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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 IMMUNE-MODULATING EFFECTS OF ECHINACEA by Anna Machala BSc, University of British Columbia, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA March 2007 © Anna Machala, 2007 ABSTRACT Rhinov i ruses ( RVs ) are the leading cause o f upper-respiratory tract infect ions in humans. T o date re lat ively l i t t le is k n o w n about the mechan ism o f R V infect ion and no cure or prevent ion exists. M o u n t i n g evidence shows that R V replicates very l itt le in its host a i rway epithel ia l cel ls leading researchers to hypothesize that the i l lness associated w i th R V infect ion is the result o f the host 's immune response, but not necessari ly R V repl icat ion. Th is study characterized R V infect ion in vitro in terms o f v i ra l repl icat ion, v i ra l R N A , and pro-inf lammatory cytokine/chemokine secretion over the course o f a typ ica l infect ion using two dist inct a i rway epithel ia l ce l l l ines ( B E A S - 2 B and A 5 4 9 ) and two different receptor-uti l iz ing R V serotypes ( R V 1 4 and R V 1 A ) . Ce l l s were infected w i th k n o w n amounts o f R V , sampled over 1 week, and assayed for infect ious v i rus, R V 1 4 R N A , and inter leukin (IL)-6 and/or IL-8 secretion. Fo r B E A S - 2 B and A 5 4 9 cel ls v i ra l repl icat ion peaked between day 1 ( D l ) and D 2 post-infection for both R V 1 4 and R V 1 A , and no s ignif icant v i ra l repl icat ion was observed after D 3 . S t imulat ion o f LL-6 and IL-8 was typ ica l l y not observed before D 2 and remained elevated up to D 7 . Overa l l , B E A S - 2 B cel ls were more susceptible to R V infect ion than A 5 4 9 , and s imi lar trends were observed for R V 1 4 and R V 1 A , except R V 1 4 fa i led to replicate in the A 5 4 9 cel ls . Furthermore, U V inact ivat ion o f both R V serotypes complete ly inhibi ted v i ra l repl icat ion and IL-6 secretion in the B E A S - 2 B mode l , suggesting the necessity o f genetical ly intact v irus to stimulate the IL-6 response. F ina l l y , the effects o f two chemica l l y dist inct Ech inacea extracts on v i ra l repl icat ion and IL-6 secretion were investigated in the B E A S - 2 B mode l . Nei ther o f the Ech inacea extracts had any effect on R V repl icat ion, nor d id they stimulate IL-6 secretion in uninfected cel ls. However , Ech inacea treatment o f R V infected cel ls s igni f icant ly affected IL-6 secretion, but a different trend was observed between R V serotypes, and for the two herb preparations. Ove ra l l , R V infect ion o f a i rway epithel ia l cel ls results in relat ively l ow levels o f R V repl icat ion but a pronounced pro -in f lammatory cytokine/chemokine response wh i ch is the l i k e l y cause o f co ld symptoms and a potential target for therapeutics. TABLE OF CONTENTS Abstract i i Table of Contents i i i List of Figures v List of Abbreviations v i i Acknowledgements v i i i Co-Authorship Statement ix Chapter 1: Introduction and Object ives 1 References 28 Chapter 2 : In Vitro Character izat ion o f Rh inov i rus Infection in A i r w a y Ep i the l i a l Ce l l s (Growth Curves ) 43 Figures 59 References 68 Chapter 3: The Effects o f U l t rav io le t Inactivated Rh inov i rus on A i r w a y Ep i the l i a l Ce l l s 73 Figures 82 References 87 Chapter 4: The Effects of Ech inacea Extracts on Rh inov i rus Infected and Uninfected A i r w a y Ep i the l i a l Ce l l s 88 Figures 97 References 99 Chapter 5: Genera l D iscuss ion and Conc lus ions 103 References 113 Appendices: Appendix A: B i ohaza rd Cert i f icate 116 Appendix B : G row th Curve Exper imenta l Des ign 117 Appendix C: E f fect o f Pur i f i ed and Unpur i f i ed Rh inov i rus on V i r a l Rep l i ca t ion and Cy tok ine/Chemok ine Secretion f rom B E A S - 2 B and A 5 4 9 Ce l l s 118 Appendix D: C e l l Counts for R V Infected and Un infec ted B E A S - 2 B and A 5 4 9 Ce l l s 121 Appendix E : H I , B E A S - 2 B and A 5 4 9 C e l l Counts at Conf luence 123 Appendix F: Rh inov i rus Stabi l i ty 124 Appendix G: Inter leukin-6 and Interleukin-8 Stabi l i ty 126 i n Appendix H: Image o f P laque Assay . . . .129 i v 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 A549 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 A549 cells over time 63 Figure 2.6: R V 1 4 R N A for H I , B E A S - 2 B and A549 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 A549 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 A549 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 Figure 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 Figure 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 R V rhinovirus C O P D chronic obstructive pu lmonary disorder U V ultraviolet I C A M - 1 intercel lular adhesion molecule-1 L D L R low density l ipoprote in receptor V L D L R very l ow density l ipoprote in receptor H I D 5 0 5 0 % human infect ious dose T C I D ™ 5 0 % tissue culture infect ious dose C P E cytopathic effects T N F tumor necrosis factor I L inter leukin N F K B nuclear factor kappa-B A T C C Amer i c an Type Cul ture Co l l e c t i on q R T - P C R quantitative real-time polymerase chain reaction E L I S A enzyme-l inked immunosorbent assay A N O V A analysis o f variance M O I mul t ip l i c i t y o f infect ion D(0-7) days after R V infect ion/inoculat ion pfu plaque fo rming units F B S fetal bov ine serum D M E M Du lbecco ' s M o d i f i e d Eag le ' s M e d i u m M E M M o d i f i e d Eag le ' s M e d i u m ACKNOWLEDGEMENTS First and foremost, I w o u l d l i ke to thank m y co-supervisors Dr . James Hudson and Dr . C o l i n Brauner for their support and mentorship. D r Hudson , thank you for sparking m y interest in the wor ld o f viruses and for your cont inued encouragement dur ing this project. D r . Brauner, thank you for taking me into your lab and a l l ow ing me to get m y hands a bit wet w i th the Ch inook . I w o u l d also l i ke to thank Dr . Robert Harr is without w h o m this thesis wou ld not be possible. I wou ld l i ke to acknowledge Dr . M a n j u Sharma and D r . Selverani V ima lanathan for their help dur ing m y project. F ina l l y , I w o u l d l ike to thank the Brauner and Nandan Labs for a l l their support over the past few years. v i i i 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. Col in Brauner) provided me with mentorship and supervision, and wi l l be listed as co-authors on the publications arising from this project. i x Chapter 1: Literature Review and Thesis Objectives INTRODUCTION The most prevalent infect ion in humans is the acute upper-respiratory tract infect ion, also k n o w n as the c o m m o n co ld (Monto , 2002). Over 100 Rh inov i ruses ( RVs ) persist in our wor ld today and are the leading cause o f such respiratory tract infect ions (Ar ruda et a l . , 1997; Johnston et a l . , 1995; van Gageldonk-Lafeber et a l . , 2005) . In healthy ind iv idua ls R V infect ions are typ ica l l y short-lived and se l f- l imi t ing; however, for susceptible groups such as infants, the e lder ly , and the immuno-compromized R V infect ion can be life-threatening. Furthermore, for people already affected by diseases such as asthma or chronic obstructive pu lmonary disorder ( C O P D ) , R V infect ion is k n o w n to cause serious exacerbations o f those condit ions (Bard in , 1992; Fraenkel et a l . , 1995; Ge rn , 2002 ; Ge rn & Busse, 1999; Greenberg, 2002 ; Grunberg & Sterk, 1999; M a l l i a et a l . , 2006 ; Message & Johnston, 2004; Seemungal , 2 0 0 1 ; Seemungal , 2000 ; Teran et a l . , 1997). In the case o f asthma, recent studies suggest that early ch i ldhood R V infect ion may even play a causative role in the development o f the disease (Gern, 2004 ; S ingh et a l . , 2007). T o date, relat ively l itt le is known about the mechan ism o f R V infect ion and no cure or prevention exists. M o u n t i n g evidence shows that R V replicates very l itt le in its host a irway epithel ia l tissues (Lopez-Souza et a l . , 2004) , leading researchers to hypothesize that the i l lness associated w i th R V infect ion is the result o f the host 's immune response to some v i ra l trigger, but not necessari ly to the level o f v i ra l repl icat ion itself. Bea r ing this in m i n d , new therapies wh i ch modulate the immune system and mitigate R V associated symptoms are becoming increas ingly interesting to scientists. One such candidate therapy is the natural herb extract Ech inacea . Ech inacea use has recently gained widespread popular i ty in Western culture, and commerc i a l products are w ide l y avai lable to consumers. A l t hough the qual i ty o f many commerc ia l formulat ions is questionable, there is growing evidence that Ech inacea has diverse effects on the immune system (Brousseau & M i l l e r , 2005 ; B rush et al . , 2006 ; Curr ie r & M i l l e r , 2000; G o e l , 2005 ; Sharma et a l . , 2006). In terms o f mit igat ing RV-related i l lness, Ech inacea may down-regulate the in f lammatory response provoked by v i ra l in fec t ion ; however, its mechanism of action is largely unknown (Sharma et al . , 2006). 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 pro-inflammatory 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 Airway Epithelial Cells) I investigated the potential viral trigger for cell inflammatory mediator secretion by using ultraviolet (UV) 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 Airway 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 Epi the l ium 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 cil ia 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 & Loosl i , 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: l ipid 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. Models of the A i r w a y Epi the l ium 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: SV40 adenovirus transformed bronchial epithelial (BEAS-2B) 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 spec ia l ized membranes wi th an air-l iquid interface produces monolayers w i th apical and basolateral sides and increases the expression o f tight junct ion proteins (B lank et a l . , 2006) . F ina l l y , w i th access to l i ve a i rway epithel ia l tissues scientists can establish pr imary cultures by processing and plat ing cel ls using spec ia l ized procedures. P r imary cultures are typ ica l l y created f rom nasal, tracheal or bronchia l tissues der ived f rom surgical procedures such as polypectomies (Donninger et a l . , 2003 ; Lopez-Souza et a l . , 2004 ; Suzuk i et a l . , 2002). Obv ious l y , such cultures are the truest in vitro models o f the a i rway epi the l ium retaining native epi the l ium characteristics most c losely . However , l i ve tissues are often d i f f i cu l t to obtain (especial ly in suff ic ient amounts), culture techniques are more sensitive and compl ica ted, and cel ls on ly a l low for 1-2 passages before undergoing terminal differentiat ion. Furthermore, pr imary tissues der ived f rom surgeries may be affected by the presence o f pre-operative drugs such as anesthetics. A l l o f the above models are susceptible to R V infect ion at least to some degree. Lopez-Souza et al. (2004) found that the least differentiated cel ls ( immorta l ized ce l l cultures) were the most vulnerable to R V infect ion and the most differentiated cel ls (pr imary cultures) were much more resistant. Undif ferent iated cultured cel ls produced vira l titers 30 to 130 t imes that o f their differentiated pr imary culture counterparts, wh i l e immor ta l i zed cel ls grown on permeable supports produced intermediate v i ra l concentrations. A s imi la r trend was observed for R V induced in f lammatory mediator secretion and suggests that undifferentiated a i rway ce l l models may represent a somewhat exaggerated response to R V infect ion. Rhinovirus ( R V ) R V s be long to the Picornaviridae f ami ly and are icosahedral w i th a protein capsid surrounding genetic material encoded in single stranded R N A (Be l la & Rossman , 1999). The i r genetic informat ion carries a posit ive polar i ty and is translated into a polyprote in upon entry into the cytoplasm. Th i s polyprote in is then automatical ly c leaved by the ce l l and further processed by v i ra l proteases, eventual ly fo rming the v i ra l capsid proteins and non-structural proteins invo l ved in the repl icat ion process (Rei thmayer et a l . , 2002) . T o date, over 100 R V serotypes have been ident i f ied based on the presence o f unique 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). Rhinovirus 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 molecule-1 ( ICAM-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 re-classified 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 pH 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 RVs) is capable of uncoating both R V s and polioviruses at normal physiological pH 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 LDLR- type 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 pH 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 So) in human fibroblasts. 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 ing 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). Rhinovirus Infection of the Ai rways 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 widespread mucosa l infect ion but as loca l ized foc i f rom where in f lammatory responses are generated. In vivo, this has been demonstrated w i th nasal b iopsies wh i ch c lear ly point to smal l ce l lu lar areas o f loca l i zed infect ion, wh i le showing few other h is to logica l abnormalit ies (Douglas et a l . , 1968; H a m o r y et a l . , 1977; W in ther et a l . , 1984). M o r e recent immunoh is tochemica l evidence also supports the f ind ing that R V infect ion o f the airways emerges in a patch-like fashion (Mosser et a l . , 2005). Mosse r et al. (2002) demonstrated that on ly 5-10% o f pr imary a i rway epithel ia l cel ls were susceptible to R V regardless o f the infect ion dose used. A r r u d a et al. (1995) also found that on ly a very smal l proport ion o f nasal epithel ia l cel ls were infected w i th R V in exper imental ly inoculated volunteers who had indeed developed co ld symptoms. Furthermore, in tissue and ce l l cultures l ike : B E A S - 2 B , A 5 4 9 , or pr imary tracheal cel ls there are no cytopathic effects (CPE ) such as: ce l l rounding, w r i nk l i ng , rupture, or death observed upon infect ion w i th R V (Johnston et a l . , 1998; S u z u k i et a l . , 2001) , although v i ra l repl icat ion can easi ly be detected. The level o f R V repl icat ion is also considered relat ively l ow wi th increas ingly differentiated cel ls produc ing decreased titers o f v i rus (Lopez-Souza et a l . , 2004). Consequent ly , there is a g row ing be l ie f that it is the pro-inf lammatory cytokines and chemokines st imulated by R V infect ion that cause the symptoms and pathogenici ty o f infect ion. Immune Response to Infection In order for R V to infect the a i rway epi the l ium several defensive barriers must first be penetrated. F i rs t l y , the v i rus must breach the mucosa l layer o f the ep i the l ium, and i f successful , the virus may adhere to the epithel ia l layer, but on l y i f it can out-compete the natural f lora o f the a irways, and evade phagocytes wh i ch are especia l ly r i ch in the lung tissues. Once attached its host receptor, v i ra l entry and repl icat ion may occur , and loca l infect ion may ensue. Once inside the ce l l , the virus is not exposed to most elements of the immune system but as v i ra l progeny are released f rom infected ce l ls , these v i r ions are confronted by the anticipatory immune response. The tissues defend themselves against potential threats such as phys ica l in jury, irritants and pathogens by