-6 secretion at 243.4 ± 9.3 p g / m L . RV1A  Replication ( F i g u r e 4.2a): S i g n i f i c a n t v i r a l r e p l i c a t i o n relative to Ohrs was observed  at 48hrs but not 96hrs (all treatments c o m b i n e d ) . A v e r a g e p f u / m L o f Ohrs treatments (all treatments c o m b i n e d ) w a s 3.6 x 10 ± 9.3 x 1 0 p f u / m L i n c r e a s i n g to 3.5 x 10 ± 7.9 x 1 0 3  1  4  2  p f u / m L at 48hrs. N o s i g n i f i c a n t differences were observed between treatments. RV1A  and IL-6 Secretion ( F i g u r e 4.2b): A h i g h l y s i g n i f i c a n t d i f f e r e n c e was f o u n d  between treatment groups. O n l y R V and R V + E t o h treatments w e r e s i g n i f i c a n t l y different f r o m a l l other treatments (83.0 ± 10.2 p g / m L ) , but they were not s i g n i f i c a n t l y different f r o m each other. Peak IL-6 concentration ( R V treatment) was 349.3 ± 49.7 p g / m L .  DISCUSSION E c h i n a c e a is the most p o p u l a r natural extract used to treat c o m m o n upper respiratory tract i n f e c t i o n s t y p i c a l l y caused b y R V i n f e c t i o n . H i s t o r i c a l l y , crude E c h i n a c e a extracts i n various f o r m s have been used to treat and/or prevent a variety o f infections (Barrett, 2 0 0 3 ) . H o w e v e r , no m o d e r n consensus on the potential health benefits o f E c h i n a c e a f o r the treatment o f R V i n f e c t i o n exists, and a p o s s i b l e m e c h a n i s m o f action is l a r g e l y u n k n o w n . R V Replication: D e p e n d i n g on the source c o n s u l t e d , the effects o f E c h i n a c e a have been d e s c r i b e d as i m m u n e - s t i m u l a t o r y , i m m u n e - p r o t e c t i v e and/or anti-viral. A l t h o u g h g r o w i n g research supports the i d e a that E c h i n a c e a ' s effects are l a r g e l y i m m u n e - m e d i a t e d the above c l a i m s are s o m e w h a t a m b i g u o u s and contribute to the c o n t r o v e r s y s u r r o u n d i n g this natural m e d i c i n e . F o r e x a m p l e , although there is some evidence that E c h i n a c e a has a v i r u c i d a l effect on viruses such as herpes s i m p l e x virus-1 ( B i n n s et a h , 2 0 0 2 ) , there is no evidence o f any effect on v i r a l r e p l i c a t i o n thus broad " a n t i - v i r a l " c l a i m s m a y be m i s l e a d i n g . In m y experiments s i g n i f i c a n t R V r e p l i c a t i o n was detected at 48hrs relative to Ohrs, but there was no differences observed between E c h i n a c e a treated and untreated c e l l s f o r either extract  93  f o r m u l a t i o n i n R V 1 4 or R V 1 A infected B E A S - 2 B cells. T h e s e data suggest that E c h i n a c e a most l i k e l y confers its p h y s i o l o g i c a l effects through an interaction w i t h the host c e l l s rather than b y a f f e c t i n g v i r a l r e p l i c a t i o n . IL-6  Secretion: IL-6 is a p r o - i n f l a m m a t o r y c y t o k i n e secreted f r o m a variety o f c e l l s ,  i n c l u d i n g a i r w a y e p i t h e l i a l c e l l s , i n response to i n j u r y and stress such as v i r a l i n f e c t i o n (Janeway, 2 0 0 5 ) . There is evidence that R V i n f e c t i o n can stimulate IL-6 secretion i n a i r w a y e p i t h e l i a l cells ( S h a r m a et a l . , 2 0 0 6 ) , and increases i n p r o - i n f l a m m a t o r y c y t o k i n e s have been l i n k e d to i n t e n s i f i e d c o l d s y m p t o m s ( G w a l t n e y et a l . , 2 0 0 3 ) . T h i s evidence is also supported b y m y f i n d i n g s , as R V and R V + E t o h treated c e l l s s h o w e d s i g n i f i c a n t l y increased IL-6 concentrations c o m p a r e d to c o n t r o l . S e v e r a l hypotheses have been postulated about h o w the i m m u n e - m o d u l a t i n g effects o f E c h i n a c e a m a y translate into health benefits. S o m e scientists have suggested that E c h i n a c e a stimulates an i n f l a m m a t o r y response, thus p r o t e c t i v e l y " h e i g h t e n i n g " the i m m u n e system (cited i n Barrett, 2 0 0 3 ) ; h o w e v e r , b e a r i n g i n m i n d that p r o - i n f l a m m a t o r y m e d i a t o r release is thought to cause c o l d s y m p t o m s further exaggerating this process seems c o u n t e r p r o d u c t i v e . A m o r e p l a u s i b l e hypothesis m a y be that E c h i n a c e a down-regulates R V i n d u c e d i n f l a m m a t o r y responses, such as IL-6 secretion, thereby r e d u c i n g c o l d s y m p t o m s . Interestingly, E c h i n a c e a treatment o f u n i n f e c t e d c e l l s f o r both experiments d i d not y i e l d any s i g n i f i c a n t IL-6 secretion relative to c o n t r o l . These results d i f f e r f r o m some p u b l i s h e d studies i n d i c a t i n g an increase o f p r o - i n f l a m m a t o r y c y t o k i n e release after treatment w i t h E c h i n a c e a ( H w a n g et a l . , 2 0 0 4 ; S h a r m a et a l . , 2 0 0 6 ) . It m a y be that the concentration o f the extracts a d m i n i s t e r e d was not sufficient to stimulate c e l l u l a r IL-6 secretion, or perhaps some o f the active c o m p o u n d s had degraded d u r i n g extract storage. O n the other h a n d , s i g n i f i c a n t differences i n IL-6 secretion were o b s e r v e d f o r both R V 1 4 and R V 1 A infected c e l l s treated w i t h E c h i n a c e a , i n d i c a t i n g that the extracts were b i o l o g i c a l l y active. C o n s i d e r i n g that crude E c h i n a c e a preparations contain m a n y i m m u n o l o g i c a l l y active c o m p o u n d s (e.g. a l k a m i d e s and c a f f e i c a c i d derivatives), it is p o s s i b l e that different constituents (or c o m b i n a t i o n s thereof) are responsible f o r E c h i n a c e a ' s contrasting effects o n R V infected and u n i n f e c t e d c e l l s . A divergent trend was observed f o r IL-6 secretion between R V 1 4 and R V 1 A . In R V 1 4 infected c e l l s , IL-6 secretion was not s i g n i f i c a n t l y different for R V + E 1 , but was  94  s i g n i f i c a n t l y elevated f r o m other R V infected treatments for R V + E 2 . H o w e v e r , i n R V 1 A infected c e l l s , E c h i n a c e a treatment resulted i n IL-6 concentrations that were s i m i l a r to c o n t r o l l e v e l s , even t h o u g h R V and R V + E t o h treatments stimulated s i g n i f i c a n t IL-6 secretion. T h e d i f f e r e n c e s observed between serotypes are d i f f i c u l t to e x p l a i n because the p h y s i o l o g i c a l m e c h a n i s m o f E c h i n a c e a is l a r g e l y u n k n o w n . S o m e studies p r o p o s e an nuclear factor kappa-B ( N F K B ) dependent p a t h w a y f o r the action o f this herbal extract, and o v e r a l l such a m e c h a n i s m is p l a u s i b l e , c o n s i d e r i n g that N F K B is i m p l i c a t e d i n b o t h p r o i n f l a m m a t o r y c y t o k i n e secretion and R V p a t h o l o g y ( Z h u et a l . , 1997). H o w e v e r , one study c o n d u c t e d b y our l a b o r a t o r y ( S h a r m a et a l . , 2 0 0 6 ) s h o w e d that E c h i n a c e a treatment o f B E A S - 2 B c e l l s resulted i n changes o f o v e r 30 t r a n s c r i p t i o n factors ( i n c l u d i n g N F K B ) , d e m o n s t r a t i n g the i n v o l v e m e n t o f c o m p l e x b i o c h e m i c a l pathways. R e c e n t research has f o u n d that a l k a m i d e s i n E c h i n a c e a extracts b i n d the c a n n a b i n o i d type-2 receptor and m a y affect N F K B t r a n s c r i p t i o n b y this p a t h w a y (Gertsch et a l . , 2 0 0 4 ; R a d u n e r et a l . , 2 0 0 6 ) . R V 1 4 and R V 1 A also u t i l i z e different receptors to infect c e l l s (intercellular adhesion molecule-1 and l o w d e n s i t y l i p o p r o t e i n receptors r e s p e c t i v e l y ) ; therefore, the cascades s i g n a l e d b y the b i n d i n g o f these receptors m a y lead to differences i n c y t o k i n e secretion. In another study, S h a r m a et al. (2006) s h o w e d increased IL-6 secretion f r o m B E A S - 2 B c e l l s u p o n E c h i n a c e a treatment alone and decreased secretion w h e n E c h i n a c e a was a d m i n i s t e r e d to R V 1 4 i n f e c t e d c e l l s . A l t h o u g h the same trend was not o b s e r v e d f o r m y R V 1 4 e x p e r i m e n t s , R V 1 A d i d support the S h a r m