two types o f immun i t y ca l led: innate and adaptive. The innate response is fast act ing, triggered w i th in 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 im 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). At 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). As a group, chemokines are 20-50% homologous, and their differences are largely due to variable positions of cysteine residues. Most chemokines are considered pro-inflammatory, 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 pro-inflammatory 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. Two 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 im 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 . IL 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 IL 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). Also , 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 ( P100/p52), R e l A (p65), Re lB , and c-Rel] is known to mediate 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, NF- IL-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. Many 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 A549 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 HeLa 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 THP-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. Many 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 anti-R V drug to be evaluated by the United States Food and Drug Administration. Another anti-R 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 immune-modulating 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 short-comings 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 bill ion 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 mill ion 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 45-1600mg 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. Also 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. As 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 of 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 of R V replication during airway infection. Many 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 of 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 N F K B ) 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 N F K B and activating transcription factor-2/cAMP responsive element binding protein-1 activation (Gertsch et al., 2004; Raduner et al., 2006; Woelkart et al., 2005). How 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 & Mil ler , 2005). Echinacea administration to aging mice has resulted in the synthesis of natural killer cells de novo in the bone marrow (Currier & Mil le 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. Also , 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 virus-receptor 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. Immune-modulating 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 General Objectives: Chapter 2 (In Vitro Character izat ion o f Rh inov i rus Infection in A i r w a y Ep i the l i a l Ce l l s -Growth Curves ) : T o characterize R V infect ion in vitro in terms o f v i ra l repl icat ion, v i ra l R N A , and ce l l secretion o f pro-inf lammatory cytokines/chemokines over the typica l course o f infect ion us ing two different receptor-uti l iz ing R V serotypes in two dist inct cultured human airway epithel ia l ce l l models. A l t hough in vitro models for R V infect ion may not m i m i c in vivo results, they are cr i t ica l to our understanding o f R V infect ion because they offer h igh ly contro l led and standardized condit ions under wh i ch R V infect ion may be investigated, w i th fewer variables than in in vivo models . A c co rd i ng l y , more subtle and mechanist ic differences may be discernable. F r o m a logist ic point o f v i ew , in vitro experiments are more approachable, less dangerous, and less cost ly than their in vivo counterparts, and thus play a cr i t ica l role i n v i ra l research. I chose to study R V infect ion in two different immor ta l i zed human a i rway epithel ia l ce l l models : a bronchia l epithel ia l ce l l mode l ( B E A S - 2 B ) , and a type II a lveolar ce l l mode l (A549) . I selected these models because they are well-characterized and w ide l y used in s imi lar R V experiments. A l t hough both ce l l l ines are der ived f rom the m i d to lower region o f the airways the B E A S - 2 B cel ls represent cel ls higher in the respiratory tract, wh i l e the alveolar cel ls are the lowest cel ls o f the respiratory ep i the l ium. In the above ce l l models I investigated the effects o f two different receptor-uti l iz ing R V serotypes: R V 1 4 (major group) and R V 1 A (minor group) in order to observe the extent of differences possible between R V serotypes. In order to address m y objectives and characterize R V infect ion, I chose to measure v i ra l repl icat ion, v i ra l R N A levels (for R V 1 4 ) , and ce l l IL-6 and IL-8 secretion at da i ly t ime intervals over the course o f a typica l infect ion (about 7 days), and termed these experiments " G r o w t h Cu rves " . V i r a l repl icat ion was assessed by plaque assay, wh i ch detects the amount o f infect ious ( fu l ly formed repl icat ing units) virus present in a given sample, and thus increases in values f rom controls indicate v i ra l repl icat ion. T o assess whether a i rway epithel ia l cel ls a l lowed on ly l ow levels o f R V repl icat ion, v i ra l titers were compared to those obtained in ident ical 24 experiments in the permiss ive HI epithel ia l ce l l l ine. H I cel ls are considered permiss ive because they a l low for the highest R V titers o f any k n o w n cel ls f o l l ow ing R V infect ion (Ar ruda et a l . , 1996). V i r a l R N A levels were assessed in both ce l l models for R V 1 4 only , as the entire R V 1 A genome sequence is not fu l l y sequenced and pr imer design is problematic . V i r a l R N A levels are indicat ive o f the number o f v i ra l genomes present in the sample, regardless of whether those genomes are present in fu l l y infect ious and repl icat ing units. Consequent ly , increases in v i ra l R N A may not mirror plaque assay results as they represent e f f i c iency in product ion o f v i ra l R N A ; funct ional or not. R N A was detected us ing quantitative real-time polymerase chain reaction (qRT-PCR ) wh i ch has been shown to be 10 times more sensitive to detect R V than convent ional P C R (Dagher et a l . , 2004). F i na l l y in f lammatory mediator secretion was measured us ing enzyme-l inked immunosorbent assays ( E L I S A s ) for specif ic cytokines and/or chemokines . In the B E A S -2B mode l , IL-6 secretion was determined, wh i le in A 5 4 9 cel ls IL-8 secretion was measured based on previous laboratory data (unpublished) wh i ch indicated pronounced secretion o f those speci f ic proteins in the respective ce l l models . A n increase in in f lammatory mediator secretion was used as an indicator for the st imulat ion o f the host in f lammatory response as is routine practice for such studies. Hypotheses: 1) R V infect ion o f a i rway epithel ia l cel ls w i l l result in R V repl icat ion and st imulat ion o f the host in f lammatory response as represented by pro-inf lammatory cytokine/chemokine secretion f rom cel ls . 2) R V infected a i rway epithel ia l cel ls w i l l not exhib i t C P E because R V infect ion results in little or no cel l death or cytotoxic i ty . 3) R V repl icat ion and R N A levels in a i rway epithel ia l cel ls w i l l be relat ively l ow as compared to permiss ive H I cel ls (by at least one order of magnitude). 4) V i r a l repl icat ion, v i ra l R N A , and ce l l pro-inf lammatory IL-6/IL-8 secretions w i l l increase post R V infect ion and gradual ly decl ine to control levels by day 7 as the infect ion is resolved. 25 5) M a x i m u m R V repl icat ion and R N A levels w i l l occur earl ier dur ing the time-course o f infect ion than peak cytokine/chemokine secretion in f lammatory mediator secretion, and cytokine/chemokine levels w i l l remain elevated longer than R V repl icat ion and R V R N A . 6) R V 1 4 and R V 1 A serotypes w i l l produce s imi la r infect ion patterns because they be long to the same virus fami ly . 7) A l v eo l a r A 5 4 9 cel ls w i l l be less susceptible to R V infect ion than bronch ia l B E A S - 2 B cel ls because they are der ived f rom lower regions o f the airways. Chapter 3 (The Ef fects U l t rav io let Inactivated Rh inov i rus on A i r w a y Ep i the l ia l Ce l l s ) : T o investigate the potential for a non-replicative v i ra l trigger for in i t iat ing the ce l lu lar in f lammatory response by expos ing bronchia l epithel ia l ( B E A S - 2 B ) cel ls to part ial ly and fu l l y U V inactivated virus and quant i fy ing subsequent v i ra l repl icat ion, v i ra l R N A and in f lammatory mediator secretion. These experiments were conducted in B E A S - 2 B cel ls for both R V 1 4 and R V 1 A . R V stocks were UV-i r rad ia ted for different lengths of t ime and those samples used dur ing infect ion procedures in order to observe the effects o f U V treated R V on repl icat ion, v ira l R N A ( R V 1 4 only ) , and IL-6 secretion in order to assess whether R V wi th damaged genetic material cou ld e l ic i t an in f lammatory response. Hypotheses: 1) The v i ra l trigger for the observed host in f lammatory response is not related to v i ra l repl icat ion, therefore R V s irradiated w i th U V C suf f ic ient ly to damage genomic R N A material but not protein structure, w i l l e l ic i t an IL-6 response when used to infect B E A S -2B cel ls . 2) A s imi lar IL-6 response f rom B E A S - 2 B cel ls w i l l occur for both R V 1 4 and R V 1 A because they be long to the same virus fami ly . Chapter 4 (The Ef fects o f Ech inacea Extracts on Rh inov i rus Infected and Uninfected A i r w a y Ep i the l ia l Ce l l s ) : T o assess the effects o f 2 chemica l l y dist inct Ech inacea extracts 26 on R V infected and uninfected B E A S - 2 B cel ls , in terms o f v i ra l repl icat ion and pro -in f lammatory IL-6 secretion, for R V 1 4 and R V 1 A . Exper iments were conducted in the B E A S - 2 B ce l l mode l w i th both R V serotypes. Ce l l s were infected w i th either R V 1 4 or R V 1 A and then treated w i th one o f two chemica l l y distinct Ech inacea extracts ( E l or E2 ) immediate ly post-infection. E l was an aqueous Ech inacea extract and E 2 an a lcohol ic tincture. Bo th extracts' chemica l prof i les were prev ious ly determined by h igh performance l i qu id chromatography (B inns et a l . , 2002). V i r a l repl icat ion was assessed at speci f ic t ime intervals for R V infected samples, and IL-6 secretion was measured in R V infected and uninfected cel ls treated w i th the Ech inacea extracts. Hypotheses: 1) Treatment o f RV- in fec ted B E A S - 2 B cel ls w i th Ech inacea w i l l not affect v i ra l repl icat ion because Ech inacea has no effect on the R V repl icat ion cyc le . 2) Treatment o f uninfected B E A S - 2 B cel ls wi th Ech inacea w i l l invoke an in f lammatory response and result in increased secretion o f IL-6. 3) Treatment o f RV- in fec ted B E A S - 2 B cel ls w i th Ech inacea w i l l inhib i t R V induced IL-6 secretion because o f comp lex interactions between Ech inacea and the v i rus infected ce l ls . 4) E l and E 2 w i l l affect IL-6 secretion dif ferent ly because they are der ived f rom distinct extract preparations and have different active constituent prof i les. 5) Ech inacea extracts w i l l have the same effects on R V 1 4 and R V 1 A because the viruses be long to the same fami ly . 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Rh inov i rus st imulat ion o f interleukin-8 in vivo and in vitro: Ro l e o f NF-kappa B. Amer i c an Journal o f Phys io logy L u n g Ce l l u l a r and M o l e c u l a r Phys io logy . 273:L814-824. 41 Z h u , Z . , W . Tang, A . Ray, Y . W u , O . Einarsson, M . L . L a n d r y , and J . M . Gwaltney, J r . 1996. Rh inov i rus st imulat ion o f interleukin-6 in vivo and in vitro. The Journal o f C l i n i c a l Investigation. 97:421-430. 42 Chapter 2: In Vitro Characterization of Rhinovirus Infection in Airway Epithelial Cells (Growth Curves)1 BACKGROUND Rhinov i ruses ( RVs ) are the leading cause o f upper respiratory tract infect ions in humans (Ar ruda et a l . , 1997). Fo r people already affected by other respiratory diseases, such as asthma or chronic obstructive pu lmonary disorder ( C O P D ) , R V infect ion can lead to dangerous exacerbations o f those condit ions (Bard in , 1992; Fraenkel et a l . , 1995; Ge rn & Busse, 1999; Grunberg & Sterk, 1999; Greenberg, 2002 ; Ha lper in , 1985; Johnston, 1993; Johnston et a l . , 1995; Johnston, 2005 ; Message, 2 0 0 1 ; Seemungal , 2000 ; Seemungal , 2001). Furthermore, in the case o f asthma R V infect ions have recently been attributed a causative role in the pathology o f the disease (S ingh et a l . , 2006). A s o f yet, no prevention or cure for R V infect ion exists and medicat ions on ly act to alleviate symptoms. The specif ic mechanism of R V infect ion is st i l l largely unknown . R V s general ly infect the upper a i rway epithel ia by direct contact w i th infected ind iv idua ls , contaminated surfaces, or large particle aerosols (Turner, 2001). However , there is g row ing evidence that the lower airways are also susceptible to R V infect ion (Fraenkel et al . , 1995; Gern et a l . , 1997; Gern et al . , 2000 ; Hayden , 2004 ; Mosse r et a l . , 2002 ; Mosse r et a l . , 2005 ; N i cho l son et a l . , 1996; Papadopoulos et a l . , 2000; Schroth et a l . , 1999). Trad i t iona l l y , it was also thought that the symptoms caused by R V infect ion: runny nose, cough, sneezing, were direct ly correlated to the levels o f v i ra l repl icat ion in the airway tissues. However , recent evidence shows that R V repl icat ion occurs at relat ively l ow levels and may not be attributable to the extent o f these symptoms (Lopez-Souza et a l . , 2004). Fo r example , Mosse r et al. (2002) showed that on ly 5-10% of pr imary a i rway epithel ia l cel ls were susceptible to R V no matter what the infect ious dose. Thus , it has been proposed that it is the host immune response to R V that causes the wide-spread symptoms o f i l lness. Fo r example , mount ing evidence suggests that R V infect ion does not manifest i tsel f as a widespread mucosa l infect ion but as smal l loca l ized foc i o f infected cel ls f rom wh i ch widespread in f lammatory responses are generated (Ar ruda et a l . , 1995; Mosse 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. At 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-uti l iz ing R V serotypes: R V 1 4 a major group intercel lular adhesion molecule-1 ( I C A M - 1 ) u t i l i z ing R V , and R V 1 A a minor group l ow density l ipoprote in receptor ( L D L R ) u t i l i z ing R V . Ce l l s were infected w i th R V 1 4 or R V 1 A at a mu l t ip l i c i t y o f infect ion (MO I ) o f 1, corresponding to 1 infect ious v i ra l particle per ce l l , and parameters such as C P E , v i ra l repl icat ion, inter leukin (IL)-6/IL-8 secretion, and R V 1 4 R N A were measured over the course o f infect ion, t yp ica l l y between days 0-7 (D0-D7) after R V inoculat ion. T w o separate G row th Curve trials were conducted for each ce l l type and each R V serotype, and addit ional ly a tandem A 5 4 9 / B E A S - 2 B G row th Curve was carried out by s imultaneously cu l tur ing, infect ing, and sampl ing those ce l l l ines, in order to a l low for a better direct compar ison. H I ce l l G row th Curves (F igure 2.1) were conducted to serve as posi t ive controls for the a i rway epithel ia l models . H I cel ls are epithel ia l cel ls k n o w n to be permiss ive to R V infect ion a l l ow ing for h igh levels o f v i ra l repl icat ion. Because R