a et al. (2006) f i n d i n g s . C o n s i d e r i n g that these are the o n l y studies i n v e s t i g a t i n g the effects o f E c h i n a c e a o n R V i n f e c t e d a i r w a y e p i t h e l i a l c e l l s , m o r e e v i d e n c e is needed before definite c o n c l u s i o n s c a n be d r a w n . E l and E 2 Echinacea Extracts: T h e q u a l i t y o f c o m m e r c i a l l y a v a i l a b l e E c h i n a c e a extracts is questionable ( G i l r o y et a l . , 2 0 0 3 ; K r o c h m a l et a l . , 2 0 0 4 ) . O n e study f o u n d that 3 9 % o f E c h i n a c e a extracts c o n t a i n e d m o r e or less E c h i n a c e a than i n d i c a t e d w h i l e 1 0 % o f the products c o n t a i n e d n o E c h i n a c e a at a l l ( G i l r o y et a l . , 2 0 0 3 ) . F u r t h e r m o r e , the best m o d e o f a d m i n i s t r a t i o n (e.g. caplet, tincture, tea) or the proper dosage is i l l - d e f i n e d . E v e n i n standardized preparations o f E c h i n a c e a , the n u m b e r and concentrations o f b i o l o g i c a l l y active constituents d i f f e r greatly due to factors such as species (E. purpurea, E. pallida, E. angustifolia), part o f plant u t i l i z e d (e.g. root, leaf, aerial) and m e t h o d o f e x t r a c t i o n (e.g. a l c o h o l i c , aqueous) ( A d i n o l f i et a l . , 2 0 0 6 ; S l o l e y et a l . , 2 0 0 1 ) . T h i s l a c k o f extract  95  h o m o l o g y certainly contributes to the c o n t r o v e r s y s u r r o u n d i n g the therapeutic benefits o f E c h i n a c e a , e s p e c i a l l y i n c l i n i c a l studies where so m a n y a d d i t i o n a l c o n f o u n d i n g factors m a y exist ( G a g n i e r et a l . , 2 0 0 6 ; Sperber et a l . , 2 0 0 4 ; T u r n e r et a l . , 2 0 0 0 ; T u r n e r et a l . , 2 0 0 5 ; W o l s k o et a l . , 2 0 0 5 ) . A s p r e v i o u s l y m e n t i o n e d , no effects o f either E l or E 2 were observed f o r either R V 1 4 or R V 1 A r e p l i c a t i o n i n the B E A S - 2 B c e l l s . In m y R V 1 A experiments both E l and E 2 suppressed R V s t i m u l a t i o n o f IL-6 secretion i n a s i m i l a r manner, w h i l e i n R V 1 4 infected c e l l s E l f a i l e d to suppress R V i n d u c e d IL-6 secretion and E 2 seemed to exaggerate the IL6 response. A g a i n , these data suggest that d i s t i n c t i v e E c h i n a c e a extracts m a y interact i n c o m p l e x w a y s w i t h v a r y i n g R V serotypes. A n ethanolic v e h i c l e c o n t r o l was i n c l u d e d to account f o r the potential n o n - E c h i n a c e a based differences between the aqueous and ethanolic extracts. N o s i g n i f i c a n t ethanolic effects w e r e observed f o r IL-6 secretion i n u n i n f e c t e d c e l l s regardless o f R V serotype, and i n R V infected c e l l s no IL-6 secretion b e y o n d that i n d u c e d b y the v i r u s c o u l d be observed f o r R V + E t o h treatments. In the R V 1 4 r e p l i c a t i o n data, a s i g n i f i c a n t d i f f e r e n c e was f o u n d between R V and R V + E t o h treatments; h o w e v e r , c o n s i d e r i n g the inherent v a r i a b i l i t y i n plaque assays, it is d o u b t f u l that this difference is b i o l o g i c a l l y relevant (see F i g u r e 4.1a). O v e r a l l little or no ethanolic effects were o b s e r v e d . It is b e c o m i n g i n c r e a s i n g l y clear that E c h i n a c e a extracts are capable o f m o d u l a t i n g i n f l a m m a t o r y c y t o k i n e secretion i n a variety o f c e l l s . M o r e o v e r , p r e v i o u s studies c o n d u c t e d b y o u r laboratory and m y e x p e r i m e n t s suggest that E c h i n a c e a interacts w i t h R V infected c e l l s d i f f e r e n t l y than w i t h uninfected c e l l s , at least i n the case o f the B E A S - 2 B c e l l l i n e ( S h a r m a et a l . , 2 0 0 6 ) . Future studies s h o u l d e x a m i n e the effects o f standardized E c h i n a c e a extracts o n various a i r w a y c e l l s infected b y different R V serotypes i n order to determine i f the observed effects can be c o n s e r v e d under various e x p e r i m e n t a l c o n d i t i o n s .  96  FIGURES a)  M M  I  RV  RV14  1 RV + E1  E 5 H ^ m m RV+E2 • RV+ETOH  H—  CL  o  en o  4  H  3  H  a,b  CO 3  CO 3 O  iL  o  T i m e After R V Infection (Hours)  b) 400  CD CL  c o  2  ca> o co  300 1  200  O  «? 100  H  E2  Etoh  RV  RV+E1 RV+E2 RV+Etoh  Treatment Figure 4.1: Effects of Echinacea extracts ( E l , E2) and ethanol on a) RV14 replication and b) IL-6 secretion in RV14 infected and uninfected BEAS-2B cells. BEAS-2B cells were infected with RV14 and known concentrations of either Echinacea extract: aqueous E l (RV+E1), or alcoholic E2 (RV+E2), 1% Ethanol (RV+Etoh) as an E2 vehicle control, or medium alone (RV). In a) an asterisk (*) indicates a significant difference (c£=0.05) in infectious virus (all treatments combined) at 48hrs or 96hrs relative to Ohrs control. Symbols that differ (a,b) indicate significant differences between treatments at 48hrs; no significant differences were found between treatments at Ohrs or 96hrs. In b) IL-6 secretion after 48 hours is shown. Symbols that differ (a,b,c) indicate a significant difference in IL-6 concentrations between treatments (n=3).  97  a)  400  H  300  200  100  E2  Eton  RV  RV + E1  R V + E 2 RV + Etoh  Treatment  Figure 4.2: Effects of Echinacea extracts ( E l , E2) and ethanol on a) RV1A replication and b) IL-6 secretion in RV1A infected and uninfected BEAS-2B cells. In a) no significant differences were found between treatments at any time (i.e. 0, 48, or 96 hours). In b) symbols that differ (a,b) indicate a significant difference in IL-6 concentrations between treatments (n=3). See Fig. 4.2 legend for further details.  98  REFERENCES Adinolfi, B., A . Chicca, E . Martinotti, M . C . Breschi, and P. Nieri 2 0 0 6 . Sequence  characterized a m p l i f i e d r e g i o n ( S C A R ) analysis o n D N A f r o m the three m e d i c i n a l Echinacea species. Fitoterapia. In press D O I : 1 0 . 1 0 1 6 j f i l o w . 2 0 0 6 . 0 9 . 0 1 2 .  Arnold, R. 1994. Interleukin 9, i n t e r l e u k i n 6, and s o l u b l e t u m o u r necrosis factor receptor type 1 release f r o m a h u m a n p u l m o n a r y e p i t h e l i a l c e l l line ( A 5 4 9 ) e x p o s e d to respiratory s y n c y t i a l v i r u s . I m m u n o l o g y . 82:126. Bardin, P. G . 1992. V i r u s e s as precipitants o f asthma s y m p t o m s . II. P h y s i o l o g y a n d m e c h a n i s m s . C l i n i c a l and E x p e r i m e n t a l A l l e r g y . 22:809. Barrett, B. P. 2 0 0 3 . M e d i c i n a l properties o f E c h i n a c e a : A c r i t i c a l r e v i e w . 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E c h i n a c e a extracts m o d u l a t e the pattern o f c h e m o k i n e and c y t o k i n e secretion i n rhinovirus-infected and u n i n f e c t e d e p i t h e l i a l c e l l s . Phytotherapy R e s e a r c h . 20:147-152. Sharma, M . , J . Arnason, and J . Hudson 2 0 0 6 . E c h i n a c e a E x t r a c t s M o d u l a t e the  p r o d u c t i o n o f m u l t i p l e transcription factors in u n i n f e c t e d cells and rhinovirus-infected  cells. Phytotherapy R e s e a r c h . 20:1074-1079.  Singh, A . M . , P. E . Moore, J . E . Gern, R. F . J . Lemanske, and T. Hartert 2 0 0 6 .  B r o n c h i o l i t i s to asthma: A r e v i e w and c a l l for studies o f gene-viral interactions in asthma  causation. A m e r i c a n J o u r n a l o f R e s p i r a t i o n and C r i t i c a l C a r e M e d i c i n e . 175:108-119.  Sloley, D., L . Urichuk, C . Tywin, R. Coutts, P. Pang, and J . Shan 2 0 0 1 . 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Solondz, R. S. Phillips, S. c. Schachter, and D. M . Eisenberg 2 0 0 5 . L a c k o f herbal supplement characterization i n p u b l i s h e d r a n d o m i z e d c o n t r o l l e d trials. T h e A m e r i c a n J o u r n a l o f M e d i c i n e . 118:1087-1093. Zhu, Z . , W . Tang, J . M . Gwaltney, Jr., Y . W u , and J . A . Elias 1997. R h i n o v i r u s s t i m u l a t i o n o f interleukin-8 in vivo and in vitro: R o l e o f N F - k a p p a B. A m e r i c a n J o u r n a l o f P h y s i o l o g y L u n g C e l l u l a r and M o l e c u l a r P h y s i o l o g y . 