V infect ion o f H I cel ls causes ce l l death and lys is (and therefore secreted cytokine/chemokine cannot be dist inguished f rom lysed ce l l mediator release) cytokine/chemokine levels were not assayed for H I cel ls . G r o w t h Curve samples were col lected on ly up to D 4 because complete ce l l death had occurred by this t ime. Fo r B E A S - 2 B cel ls (Figures 2.2 and 2.3): C P E , R V 1 4 v i ra l repl icat ion, v i ra l R N A , and IL-6 secretion were assessed for infect ions w i th both R V serotypes. IL-6 was assayed as an indicatory in f lammatory mediator in the B E A S - 2 B mode l because previous laboratory data (not shown) indicated a good response for IL-6 secretion f rom R V infected B E A S - 2 B cel ls . Fo r the A 5 4 9 ce l l mode l (Figures 2.4 and 2.5): C P E , v i ra l repl icat ion, v i ra l R N A , and IL-8 secretion were assessed for both R V serotypes. Here, IL-8 was chosen as an in f lammatory indicator based on previous data (not shown) wh i ch suggested a pronounced IL-8 response in A 5 4 9 cel ls compared to a weak IL-6 response. F ina l l y , for the tandem A 5 4 9 / B E A S - 2 B (Figures 2.7-2.9) the same parameters were assessed, inc lud ing both IL-6 and IL-8 secretion and R V 1 4 R N A . Cell Culture: The S V 4 0 adenovirus transformed human bronchia l epithel ia l ce l l l ine ( B EAS-2B ) was obtained f rom the Amer i c an Type Cul ture Co l l e c t i on ( A T C C ) (Rockv i l l e , M D ) and cultured in 7 5 m m 2 f lasks in 50:50 Du lbecco ' s M o d i f i e d Eag le ' s M e d i u m ( D M E M ) and H a m ' s F12 wi th 1 0 % endotoxin free fetal bov ine serum (FBS ) . Cu l ture 45 reagents were obtained f rom Invitrogen (Vancouver , Canada) . Ce l l s were passaged week ly and incubated at 35-37°C wi th 5 % carbon d iox ide in 9 5 % air. A 5 4 9 cel ls , a transformed alveolar epi thel ia l c e l l l ine , were also cul tured under s imi la r condi t ions but in D M E M and 5 % F B S . F ina l l y , RV-sens i t i ve H I cel ls ( f rom A T C C ) were cultured under the same condit ions w i th D M E M and 5 % F B S . Viruses: Bo th R V 1 4 and R V 1 A were obtained f rom the A T C C . R V s were propagated by infect ing H I cel ls grown to conf luence in 7 5 m m 2 f lasks conta in ing D M E M , a l l ow ing for f u l l C P E . Cell-free culture f lu id was harvested when C P E were at a m a x i m u m by centr i fugation at 10,000 x g for 20 minutes at 4°C. Th is centr i fugation c lar i f ied the virus suspension f rom bacteria and ce l l debris. The stock virus suspension was a l iquoted into cryov ia ls and stored at -80°C for exper imental use. Ti ter o f v i ra l stock was determined by v i ra l plaque assay (see be low) o f serial ly di luted stock, and expressed in plaque forming units (pfu) per m L . P fu represent the number o f infect ious R V particles present in a k n o w n vo lume of sample. Fo r experiments, al iquoted stock virus was rapid ly thawed at 37°C and vortexed pr ior to use. Because such c lar i f ied v i ra l stocks contain H I ce l l remnants (e.g. soluble proteins, organelles) control experiments were conducted to con f i rm that any observed changes in IL-6 and IL-8 secretion were due to virus, and not some other ce l lu lar component present in the inocu la (Append ix C ) . V i r a l Infections and Growth Curves: H I , B E A S - 2 B , or A 5 4 9 cel ls were grown unti l f reshly confluent under standardized condit ions in sterile 6-well plates. Once cel ls reached conf luence it was assumed that ce l l number d id not change s igni f icant ly over the course o f experiments for either control o f R V infected cultures. Th i s assumption was conf i rmed exper imenta l ly (Append ix D ) . C e l l number per w e l l (6-well plate) at conf luence was pre-determined for ce l l l ines (Append ix E ) and used to calculate v i ra l dose. D u r i n g infect ion, cel ls were inoculated w i th R V 1 4 or R V 1 A at an M O I = l , or mock infected w i th med ium, and incubated at 35°C for 1 hour. A f te r infect ion, cel ls were washed 3 times w i th l m L o f med ium to remove any exogenous v i rus , and incubated at 35°C i n 3 m L o f fresh med ium and 1 % F B S . Washes were assayed to con f i rm virus remova l (not shown). Samples were col lected at the same time dai ly between DO ( immediate ly post-infection and washes) and 46 D 7 . Samp l ing consisted o f first r emov ing l m L o f supernatant f rom appropriate we l l s , centr i fuging at 1000 x g to remove ce l lu lar debris, and f reezing samples at -20°C for future cytokine/chemokine assays. Ce l l s f rom R V infected samples were scraped into the remainder o f the m e d i u m (2mL) , pipetted into c ryov ia ls , and stored at -80°C for plaque assays. F i na l l y , for quantitative real-time polymerase chain reaction ( qRT-PCR ) designated samples, med ium was removed, ce l l monolayers washed twice w i th sterile phosphate buffered sal ine, and l m L o f T R I Z O L reagent was added to each we l l , a l l ow ing for ce l l lys is to occur. Th i s suspension was stored in Eppendor f tubes at -80°C for R N A extract ion. Cont ro l experiments were conducted in order to assess the stabi l i ty o f the R V s and IL-6/IL-8 under exper imental condit ions (Appendices F and G ) . Plaque Assays (See Append ix H for image) : V i r a l infect iv i ty and repl icat ion were measured by plaque assays in permiss ive H I cel ls . P rev ious ly f rozen R V infected ce l l samples f rom G rowth Curves were rapid ly frozen and thawed twice at 37°C to rupture cel ls and release v irus. These samples were then serial ly di luted and used to infect permiss ive H I cel ls in duplicate where 0 .75mL o f sample was added onto freshly conf luent H I cel ls grown in 6-well trays, and a l lowed incubated for 1 hour at 35°C. A f te r in fec t ion , the inocu la were aspirated and replaced wi th a 50:50 l i qu id mixture o f 2x M E M (with 5 % F B S ) and 1% sterile agarose in d H 2 0 . The agarose was a l lowed to so l id i f y at room temperature, and plates were incubated at 35°C for 4 days. A s the virus repl icated in infected ce l ls , lys is occurred in the H I cel ls fo rming round areas o f ce l l death ca l led "p laques" . Once incubat ion was complete plates were f ixed w i th 3 % formaldehyde in phosphate buffered sal ine, agarose was removed, and H I cel ls were stained w i th 1 % crystal v iolet in d H 2 0 to reveal the clear unstained plaques. Plaques were counted and reported as pfu/mL (infectious v i r ions/mL) . A n increase in infect ious virus over t ime (relative to DO) indicated v i ra l repl icat ion. Cytokines and Chemokines: IL-6 and IL-8 were assayed in duplicate f r om thawed G row th Curve supernatant samples according to standard protocols prov ided by enzyme-l inked immunosorbent assay ( E L I S A ) commerc ia l kits f rom Immunotools (Fr iesoythe, Germany) . Absorbenc ies were determined by an E L I S A plate reader (Pasteur Diagnost ics 47 L P 4 0 0 ) at a 4 5 0 n m wavelength. H i g h l y concentrated samples were di luted w i th med ium and re-assayed to fa l l w i th in the standard assay range wh i ch was f rom 0-450 pg/mL for both IL-6 and IL-8. Sens i t iv i ty o f the E L I S A s was 4 pg/mL. RNA Extrac t ion: R N A was extracted f o l l ow ing standard T R I Z O L reagent protocol and reconstituted in 5 0 p 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 for R V 1 4 standard curves was extracted f r om stock R V 1 4 suspensions us ing the Q I A G E N R N e a s y k i t (Miss issauga, O N ) . qRT-PCR: q R T - P C R was conducted for R V 1 4 samples. Pr imers were designed us ing the k n o w n R V 1 4 genetic sequence f rom the Nat iona l Center for B io techno logy Information Database and purchased f rom Operon (Huntsv i l le , A L ) . Pr imers were designed w i th the f o l l ow ing sequences: forward 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 238 base pair product. Pr imers were tested by convent ional two-step P C R of k n o w n R V infected samples, and P C R products conf i rmed by gel electrophoresis (not shown). Fo r R V 1 4 R N A samples a one-step q R T - P C R k i t f rom Q I A G E N (Miss issauga, O N ) was used. The P C R mixture contained: 2 5 u L M a s t e r M i x (HotStarTaq D N A Polymerase, Quant iTect S Y B R Green Buf fe r , S Y B R Green I dye, 1 .5mM M g C 1 2 , 2 0 0 u M each d N T P ) , 2 .5uL o f each pr imer, 0.5 u L R T m i x (Omniscr ipt and Sensiscr ipt Reverse Transcriptases), and l O u L o f the R N A template to a f inal vo lume o f 50uL . Standard curve R V concentrations were determined spectrophotometr ical ly and ser ia l ly d i luted at k n o w n concentrations. Dupl i ca te reactions were carr ied out in an M J Research D N A Eng ine Opt i con Cont inuous F luorescence Detector (OP000537) programmed to: incubate at 42°C for 55min , 94°C for 5m in , 94°C for 30 sec, 55°C for 30 sec, 72°C for l m i n , repeat for 35cyc les , 72°C for l O m i n , mel t ing curve f rom 55°C to 95°C read every 0.5°C, incubate 72°C for l O m i n , and 4 °C forever. Data were analyzed us ing Opt i con M o n i t o r analysis software vers ion 1.07. Statistics: G row th Curve infect ious virus (replication) data (n=4 for each day per trial) were subjected to Kruska l-Wa l l i s ranks analysis o f variance ( A N O V A ) w i th day as a 48 factor, f o l l owed by post hoc mul t ip le comparisons Dunnet ' s tests in order to compare da i ly data relative to DO controls. Non-parametric testing was employed because data d id not satisfy parametric assumptions (normality and/or equal variances) and cou ld not be transformed. A s ignif icant increase in infect ious virus over t ime (relative to DO) indicated v i ra l repl icat ion. E L I S A cytokine/chemokine data (n=3 per day for each treatment) were analyzed us ing two-way A N O V A s wi th day and treatment (control uninfected and R V infected cel ls) as factors and post-hoc mul t ip le compar isons Dunnet ' s tests w i th da i ly • control treatments as controls. In the case o f B E A S - 2 B / A 5 4 9 tandem G r o w t h Curves , a 2-way A N O V A was per formed for infect ious virus data us ing day and treatment as factors fo l l owed by a post-hoc Dunnet ' s test us ing DO as a contro l . Fo r IL-6 and IL-8 data a 3-way A N O V A was performed w i th : day, treatment, and ce l l type as factors and a post-hoc Dunnet ' s test w i th da i ly contro l treatments as controls. R N A data (n=2 per ce l l type for each day) were analyzed by 2-way A N O V A wi th day and ce l l type as factors and post-hoc Tukey tests. A l l data were analyzed using S i gma Stat 3.0 software and statistical s igni f icance was set at a=0.05. S igni f icant differences correspond to p<0.05, and h igh ly s ignif icant differences correspond to p<0.001. Results are presented as mean ± standard error o f the mean. RESULTS For al l G r o w t h Curve experiments trials were s igni f icant ly different f rom each other (data cou ld not be pooled) . H I Cells (Figure 2.1): C P E : Fo r HI cel ls infected w i th R V 1 4 C P E began to occur on D l , and manifested themselves as rounded cel ls wh i ch eventual ly became detached f rom the substratum. F u l l C P E and ce l l death was observed for a l l samples and usual ly complete by D 4 . HI cel ls infected w i th R V 1 A also showed C P E result ing in complete ce l l death by the D 3 or D 4 . Anecdota l l y , the occurrence of C P E seemed to occur faster w i th R V 1 A than R V 1 4 . R V 1 4 Replicat ion (F igure 2.1a): Fo r both trials, s ignif icant v i ra l repl icat ion (relative to DO) occurred on D l and D 2 . Fo r trial 1, m a x i m u m infect ious virus was observed on D l (at 49 1.8 x 10 7±2.8 x 10 6 p fu/mL) , and for tr ial 2 infect ious virus also peaked s igni f icant ly on D l at 4.4 x 10 6 ± 1.1 x 1 0 s pfu/mL. RV1A Replication (F igure 2.1b): A g a i n , for both trials, s ignif icant R V repl icat ion (relative to DO) was observed on D l and D 2 . Peak v i ra l titers were measured on D l (3.7 x 10 7 ± 2.4 x 1 0 6 p f u / m L and 1.9 x 10 7± 1.2 x 10 6 p fu/mL respectively) . BEAS-2B Cells (Figures 2.2 and 2.3): CPE: U p o n infect ion o f B E A S - 2 B cel ls w i th R V 1 4 or R V 1 A , no C P E were observed at any point dur ing G row th Curves . RV14 Replication (F igure 2.2a): Fo r tr ial 1, no s ignif icant repl icat ion was observed relative to DO. Peak v i ra l titers occurred on D l (4.4 x 10' ± 1.3 x 10 pfu/mL) , dec l in ing to 1.7 x 10 2 ± 2.9 x 10 1 p fu/mL by D 7 . In trial 2, s ignif icant v i ra l repl icat ion (relative to DO) occurred on D l at 2.2 x 10 4 ±1.2 x 10 3 pfu/mL. RV14 and IL-6 Secretion (Figures 2.2b and c ) : Fo r trial 1 (F igure 2.2b), no signif icant differences were observed between uninfected (control) and R V 1 4 infected cel ls at any t ime post-infection. F o r trial 2 (Figure 2.2c), IL-6 secretion in R V 1 4 infected cel ls was h igh ly s ignif icant when compared to uninfected controls on D 3 , D 5 , and D 7 post-infection. Peak IL-6 concentrat ion was observed on D 7 at 3517.9 ± 993.8, and on D 3 IL-6 concentration in R V infected cel ls was nearly 1375 times its contro l . RV1A Replication (F igure 2.3a): Fo r tr ial 1, v i ra l repl icat ion was s igni f icant ly different f rom DO on D 2 and D 3 . Peak v i ra l titers o f 7.3 x 10 4 ± 2.6 x 10 3 p fu/mL were observed on D 2 . In trial 2, s ignif icant v i ra l repl icat ion occurred on D l relative to DO, wh i ch represented the peak R V titer at 2.9 x 10 4± 1.4 x 10 3 pfu/mL. RV1A and IL-6 secretion (F igures 2.3b and c): In tr ia l 1, IL-6 concentrat ion (F igure 2.3b) was s igni f icant ly different between treatments oh D 3 , D 5 , and D 7 . Peak IL-6 concentration occurred on D 7 at 2434.3 ± 215.3 pg/mL wh i ch represented an 8-fold increase over its contro l . 50 For trial 2 (Figure 2.3c), there was a signif icant difference between uninfected and R V 1 A infected ce l ls on D 3 , D 5 , and D 7 . Peak IL-6 concentrations were measured on D 7 at 4224.3 ± 530.0 pg/mL, wh i ch was 570 times contro l levels. A549 Cells (Figures 2.4 and 2.5): CPE: N o C P E or ce l l death was observed at any point, D 0 - D 7 , for A 5 4 9 cel ls infected w i th R V 1 4 o r R V 1 A . RV14 Replication (F igure 2.4a): Fo r both trials, no signif icant R V 1 4 repl icat ion occurred relative to DO. RV14 and IL-8 Secretion (Figures 2.4b and c ) : In the first tr ial (Figure 2.4b), no signif icant differences were observed in IL-8 secretion between treatments. Fo r trial 2 (Figure 2.4c), there were statistical ly s ignif icant differences between treatments on D l , D 2 , D 3 , and D 5 . Peak IL-8 concentration was observed on D 5 at 302.3 ± 39.5 pg/mL, wh i le on D 2 the largest increase relative to control was measured (5-fold). RV1A Replication (F igure 2.5a): Fo r the first tr ia l , a s ignif icant difference in infect ious v i rus was observed on D l and D 2 relative to DO. Peak v i ra l repl icat ion was measured on D l w i th 3.7 x 10 s ± 6.9 x 10 3 pfu/mL. In tr ial 2, a signif icant difference in v i ra l repl icat ion was also observed on D l relative to DO. Peak R V 1 A titers were measured on this day at 5.6 x 10 4± 1.3 x 10 4 pfu/mL. RV1A and IL-8 Secretion (Figures 2.5b and c ) : Fo r tr ial 1 (Figure 2.5b), IL-8 concentrations were h igh ly s ignif icant between treatments on D l , D 2 , D 3 , and D 6 . Peak IL-8 concentration was observed on D 6 at 769.0 ± 57.4 pg/mL; however , the largest increase in IL-8 relative to control was measured on D l (3.5-fold). In trial 2 (Figure 2.5c), there was a h igh ly s ignif icant increase in IL-8 secretion between treatments for D 2 , D 3 , and D 6 . Peak IL-8 concentration was 1864.9 ± 25.3 pg/mL on D 6 , and the largest increase in IL-8 secretion relative to its control was observed on D 2 (5.2 t imes). Growth Curve RV14 RNA Levels (F igure 2.6): There is a statist ical ly s igni f icant difference in R N A levels between H I ce l ls and both B E A S - 2 B and A 5 4 9 cel ls (all days combined) . H I cel ls produced increasing R N A levels to 51 a peak o f 2.8 x 10 5 ±1.4xl0 4 pg/well at D 3 , and al l time-points after DO were in the 10 3 " 5 pg range. Fo r B E A S - 2 B cel ls peak R V 1 4 R N A levels occurred on D 3 measur ing 17.7 ± 7.2 pg/well and then decreasing to 0.005 ± 0.001 pg/well by D 7 . Fo r A 5 4 9 cel ls , peak R V 1 4 R N A was measured on DO at 1.7 x 10"6± 6.7 x 10" 7 pg/wel l , and then dec l ined to 2.3 x 10 " 1 0 ± 7.8 x 10" 1 1 pg/well on D l , after wh i ch R V 1 4 R N A was undetectable for D 2 , D 3 and D 6 . W i t h i n the HI ce l ls , R V 1 4 R N A is s igni f icant ly higher on D 2 relative to DO. For B E A S - 2 B and A 5 4 9 cel ls there were no signif icant differences observed w i th in the respective ce l l types when compared to DO. BEAS-2B/A549 Tandem (Figures 2.7-2.9): C P E : N o C P E or ce l l death was observed in any o f the samples infected w i th either R V 1 4 or R V 1 A for B E A S - 2 B or A 5 4 9 cel ls . RV14 Replication (F igure 2.7a): Rep l i ca t ion levels for B E A S - 2 B cel ls were s igni f icant ly different f rom A 5 4 9 cel ls on D l , D 2 and D 3 . W i t h i n B E A S - 2 B cel ls there was a signif icant difference observed on D l and D 2 relative to DO. Peak v i ra l titer was measured on D l at 1.6 x 10 4± 9.6 x 10 2 p fu/mL . W i t h i n A 5 4 9 cel ls there were no statist ical ly s ignif icant differences in R V 1 4 repl icat ion. IL-8 Secretion (F igure 2.7b): 11-8 concentrations were h igh ly s ignif icant between B E A S -2B and A 5 4 9 cel ls (all days combined) ; however, no signif icant differences between infected and uninfected cel ls were observed in either ce l l l ine for IL-8 secretion. The m a x i m u m LL-6 concentrat ion measured was 327.1 ±42.3 pg/mL in the B E A S - 2 B cel ls on D 7 . IL-6 Secretion (F igure 2.7c): A g a i n mediator secretion was h igh l y s ignif icant between B E A S - 2 B and A 5 4 9 cel ls (al l days combined) , but there were no differences observed between treatments. M a x i m u m IL-6 concentration in B E A S - 2 B cel ls was measured on D l at 27.4 ± 2.8 pg/mL. RV1A Viral Replication (Figure 2.8a): Rep l i ca t ion levels in B E A S - 2 B cel ls were h igh ly s ignif icant compared to A 5 4 9 cel ls as observed on D l and D 2 . W i t h i n B E A S - 2 B cel ls h igh ly s ignif icant increases in v i ra l repl icat ion were measured on D l and D 2 . Peak v i ra l repl icat ion (relative to DO) occurred on D l at 3.4 x 10 4± 3.6 x 10 3 p fu/mL . W i t h i n A 5 4 9 cel ls no s ignif icant differences in R V 1 A repl icat ion were observed, a l though a typica l 52 G r o w t h Curve trend was observed w i th peak v i ra l titer o f 4.5 x 10 3 ± 1.4 x 1 0 2 p fu/mL on D l . IL-8 Secretion (F igure 2.8b): IL-8 concentrations were s igni f icant ly different between B E A S - 2 B and A 5 4 9 cel ls (all days combined) . W i t h i n the B E A S - 2 B cel ls those infected w i th R V were h igh ly s ignif icant f rom their respective controls for D 2 , D 3 , D 5 , and D 7 . Peak IL-8 concentrat ion was measured on D 5 at 2236.7 ± 60.4 pg/mL. The same trend was observed for A 5 4 9 cel ls where there was a h igh l y s ignif icant difference between treatments for D 2 , D 3 , D 5 , and D 7 and m a x i m u m LL-8 concentration was measured on D 3 (1966.7 ± 83.3 pg/mL). IL-6 Secretion (F igure 2.8c): Di f ferences between B E A S - 2 B and A 5 4 9 cel ls were h igh ly s ignif icant (al l days combined) . W i t h i n B E A S - 2 B cel ls , treatments were s igni f icant ly different on D 3 and h igh ly s ignif icant on D 7 . Peak LL-6 concentrat ion for B E A S - 2 B was 2615 ± 124.1 pg/mL wh i ch was 53 times its contro l . In A 5 4 9 cel ls both D 3 and D 7 were h igh ly s ignif icant between treatments. Peak IL-6 concentration for A 5 4 9 cel ls was measured on D 7 at 1114.7 ± 105.5 pg/mL wh i ch represented the m a x i m u m increase relative to control at 124 times. B E A S - 2 B / A 5 4 9 Tandem R V 1 4 R N A (Figure 2.9): There was a statist ical ly s ignif icant difference in R V 1 4 R N A levels between B E A S - 2 B and A 5 4 9 cel ls as observed on D l and D 2 . Fo r B E A S - 2 B peak R N A levels were measured on D 2 at 11.6 ± 5.8 pg/wel l , wh i le peak R V 1 4 R N A in A 5 4 9 cel ls was observed on DO at 4.0 x 10~2 ± 9.4 x 10~3 pg/wel l . However , w i th in ce l l l ines no s igni f icant ly differences were observed relative to DO. DISCUSSION R V infect ion o f the a i rway epithel ia l ce l ls ( B E A S - 2 B and A 5 4 9 ) resulted in signif icant v i ra l repl icat ion and st imulat ion o f cytokine/chemokine secretion in the overwhe lming majority o f experiments, certainly support ing the g row ing consensus that R V infect ion does provoke an in f lammatory response in its host cel ls. C P E : Prev ious studies of in vitro ce l l l ine experiments, cultured pr imary ce l ls , and b iopsy der ived a i rway epithel ia l tissues indicated that no C P E or ce l l death was observed dur ing 53 R V infect ion (Mosser et a l . , 2002 ; Gr i ego et al 2000, Lopez-Souza et a l , 2004). Fo r example , Jang et al. (2005) found that nasal turbinate mucosa infected w i th R V 1 6 d id not cause any observable damage to the pseudostratif ied co lumnar ep i the l ium, basement membrane, or c i l i a . M y results also support these f indings since no C P E or ce l l death were observed for either the B E A S - 2 B or A 5 4 9 ce l l l ines at any point dur ing G row th Curve experiments for either R V serotype. In contrast, a l l permiss ive HI cel ls infected w i th R V exhib i ted C P E by D l and complete ce l l death by D 4 (a typica l ly t ic cycle) . These observations suggest that R V has some unknown mechanism o f cross ing out the p lasma membrane without rupturing the ce l l , or alternatively, a minute fract ion o f cel ls lyse, and these l ow loca l i zed levels o f R V are suff ic ient to induce a pronounced immune response. It may be that both the above mechanisms do occur, and act synergist ica l ly to e l ic i t the observed in f lammatory response. L o w levels of Replicat ion Compared to H I Cells: There is growing consensus that R V replicates at very l ow levels in the a i rway epithel ia l tissues. W h e n compar ing m y G r o w t h Curve data between H I ce l ls and the a i rway ce l l models this hypothesis certainly seems to be supported. HI cel ls produced peak R V titers at 3-4 orders o f magnitude higher than B E A S - 2 B cel ls , and this trend was general ly more exaggerated for the A 5 4 9 ce l l mode l . E ven taking into account the 1.4-fold greater HI ce l l number at conf luence (see A p p e n d i x E ) the levels o f R V repl icat ion remain impressive. What makes the a i rway cel ls less susceptible to R V infect ion than the permiss ive cel ls remains unknown . It is feasible that the a i rway cel ls a l low less RV-receptor b ind ing and entry than H I cel ls because o f fewer surface I C A M - 1 or L D L R receptors. S ince R V receptor expression is a h igh ly dynamic process this hypothesis is d i f f i cu l t to test. There is evidence that R V infect ion causes a rapid up-regulation in the surface expression o f I C A M - 1 (Grunberg, 2000; Papi & Johnston, 1999; Wh i teman et a l . , 2003 ; W in ther et a l . , 2002) , but perhaps this process is not as eff ic ient in the a i rway cel ls when compared to permiss ive cel ls . T a k i n g the G r o w t h Curve data into considerat ion, it seems that H I cel ls may a l low for increased v i ra l passage across the ce l l membrane. DO levels in H I cel ls are on the order o f 10 4 , wh i le the a i rway cel ls DO values are lower , even bear ing in m ind in i t ia l ce l l numbers and v i ra l dose. Furthermore, regardless o f infect ion suscept ibi l i ty , a i rway epithel ia l cel ls may on l y a l low 54 for l im i ted R V repl icat ion. Th is cou ld potential ly occur by an active mechan ism, or passive ly due to s lower and/or less eff ic ient repl icat ion machinery. G r o w t h Curve Replicat ion Trends: In the vast majority o f the G row th Curve repl icat ion data a s ignif icant leve l o f v i ra l repl icat ion was observed between D l and D 2 (relative to DO), w i th the majority o f peak titers be ing measured on D l . F o l l o w i n g the increases in R V repl icat ion titers tended to gradual ly decrease to starting levels and were not s igni f icant ly different f rom DO after D 2 . H o w these cultured cel ls manage to resolve R V infect ion is st i l l largely a mystery. In vivo, in f lammatory mediators recruit and activate white b lood cel ls to engul f pathogens, and ant ibody format ion is thought to ult imately r id the body o f infect ion (Janeway, 2005). H o w e v e r the a i rway ce l l models also seem to be capable o f mit igat ing R V infect ion without the aid o f immune cel ls . It is possible that the epithel ia l ce l l is able to recognize v i ra l repl icat ion by some intra-cellular immune process and down-regulate its genomic machinery in response to R V . There is emerging evidence o f intra-cellular immun i t y against retroviruses, and furthermore, some eukaryotic cel ls are capable of b lo ck ing polyc is t ronic m R N A translation (Fire, 2005 ; Zheng et a l . , 2005). R V induced cytokine/chemokine secretion f r om affected cel ls may also alert ne ighbour ing ce l ls to decrease suscept ibi l i ty to R V infect ion. Th is cou ld potential ly be accompl ished by affect ing the transcriptional cascades needed by R V . The question remains whether R V infects many cel ls in the monolayer a l l produc ing l ow R V titers, or i f on ly a few cel ls become infected each repl icat ing R V at high levels. Mosse r et al. (2005) found that in b iopsy derived tissues on ly 5-10% o f the cel ls were infected regardless o f v i ra l dose, and h is to log ica l l y R V infect ion manifested itself as smal l loca l ized clusters o f infected cel ls . However , it remains unclear i f v i ra l entry is even necessary to induce an in f lammatory response, arguably a mere RV-receptor or ce l l interaction is suff icient to trigger cytokine/chemokine secretion. Fo r example , one study found evidence o f cytokine/chemokine st imulat ion by ultraviolet ( U V ) inactivated R V (Johnston et a l . , 1998). In such a case, R V repl icat ion levels and the number o f infected cel ls may be complete ly irrelevant cons ider ing that R V cou ld stimulate epithel ia l cel ls to secrete in f lammatory cytokines/chemokines irrespective o f R V cross ing the p lasma membrane. A t the same t ime, it is clear that R V does infect at least some cel ls and s igni f icant ly replicate in the a i rway ep i the l ium. Fo r a majori ty o f patients presenting w i th 55 self-diagnosed colds R V can be consistently isolated f rom a i rway lavages and tissues (Ar ruda et a l . , 1997; Johnston et a l . , 1995; van Gageldonk-Lafeber et a l . , 2005). Perhaps R V repl icat ion is not the cause o f co ld symptoms but plays a cruc ia l role in the propagation o f the infect ion by causing the release o f new v i ra l progeny onto yet unaffected cel ls and generating an addit ive in f lammatory response. Immune Response Hypothesis: Because o f the observed l ow levels o f R V repl icat ion, there is a grow ing hypothesis that the i l lness associated w i th R V infect ion is init iated by the v i rus, but propagated and ampl i f i ed by the host immune system through the secretion of pro-inf lammatory cytokines and chemokines l i ke IL-6 and IL-8. Such a mechanism is very different f rom many viruses where ce l l death caused by v i ra l repl icat ion is the source o f virus pathology. Johnston et al. (1998) found a pro longed release o f IL-8 up to 120 hours in the pu lmonary ep i the l ium to l ow doses o f R V 9 even though repl icat ion peaked at 24 hours. Gern et al. (2000) also found increased IL-8 secretion in response to R V 1 6 in vivo w i th no correlat ion to quantities o f v i ra l shedding. In exper imental colds rhinorrhea symptoms usual ly peaked on D 2 whi le in natural colds the m a x i m u m occurred on D 3 , but throat and cough symptoms peaked closer to D 4 , indicat ing increasing symptoms we l l beyond m a x i m u m v i ra l repl icat ion (Gwal tney et a l . , 2003). In an in vitro context, m y cytokine/chemokine data also support these f indings. A s prev ious ly descr ibed, peak v i ra l titers were consistently measured between D l and D 2 for a l l ce l l models w i th no s ignif icant v i ra l repl icat ion f rom D 3 onward. However , elevated cytokine/chemokine secretions and m a x i m u m increases when compared to respective controls were typ ica l l y observed later in infect ion (often D2-7). A l s o cytok ine and chemokine secretion remained elevated we l l beyond detectable R V repl icat ion. The mechan ism leading f rom R V interaction/infection w i th its host ce l l to the secretion o f pro-inf lammatory cytokines/chemokines (such as IL-6 and IL-8) is not we l l described. IL-6 and IL-8 transcript ion is contro l led by various transcription factors inc lud ing nuclear factor k a p p a - B ( N F K B ) (Caamano & Hunter, 2002 ; O l i ve i r a et a l . , 1994). N F K B is a cytosol ic protein, norma l l y bound by inhibi tors , wh i ch becomes unbound in response to various stressors and binds its promoter sites w i th in the nucleus result ing in the increased transcription o f various cytokines/chemokines (Caamano & Hunter, 2002) . There is some evidence that R V infect ion is mediated through an N F K B mechan ism result ing in the up-56 regulat ion o f in f lammatory mediator secretion (Spurrel l et a l . , 2005 ; Z h u et a l . , 1997). However , Sharma et al. (2006) showed elevated expression o f over 30 transcription factors ( inc luding N F K B ) upon R V infect ion, thus c lear ly the R V mechanism remains poor ly understood. Cell Model and R V Serotype Comparisons: A direct compar ison o f the two a irway epithel ia l models showed s igni f icant ly more R V repl icat ion, R N A , and cytokine/chemokine secretion in B E A S - 2 B cel ls than observed in A 5 4 9 cel ls regardless o f R V serotype. A cause for this increased susceptibi l i ty o f B E A S - 2 B cel ls to infect ion is not k n o w n but may be attributable to increased expression o f R V receptors, and/or a more eff ic ient or faster repl icat ion cyc le . Whether this increased B E A S - 2 B suscept ib i l i ty is representative o f cel ls occurr ing higher in the airways is d i f f i cu l t to say cons ider ing that cultured cel ls may not represent the native a i rway epi the l ium adequately, and overal l both ce l l l ines are der ived f r om the m i d to lower respiratory tract. Furthermore, A 5 4 9 cel ls represent surfactant secreting lung cel ls wh i ch are intr ins ica l ly different f rom bronchia l epithel ia l tissues. However , it seems that at least in vitro both bronchia l and alveolar-derived cel ls can be infected by R V and initiate in f lammatory responses. Th i s evidence further supports g row ing evidence that lower a i rway cel ls are vulnerable to R V infect ion (Gern et a l . , 1997; Hayden , 2004) , and is important in the pathology o f diseases such as asthma and C O P D wh i ch are considered lower a i rway diseases. R V 1 A produced more pronounced effects than R V 1 4 in terms o f v i ra l repl icat ion and IL-6/IL-8 secretion for both B E A S - 2 B and A 5 4 9 cel ls , and addi t ional ly R V 1 4 fai led to replicate in the A 5 4 9 cel ls . Th is may be due to differential expression o f L D L R receptor versus I C A M - 1 receptors but cou ld also be caused by intr insic differences between the two serotypes. The R V 1 4 genome has been fu l l y sequenced (Stanway et a l . , 1984); however, on ly certain port ions o f the R V 1 A genome are k n o w n thus sequence homo logy between the two is undetermined. R V studies may ut i l ize any o f 100+ serotypes and although serotypes are documented the potential differences amongst them are largely ignored. M y data suggest that serotype select ion may be an important factor for R V research. R V 1 4 experiments were also m u c h more variable in their IL-6 and IL-8 responses than R V 1 A . Fo r some G row th Curves R V 1 4 fa i led to stimulate a pro-inf lammatory response (Figure 2.2b), wh i l e in other exper imenta l ly identical experiments IL-6/IL-8 st imulat ion was h igh ly 57 signif icant (F igure 2.2c). The source o f this var iab i l i ty is unknown , but perhaps the R V 1 4 v i ra l dose was near some threshold leve l for tr iggering a cytokine/chemokine response in a i rway epithel ia l cel ls . Ove ra l l , the B E A S - 2 B / R V 1 A is the most consistent mode l for R V infect ion o f the a irways. R V infect ion of cultured a i rway epithel ia l cel ls results in relat ively l ow levels o f v i ra l repl icat ion accompanied by a prolonged secretion o f cytokines and chemokines such as IL-6 and IL-8. Further research should focus on the speci f ic l inks between R V infect ion and the e l ic i ted a i rway epithel ia l in f lammatory response in order to further define the mechanisms invo lved . 