273:L814-824. Zhu, Z . , W . Tang, A . Ray, Y . W u , O . Einarsson, M . L . Landry, and J . M . Gwaltney, Jr. 1996. R h i n o v i r u s s t i m u l a t i o n o f interleukin-6 in vivo and in vitro. T h e J o u r n a l o f C l i n i c a l Investigation. 97:421-430.  102  CHAPTER 5: General Discussion and Conclusions B i o l o g i c a l Relevance of Studies: R h i n o v i r u s ( R V ) i n f e c t i o n is the most frequent acute i l l n e s s i n h u m a n s , although s u r p r i s i n g l y little is k n o w n about its pathogenesis, and its role i n the exacerbations o f serious respiratory diseases has o n l y recently been reported. E v e n i n healthy i n d i v i d u a l s , R V i n f e c t i o n impacts not o n l y o u r sense o f w e l l - b e i n g but often results i n m i s s e d days f r o m w o r k . F o r e x a m p l e , F e n d r i c k et al. (2003) reported 5 0 0 m i l l i o n i n c i d e n c e s o f n o n - i n f l u e n z a v i r a l respiratory i n f e c t i o n s per year i n the U n i t e d States w i t h an annual estimated cost burden o f o v e r 4 0 b i l l i o n d o l l a r s . M o r e o v e r , the e m e r g i n g role o f R V i n f e c t i o n i n the m o r b i d i t y and m o r t a l i t y o f diseases such as asthma and c h r o n i c obstructive p u l m o n a r y disorder ( C O P D ) illustrates the c l i n i c a l s i g n i f i c a n c e o f an i n f e c t i o n w h i c h m a y have been p r e v i o u s l y c o n s i d e r e d more nuisance than danger. Johnston et al. (1995) demonstrated that u p to 8 5 % o f asthma exacerbations i n 292 c h i l d r e n were associated w i t h v i r a l i n f e c t i o n , and p i c o r n a v i r u s e s ( R V s and enteroviruses) accounted f o r c l o s e to 6 6 % o f those i n f e c t i o n s . A study b y K h e t s u r i a n i et al. (2005) f o u n d that over 6 3 % o f asthma exacerbations were associated w i t h viruses, o f w h i c h 6 0 % were i d e n t i f i e d s p e c i f i c a l l y as R V s . F u r t h e r m o r e , e v i d e n c e suggests that patients w i t h moderate to severe C O P D are more susceptible to R V i n f e c t i o n than their healthy counterparts, and R V i n f e c t i o n causes m o r e severe s y m p t o m s i n C O P D patients than i n healthy people (Greenberg et a l . , 2 0 0 0 ) . A d d i t i o n a l l y , acute respiratory i n f e c t i o n s are the l e a d i n g cause o f infant m o r t a l i t y , w h i c h w a s i n i t i a l l y attributed to the i n f l u e n z a and respiratory s y n c y t i a l viruses; h o w e v e r , a recent study o f 263 c h i l d r e n under the age o f 12 months f o u n d that almost 5 0 % o f the viruses isolated d u r i n g upper and l o w e r acute respiratory tract i n f e c t i o n s were R V s ( K u s e l et a l . , 2 0 0 6 ) . F i n a l l y , R V i n f e c t i o n has also been i m p l i c a t e d i n cases o f v i r a l p n e u m o n i a , co-infections w i t h bacterial p n e u m o n i a , and c o m p l i c a t i o n s i n l u n g transplant recipients ( F a l s e y & W a l s h , 2 0 0 6 ; K a i s e r et a l . , 2 0 0 6 ; L e h t i n e n et a l . , 2 0 0 6 ) . U n d e r s t a n d i n g R V i n f e c t i o n i s b e c o m i n g i n c r e a s i n g l y important to scientists e s p e c i a l l y c o n s i d e r i n g the g r o w i n g research i n d i c a t i n g that R V plays an important role i n the p a t h o l o g y o f m a n y other serious c o n d i t i o n s . T h e e m e r g i n g e v i d e n c e that R V illness m a y be l a r g e l y caused b y a host i n f l a m m a t o r y response and not necessarily b y v i r a l r e p l i c a t i o n is important f r o m both a basic v i r o l o g i c a l  103  and c l i n i c a l therapeutic perspective. T h e R V m e c h a n i s m m a y illustrate a u n i q u e v i r a l " s t r a t e g y " where R V is able to incite illness w h i l e c a u s i n g little or no c e l l damage or death. T o date, it is u n k n o w n whether R V r e p l i c a t i o n is necessary i n order to cause illness. A l t h o u g h R V s y m p t o m s have been l i n k e d to increases i n p r o - i n f l a m m a t o r y m e d i a t o r secretion, some scientists suggest that v i r a l r e p l i c a t i o n m a y be the trigger f o r this i n f l a m m a t o r y response ( G w a l t n e y et a l . , 2 0 0 3 ) , w h i l e c o n v e r s e l y , l i m i t e d e v i d e n c e has s h o w n that n o n i n f e c t i o u s virus m a y be capable o f p r o v o k i n g p r o - i n f l a m m a t o r y c y t o k i n e secretion in vitro (Johnston et a l . , 1998). F u r t h e r m o r e , the e m e r g i n g i m p l i c a t i o n o f the host i m m u n e response as the cause o f R V - a s s o c i a t e d illness has i n s p i r e d scientists to investigate R V treatments, such as E c h i n a c e a , w h i c h are thought to mediate s y m p t o m s l a r g e l y through host i m m u n i t y .  G e n e r a l D i s c u s s i o n : M y studies investigated the relationships between R V r e p l i c a t i o n and p r o - i n f l a m m a t o r y c y t o k i n e / c h e m o k i n e secretion f o r t w o different r e c e p t o r - u t i l i z i n g R V serotypes ( R V 1 4 and R V 1 A ) u s i n g t w o distinct a i r w a y e p i t h e l i a l c e l l m o d e l s ( B E A S - 2 B and A 5 4 9 ) . I also investigated the necessity o f R V r e p l i c a t i o n i n s t i m u l a t i n g an i n f l a m m a t o r y response b y assessing the a b i l i t y o f n o n i n f e c t i o u s virus to stimulate an i n t e r l e u k i n (IL)-6 response i n the B E A S - 2 B m o d e l . F i n a l l y , I studied the effects o f t w o c h e m i c a l l y characterized E c h i n a c e a extracts on both v i r a l r e p l i c a t i o n and p r o - i n f l a m m a t o r y IL-6 secretion. F o r the G r o w t h C u r v e experiments (Chapter 2), I f o u n d that s i g n i f i c a n t R V r e p l i c a t i o n o c c u r r e d i n both B E A S - 2 B and A 5 4 9 cells between day 1 ( D l ) and D 2 post-infection; h o w e v e r , r e p l i c a t i o n was no longer detectable after D 3 i n all cases. F u r t h e r m o r e , the levels o f v i r a l r e p l i c a t i o n were r e l a t i v e l y l o w w h e n c o m p a r e d to the p e r m i s s i v e H I cells w h i c h p r o d u c e d R V titers 3-4 orders o f m a g n i t u d e h i g h e r than either o f the a i r w a y epithelial c e l l m o d e l s . F o r e x a m p l e , H I c e l l peak titers reached 1 0 ~ p f u / m L w h i c h corresponds to 7  8  a p p r o x i m a t e l y 10-100 i n f e c t i o u s R V particles present per c e l l . H o w e v e r , peak R V titers f o r B E A S - 2 B and A 5 4 9 c e l l s t y p i c a l l y represented 0-0.1 i n f e c t i o u s v i r a l particles present per c e l l . F u r t h e r m o r e , R V 1 4 d i d not appear to replicate i n the A 5 4 9 c e l l s . R V 1 4 R N A levels also supported the r e p l i c a t i o n data, where H I cells p r o d u c e d levels o f v i r a l R N A m u c h h i g h e r than i n either o f the a i r w a y m o d e l s . T h i s e v i d e n c e supports the hypothesis that R V  104  replicates at l o w levels i n the a i r w a y e p i t h e l i a l c e l l s , and these trends w e r e supported f o r both a i r w a y m o d e l s and R V serotypes. H o w e v e r it is unclear i f the m a j o r i t y o f the cells were infected w i t h R V each p r o d u c i n g l o w levels o f v i r u s , or whether a s m a l l n u m b e r o f cells replicated the virus at v e r y h i g h levels. A d d i t i o n a l l y , as h y p o t h e s i z e d , no cytopathic effects ( C P E ) w e r e o b s e r v e d i n any o f the experiments i n v o l v i n g a i r w a y e p i t h e l i a l c e l l s , suggesting that v i r a l p r o g e n y either have some u n k n o w n m e c h a n i s m o f c r o s s i n g the p l a s m a m e m b r a n e w i t h o u t l y s i n g c e l l s , or perhaps a v e r y s m a l l n u m b e r o f cells ruptured r e l e a s i n g their v i r a l p r o g e n y . In vivo, M o s s e r et al. (2002 and 2005) f o u n d i m m u n o h i s t o c h e m i c a l evidence o f patchy R V i n f e c t i o n a f f e c t i n g 5 - 1 0 % o f the cells w i t h f e w or n o abnormalities o f the a i r w a y e p i t h e l i a l tissues, suggesting that perhaps o n l y a s m a l l p r o p o r t i o n o f cells are affected b y R V . F o r the majority o f experiments R V was also f o u n d to stimulate p r o - i n f l a m m a t o r y IL-6 and/or IL-8 secretion and these elevated secretions were t y p i c a l l y observed after the second day post-infection and persisted w e l l b e y o n d s i g n i f i c a n t R V r e p l i c a t i o n . T h i s e v i d e n c e supports the hypotheses that R V w o u l d stimulate p r o - i n f l a m m a t o r y c y t o k i n e / c h e m o k i n e secretion and that peak secretions w o u l d o c c u r later and persist longer than peak r e p l i c a t i o n and v i r a l R N A levels. T h e s e data also demonstrate that R V triggers a p r o n o u n c e d i n f l a m m a t o r y response f r o m cells and mirrors in vivo research w h e r e R V i n d u c e d a p r o l o n g e d i n f l a m m a t o r y response f r o m a i r w a y cells w h i c h was l i n k e d to c o l d s y m p t o m s independent o f v i r a l s h e d d i n g ( G w a l t n e y et a l . , 2 0 0 3 ) . In contrast to what was h y p o t h e s i z e d , ultraviolet ( U V ) i n a c t i v a t i o n (Chapter 3) o f R V 1 4 and R V 1 A c o m p l e t e l y i n h i b i t e d both v i r a l r e p l i c a t i o n and the a b i l i t y to stimulate an IL-6 response suggesting that g e n e t i c a l l y intact v i r u s is necessary to trigger IL-6 secretion, at least i n the B E A S - 2 B m o d e l . T h u s it m a y be the case that v i r a l r e p l i c a t i o n is the indirect cause o f R V s y m p t o m s b y s t i m u l a t i n g the host c e l l to secrete i n f l a m m a t o r y c y t o k i n e s / c h e m o k i n e s , a l t h o u g h the m e c h a n i s m is not yet k n o w n . F u r t h e r m o r e i n A p p e n d i x C , I c o n f i r m e d that i n f e c t i o u s v i r u s was necessary and responsible f o r the observed IL-6 and IL-8 s t i m u l a t i o n , and that this response c o u l d not be i n d u c e d b y other c e l l u l a r c o m p o n e n t s (e.g. s o l u b l e proteins and organelles) present i n the R V i n o c u l a . In c o m p a r i n g the c e l l m o d e l s , B E A S - 2 B cells were f o u n d to be m o r e susceptible to R V i n f e c t i o n than the A 5 4 9 c e l l s . T h e y p r o d u c e d s i g n i f i c a n t l y h i g h e r titers o f b o t h R V 1 4 and  105  R V 1 A observed between D l and D 3 post-infection and h i g h e r levels o f R V 1 4 R N A w e r e detectable i n B E A S - 2 B o n D l and D 2 i n tandem B E A S - 2 B / A 5 4 9 cultures. F u r t h e r m o r e , R V 1 4 f a i l e d to replicate i n A 5 4 9 c e l l s although i n one case an increased IL-8 response was evident. A l t h o u g h these results d i d not support the i n i t i a l hypothesis, the differences observed between c e l l m o d e l s m a y not be s u r p r i s i n g c o n s i d e r i n g that type-II surfactant secreting a l v e o l a r cells ( A 5 4 9 ) are i n t r i n s i c a l l y different f r o m b r o n c h i a l cells ( B E A S - 2 B ) . These c u l t u r e d c e l l lines most l i k e l y reflect undifferentiated basal-type c e l l s ; h o w e v e r , the A 5 4 9 m a y retain p h y s i o l o g i c a l differences such as a decreased n u m b e r o f I C A M - 1 receptors relative to B E A S - 2 B c e l l s . In general, c o n s i d e r i n g that these c e l l lines are d e r i v e d f r o m the m i d to l o w e r a i r w a y s , m y studies support the g r o w i n g e v i d e n c e that both upper and l o w e r a i r w a y e p i t h e l i a l cells are susceptible to R V i n f e c t i o n ( M o s s e r et a l . , 2 0 0 5 ; P a p a d o p o u l o s et a l . , 2 0 0 0 ; Z h u et a l . , 1996). A l t h o u g h both R V 1 4 and R V 1 A p r o d u c e d s i m i l a r trends i n r e p l i c a t i o n and p r o i n f l a m m a t o r y m e d i a t o r release, some differences were observed between serotypes. A s p r e v i o u s l y m e n t i o n e d , R V 1 4 f a i l e d to replicate s i g n i f i c a n t l y i n the A 5 4 9 c e l l s . A d d i t i o n a l l y , i n some cases there was an apparent u n c o u p l i n g between R V 1 4 r e p l i c a t i o n and s t i m u l a t i o n o f IL-6 and/or IL-8 release. F o r e x a m p l e , i n a f e w instances, although R V 1 4 r e p l i c a t i o n was evident there was no difference observed between c o n t r o l and R V infected IL-6 or IL-8 c e l l secretions (Figures 2.7b and c). In one A 5 4 9 case, a s i g n i f i c a n t increase i n IL-8 secretion was observed although no s i g n i f i c a n t r e p l i c a t i o n was detectable ( F i g u r e 2.4c). F i n a l l y , d u r i n g the first trial o f the U V e x p e r i m e n t s , R V 1 4 f a i l e d to stimulate any IL-6 response f r o m B E A S - 2 B cells e v e n though s i g n i f i c a n t v i r a l r e p l i c a t i o n was measured (Figures 3.1 a and b). T h e cause o f this d i s c r e p a n c y remains u n c l e a r ; it m a y be that i n some cases elevated c o n t r o l c y t o k i n e / c h e m o k i n e secretions m a y have m a s k e d observable differences i n R V infected c e l l s . These elevated c o n t r o l levels m a y be attributable to changes i n c e l l p h y s i o l o g y w i t h passage number, or p o s s i b l y some other c o n f o u n d i n g factor. H o w e v e r , it seems c u r i o u s that this d i s c r e p a n c y c o u l d o n l y be observed i n R V 1 4 e x p e r i m e n t s . D u r i n g t a n d e m experiments (where b o t h R V 1 4 and R V 1 A designated cells w e r e plated s i m u l t a n e o u s l y ) R V 1 A infected cells s h o w e d clear increases i n IL-6 and IL-8 secretion relative to c o n t r o l w h i l e R V 1 4 d i d not, e v e n though c o n t r o l levels f o r A 5 4 9 cells were s i m i l a r between R V serotypes. It m a y be that the v i r a l dose f o r R V 1 4  106  m a y be near some th re s h o l d f o r e l i c i t i n g a c y t o k i n e / c h e m o k i n e response w h i c h c o u l d be i n f l u e n c e d b y other factors such as c e l l age. O r perhaps R V r e p l i c a t i o n and IL-6/IL-8 responses are c o n t r o l l e d b y different m e c h a n i s m s w h i c h m a y or m a y not be related. F o r R V 1 A a consistent trend was o b s e r v e d f o r both serotypes and c e l l lines res ul ti ng i n s i g n i f i c a n t v i r a l r e p l i c a t i o n and IL-6/IL-8 release i n a l l cases. R e g a r d l e s s o f this observed d i s c r e p a n c y , it is clear that R V i n f e c t i o n o f the a i r w a y c e l l s is capable o f i n d u c i n g v i r a l r e p l i c a t i o n and the secretion o f p r o - i n f l a m m a t o r y c y t o k i n e s and c h e m o k i n e s f o r both intercellular adhesion molecule-1 ( I C A M - 1 ) and l o w density l i p o p r o t e i n receptor ( L D L R ) u t i l i z i n g R V serotypes. T h e treatment o f B E A S - 2 B cells w i t h E c h i n a c e a extracts also y i e l d e d interesting results (Chapter 4). N e i t h e r the aqueous p o l y s a c c h a r i d e - r i c h ( E l ) nor the a l c o h o l i c a l k a m i d e - r i c h (E2) E c h i n a c e a extracts had any observable effect o n R V 1 4 or R V 1 A r e p l i c a t i o n . A l t h o u g h the presence o f c i c h o r i c a c i d i n E c h i n a c e a has been s h o w n to have s o m e v i r u c i d a l effects on viruses such as herpes s i m p l e x virus-1 ( B i n n s et a l . , 2 0 0 2 ) , the lack o f i m p a c t o n R V r e p l i c a t i o n further supports the n o t i o n that E c h i n a c e a exerts most o f its effect through the host i m m u n e response. N o effect o f E c h i n a c e a on IL-6 secretion was o b s e r v e d i n u n i n f e c t e d B E A S - 2 B c e l l s . T h i s result is s u r p r i s i n g c o n s i d e r i n g that m a n y studies have reported s t i m u l a t o r y effects o f E c h i n a c e a o n v a r i o u s i m m u n e c e l l s and a i r w a y epithelial c e l l s ( B r u s h et a l . , 2 0 0 6 ; B r o u s s e a u & M i l l e r , 2 0 0 5 ; C u r r i e r & M i l l e r , 2 0 0 0 ; G o e l et a l . , 2 0 0 5 ; M o z z a r o n i et a l . , 2 0 0 5 ; S a s a g a w a et a l . , 2 0 0 6 ; S h a r m a et a l . , 2 0 0 6 ) . It m a y be that the extracts used w e r e not concentrated e n o u g h to produce such s t i m u l a t o r y effects, or s o m e o f the active constituents had degraded d u r i n g storage. H o w e v e r , there was a clear effect o f E c h i n a c e a w h e n a d m i n i s t e r e d to R V infected c e l l s , a l t h o u g h the results were m a r k e d l y different between R V