58 FIGURES a) Figure 2.1: Effect of a) RV14 and b) RV1A on viral replication over time in HI cells. Each trial represents n=4. An asterisk (*) indicates a significant difference (ct=0.05) in infectious virus relative to DO within a given trial. 59 a) b) c) 6000 5000 Q _ c o 2 3000 CD 6000 5000 O) -2= 4000 S 3000 cz CD O 2 0 0 0 H 1000 •{ 0 1 2 3 4 5 6 7 Time After RV Infection (Days) 4000 i 2 0 0 0 1 0 0 0 H 0 1 2 3 5 7 Time After RV Infection (Days) Control Trial 2: BEAS-2B/RV14 F = l RV14 * 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 RV 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) c) 6000 -5 5000 a. § 4000 •£ 3000 a> o o 2000 CD - 1000 Control RV1A Trial 1: BEAS-2B/RV1A J L I i 0 1 2 3 5 7 Time After RV Infection (Days) 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) b) E £ 6 a o o> 2 5 3 g o A549/RV1A Trial 1 Trial 2 0 1 2 3 4 5 6 Time After RV Infection (Days) 2500 c) E 2000 -j a. o 1500 g 1000 c o O 500 Control RV1A Trial 1: A549/RV1A I i 0 1 2 3 6 Time After RV Infection (Days) 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 RNA for H I , BEAS-2B, and A549 cells over time. Daily RV14 RNA levels were measured for each cell line (n=2 each). An asterisk (*) indicates a significant difference (ct=0.05) in RV14 RNA within a given cell type relative to its DO. There is a significant difference in RNA 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) b) 0 1 2 3 4 5 6 7 Time After RV Infection (Days) 500 C ) E 400 ] 300 g 200 -I c o O °? 100 Control RV14 BEAS-2B IL-8/RV14 A549 - F *F W1' 0 2 3 5 7 0 2 3 5 7 Time After RV Infection (Days) 500 E 400 Control RV14 IL-6/RV14 300 200 c o O CD 100 BEAS-2B A549 L0_ JLEL 1 3 7 1 3 7 Time After RV 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 RV 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). 6 5 c) 4000 E "Bi 3000 o. c g 2 2000 c CD o c o ° 1000 co Control RV1A BEAS-2B IL-8/RV1A A549 J , l l 0 2 3 5 7 0 2 3 5 7 Time After RV Infection (Days) 4000 E "Q, 3000 g 2 2000 <D o c o O CD 1000 -\ Control RV1A BEAS-2B IL-6/RV1A A549 J L 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 RV infected and uninfected controls (n=3 each) at a given time. 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Amer i c an Journal o f Respirat ion and Cr i t i ca l Care Med i c i ne . 155:1362-1366. Turner, R. B. 1998. Assoc ia t ion between interleukin-8 concentration in nasal secretions and severity o f symptoms o f exper imental rh inov i rus colds. C l i n i c a l Infectious Diseases. 26: 840-846. Turner, R. B. 2001 . Rev i ew : The treatment of rh inov i rus infect ions: Progress and potential . An t i v i r a l Research. 49:1-14. van Gageldonk-Lafeber, A., M . Heijnen, A. Bareids, M . Peters, S. van der Plas, and B. Wilbrink 2005. A case-control study o f acute respiratory tract infect ion in general practice patients in the Netherlands. 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 2003. H u m a n rhinovirus select ively modulates membranous and soluble forms o f its intercel lular adhesion molecule-1 ( I C A M - 1 ) receptor to promote epithel ia l ce l l infect iv i ty . Journal o f B i o l o g i c a l Chemist ry . 278:11954-11961. 71 Winther, B . , E . Arruda, T. J . Witek, S. D . Marlin, M . M . Tsianco, D . J . Innes, and F. G . Hayden 2002 . Express ion o f I C A M - 1 in nasal ep i the l ium and levels o f soluble I C A M - 1 in nasal lavage f l u id dur ing human experimental rh inovirus infect ion. A rch i ves o f Oto laryngolo lgy- Head & N e c k Surgery. 128:131-136. Zheng, Y . H . , and M . Peterlin 2005. Intracellular immun i t y to H I V - 1 : N e w l y def ined retroviral battles inside infected cel ls . Ret rov i ro logy . 2:25-38. Zhu, Z. , W. Tang, J . M . Gwaltney, Jr., Y . Wu, and J . A . Elias 1997. Rh inov i rus st imulat ion o f interleukin-8 in vivo and in vitro: Ro l e o f NF-kappa B. A m e r i c a n Journal Phys io logy L u n g Ce l l u l a r and M o l e c u l a r Phys io logy . 273:L814-824. Zhu, Z. , W. Tang, A. Ray, Y. Wu, O. Einarsson, M . L . Landry, and J . M . Gwaltney, Jr. 1996. Rh inov i rus st imulat ion o f interleukin-6 in vivo and in vitro. The Journal 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 Despite the fact that rh inov i rus ( R V ) colds are the most prevalent i l lness in humans, very l itt le is k n o w n about R V pathogenesis. S ince evidence suggests that R V s cause l itt le or no damage to their host tissues and replicate at re lat ively l ow levels w i th in the host, it has been postulated that the symptoms o f c o m m o n colds are the result o f a host in f lammatory response characterized by the secretion o f pro-inf lammatory cytokines (and chemokines) such as inter leukin (IL)-6, and not R V repl icat ion per se ( Lopez-Souza et a l . , 2004 ; Mosse r et a l , 2005 ; Mosse r et a l . , 2002). Furthermore, a l ink has been demonstrated between the severity o f co ld symptoms (e.g. rhinorrhea, sore throat, cough ing , malaise) and increases in in f lammatory mediator secretion (Gwal tney et a l . , 2003) . If repl icat ion is not the cause o f R V pathology then it is conceivable that R V triggers an in f lammatory response in its host a i rway epithel ia l cel ls by some virus-cel l interaction without the necessity o f R V repl icat ion or passage across the p lasma membrane. One method for invest igat ing this hypothesis is by us ing ultraviolet ( U V ) inactivated R V for exper imental infect ions. There is evidence that U V C (260nm) irradiat ion o f R V first affects the v i ra l nuc le ic ac id site before impact ing protein structures. Consequent ly U V exposure causes infect iv i ty to be lost before observed changes in antigen spec i f ic i ty (Hughes et al . , 1979). U V pr imar i l y causes damage in the fo rm of cyc lobuty lpy r im id ine dimers and photoproducts wh i ch inh ib i t the repl icat ion o f genetic material (Myatt et a l . , 2003). If noninfect ious R V is capable o f st imulat ing an in f lammatory response in host ce l ls , then this supports the idea o f a non-replicative trigger for R V pathology. V e r y few studies have investigated such a hypothesis; however , there is l im i ted evidence that inact ivated R V may retain some o f its cytokine st imulat ing abi l i t ies. Fo r example , Johnston et al. (1998) found that 30 minute U V C irradiat ion o f R V 9 complete ly inhib i ted v i ra l repl icat ion in A 5 4 9 cel ls , but on ly reduced IL-8 secretion by about one half. On the other 2 A version of this chapter will be submitted for publication. Machala, A . M . , Harris, R.A., Brauner, C.J., Hudson, J.B. 73 hand, Gr i ego et al. (2000) found that U V treated R V d id not induce much IL-6 or IL-8 secretion in B E A S - 2 B cel ls beyond control values. It is unclear whether noninfect ious R V is capable o f e l i c i t ing an immune response in airway epithel ia l cel ls . Th i s study investigated the abi l i ty o f UV-treated R V to stimulate IL-6 secretion in the bronch ia l B E A S - 2 B ce l l l ine. MATERIALS AND METHODS A l l v i ra l , ce l l culture and molecular work was conducted under sterile condit ions in a type II biosafety cabinet. A l l protocols were pre-approved by the U B C biosafety committee in certif icate H04-0061 (Append ix A ) . Experiments: Cu l tured B E A S - 2 B cel ls were inoculated w i th either R V 1 4 or R V 1 A prev ious ly treated w i th U V C for exposure t imes of 2, 5, 10, 15 and 30 minutes ( U V 2 , U V 5 , U V 1 0 , U V 1 5 , and U V 3 0 respectively) , or w i th untreated R V ( N O U V 0 , N O U V 3 0 ) . Infectious v i rus, IL-6 secretion, and v i ra l R N A were measured at t ime=0 (Ohrs) immediate ly post-infection, and 48 hours later (48hrs). A l though the nucle ic acid site is thought to be affected by U V first, the exact t iming o f nucle ic ac id and protein disrupt ion is largely unknown thus R V s were exposed to U V for different lengths of t ime to produce noninfect ious virus wh i ch retained as many o f its other characteristics (e.g. antigenic activity) as possible. T w o separate trials were conducted for each R V serotype (n=3 each). Cel l Cul ture : The S V 4 0 adenovirus transformed human bronchia l epithel ia l ce l l l ine ( B EAS-2B ) was obtained f rom the Amer i c an Type Cul ture Co l l ec t ion ( A T C C ) (Rockv i l l e , M D ) and cultured in 7 5 m m 2 f lasks in 50:50 Du lbecco ' s M o d i f i e d Eag le ' s M e d i u m ( D M E M ) and H a m ' s F12 wi th 1 0 % endotoxin free fetal bov ine serum (FBS ) . Cul ture reagents were obtained f r om Invitrogen (Vancouver , Canada) . Ce l l s were t ryps in ized and passaged week l y and incubated at 35-37°C wi th 5 % carbon d iox ide in 9 5 % air. R V -sensitive H I cel ls ( f rom A T C C ) were cultured under the same condit ions w i th D M E M and 5 % F B S . 74 Viruses: Bo th R V 1 4 and R V 1 A were obtained f rom the A T C C . R V s were propagated by infect ing HI cel ls grown to conf luence in 7 5 m m 2 f lasks conta in ing D M E M , a l l ow ing for fu l l cytopathic effects (CPE ) . Cell-free culture f l u id was harvested when C P E were at a m a x i m u m by centr i fugation at 10,000 x g for 20 minutes at 4 °C. The stock virus suspension was al iquoted into c ryov ia ls and stored at -80°C for exper imental use. T i ter o f v i ra l stock was determined by v i ra l plaque assay (see be low) of ser ial ly di luted stock, and expressed in plaque fo rming units (pfu) per m L . P fu represent the number o f infect ious R V particles present in a known vo lume o f sample. F o r experiments, al iquoted stock virus was rapid ly thawed at 37°C and vortexed pr ior to use. Because such c lar i f ied v i ra l stocks contain H I ce l l remnants (e.g. soluble proteins, organelles) control experiments were conducted to con f i rm that any observed changes in IL-6 and IL-8 secretion were due to virus and not some other component present in the inocu la (Append ix C ) . Preparation of U V inactivated virus: Stock suspensions o f either R V 1 4 or R V 1 A wi th k n o w n v i ra l titers (10 8 pfu/mL) and equal vo lumes were placed in single-wel l culture plates (l ids removed) and irradiated by a 260nm U V C l ight source placed 10 c m away in a dark room. Plates were gently agitated throughout U V C exposure. A t specif ic durations o f U V treatment: 2, 5, 10, 15 and 30 minutes ( U V 2 , U V 5 , U V 1 0 , U V 1 5 , and U V 3 0 respectively) , equal vo lumes o f R V stocks were removed, placed in cryov ia ls , and immedia te ly stored at -80°C. Suff ic ient stock R V vo lume was used to avo id dry ing o f samples dur ing U V exposure. Cont ro l R V stocks were prepared s imultaneously under the same condit ions except shielded f rom l ight exposure in order to control for R V stabi l i ty over the 30 minute sampl ing per iod. The control samples were col lected just pr ior to U V treatment ( N O U V 0 ) , and at the 30 minute endpoints ( N O U V 3 0 ) . Infections with U V inactivated RV: B E A S - 2 B cel ls were plated into 6-well trays and grown unt i l f reshly confluent. Once cel ls reached conf luence it was assumed that ce l l number d id not change s igni f icant ly over the course o f experiments for either control or R V infected cultures. Th i s assumption was conf i rmed exper imental ly (Append ix D ) . C e l l number per we l l (6-well plate) at conf luence was pre-determined for ce l l l ines (Append ix E ) and used to calculate v i ra l dose. R V inocu la were prepared in culture m e d i u m (no F B S ) 75 f r om the above descr ibed 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 th equivalent amounts o f v i rus, based on a p re-UV treatment v i ra l dose o f M O I = l . A t infect ion, med ium was aspirated f rom cel ls and replaced wi th l m L of the appropriate UV-treated R V inocu la , or w i th med ium alone (control) , and cel ls were incubated for 1 hour at 35°C. A f te r infect ion, inocu la were aspirated and cel ls washed 3 times w i th l m L culture med ium to remove exogenous virus (virus remova l exper imenta l ly conf i rmed, not shown). F o l l o w i n g this, 3 m L o f fresh med ium ( 1 % F B S ) was added to the cel ls and one subset o f the plates was sampled at Ohrs wh i le the remainder were incubated at 35°C and sampled at 48hrs. Samp l ing consisted of first r emov ing l m L o f supernatant f rom appropriate we l ls , centr i fuging at 1000 x g to remove ce l lu lar debris, and f reezing samples at -20°C for future IL-6 assays. Ce l l s f rom R V infected samples were scraped into the remainder o f the med ium (2mL) , pipetted into c ryov ia ls , and stored at -80°C for plaque assays. F i na l l y , for quantitative real-time polymerase chain reaction ( qRT-PCR ) designated samples, med ium was removed, ce l l monolayers washed twice w i th sterile phosphate buffered sal ine, and l m L o f T R I Z O L reagent was added to each we l l a l l ow ing for ce l l lys is to occur. Th i s suspension was stored in Eppendor f tubes at -80°C for subsequent R N A extract ion. Plaque Assays (See Append ix H for image) : V i r a l infect iv i ty and repl icat ion were measured by plaque assay in permiss ive HI cel ls. Once al l the ce l l scraping samples were col lected, they were rapid ly frozen and thawed at 37°C twice, to rupture cel ls and release v irus. These samples were used at serial d i lut ions to infect the permiss ive HI cel ls in dupl icate. 