serotypes. F o r R V 1 4 , E l d i d not have any s i g n i f i c a n t effect on R V infected c e l l s , w h i l e E 2 treatment stimulated further IL-6 secretion f r o m R V 1 4 infected c e l l s . H o w e v e r , both E l and E 2 treatment o f R V 1 A infected cells c o m p l e t e l y i n h i b i t e d IL-6 secretion. If E c h i n a c e a confers its health benefit b y i n h i b i t i n g p r o i n f l a m m a t o r y c y t o k i n e secretion (and therefore c o l d s y m p t o m s ) then i n this case a health benefit m a y be observed d u r i n g R V 1 A i n f e c t i o n , but not for R V 1 4 , w h e r e E 2 c o u l d a r g u a b l y exaggerate the i n f l a m m a t o r y response. B e a r i n g this i n m i n d , the e v a l u a t i o n o f  107  E c h i n a c e a treatment o f R V infected cells s h o u l d c o n s i d e r not o n l y the active constituent p r o f i l e o f the herb extracts but also the R V serotype responsible f o r i n f e c t i o n . T h e strength o f m y research is the characterization o f R V i n f e c t i o n for t w o different receptor-utilizing serotypes i n t w o distinct a i r w a y e p i t h e l i a l c e l l m o d e l s o v e r the course o f a t y p i c a l i n f e c t i o n . I have demonstrated that R V i n f e c t i o n results i n s i g n i f i c a n t v i r a l r e p l i c a t i o n and p r o - i n f l a m m a t o r y c y t o k i n e / c h e m o k i n e s t i m u l a t i o n through both the I C A M 1 and L D L R p a t h w a y s ; h o w e v e r , the s p e c i f i c trends observed are dependent o n both c e l l m o d e l and R V serotype c h o i c e . M o s t p r i o r R V studies o f this nature have been carried out b y c h o o s i n g one s p e c i f i c c e l l type and one R V serotype and s a m p l i n g o n l y at l i m i t e d time intervals. M o r e o v e r , in c o m p a r i n g R V studies, m a n y researchers treat R V serotype and c e l l c h o i c e as l a r g e l y redundant factors, but m y f i n d i n g s suggest that such b r o a d c o m p a r i s o n s m a y be o f questionable v a l i d i t y . I have also supported the f i n d i n g s o f S h a r m a et al. (2006), where IL-6 secretion f r o m a i r w a y e p i t h e l i a l c e l l s stimulated b y E c h i n a c e a treatment was different between R V infected a n d u n i n f e c t e d c e l l s , suggesting the i n v o l v e m e n t o f l a r g e l y u n k n o w n but c o m p l e x b i o c h e m i c a l pathways.  Overall Significance and Future Studies M y G r o w t h C u r v e e x p e r i m e n t s strengthen the g r o w i n g consensus that R V i n f e c t i o n causes an increase i n p r o - i n f l a m m a t o r y c y t o k i n e secretion that can be o b s e r v e d b e y o n d peak v i r a l r e p l i c a t i o n . I demonstrated this effect under c o n t r o l l e d c o n d i t i o n s for t w o different a i r w a y e p i t h e l i a l c e l l m o d e l s and f o r t w o different receptor-utilizing R V serotypes. T h e s e results offer a m o r e c o m p r e h e n s i v e e x p e r i m e n t a l e x a m i n a t i o n than p r e v i o u s l y demonstrated and s h o w that R V r e p l i c a t i o n and s t i m u l a t i o n o f c e l l p r o i n f l a m m a t o r y c y t o k i n e / c h e m o k i n e secretion occurs f o r both the I C A M - 1 and L D L R pathways. Future studies s h o u l d c o n s i d e r u t i l i z i n g other a i r w a y e p i t h e l i a l c e l l m o d e l s and p r i m a r y cultures, as w e l l as a d d i t i o n a l R V serotypes. T h e U V experiments supported p r e v i o u s f i n d i n g s i n B E A S - 2 B cells that g e n e t i c a l l y intact v i r u s is necessary to i n d u c e a p r o - i n f l a m m a t o r y c y t o k i n e response f o r both R V 1 4 and R V 1 A ( G r i e g o et a l . , 2 0 0 0 ; P a p a d o p o u l o s et a l . , 2 0 0 1 ) . F u r t h e r m o r e , m y results demonstrated that quantitative r e a l time p o l y m e r a s e c h a i n reaction ( q R T - P C R ) is capable o f d i f f e r e n t i a t i n g between U V treated and untreated R V 1 4 , an issue w h i c h is i n dispute i n the literature ( M a et a l . , 1 9 9 4 ;  108  Myatt et a l , 2003). Further studies should consider the effect of U V treated R V on the secretion of other chemokines and cytokines, and should also be conducted in the A549 cell model where some evidence of noninfectious R V stimulating IL-8 secretion exists (Johnston et al., 1998). M y Echinacea experiments support the evidence of Sharma et al. (2006) that the effects of Echinacea on cytokine/chemokine secretion in airway epithelial cells differ between R V infected and uninfected cells. Therefore, future research should concentrate on interactions between R V (and possibly other pathogen) infected cells and Echinacea extracts in addition to investigating this herbal extract's effects on uninfected "healthy" cells. Furthermore, considering the different trends observed between the two R V serotypes, it would be important to study the effects of Echinacea on cells infected with various R V serotypes, as it is possible that this extract may only confer protection against some R V s . The development of cold preventions and therapeutics has proved itself a difficult task for scientists. Since R V cold symptoms have been linked to pro-inflammatory cytokine and chemokine secretion, immune-modulating compounds such as Echinacea may offer relief from symptoms (at least for some R V serotype infections) by inhibiting the release of these mediators. Although there is growing consensus that R V replication is not the cause of cold symptoms and that the virus causes little or no damage to the airway epithelium, it remains unclear whether R V replication is necessary to trigger the inflammatory response. Some evidence (including my own) indicates that genetically intact infectious R V is necessary to stimulate pro-inflammatory cytokine/chemokine secretion. Furthermore, in studies where noninfectious R V was capable of eliciting cytokine/chemokine secretion, those observed secretions were still not as pronounced as measured in their infectious R V control counterparts. For example, Johnston et al. (1998) found that U V treated (noninfectious) R V 9 only stimulated 50% of the IL-8 secretion measured in infectious R V 9 controls, thus even in this case infectious virus may have been necessary to induce at least part of the I L 8 response. Considering the above, the best R V drug formulation could potentially be a combination of immune-modulating compounds, such as Echinacea in addition to anti-viral compounds such as Tremacamra and Pleconaril (Turner et al., 1999; Zhang et al., 2004). The timing and mode of administration of such a drug would also be important in view Of the relatively rapid onset of R V infection. Pills and capsules, which must first be  109  m e t a b o l i z e d and b i o l o g i c a l l y a v a i l a b l e i n the b l o o d s t r e a m , m a y be o f lesser benefit than preparations that c o u l d b e a p p l i e d d i r e c t l y to the affected tissues (e.g. nasal sprays). O v e r a l l , the d e v e l o p m e n t o f an effective cure f o r the c o m m o n c o l d w o u l d u n i v e r s a l l y benefit a l l people, i m p r o v i n g the w e l l - b e i n g o f the healthy, and p o t e n t i a l l y s a v i n g the l i v e s o f the vulnerable.  Conclusions D e s c r i b e d b e l o w are the general c o n c l u s i o n s that can be d r a w n f r o m this thesis i n relation to the objectives and hypotheses p r o p o s e d i n C h a p t e r 1.  Chapter 2 (In Vitro Characterization of Rhinovirus Infection in Airway Epithelial Cells - Growth Curves): 1.  A s h y p o t h e s i z e d , R V i n f e c t i o n d i d not cause any observable c e l l death o r C P E i n B E A S - 2 B o r A 5 4 9 c e l l s supporting e v i d e n c e that R V i s not c y t o t o x i c to a i r w a y epithelial cells.  2.  A s h y p o t h e s i z e d , R V replicated at r e l a t i v e l y l o w levels i n a i r w a y e p i t h e l i a l cells (0 to 0.1 i n f e c t i o u s v i r u s particles per cell) w h e n c o m p a r e d to p e r m i s s i v e H I cells (1-100 infectious v i r u s particles per c e l l ) . F u r t h e r m o r e , R V 1 4 f a i l e d to replicate i n the alveolar A 5 4 9 cells.  3.  S i g n i f i c a n t R V r e p l i c a t i o n was observed between D l and D 2 post-infection f o r all c e l l lines ( H I , B E A S - 2 B and A 5 4 9 ) . N o s i g n i f i c a n t r e p l i c a t i o n was detected f r o m D 3 o n w a r d . T h e R V r e p l i c a t i o n trends observed supported the hypothesis that v i r a l r e p l i c a t i o n w o u l d peak post-infection and g r a d u a l l y d e c l i n e to c o n t r o l levels over time.  4.  R V i n f e c t i o n stimulated p r o - i n f l a m m a t o r y IL-6 and IL-8 secretion f r o m a i r w a y e p i t h e l i a l c e l l lines. M a x i m u m c y t o k i n e / c h e m o k i n e levels w e r e t y p i c a l l y measured between D 2 and D 7 a n d , once stimulated, u s u a l l y r e m a i n e d elevated (relative to control) o v e r the course o f i n f e c t i o n . IL-6 and IL-8 levels d i d not t y p i c a l l y d e c l i n e b y D 7 as was o r i g i n a l l y h y p o t h e s i z e d .  5.  C o n s i d e r i n g that R V r e p l i c a t i o n was no l o n g e r detectable after D 2 , m y results support the hypothesis that it is the p r o - i n f l a m m a t o r y c y t o k i n e s / c h e m o k i n e s that are responsible for c o l d s y m p t o m s w h i c h t y p i c a l l y last f o r at least one week.  110  6.  A s h y p o t h e s i z e d , the b r o n c h i a l B E A S - 2 B cells were m o r e susceptible to R V i n f e c t i o n than the alveolar A 5 4 9 c e l l s . T h i s m a y suggest that b r o n c h i a l cells d e r i v e d f r o m higher i n the a i r w a y e p i t h e l i u m are m o r e susceptible to i n f e c t i o n than a l v e o l a r c e l l s .  7.  In contrast to what was h y p o t h e s i z e d , R V 1 A p r o d u c e d m o r e p r o n o u n c e d effects than R V 1 4 i n terms o f v i r a l r e p l i c a t i o n and IL-6/IL-8 secretion i n the a i r w a y e p i t h e l i a l cells suggesting substantial differences between R V serotypes.  Chapter 3 (The Effects Ultraviolet Inactivated Rhinovirus on Airway Epithelial Cells): 8.  C o n t r a r y to what was h y p o t h e s i z e d , U V treatment o f R V 1 4 and R V 1 A c o m p l e t e l y i n h i b i t e d v i r a l r e p l i c a t i o n and IL-6 secretion i n B E A S - 2 B c e l l s , i n d i c a t i n g that g e n e t i c a l l y intact (infectious) v i r u s was necessary to stimulate this response.  Chapter 4 (The Effects of Echinacea Extracts on Rhinovirus Infected and Uninfected Airway Epithelial Cells): 9.  A s h y p o t h e s i z e d , E c h i n a c e a treatment had no effect o n R V r e p l i c a t i o n suggesting that E c h i n a c e a does not affect the R V r e p l i c a t i o n c y c l e .  10. In contrast to w h a t was h y p o t h e s i z e d , E c h i n a c e a d i d not stimulate IL-6 secretion f r o m uninfected B E A S - 2 B c e l l s ; h o w e v e r , as h y p o t h e s i z e d E c h i n a c e a d i d affect IL-6 secretion i n R V infected cells suggesting c o m p l e x interactions between E c h i n a c e a extracts and R V infected c e l l s . 11. U n l i k e the o r i g i n a l hypothesis, different effects were observed f o r R V 1 4 and R V 1 A ; therefore, E c h i n a c e a m a y not confer benefit against a l l R V s . 12. A s h y p o t h e s i z e d , the t w o distinct E c h i n a c e a extracts p r o d u c e d different IL-6 results i n d i c a t i n g that the effects o f E c h i n a c e a m a y depend o n their c h e m i c a l p r o f i l e s .  U n d e r s t a n d i n g the p a t h o g e n i c i t y o f R V i n f e c t i o n i s c r u c i a l i n treating the c o m m o n c o l d and further e l u c i d a t i n g its role i n respiratory disease. F u r t h e r m o r e , the e v a l u a t i o n o f c o l d treatments, such as E c h i n a c e a , helps the p u b l i c and health practitioners m a k e m o r e  111  i n f o r m e d treatment d e c i s i o n s . M y in vitro characterization o f R V i n f e c t i o n strengthens the current s c i e n t i f i c k n o w l e d g e o f this v i r a l i n f e c t i o n , and such studies offer a b a c k b o n e f r o m w h i c h further in vivo studies and c l i n i c a l trials m a y be d e v e l o p e d .  112  REFERENCES Binns, S., J . Hudson, S. 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Quantitative and qualitative analysis o f r h i n o v i r u s i n f e c t i o n i n b r o n c h i a l tissues. A m e r i c a n J o u r n a l o f R e s p i r a t i o n and C r i t i c a l C a r e M e d i c i n e . 171:645-651.  Myatt, T . A . , S. L . Johston, S. Rudnick, and D. K . Milton 2 0 0 3 . A i r b o r n e r h i n o v i r u s  detection and effect o f ultraviolet irradiation o n detection b y a semi-nested R T - P C R assay.  B i o M e d C e n t r a l P u b l i c H e a l t h . 3:5-13.  114  Papadopoulos, N . G . , P. J . Bates, P. G . Bardin, A . Papi, S. H . Leir, D. J ; Frenkel, J . Meyer, P. M . Lackie, G . Sanderson, S. T . Holgate, and S. L . Johnston 2 0 0 0 .  R h i n o v i r u s e s infect the l o w e r a i r w a y s . T h e J o u r n a l o f Infectious Diseases. 181:1875-84. Papadopoulos, N . G . , A . Papi, J . Meyer, L . A . Stanciu, S. Salvi, S. T. Holgate, and S. L . Johnston 2 0 0 1 . 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E f f i c a c y o f T r e m a c a m r a , a s o l u b l e intercellular adhesion m o l e c u l e 1, f o r e x p e r i m e n t a l r h i n o v i r u s i n f e c t i o n : A r a n d o m i z e d c l i n i c a l trial. J o u r n a l o f the A m e r i c a n M e d i c a l A s s o c i a t i o n . 281:1797-1804. Zhang, Y . , A . A . Simpson, R. M . Ledford, C . M . Bator, S. Chakravarty, G . A . Skochko, T . M . Demenczuk, A . Watanyar, D. C . Pevear, and M . G . Rossmann 2 0 0 4 . Structural and v i r o l o g i c a l studies o f the stages o f virus r e p l i c a t i o n that are affected b y a n t i r h i n o v i r u s c o m p o u n d s . J o u r n a l o f V i r o l o g y . 78:11061-11069. Zhu, Z . , W . Tang, A . Ray, Y . W u , O . Einarsson, M . L . Landry, and J . M . Gwaltney, J r . 1996. R h i n o v i r u s s t i m u l a t i o n o f interleukin-6 in vivo and in vitro. T h e J o u r n a l o f C l i n i c a l investigation. 97:421-430.  115  APPENDIX B: Growth Curves Experimental Design  Cells grown to confluence  I Cell media aspirated and replaced with RV inocula or medium alone (control):  O 0 O O©©  ©OO  o o o  © o o  o o o o o o  o o o © o o  o o o o o ©  o o o o o o  o o o  o o o  o o o  o o o  o o o  Grey=RV Infected Black=Controls  Infection: 1 hour at 35°C  Washes (to remove exogenous RV)  I Replace medium (1% FBS) and incubate (35°C)  i At given time-point (D0-D7) take samples:  I  e.g. at D 2 (remove 2 plates)  1. IL-6/IL-8: Supernatants removed, centrifuged, and stored at -20°C for ELISAs (n=3 controls, n=3 RV)  2. Infectious Virus: RV infected  cells scraped into medium and stored at -80°C for plaque assays (n=4)  3. RV14 RNA: Media aspirated, cells washed with phosphate buffered saline, TRIZOL reagent added, stored at -80°C for RNA extraction (n=2) Samplei  •  •  Collection Legend IL-6/IL-8 Infectious virus RV14 RNA  117  APPENDIX C: Effect of Purified and Unpurified Rhinovirus Inocula on Viral Replication and Cytokine/Chemokine Secretion from BEAS-2B and A 5 4 9 Cells. R V stock solutions used i n a l l experiments were harvested f r o m l y s e d H I c e l l s w h i c h were c l a r i f i e d b y centrifugation at 10,000 x g i n order to r e m o v e c e l l u l a r debris a n d produce concentrated R V c o n t a i n i n g stocks suspended culture m e d i u m . H o w e v e r , these R V stocks were not p u r i f i e d f r o m other s m a l l c e l l u l a r components (e.g. s o l u b l e proteins, organelles) w h i c h c o u l d have potentially affected c y t o k i n e / c h e m o k i n e secretion, independent f r o m v i r u s , w h e n administered i n R V i n o c u l a d u r i n g experiments. T h e r e f o r e , the effects o f p u r i f i e d a n d u n p u r i f i e d R V o n IL-6 a n d IL-8 secretion were c o m p a r e d i n order to c o n f i r m that the use o f u n p u r i f i e d R V stocks i n experiments was v a l i d , and that any observed effects o n c y t o k i n e and/or c h e m o k i n e secretion were a result o f virus o n l y (and not other H I residual c e l l u l a r components).  Methods:  R V 1 4 and R V 1 A stocks (harvested f r o m H I cells) were aliquoted into sterile  tubes ( 0 . 5 m L each) a n d ultracentrifuged under v a c u u m at 100,000 x g i n order to pellet the v i r u s . These virus pellets were then either re-suspended i n their o r i g i n a l culture m e d i u m (unpurified) o r fresh m e d i u m (purified). T h e p u r i f i e d and u n p u r i f i e d R V stocks were then used to infect B E A S - 2 B a n d A 5 4 9 cells ( M O I = l ) u s i n g standard i n f e c t i o n procedures (n=6). T h e effects o f unpurified/purified R V o n v i r a l r e p l i c a t i o n a n d IL-6 (for B E A S - 2 B ) or IL-8 (for A 5 4 9 ) secretion after 48 hours were ascertained b y plaque assays and E L I S A s .  Results: T h e r e  were n o statistically s i g n i f i c a n t (a=0.05, 1-way A N O V A s and post-hoc  T u k e y tests) differences f o u n d between p u r i f i e d and u n p u r i f i e d R V i n o c u l a o n infectious virus i n B E A S - 2 B o r A 5 4 9 cells 4 8 hours post-infection (Figures C I and C 2 ) . F u r t h e r m o r e , there were n o s i g n i f i c a n t differences (a=0.05, 1-way A N O V A s and post-hoc T u k e y tests) f o u n d i n IL-6 o r IL-8 s t i m u l a t i o n i n the B E A S - 2 B and A 5 4 9 cells respectively between p u r i f i e d and u n p u r i f i e d R V i n o c u l a 48 hours post-infection (Figures C 3 and C 4 ) .  118  1Q.  6  O  o  •G 4  RV14  RV1A  RV Serotype Figure CI: Effect of purified and unpurified RV14 and R V 1 A on infectious rhinovirus in BEAS-2B cells 48 hours post-infection. R V stocks were pelleted by ultracentrifugation and virus was either re-suspended in fresh medium (pure) or in its original medium (unpure). No statistical differences (a=0.05) in infectious virus were observed between purified and unpurified R V inocula for either RV14 or R V 1 A (n=6).  o 4H  >  CO  o  r> 3 j  RV14  RV1A  RV Serotype Figure C2: Effect of purified and unpurified RV14 and R V 1 A on nfectious rhinovirus in A549 cells 48 hours post-infection. See Fig. C2 legend for further details.  119  400 BEAS-2B/IL-6  mam Pure 05133 Unpure E 300 O) Q. C  o I 200  cd> o oc  O  <o 100  •  Bl RV14  I  RV1A  RV Serotype  0  Figure C3: Effect of purified and unpurified RV14 and R V 1 A on IL-6 secretion from BEAS-2B cells 48 hours post-infection. R V stocks were pelleted by ultracentrifugation and virus was either re-suspended in fresh medium (pure) or in its original medium (unpure). No significant differences (a=0.05) in IL-6 secretion were observed between purified and unpurified R V inocula for either RV14 or R V 1 A (n=6).  A549/IL-8  RV14  RV1A R V Serotype  Figure C4: Effect of purified and unpurified RV14 and R V 1 A on IL-8 secretion from A549 cells 48 hours post-infection. See Fig. C3 legend for further details.  C o n c l u s i o n : There is n o difference observed i n : i n f e c t i o u s v i r u s (replication), IL-6 secretion f r o m B E A S - 2 B c e l l s , or IL-8 secretion f r o m A 5 4 9 cells between u n p u r i f i e d o r p u r i f i e d R V 1 4 and R V 1 A i n o c u l a . T h e r e f o r e , the effects o n c y t o k i n e / c h e m o k i n e secretion observed i n both c e l l lines are attributable to the v i r u s and not other H I c e l l proteins and/or remnants d e r i v e d f r o m R V harvesting p r o t o c o l s . O v e r a l l , the use o f u n p u r i f i e d (clarified) R V stocks as i n o c u l a is e x p e r i m e n t a l l y v a l i d .  120  APPENDIX D: Cell Counts for Rhinovirus Infected and Uninfected B E A S 2B and A 5 4 9 Cells. C e l l counts were c o n d u c t e d f o r u n i n f e c t e d a n d R V infected A 5 4 9 a n d B E A S - 2 B c e l l s o n D O , D 2 , a n d D 7 post-infection i n order to assess whether R V i n f e c t i o n h a d any effect o n c e l l g r o w t h a n d n u m b e r , a n d to c o n f i r m that c e l l numbers r e m a i n e d r e l a t i v e l y u n c h a n g e d once cultures h a d reached c o n f l u e n c e . T h i s e x p e r i m e n t w a s carried out to c o n f i r m that changes i n c e l l n u m b e r were not c o n t r i b u t i n g factors i n R V r e p l i c a t i o n , R V 1 4 R N A , a n d c y t o k i n e / c h e m o k i n e data. M e t h o d s : B E A S - 2 B a n d A 5 4 9 cells were g r o w n to c o n f l u e n c e under standardized c o n d i t i o n s (see C h a p t e r 2) i n 2 4 w e l l plates. Supernatants were then aspirated and i n o c u l a of: R V 1 4 , R V 1 A or m e d i u m alone were added a n d a l l o w e d to infect f o r 1 h o u r at 35°C. A f t e r i n f e c t i o n , cells w e r e w a s h e d w i t h D M E M a n d fresh culture m e d i u m w a s added to the w e l l s ( 1 % F B S ) . A t D O , D 2 , a n d D 7 post-infection cells were t r y p s i n i z e d , suspended i n l m L o f fresh m e d i u m , a n d counted b y h e m a c y t o m e t e r ( w i t h trypan b l u e e x c l u s i o n f o r dead cells) w i t h a l O u L l o a d i n g v o l u m e (n=6). R e s u l t s : T h e r e were n o statistical differences (a=0.05, 2 w a y A N O V A s , D u n n e t ' s Test) f o u n d i n n u m b e r o f c e l l s between any o f the treatments ( C o n t r o l , R V 1 4 , or R V 1 A ) at a n y o f the s a m p l i n g time-points f o r either the B E A S - 2 B or A 5 4 9 c e l l lines (Figures D l a n d D 2 ) . F u r t h e r m o r e , c e l l n u m b e r r e m a i n e d f a i r l y constant f r o m D O to D 7 i n R V infected a n d c o n t r o l c e l l s . T h e r e were v e r y f e w dead cells present a n d supernatants c o n t a i n e d v i r t u a l l y no c e l l s . A n e c d o t a l l y , the culture m e d i u m f o r R V 1 4 a n d R V 1 A i n f e c t e d cells d i d appear more a c i d i c than that o f the uninfected B E A S - 2 B a n d A 5 4 9 c e l l s .  121  CD  £  7H  Control RV14 RV1A  BEAS-2B  6  CD  E  o O  4H  CD  O  0  2  7  Time After Infection (Days)  Figure D l : Cell counts for rhinovirus infected and uninfected BEAS-2B cells over time. No significant differences (a=0.05) in cell number were found between uninfected and R V infected cells at any time (n=6).  E  5  3  o O a> O 0  2  7  Time After RV Infection (Days)  Figure D2: Cell counts for rhinovirus infected and uninfected alveolar epithelial A549 cells over time. See Fig. D l legend for further details.  C o n c l u s i o n : Infection o f a i r w a y e p i t h e l i a l cells w i t h R V 1 4 o r R V 1 A had n o effect o n c e l l n u m b e r w h e n c o m p a r e d to controls and c e l l n u m b e r r e m a i n e d quite constant f o r the duration o f the s a m p l i n g p e r i o d f o r both B E A S - 2 B and A 5 4 9 c e l l l i n e s . T h e r e f o r e , changes i n c e l l n u m b e r d i d not affect to R V r e p l i c a t i o n , R V 1 4 R N A , o r c y t o k i n e / c h e m o k i n e secretion data i n m y e x p e r i m e n t a l designs.  122  APPENDIX E: H I , BEAS-2B, and A 5 4 9 Cell Counts at Confluence C e l l counts i n 6-well plates at c o n f l u e n c e f o r H I , A 5 4 9 , and B E A S - 2 B c e l l lines were c o n d u c t e d i n order determine c a l c u l a t i o n o f v i r a l i n f e c t i o n dose ( M O I = l ) f o r R V i n f e c t i o n experiments. M e t h o d s : C e l l s were g r o w n under standardized c o n d i t i o n s ( 3 m L D M E M o r 5 0 : 5 0 D M E M / F 1 2 w i t h 5 - 1 0 % F B S ) at 35°C f o r u n t i l f r e s h l y c o n f l u e n t i n 6-well plates. A f t e r 48 hours, supernatants were aspirated, cells t r y p s i n i z e d , d i l u t e d 5:1 i n culture m e d i u m and c o u n t e d b y hemacytometer (n=5) w i t h trypan blue e x c l u s i o n to stain dead cells w i t h a l o a d i n g v o l u m e o f l O u L . R e s u l t s ( F i g u r e E l ) : C e l l count at c o n f l u e n c e w a s s i g n i f i c a n t l y (a=0.05) h i g h e r i n H I cells b y 1.4-fold c o m p a r e d to B E A S - 2 B and A 5 4 9 cells but n o difference i n c e l l n u m b e r w a s observed between the a i r w a y c e l l lines (1-way A N O V A and post-hoc T u k e y test). C e l l counts were ( i n n u m b e r o f cells/well): H l = 1 . 9 x 1 0 ±3.3 x 1 0 , B E A S - 2 B = 1 . 3 x 1 0 ± 8.2 6  4  6  x 1 0 and A 5 4 9 = 1 . 4 x 1 0 ± 8.5 x 1 0 . A l l c e l l counts were at an order o f m a g n i t u d e o f 1 0 4  6  4  cells per w e l l . R V i n f e c t i o n doses o f 1 infectious v i r a l particle per c e l l ( M O I = l ) i n a l l R V i n f e c t i o n experiments were based o n these c e l l counts.  BEAS-2B  A549  Cell Type  Figure E l : Cell counts for uninfected H I , BEAS-2B and A549 cells at confluence. An asterisk (*) indicates that HI cell counts at confluence were significantly higher (a=0.05) than BEAS-2B and A549 cell types, although all cell numbers were at a 10 order of magnitude. 6  123  6  APPENDIX F: Rhinovirus Stability T h e stability o f R V 1 4 a n d R V 1 A under t y p i c a l e x p e r i m e n t a l c o n d i t i o n s w a s ascertained, i n order to determine whether i n f e c t i o u s v i r u s assayed at g i v e n G r o w t h C u r v e s a m p l i n g times ( D 0 - D 7 ) represented d a i l y secretions o r a c c u m u l a t e d R V secretions u p to sampling day. M e t h o d s : R V 1 4 and R V 1 A stocks d i l u t e d w i t h culture m e d i u m ( D M E M ) a n d 1 % F B S to an i n i t i a l starting concentration o f 1 0 " p f u / m L a n d a l i q u o t e d into sterile 6-well trays ( 3 m L 4  5  o f R V s o l u t i o n per w e l l ) i n order to m o c k t y p i c a l G r o w t h C u r v e c o n d i t i o n s (n=4). S a m p l e s were taken d a i l y , f r o z e n at -80°C, and assayed f o r infectious virus (pfu/mL). R e s u l t s : F o r both R V 1 4 ( F i g u r e F I ) a n d R V 1 A ( F i g u r e F 2 ) i n f e c t i o u s v i r u s decreased s i g n i f i c a n t l y (a=0.05, 1-way A N O V A s a n d post-hoc T u k e y tests) f r o m d a y to d a y u n t i l it was n o l o n g e r detectable o n D 4 f o r R V 1 4 , a n d s o m e w h e r e between D 4 a n d D 7 f o r R V 1 A . H o w e v e r , the i n i t i a l concentration o f R V 1 A (DO) w a s h i g h e r than f o r R V 1 4 p r o b a b l y a c c o u n t i n g f o r t i m e d i f f e r e n c e i n degradation. O v e r a l l , i n f e c t i o u s v i r u s decreased b y 1-2 orders o f m a g n i t u d e d a i l y f o r both R V 1 4 a n d R V 1 A .  1  2  4  Time (Days)  Figure FI: RV14 Stability at 35°C under mock Growth Curve conditions over 7 days. Symbols that differ (a, b, c, d) indicate significant differences (a=0.05) in infectious virus between sampling times (n=4). Infectious virus was undetectable at D4 and D7.  124  7 •~ 6  E  "3 D.  5  o  4  o  to 3  I  3 1  RV1A  a  b J 1  Jjjiijl  c {  2  ^ *  d  o CD  I  1 0  0  1  2  SB  e  4  7  Time (Days)  Figure F2: RV1A Stability at 35°C under typical experimental conditions over 7 days. Symbols that differ (a, b, c, d, e) indicate significant differences (a=0.05) in infectious virus between sampling times (n=4). Infectious virus was undetectable on D7.  C o n c l u s i o n : In culture m e d i u m , R V 1 4 and R V 1 A stock solutions u n d e r g o d a i l y degradation o f 1-2 orders o f m a g n i t u d e . H o w e v e r , R V remains detectable f o r at least 3 days under these c o n d i t i o n s d e p e n d i n g o n i n i t i a l R V concentrations. T h e r e f o r e , G r o w t h C u r v e r e p l i c a t i o n data f o l l o w i n g the i n i t i a l peak i n infectious v i r u s (usually around D l ) c o u l d represent g r a d u a l l y d e g r a d i n g v i r u s s y n t h e s i z e d o n D l , or a c o m b i n a t i o n o f a c c u m u l a t e d and n e w l y synthesized R V . F u r t h e r m o r e , this e x p e r i m e n t does not take into account the p o s s i b i l i t y that R V contained w i t h i n c e l l s is protected f r o m the degradation observed i n the e x t r a c e l l u l a r e n v i r o n m e n t . A d d i t i o n a l l y , i n culture systems c e l l s m a y secrete factors w h i c h facilitate the degradation o f R V b e y o n d what can be observed i n culture m e d i u m alone.  125  APPENDIX G: Interleukin-6 and Interleukin-8 Stability T h e stability o f IL-6 and IL-8 under t y p i c a l e x p e r i m e n t a l c o n d i t i o n s w a s assessed i n order to determine whether c y t o k i n e / c h e m o k i n e assayed at g i v e n time-points ( D 0 - D 7 ) i n experiments represented d a i l y secretions o r a c c u m u l a t e d IL-6/IL-8 secretions u p to s a m p l i n g day. M e t h o d s : C y t o k i n e secretion w a s stimulated i n B E A S - 2 B cells b y i n f e c t i o n w i t h R V 1 A . A f t e r 7 2 hours supernatants w e r e c o l l e c t e d , c o m b i n e d , centrifuged at 1000 x g to r e m o v e c e l l u l a r debris, re-distributed to sterile 6-well plates ( 3 m L per plate), and i n c u b a t e d at 35°C. S a m p l e s were c o l l e c t e d i m m e d i a t e l y (DO) and seven days after re-distribution ( D 7 ) , stored at -20°C, and assayed b y E L I S A (n=3). R e s u l t s : B o t h IL-6 ( F i g u r e G l ) and IL-8 ( F i g u r e G 2 ) were stable under the g i v e n e x p e r i m e n t a l c o n d i t i o n s u p to D 7 post-incubation at 35°C (a=0.05, 1-way A N O V A a n d post-hoc D u n n e t ' s test).  300 250 H & 200 S 150  c o  o  100  1  CD  -  50  1 0  7 Time (Days)  Figure G l : IL-6 stability at 35°C under typical experimental conditions over 7 days. No significant differences (a^O.05) in IL-6 concentration were observed between DO and D7 (n=3).  126  800  IL-8  E  600  1  Q. J5 400 1 c o o c o O co 200 H  Time (Days)  Figure G 2 : 1 1 - 8 stability at 35°C under typical experimental conditions over 7 days. See Fig. Gl legend for further details.  C o n c l u s i o n : E x p e r i m e n t a l l y measured IL-6 and IL-8 secretions most l i k e l y represent a c c u m u l a t e d c y t o k i n e / c h e m o k i n e levels u p to the s p e c i f i e d day o f s a m p l i n g (unless the cells secrete factors w h i c h degrade IL-6 and IL-8). T h i s m o d e l is the t y p i c a l m o d e l used f o r c y t o k i n e / c h e m o k i n e secretion. A l t e r n a t i v e l y , one c o u l d r e m o v e and replace the m e d i u m i n c e l l cultures d a i l y ; h o w e v e r , this is not u s u a l l y done because o f the p r o b l e m s that it m a y cause. F o r e x a m p l e , d a i l y r e m o v a l o f supernatant w o u l d also r e m o v e a n y secreted v i r u s i n the m e d i u m thus interfering w i t h r e p l i c a t i o n data samples. F u r t h e r m o r e , the a d d i t i o n o f fresh d a i l y m e d i u m m a y disturb c e l l s , and a d d i t i o n o f new nutrients m a y encourage c e l l g r o w t h and other c e l l u l a r changes. M o d i f i c a t i o n o f the data to represent " d a i l y " secretions b y subtracting values f r o m the p r e v i o u s s a m p l i n g d a y was d o n e ; h o w e v e r , for the most part this d i d not affect o v e r a l l trends. F i g u r e G 3 shows an e x a m p l e o f such a data m o d i f i c a t i o n for trial 1 IL-6 secretion o f the R V 1 A / B E A S - 2 B G r o w t h C u r v e ( o r i g i n a l l y F i g u r e 2.3b). T h e o v e r a l l trend is s i m i l a r w i t h m a x i m u m IL-6 secretion o c c u r r i n g o n D 5 and D 7 ; h o w e v e r , c o n s i d e r i n g that these s a m p l i n g times m a y represent a c c u m u l a t e d IL-6 over 2 days the actual d a i l y secretion w o u l d l i k e l y be l o w e r ( p o s s i b l y h a l f this value). T h i s c o u l d result i n a m u c h less p r o n o u n c e d effect o n D 5 and D 7 ; h o w e v e r , these values w o u l d r e m a i n the m a x i m u m increases i n IL-6 secretion relative to their controls.  >  127  2000  1500  w  1000  co Q  500  Trial 1: BEAS-2B/RV1A  Control RV  H  i  o > CD  T  -500 1  2  3  5  7  Time After RV Infection (Days)  Figure G3: Modification of Trial 1 BEAS-2B Growth Curve IL-6 data to show "daily" (day - previous day) secretion. R V and control IL-6 data from the trial 1 BEAS-2B Growth Curve with R V 1 A (originally Figure 2.3b) were modified by subtracting each daily value from its corresponding previous sampling time in order to show secretions for a particular day (versus accumulated IL-6 up to sampling point). An asterisk (*) indicates a significant difference (a=0.05) in IL-6 secretion between treatments at a particular time.  128  APPENDIX H: Image of Plaque Assay  Figure HI: Image of representative plaque assay. This image depicts a completed plaque assay which was treated with cell-staining 1 % crystal violet in dH20. Viral plaques appear as clear unstained circular areas while intact cells appear dark. One plaque corresponds to one infectious virus particle present in the assayed sample. Plaques per well were counted (multiplied by a dilution factor if applicable) and reported as pfu/mL.  129  

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