0 .75mL o f sample was added onto freshly conf luent H I cel ls g rown in 6-well trays and a l lowed to infect for 1 hour at 35°C. A f t e r in fect ion, the inocu la were aspirated and replaced wi th a 50:50 l i qu id mixture o f 2x M E M (with 5 % F B S ) and 1 % agarose gel . The agarose gel was a l lowed to so l id i f y at r oom temperature, and then the plates were incubated at 35°C for 4 days. A s the virus repl icated in infected ce l ls , lys is occurred in the HI cel ls fo rming round areas o f ce l l death ca l led "p laques " . Once incubat ion was complete, plates were f i xed w i th 3 % formaldehyde, agarose was removed, and cel ls stained wi th crystal v iolet to reveal the clear unstained plaques. These plaques were then counted and 76 reported as p fu/mL ( infectious v ir ions) present in the sample. A n increase i n infect ious virus over t ime (relative to DO) indicated v i ra l repl icat ion. Cytokines: IL-6 was assayed in duplicate f rom supernatant samples us ing standard protocol prov ided by commerc i a l l y avai lable enzyme-l inked immunosorbent assay ( E L I S A ) kits f rom Immunotools (Friesoythe, Germany) . Absorbenc ies were read on an E L I S A plate reader (Pasteur Diagnost ics L P 4 0 0 ) at a 4 5 0 n m wavelength. H i g h l y concentrated samples were di luted w i th med ium and re-assayed to fa l l w i th in the standard assay range wh i ch was f rom 0-450 pg/mL. Sensi t iv i ty o f the assay was 4 pg/mL. R N A Extrac t ion: R N A was extracted f o l l ow ing standard T R I Z O L reagent protocol and reconstituted in 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 for R V 1 4 standard curves was extracted f r om stock R V 1 4 suspensions us ing the Q I A G E N R N e a s y k i t (Miss issauga, O N ) . q R T - P C R : q R T - P C R was conducted for R V 1 4 samples. Pr imers were designed us ing the k n o w n R V 1 4 genetic sequence f r om the Nat iona l Center for B io techno logy Informat ion database and purchased f rom Operon (Huntsv i l le , A L ) . Pr imers were designed wi th the f o l l ow ing sequences: forward 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 238 base pair product. Pr imers were tested by convent ional two-step P C R of known R V infected samples, and P C R products conf i rmed by gel electrophoresis (not shown). Fo r R V 1 4 R N A samples a one-step q R T - P C R k i t f rom Q I A G E N (Miss issauga, O N ) was used. The P C R mixture contained: 2 5 u L M a s t e r M i x (HotStarTaq D N A Polymerase, Quant iTect S Y B R Green Buf fe r , S Y B R Green I dye, 1 .5mM M g C 1 2 , 2 0 0 u M each d N T P ) , 2 .5uL o f each pr imer, 0.5 u L R T m i x (Omniscr ipt and Sensiscr ipt Reverse Transcriptases), and l O u L o f the R N A template to a f ina l vo lume of 5 0 u L . Standard curve R V concentrations were determined spectrophotometr ical ly and ser ia l ly d i luted at k n o w n concentrations. Fo r standards and samples, duplicate reactions were carr ied out in a M J Research D N A Eng ine Opt i con Cont inuous Fluorescence Detector (OP000537) programmed to: incubate at 42°C for 55min , 94°C for 5m in , 94°C for 30 sec, 55°C for 30 77 sec, 72°C for l m i n , repeat for 35cyc les , 72°C for l O m i n , mel t ing curve f r om 55°C to 95°C read every 0.5°C, incubate 72°C for l O m i n , and 4 °C forever. Data were analyzed using Opt i con M o n i t o r analysis software vers ion 1.07. Statistics: Infectious v i rus (replication) was analyzed by two-way analysis o f variance ( A N O V A ) w i th t ime and treatment as factors, and post-hoc Dunnet ' s test w i th Ohrs as a contro l . IL-6 data for each serotype were subjected to a 1-way A N O V A wi th treatment as a factor and post-hoc Dunnet ' s test to compare treatments w i th control (C) . R N A data were analyzed by one-way A N O V A wi th treatment as a factor and post-hoc Dunnet ' s test w i th untreated R V ( N O U V O ) as a contro l . A l l data were analyzed us ing S i gma Stat 3.0 software and statistical s igni f icance was set at a=0.05. Results are presented as mean ± standard error o f the mean. A l l statistical differences were considered s ignif icant when p < 0.05; however, in most cases, when differences were found they were h igh ly s ignif icant (p<0.001). RESULTS C P E : N o C P E were observed in the B E A S - 2 B cel ls for any o f the contro l , R V infected, or UV-treated R V infected samples. RV14 Replication (F igures 3.1a and 3.2a): There was a h igh ly s ignif icant difference between trial 1 and 2; however, the trends observed were the same. Fo r both experiments, h igh ly s ignif icant v i ra l repl icat ion was measured f rom Ohrs to 48hrs for both the N O U V O and N O U V 3 0 samples. However , no s ignif icant differences were found between N O U V O and N O U V 3 0 samples for either sample t ime (i.e. Ohrs or 48hrs) , ind icat ing that no measurable (non-UV) R V 1 4 degradation had occurred dur ing preparation o f U V irradiated virus. Furthermore, no s ignif icant R V repl icat ion was observed in B E A S - 2 B cel ls infected w i th any o f the U V treated ( U V 2 - U V 3 0 ) samples demonstrat ing a complete inh ib i t ion o f R V 1 4 repl icat ion by U V . RV14 and IL-6 Secretion (Figures 3.1b and 3.2b): The differences between tr ial 1 and 2 were h igh ly s ignif icant, and trends observed were not s imi lar . In the first t r ia l , R V 1 4 fa i led to stimulate an IL-6 response f rom B E A S - 2 B cel ls when compared to contro l (C) . A l t hough there was some statistical differences found ( N O U V O and U V 5 ) , cons ider ing the 78 very l ow concentrations o f IL-6 ( m a x i m u m 40.0 ± 8.0 pg/mL) , it is h igh ly doubtfu l that this was o f any b io log i ca l relevance. However , in trial 2, R V 1 4 induced h igh ly s ignif icant IL-6 secretion in the cel ls 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 st imulated IL-6 secretion in B E A S - 2 B cel ls . RV1A Replication (Figures 3.3a and 3.4a): There was also a h igh ly s ignif icant statistical difference between trials 1 and 2; however, the trends observed were again the same between experiments and the same as seen for R V 1 A repl icat ion. There was a h igh ly s ignif icant difference f rom Ohrs to 48hrs for N O U V O and N O U V 3 0 , indicat ing R V repl icat ion. However , no signif icant differences were found between N O U V O and N O U V 3 0 samples at either Ohrs or 48hrs, ind icat ing that no measurable (non-UV) R V 1 A degradation had occurred dur ing preparation o f U V irradiated virus. Furthermore, no s igni f icant R V repl icat ion was observed in B E A S - 2 B ce l ls infected w i th any o f the U V treated ( U V 2 - U V 3 0 ) samples, demonstrat ing a complete inh ib i t ion o f R V 1 A repl icat ion by U V . RV1A and IL-6 Secretion (Figures 3.3b and 3.4b): A h igh ly s igni f icant difference was found between trials 1 and 2, but w i th the same trends as prev ious ly descr ibed. A g a i n the N O U V O and N O U V 3 0 treatments produced h igh ly s ignif icant differences when compared to control (C) . None o f the UV-i r rad iated R V 1 A treatments st imulated s ignif icant IL-6 secretion f rom cel ls when compared to controls. RV14 RNA (F igure 3.5): There was a h igh l y s ignif icant difference between the U V untreated ( N O U V O ) R V 1 4 R N A treatment, wh i ch contained 14 pg/well o f v i ra l R N A , and al l other treatments. Th i s indicated damage to the v i ra l genetic material for a l l o f the U V treated samples. DISCUSSION The postulate that R V s may provoke the secretion o f pro-inf lammatory cytokines such as IL-6 without actual ly infect ing a i rway epithel ia l cel ls is certainly provocat ive. A l though cytokine secretion has been l inked more c lose ly to co ld symptoms than R V repl icat ion (Gwal tney et a l . , 2003) , there is no evidence that noninfect ious virus is actual ly capable o f causing i l lness. V e r y few studies have investigated the effects of U V inact ivated R V on a irway epithel ia l ce l ls , and those publ ished have y ie lded inconc lus ive results. Fo r example , 79 Johnston et al. (1998) found that 30 minute U V inact ivat ion o f R V 9 inhib i ted v i ra l repl icat ion complete ly but on ly reduced IL-8 secretion by hal f in A 5 4 9 cel ls . However , both Gr i ego et al. (2000) and Papadopolous et al. (2001) demonstrated that U V inact ivat ion o f R V l b and R V 3 9 halted both R V repl icat ion and IL-8, IL-6, and R A N T E S secretion f rom B E A S - 2 B cel ls . M y results support the latter two studies, as no increased IL-6 secretion was observed for U V inactivated R V 1 4 or R V 1 A for any U V exposure t ime. E v e n 2 minutes o f U V C exposure was suff ic ient to arrest both v i ra l repl icat ion and IL-6 secretion f r om the B E A S - 2 B cel ls . Hughes et al. (1979) showed that U V C treatment o f R V 1 7 and R V 4 0 inact ivated the v i ra l nucle ic ac id in less than 10 seconds for di lute virus preparations and up to 90 seconds for more concentrated samples. However , the same study demonstrated that antigenic act iv i ty (provocat ion o f ant ibody formation) o f R V cou ld be retained wi th at least 13 minutes o f U V exposure. It is possible that the capacity to induce antibody format ion is retained longer than the abi l i ty to stimulate an in f lammatory response since white b lood cel ls may recognize R V more effect ive ly than the a i rway epithel ia l cel ls . M y experiments also showed a s ignif icant difference in R V 1 4 R N A for U V treated samples when compared to untreated samples. Th i s result is interesting because there have been conf l i c t ing reports suggesting that U V treatment o f po l iov i rus and R V may or may not affect P C R assays ( M a et a l . , 1994; Mya t t et a l . , 2003). The reason for the discrepancy between the Johnston et al. (1998) study and the other studies ( inc luding this one) is not clear. The choice of ce l l l ine cou ld be the source o f the difference since on ly Johnston et al. (1998) used A 5 4 9 cel ls (versus B E A S - 2 B ) . Moreove r , R V serotype differences cou ld also contribute to the observed differences, for example the R V 9 capsid may be more resistant to U V damage than other R V s . S ince U V inact ivat ion of R V is also affected by in i t ia l v irus concentration and U V intensity, it is possible that even the U V 2 v irus treatment had both non-functional genetic material and protein structure wh i ch cou ld not provoke an IL-6 response. F ina l l y , the choice o f the cytok ine or chemokine measured may affect results, bearing in m ind that numerous interacting in f lammatory mediators are released f rom airway cel ls in response to R V infect ion. Interestingly, for one o f the R V 1 4 experiments no IL-6 response was observed for untreated R V even though signif icant v i ra l repl icat ion was apparent. Th i s same phenomenon was observed in previous experiments (see Chapter 2) ; however , the 80 explanat ion remains unclear. Perhaps the R V 1 4 dosage is near some threshold value for e l i c i t ing a cy tok ine response f rom the cel ls . In conc lus ion , these experiments demonstrated that U V inactivated R V was not capable o f e l i c i t ing IL-6 secretion f rom B E A S - 2 B cel ls for either R V 1 4 or R V 1 A . Th i s suggests that R V infect ion and/or repl icat ion may be necessary to the pathology o f the c o m m o n co ld . 81 FIGURES a) E 3 4 Q. O O CO 3 CO O 0 ) 1 a i a.y Trial 1: RV14 mam Ohrs (aajsi 48hrs a,y a,y a,y NO UVO NOUV30 UV2 UV5 UV10 UV15 UV30 Treatment b) 600 N O UVO N O U V 3 0 UV2 UV5 Treatment UV10 UV15 U V 3 0 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, UV5, 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) E "3 4 H — Q. O O) O co 3 3 CO 3 O € 2 CD Trial 2: RV14 Ohrs [-""I 48hrs a,y a.y a,y b) NO UVO NOUV30 UV2 UV5 UV10 UV15 UV30 Treatment 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) 3 5 o o 4 co 3 co 3 O O CD b) a i l | • 1 ii j; 1 a.y a.y Trial 1: RV1A wmm Ohrs ZEEB 48hrs a.y a.y ii I IB I a.y I NO UVO NO UV30 UV2 UV5 UV10 UV15 UV30 Treatment - i — — — i 1 — ' — ' — i — ' — ' — i — ' — ' — i — ' — • — \ — * — ' — i — C 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) b) NO UVO NO UV30 UV2 UV5 UV10 UV15 UV30 Treatment 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 UV) , and RV14 pre-treated with U V C for 2, 5, 15 or 30 minutes (UV2, UV5 , UV15, and UV30 respectively). An asterisk (*) indicates a significant difference (a=0.05) in RV14 RNA 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 2000. Ro l e o f p38 mitogen-activated protein kinase in rhinovirus-induced cytok ine product ion by bronchia l epithel ia l ce l ls . The Journal o f Immunology . 165:5211-5220. Gwaltney, J . M . , J . O. Hendley, and J . T. Patrie 2003. S ymp tom severity patterns in exper imental c o m m o n colds and their usefulness in t im ing onset o f i l lness in natural co lds. C l i n i c a l Infectious Diseases. 36:714-23. Hughes, J . H. , M . Mitchell, and V. Hamparian 1979. Rh inov i ruses : K ine t i cs o f ultraviolet inact ivat ion and effects o f U V and heat on immunogenic i ty . A r ch i ves 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 rh inovirus infect ion induces a pro longed release o f IL-8 in pulmonary ep i the l ium. The Journal o f Immunology . 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 2004. Resistance o f differentiated human a i rway epi the l ium to infect ion by rh inovirus. A m e r i c a n Journal o f Phys io logy L u n g Ce l l u l a r and Mo l e cu l a r Phys io logy . 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 po l iov i rus inact ivat ion by disinfectants. A p p l i e d and Env i ronmenta 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 2002. S im i l a r frequency o f rhinovirus-infect ible cel ls in upper and lower a i rway ep i the l ium. Journal 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 2005. Quantitat ive and qualitat ive analysis o f rh inovirus infect ion in bronchia l tissues. The Amer i c an Journal o f Respirat ion and Cr i t i ca l Care Med i c i ne . 171:645-651. Myatt, T. A. , S. L . Johston, S. Rudnick, and D. K. Milton 2003. A i rbo rne rh inov i rus detection and effect o f ultraviolet i rradiat ion on detection by a semi-nested R T - P C R assay. B i o M e d Centra l Pub l i c Hea l th . 3:5-12. Papadopoulos, N. G. , A. Papi, J . Meyer, L . A. Stanciu, S. Salvi, S. T. Holgate, and S. L . Johnston 2001. Rh inov i rus infect ion up-regulates eotaxin and eotaxin-2 expression in bronchia l epithel ia l ce l ls . C l i n i c a l and Exper imenta l A l l e rgy . 31:1060-1066. 87 CHAPTER 4: The Effects of Echinacea Extracts on RV Infected and Uninfected Airway Epithelial Cells3 BACKGROUND Rh inov i rus ( R V ) infect ions o f a i rway epithel ia l cel ls are the most important cause o f the c o m m o n co ld (Monto , 2002). Fo r people already affected by respiratory diseases such as asthma and chronic obstructive pu lmonary disorder ( C O P D ) , R V infect ion may cause dangerous exacerbations of those condit ions (Bard in , 1992; Ge rn & Busse, 1999; Gern , 2002 ; Grunberg et a l . , 1997; Grunberg & Sterk, 1999; Ha lper in , 1985; Johnston et a l . , 1995; S ingh et a l . , 2006) . Surpr is ing ly , R V s replicate at relat ively l ow levels in the a i rway epithel ia l tissues, and mount ing evidence suggests that co ld symptoms are the result o f a pronounced host in f lammatory response to infect ion characterized by the secretion o f p ro -in f lammatory cytokines such as inter leukin (IL)-6, and not R V repl icat ion (Gwal tney, 2002 ; Grunberg et a l . , 1997; Hend ley & Gwa l tney , 2004 ; Lopez-Souza et a l . , 2004) . Furthermore, in vivo studies have l i nked increases in pro-inf lammatory cytok ine secretion to increases in severity o f co ld symptoms (Gwal tney et a l . , 2003). A i r w a y epithel ia l ce l ls , both in vivo and in vitro, have demonstrated the abi l i ty to secrete various cytokines ( inc luding IL-6) in response to environmental stresses inc lud ing R V infect ion (A rno ld , 1994; Be rg et a l . , 2004 ; Johnston et a l . , 1998; Lopez-Souza et a l . , 2004 ; Sharma et a l . , 2006 ; Spannhake et a l . , 2002 ; T a k i z a w a et a l . , 2000 ; et a l . , 1998; Z h u et a l . , 1997; Z h u et al . , 1996). N o cure or prevention for R V infect ion exists and most drugs on ly act to al leviate symptoms. Vacc ines have been d i f f i cu l t to develop because over 100 poor ly cross-neutra l iz ing serotypes o f R V persist. Scientists have developed var ious R V anti-viral compounds , such as P leconar i l and Rupr in t r i v i r , wh i ch prevent R V repl icat ion or entry across the p lasma membrane (Hayden et a l . , 2003 ; Zhang et a l . , 2004). However , cons ider ing the poss ib i l i t y that R V may stimulate cytokine secretion f rom cel ls without the necessity o f R V repl icat ion or ce l l entry, anti-viral development may be fut i le. 3 A version of this chapter will be submitted for publication. Machala, A . M . , Harris, R.A., Brauner, C.J., Hudson, J.B. 88 A g row ing number o f researchers are invest igat ing the abi l i ty o f immune-modulat ing compounds to mitigate RV-associated symptoms. Fo r example , compounds capable o f down-regulat ing pro-inf lammatory cytokine secretion cou ld conce ivab ly reduce co ld symptoms. Ech inacea is a popular natural herb extract wh i ch is thought to have immune-modulat ing effects. There is evidence that Ech inacea has st imulatory effects on macrophages, monocytes, T lymphocytes , natural k i l l e r ce l ls , and epithel ia l ce l ls , result ing in increased pro-inf lammatory cytokine release (Brousseau & M i l l e r , 2005 ; B rush et a l . , 2006; Cur r ie r & M i l l e r , 2000 ; G o e l , 2005 ; M o z z a r o n i et a l . , 2005 ; Sasagawa et a l . , 2006 ; Sharma et a l , 2006). However , Sharma et al. (2006) showed that when Ech inacea was administered to R V infected a irway epithel ia l ce l ls , RV- induced cytok ine secretion was inh ib i ted, suggesting a more complex interaction between the herb extract and virus infected cel ls than that observed in uninfected cel ls . C l i n i c a l trials invest igat ing Ech inacea have y ie lded inconc lus ive results (Goe l et a l . , 2004; Sperber et a l . , 2004 ; Turner et a l . , 2005 ; Turner et a l . , 2000) , and the qual i ty o f many commerc ia l formulat ions is questionable (G i l roy et a l . , 2003 ; K r o c h m a l et a l . , 2004) . Standardized in vitro studies are needed to further elucidate the effects of Ech inacea in R V infected a i rway epithel ia l cel ls . Th is study a imed to assess the effect of two chemica l l y dist inct Ech inacea extracts on cultured bronchia l epithel ia l cel ls ( B E A S - 2 B ) infected w i th R V 1 4 or R V 1 A . V i r a l repl icat ion and IL-6 secretion were measured in order to address the hypothesis that Ech inacea is immune-modulatory and stimulates IL-6 in uninfected ce l ls , but inhibi ts R V -induced cytokine secretion in cultured a irway epithel ia l cel ls . MATERIALS AND METHODS A l l v i ra l , ce l l culture and molecu lar work was conducted under sterile condit ions in a type II biosafety cabinet. A l l protocols were pre-approved by the U B C biosafety committee in certif icate H04-0061 (Append ix A ) . C e l l C u l t u r e : The S V 4 0 adenovirus transformed human bronchia l epithel ia l ce l l l ine ( B EAS-2B ) was obtained f rom the Amer i c an Type Cul ture Co l l e c t i on ( A T C C , R o c k v i l l e , M D ) and cultured in 7 5 m m 2 f lasks in 50:50 Du lbecco ' s M o d i f i e d Eag le ' s M e d i u m 89 ( D M E M ) and H a m ' s F12 wi th 1 0 % endotoxin free fetal bov ine serum (FBS ) . Cul ture reagents were obtained f rom Invitrogen (Vancouver , Canada) . Ce l l s were passaged week l y and incubated at 35-37°C wi th 5 % carbon d iox ide in 9 5 % air. RV-sens i t i ve H I cel ls ( f rom A T C C ) were cultured under the same condit ions w i th D M E M and 5 % F B S . Echinacea Extracts: T w o commerc ia l preparations ( E l and E2 ) were analyzed for their major constituents. E l was a spray dr ied expressed ju ice extract o f the aerial parts o f E. purpurea (accession number U O 1 9 1 8 0 ) . E l was r i ch in water extractable polysacchar ides, w i th a total extractable polysacchar ide content o f 2 3 . 7 % w/w (Sharma et a l . , 2006) . E 2 was a 5 5 % ethanolic tincture f rom E. purpurea roots (1:9 w/v). H i g h performance l i qu id chromatography analysis o f the E 2 tincture showed the presence o f a lkamides and caffeic acid derivatives (caftaric ac id 59.5ug/mL, chlorogenic acid 19.3pg/mL, caffe ic ac id 2.4pg/mL, cynarine Opg/mL, echinacoside Opg/mL, c i chor ic ac id 37ug/mL and tetraene a lky lamides 80.5ug/mL) . Bo th o f these extracts were analyzed prev ious ly by B inns et al. (2002) and prov ided to our laboratory. Extracts were f i l tered at 0 .2pm (pre-characterization), di luted in culture med ium, and stored at -20°C. Viruses: B o t h R V 1 4 and R V 1 A were obtained f rom the A T C C . R V s were propagated by infect ing H I cel ls grown to conf luence in 7 5 m m 2 f lasks conta in ing D M E M , a l l ow ing for fu l l cytopathic effects (CPE ) . Cell-free culture f l u id was harvested when C P E were at a m a x i m u m by centr i fugation at 10,000 x g for 20 minutes at 4 °C. The stock v irus suspension was al iquoted into c ryov ia ls and stored at -80°C for experimental use. T i ter o f v i ra l stock was determined by v i ra l plaque assay (see be low) o f ser ia l ly d i luted stock, and expressed in plaque fo rming units (pfu) per m L . P fu represent the number o f infect ious R V particles present in a k n o w n vo lume o f sample. Fo r experiments, al iquoted stock virus was rap id ly thawed at 37°C and vortexed pr ior to use. Because such c lar i f ied v i ra l stocks contain H I ce l l remnants (e.g. soluble proteins, organelles) control experiments were conducted to con f i rm that any observed changes in IL-6 and IL-8 secretion were due to virus and not some other ce l lu lar component present in the inocu la (Append ix C ) . 90 Viral Infections: B E A S - 2 B cel ls were cultured in 6-well plates unt i l freshly conf luent in 50:50 D M E M : H a m ' s F12 m i x wi th 1 0 % F B S . Once cel ls reached conf luence it was assumed that ce l l number d id not change s igni f icant ly over the course o f experiments for either control o f R V infected cultures. Th is assumption was conf i rmed exper imenta l ly (Append ix D ) . C e l l number per we l l (6-well plate) at conf luence was pre-determined for ce l l l ines (Append ix E ) and used to calculate v i ra l dose. P r io r to infect ions media was aspirated and replaced w i th 0 .75mL o f R V 1 A or R V 1 4 inocu la at a mu l t ip l i c i t y o f infect ion ( M O I ) = l , or mock infected w i th med ium. Ce l l s were incubated at 35°C for 1 hour. F o l l o w i n g infect ion inocu la were aspirated and cel ls were washed 3 times w i th l m L of med ium in order to remove exogenous virus (virus remova l exper imenta l ly conf i rmed, not shown). F o l l o w i n g this, fresh culture med ium conta in ing either: 50pg/mL o f E l , 1:50 E2/medium d i lu t ion , 0 . 9 % ethanol (E2 vehic le control ) , or med ium alone (control) was added to wel ls , and cel ls were incubated at 35°C i n a 5 % carbon d iox ide incubator. Samples were col lected at the same t ime immediate ly post-infection and washes (Ohrs), and 48 hours (48hrs), and 96 hours (96hrs) later. Samp l ing consisted o f first r emov ing l m L o f supernatant f rom appropriate we l ls , centr i fuging at 1000 x g to remove ce l lu lar debris, and freezing samples at -20°C for future cytokine/chemokine assays. Ce l l s f rom R V infected samples were scraped into the remainder o f the med ium (2mL) , pipetted into c ryov ia ls , and stored at -80°C for plaque assays. Plaque Assays: V i r a l infect iv i ty and repl icat ion were measured by plaque assays in permiss ive H I cel ls . P rev ious ly f rozen R V infected ce l l samples f rom G r o w t h Curves were rapid ly frozen and thawed twice at 37°C to rupture cel ls and release v irus. These samples were then ser ia l ly d i luted and used to infect permiss ive H I cel ls in duplicate where 0 .75mL o f sample was added onto freshly conf luent H I cel ls grown i n 6-well trays and a l lowed incubated for 1 hour at 35°C. A f te r infect ion the inocu la were aspirated and replaced wi th a 50:50 l i qu id mixture o f 2x M E M (with 5 % F B S ) and 1 % sterile agarose in d H 2 0 . The agarose was a l lowed to so l id i fy at room temperature, and plates were incubated at 35°C for 4 days. A s the virus replicated in infected ce l ls , lys is occurred in the H I cel ls fo rming round areas o f ce l l death ca l led "p laques" . Once incubat ion was complete , plates were f i xed w i th 3 % formaldehyde in phosphate buffered sal ine, agarose was removed, and H I 91 cel ls were stained wi th 1 % crystal v iolet in d H 2 0 to reveal the clear unstained plaques. Plaques were counted and reported as pfu/mL. A n increase in infect ious virus over time (relative to DO) indicated v i ra l repl icat ion. C y t o k i n e s IL-6 was assayed f rom supernatant samples us ing standard protocol prov ided by commerc i a l l y avai lable enzyme-l inked immunosorbent assay ( E L I S A ) kits f rom Immunotools (Fr iesoythe, Germany) . Absorbenc ies were read on an E L I S A plate reader (Pasteur Diagnost ics L P400 ) at a 4 5 0 n m wavelength. H i g h l y concentrated samples were di luted w i th med ium and re-assayed to fa l l w i th in the standard assay range wh i ch was f rom 0-450 pg/mL. Sensi t iv i ty o f the assay was 4 pg/mL. Sta t i s t i c s : Infectious virus (replication) data (n=3 per treatment for each sampl ing time) were analyzed by two-way analysis o f variance ( A N O V A ) wi th day and treatment as factors, f o l l owed by post-hoc Tukey tests. Fo r IL-6 secretion (n=3 for each treatment) a one-way A N O V A w i th treatment as a factor was performed wi th post-hoc Tukey tests in order to compare a l l treatments. A l l data were analyzed us ing S i gma Stat 3.0 software and statistical s igni f icance was set at a=0.05. S igni f icant statistical differences correspond to p<0.05, and h igh ly s ignif icant statistical differences correspond to p<0.001. Results are presented as mean ± standard error o f the mean. RESULTS C P E : N o C P E were observed for any of the samples inc lud ing al l R V infected ce l ls , E l and E 2 treated ce l l s , and R V + E c h i n a c e a treated combinat ions . R V 1 4 R e p l i c a t i o n (F igure 4.1a): There was a h igh ly s ignif icant difference in infect ious virus between Ohrs and 48hrs (al l treatments combined) indicat ing v i ra l repl icat ion, but no v i ra l repl icat ion was detected at 96hrs (relative to Ohrs). Average o f Ohrs treatment p fu/mL values (al l treatments combined) was 3.6 x 10 3 ± 9.3 x 10 1 p fu/mL, increasing to 3.5 x 10 4 ± 7.9 x 10 2 p fu/mL at 48hrs. In compar ing treatments, on ly R V and R V + E t o h were s igni f icant ly different, and this difference was on ly observed at 48hrs, but was h igh ly s ignif icant. 92 RV14 and IL-6 Secretion (Figure 4.1b): There is a h igh ly s ignif icant difference between treatment groups. N o n e o f the uninfected ce l l treatments (C , E l , E 2 , Etoh) were s igni f icant ly different f r om 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 found to be s igni f icant ly different f rom uninfected cel ls . W i t h i n R V infected cel ls there were no signif icant differences between R V , R V + E 1 or R V + E t o h ; however, there was a signif icant difference between R V + E 2 and al l other treatments. R V + E 2 represented peak IL-6 secretion at 243.4 ± 9.3 pg/mL. RV1A Replication (F igure 4.2a): S ign i f icant v i ra l repl icat ion relative to Ohrs was observed at 48hrs but not 96hrs (al l treatments combined) . Average p fu/mL of Ohrs treatments (all treatments combined) was 3.6 x 10 3± 9.3 x 10 1 p fu/mL increasing to 3.5 x 10 4± 7.9 x 10 2 pfu/mL at 48hrs. N o signif icant differences were observed between treatments. RV1A and IL-6 Secretion (Figure 4.2b): A h igh ly s ignif icant difference was found between treatment groups. O n l y R V and R V + E t o h treatments were s igni f icant ly different f rom a l l other treatments (83.0 ± 10.2 pg/mL) , but they were not s igni f icant ly different f rom each other. Peak IL-6 concentration ( R V treatment) was 349.3 ± 49.7 pg/mL. DISCUSSION Ech inacea is the most popular natural extract used to treat c o m m o n upper respiratory tract infect ions typ ica l l y caused by R V infect ion. H i s to r i ca l l y , crude Ech inacea extracts in various forms have been used to treat and/or prevent a variety o f infect ions (Barrett, 2003). However , no modern consensus on the potential health benefits o f Ech inacea for the treatment o f R V infect ion exists, and a possible mechanism o f act ion is largely unknown. R V Replication: Depend ing on the source consulted, the effects o f Ech inacea have been described as immune-st imulatory, immune-protect ive and/or anti-viral. A l t hough growing research supports the idea that Ech inacea 's effects are largely immune-mediated the above c la ims are somewhat ambiguous and contribute to the controversy surrounding this natural medic ine . Fo r example , although there is some evidence that Ech inacea has a v i ruc ida l effect on viruses such as herpes s implex virus-1 (B inns et ah, 2002) , there is no evidence o f any effect on v i ra l repl icat ion thus broad "ant i-v i ra l " c la ims may be mis lead ing . In m y experiments s ignif icant R V repl icat ion was detected at 48hrs relative to Ohrs, but there was no differences observed between Ech inacea treated and untreated cel ls for either extract 93 formulat ion in R V 1 4 or R V 1 A infected B E A S - 2 B cel ls. These data suggest that Ech inacea most l i ke l y confers its phys io log ica l effects through an interaction w i th the host cel ls rather than by affect ing v i ra l repl icat ion. IL-6 Secretion: IL-6 is a pro-inf lammatory cytok ine secreted f rom a variety o f ce l ls , inc lud ing a i rway epithel ia l ce l ls , in response to in jury and stress such as v i ra l infect ion (Janeway, 2005) . There is evidence that R V infect ion can stimulate IL-6 secretion in a i rway epithel ia l cel ls (Sharma et a l . , 2006) , and increases in pro-inf lammatory cytokines have been l i nked to intensif ied co ld symptoms (Gwal tney et a l . , 2003). Th i s evidence is also supported by m y f ind ings, as R V and R V + E t o h treated cel ls showed s igni f icant ly increased IL-6 concentrations compared to contro l . Several hypotheses have been postulated about how the immune-modulat ing effects o f Ech inacea may translate into health benefits. Some scientists have suggested that Ech inacea stimulates an in f lammatory response, thus protect ively "he igh ten ing " the immune system (cited in Barrett, 2003) ; however, bear ing in m i n d that pro-inf lammatory mediator release is thought to cause co ld symptoms further exaggerating this process seems counterproductive. A more plausible hypothesis may be that Ech inacea down-regulates R V induced in f lammatory responses, such as IL-6 secretion, thereby reducing co ld symptoms. Interestingly, Ech inacea treatment o f uninfected cel ls for both experiments d id not y ie ld any s ignif icant IL-6 secretion relative to contro l . These results dif fer f rom some publ ished studies indicat ing an increase o f pro-inf lammatory cytokine release after treatment w i th Ech inacea (Hwang et a l . , 2004 ; Sharma et a l . , 2006). It may be that the concentration o f the extracts administered was not suff ic ient to stimulate ce l lu lar IL-6 secretion, or perhaps some o f the active compounds had degraded dur ing extract storage. O n the other hand, s ignif icant differences in IL-6 secretion were observed for both R V 1 4 and R V 1 A infected cel ls treated w i th Ech inacea , indicat ing that the extracts were b io log i ca l l y active. Cons ider ing that crude Ech inacea preparations contain many immuno log i ca l l y active compounds (e.g. a lkamides and caffe ic ac id derivat ives) , it is possible that different constituents (or combinat ions thereof) are responsible for Ech inacea 's contrasting effects on R V infected and uninfected cel ls . A divergent trend was observed for IL-6 secretion between R V 1 4 and R V 1 A . In R V 1 4 infected ce l ls , IL-6 secretion was not s igni f icant ly different for R V + E 1 , but was 94 s igni f icant ly elevated f r om other R V infected treatments for R V + E 2 . Howeve r , in R V 1 A infected ce l ls , Ech inacea treatment resulted in IL-6 concentrations that were s imi la r to contro l levels , even though R V and R V + E t o h treatments st imulated signif icant IL-6 secretion. The differences observed between serotypes are d i f f i cu l t to exp la in because the phys io log ica l mechan ism o f Ech inacea is largely unknown . Some studies propose an nuclear factor kappa-B ( N F K B ) dependent pathway for the action o f this herbal extract, and overa l l such a mechan ism is p lausible , consider ing that N F K B is impl i ca ted in both pro -in f lammatory cytokine secretion and R V pathology (Zhu et a l . , 1997). However , one study conducted by our laboratory (Sharma et a l . , 2006) showed that Ech inacea treatment o f B E A S - 2 B cel ls resulted in changes o f over 30 transcript ion factors ( inc lud ing N F K B ) , demonstrat ing the invo lvement o f complex b iochemica l pathways. Recent research has found that a lkamides in Ech inacea extracts b ind the cannabinoid type-2 receptor and may affect N F K B transcript ion by this pathway (Gertsch et a l . , 2004; Raduner et a l . , 2006). R V 1 4 and R V 1 A also ut i l ize different receptors to infect cel ls ( intercel lular adhesion molecule-1 and low density l ipoprote in receptors respect ively) ; therefore, the cascades signaled by the b ind ing o f these receptors may lead to differences in cy tok ine secretion. In another study, Sharma et al. (2006) showed increased IL-6 secretion f rom B E A S - 2 B cel ls upon Ech inacea treatment alone and decreased secretion when Ech inacea was administered to R V 1 4 infected cel ls . A l t hough the same trend was not observed for m y R V 1 4 experiments, R V 1 A d id support the Sharma et al. (2006) f indings. Cons ide r ing that these are the on l y studies invest igat ing the effects o f Ech inacea on R V infected a i rway epithel ia l ce l ls , more evidence is needed before definite conc lus ions can be drawn. E l and E2 Echinacea Extracts: The qual i ty of commerc i a l l y avai lable Ech inacea extracts is questionable (G i l roy et a l . , 2003 ; K r o c h m a l et a l . , 2004). One study found that 3 9 % o f Ech inacea extracts contained more or less Ech inacea than indicated wh i l e 1 0 % of the products contained no Ech inacea at a l l (G i l roy et a l . , 2003) . Furthermore, the best mode o f administrat ion (e.g. caplet, t incture, tea) or the proper dosage is i l l-def ined. E ven in standardized preparations o f Ech inacea , the number and concentrations o f b io log i ca l l y active constituents di f fer greatly due to factors such as species (E. purpurea, E. pallida, E. angustifolia), part o f plant ut i l ized (e.g. root, leaf, aerial) and method o f extract ion (e.g. a lcoho l i c , aqueous) ( A d i n o l f i et a l . , 2006 ; S lo ley et a l . , 2001). Th is lack o f extract 95 homology certainly contributes to the controversy surrounding the therapeutic benefits o f Ech inacea , especia l ly in c l in i ca l studies where so many addit ional confound ing factors may exist (Gagnier et a l . , 2006 ; Sperber et a l . , 2004 ; Turner et a l . , 2000 ; Turner et a l . , 2005 ; W o l s k o et a l . , 2005). A s prev ious ly ment ioned, no effects of either E l or E 2 were observed for either R V 1 4 or R V 1 A repl icat ion in the B E A S - 2 B cel ls . In m y R V 1 A experiments both E l and E 2 suppressed R V st imulat ion o f IL-6 secretion in a s imi la r manner, wh i le in R V 1 4 infected cel ls E l fa i led to suppress R V induced IL-6 secretion and E 2 seemed to exaggerate the IL-6 response. A g a i n , these data suggest that dist inct ive Ech inacea extracts may interact in complex ways wi th vary ing R V serotypes. A n ethanolic vehic le contro l was inc luded to account for the potential non-Echinacea based differences between the aqueous and ethanolic extracts. N o s ignif icant ethanolic effects were observed for IL-6 secretion in uninfected cel ls regardless o f R V serotype, and in R V infected cel ls no IL-6 secretion beyond that induced by the virus cou ld be observed for R V + E t o h treatments. In the R V 1 4 repl icat ion data, a s ignif icant difference was found between R V and R V + E t o h treatments; however, cons ider ing the inherent var iabi l i ty in plaque assays, it is doubtful that this difference is b io log i ca l l y relevant (see F igure 4.1a). Overa l l l itt le or no ethanolic effects were observed. It is becoming increas ingly clear that Ech inacea extracts are capable o f modulat ing in f lammatory cytokine secretion in a variety o f ce l ls . Moreover , previous studies conducted by our laboratory and m y experiments suggest that Ech inacea interacts w i th R V infected cel ls di f ferent ly than w i th uninfected ce l ls , at least in the case o f the B E A S - 2 B ce l l l ine (Sharma et a l . , 2006). Future studies should examine the effects o f standardized Ech inacea extracts on various a i rway cel ls infected by different R V serotypes in order to determine i f the observed effects can be conserved under various experimental condi t ions. 96 FIGURES a) E H— C L o en o CO 3 CO 3 O o b) 5 H 4 H 3 H M M RV I 1 RV + E1 ^ m m RV+E2 RV14 • RV+ETOH a,b iL Time After RV Infection (Hours) 400 300 1 CD CL c o 2 200 c a> o c o 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 Treatment RV + E1 RV+E2 RV + Etoh 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. 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Wu, O. Einarsson, M . L . Landry, and J . M . Gwaltney, Jr. 1996. Rh inov i rus st imulat ion o f interleukin-6 in vivo and in vitro. The Journal o f C l i n i c a l Investigation. 97:421-430. 102 CHAPTER 5: General Discussion and Conclusions Biological Relevance of Studies: Rh inov i rus ( R V ) infect ion is the most frequent acute i l lness in humans, although surpr is ingly l itt le is known about its pathogenesis, and its role in the exacerbations of serious respiratory diseases has on ly recently been reported. E ven in healthy ind iv idua ls , R V infect ion impacts not on ly our sense o f wel l-being but often results in missed days f rom work . Fo r example , Fendr ick et al. (2003) reported 500 m i l l i o n incidences o f non-inf luenza v i ra l respiratory infect ions per year i n the Un i ted States w i th an annual estimated cost burden o f over 40 b i l l i on dol lars. Moreove r , the emerging role o f R V infect ion in the morb id i ty and mortal i ty o f diseases such as asthma and chronic obstructive pu lmonary disorder ( C O P D ) il lustrates the c l in i ca l s igni f icance o f an infect ion wh i ch may have been prev ious ly considered more nuisance than danger. Johnston et al. (1995) demonstrated that up to 8 5 % o f asthma exacerbations in 292 ch i ldren were associated w i th v i ra l in fect ion, and picornaviruses ( R V s and enteroviruses) accounted for close to 6 6 % o f those infect ions. A study by Khetsur ian i et al. (2005) found that over 6 3 % o f asthma exacerbations were associated w i th viruses, o f wh i ch 6 0 % were ident i f ied spec i f ica l ly as R V s . Furthermore, evidence suggests that patients w i th moderate to severe C O P D are more susceptible to R V infect ion than their healthy counterparts, and R V infect ion causes more severe symptoms in C O P D patients than in healthy people (Greenberg et al . , 2000) . Add i t i ona l l y , acute respiratory infect ions are the leading cause o f infant mortal i ty, wh i ch was in i t i a l l y attributed to the in f luenza and respiratory syncyt ia l v iruses; however, a recent study o f 263 chi ldren under the age o f 12 months found that almost 5 0 % o f the viruses isolated dur ing upper and lower acute respiratory tract infect ions were R V s (Kuse l et a l . , 2006) . F ina l l y , R V infect ion has also been impl i ca ted in cases o f v i ra l pneumonia , co-infections w i th bacterial pneumonia , and compl icat ions in lung transplant recipients (Falsey & W a l s h , 2006; Ka i se r et a l . , 2006 ; Leht inen et a l . , 2006). Understanding R V infect ion is becoming increas ingly important to scientists especia l ly cons ider ing the g row ing research indicat ing that R V plays an important role in the pathology of many other serious condit ions. The emerging evidence that R V i l lness may be largely caused by a host in f lammatory response and not necessari ly by v i ra l repl icat ion is important f rom both a basic v i ro log i ca l 103 and c l in i ca l therapeutic perspective. The R V mechan ism may il lustrate a unique v i ra l "strategy" where R V is able to incite i l lness wh i l e causing l itt le or no ce l l damage or death. T o date, it is unknown whether R V repl icat ion is necessary in order to cause i l lness. A l t hough R V symptoms have been l inked to increases in pro-inf lammatory mediator secretion, some scientists suggest that v i ra l repl icat ion may be the trigger for this in f lammatory response (Gwal tney et a l . , 2003) , wh i l e conversely , l im i ted evidence has shown that noninfect ious virus may be capable o f p rovok ing pro-inf lammatory cytokine secretion in vitro (Johnston et a l . , 1998). Furthermore, the emerging imp l i ca t ion o f the host immune response as the cause o f RV-associated i l lness has inspired scientists to investigate R V treatments, such as Ech inacea , wh i ch are thought to mediate symptoms largely through host immuni ty . G e n e r a l D i s c u s s i o n : M y studies investigated the relat ionships between R V repl icat ion and pro-inf lammatory cytokine/chemokine secretion for two different receptor-uti l iz ing R V serotypes ( R V 1 4 and R V 1 A ) us ing two distinct a i rway epithel ia l ce l l models ( B E A S - 2 B and A 5 4 9 ) . I also investigated the necessity o f R V repl icat ion in st imulat ing an in f lammatory response by assessing the abi l i ty o f noninfect ious virus to stimulate an inter leukin (IL)-6 response in the B E A S - 2 B mode l . F ina l l y , I studied the effects of two chemica l l y characterized Ech inacea extracts on both v i ra l repl icat ion and pro-inf lammatory IL-6 secretion. Fo r the G r o w t h Curve experiments (Chapter 2), I found that s ignif icant R V repl icat ion occurred in both B E A S - 2 B and A 5 4 9 cel ls between day 1 ( D l ) and D 2 post-infect ion; however, repl icat ion was no longer detectable after D 3 in al l cases. Furthermore, the levels o f v i ra l repl icat ion were relat ively l ow when compared to the permiss ive H I cel ls wh i ch produced R V titers 3-4 orders o f magnitude higher than either o f the a i rway epithel ial ce l l models . Fo r example , H I ce l l peak titers reached 10 7 ~ 8 pfu/mL wh i ch corresponds to approximate ly 10-100 infect ious R V particles present per ce l l . However , peak R V titers for B E A S - 2 B and A 5 4 9 cel ls typ ica l ly represented 0-0.1 infect ious v i ra l particles present per ce l l . Furthermore, R V 1 4 d id not appear to replicate in the A 5 4 9 cel ls . R V 1 4 R N A levels also supported the repl icat ion data, where H I cel ls produced levels o f v i ra l R N A much higher than in either o f the a i rway models . Th is evidence supports the hypothesis that R V 104 replicates at l ow levels i n the a i rway epithel ia l ce l ls , and these trends were supported for both a i rway models and R V serotypes. Howeve r it is unclear i f the major i ty o f the cel ls were infected w i th R V each produc ing l ow levels o f v i rus, or whether a smal l number o f cel ls repl icated the virus at very h igh levels. Add i t i ona l l y , as hypothesized, no cytopathic effects (CPE ) were observed in any of the experiments i n vo l v i ng a irway epithel ia l ce l ls , suggesting that v i ra l progeny either have some unknown mechanism o f cross ing the p lasma membrane without l ys ing ce l ls , or perhaps a very smal l number o f cel ls ruptured releasing their v i ra l progeny. In vivo, Mosse r et al. (2002 and 2005) found immunoh is tochemica l evidence o f patchy R V infect ion affect ing 5-10% o f the cel ls w i th few or no abnormalit ies o f the a i rway epithel ia l tissues, suggesting that perhaps on ly a smal l proport ion o f cel ls are affected by R V . Fo r the majority o f experiments R V was also found to stimulate pro-inf lammatory IL-6 and/or IL-8 secretion and these elevated secretions were typ ica l l y observed after the second day post-infection and persisted we l l beyond signif icant R V repl icat ion. Th i s evidence supports the hypotheses that R V w o u l d stimulate pro-inf lammatory cytokine/chemokine secretion and that peak secretions wou ld occur later and persist longer than peak repl icat ion and v i ra l R N A levels. These data also demonstrate that R V triggers a pronounced in f lammatory response f rom cel ls and mirrors in vivo research where R V induced a pro longed in f lammatory response f rom airway cel ls wh i ch was l inked to co ld symptoms independent o f v i ra l shedding (Gwal tney et a l . , 2003) . In contrast to what was hypothes ized, ultraviolet ( U V ) inact ivat ion (Chapter 3) o f R V 1 4 and R V 1 A complete ly inhib i ted both v i ra l repl icat ion and the abi l i ty to stimulate an IL-6 response suggesting that genetical ly intact v i rus is necessary to trigger IL-6 secretion, at least in the B E A S - 2 B mode l . Thus it may be the case that v i ra l repl icat ion is the indirect cause o f R V symptoms by st imulat ing the host ce l l to secrete in f lammatory cytokines/chemokines, a l though the mechanism is not yet known . Furthermore in Append ix C , I conf i rmed that infect ious virus was necessary and responsible for the observed IL-6 and IL-8 st imulat ion, and that this response cou ld not be induced by other ce l lu lar components (e.g. soluble proteins and organelles) present in the R V inocula . In compar ing the ce l l models , B E A S - 2 B cel ls were found to be more susceptible to R V infect ion than the A 5 4 9 cel ls . They produced s igni f icant ly higher 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 higher levels o f R V 1 4 R N A were detectable in B E A S - 2 B on D l and D 2 in tandem B E A S - 2 B / A 5 4 9 cultures. Furthermore, R V 1 4 fa i led to replicate in A 5 4 9 cel ls although in one case an increased IL-8 response was evident. A l t hough these results d id not support the in i t ia l hypothesis, the differences observed between ce l l models may not be surpr is ing cons ider ing that type-II surfactant secreting alveolar cel ls (A549) are int r ins ica l ly different f rom bronchia l cel ls ( B EAS-2B ) . These cultured ce l l l ines most l i ke l y reflect undifferentiated basal-type ce l l s ; however, the A 5 4 9 may retain phys io log ica l differences such as a decreased number of I C A M - 1 receptors relative to B E A S - 2 B cel ls . In general, cons ider ing that these ce l l l ines are der ived f rom the m i d to lower a irways, m y studies support the g row ing evidence that both upper and lower a i rway epithel ia l cel ls are susceptible to R V infect ion (Mosser et a l . , 2005 ; Papadopoulos et a l . , 2000; Z h u et a l . , 1996). A l t hough both R V 1 4 and R V 1 A produced s imi lar trends in repl icat ion and pro -in f lammatory mediator release, some differences were observed between serotypes. A s prev ious ly ment ioned, R V 1 4 fa i led to replicate s igni f icant ly in the A 5 4 9 cel ls . Add i t i ona l l y , in some cases there was an apparent uncoup l ing between R V 1 4 repl icat ion and st imulat ion o f IL-6 and/or IL-8 release. Fo r example , in a few instances, although R V 1 4 repl icat ion was evident there was no difference observed between control and R V infected IL-6 or IL-8 ce l l secretions (Figures 2.7b and c). In one A 5 4 9 case, a s ignif icant increase in IL-8 secretion was observed although no signif icant repl icat ion was detectable (Figure 2.4c). F ina l l y , dur ing the first tr ial o f the U V experiments, R V 1 4 fa i led to stimulate any IL-6 response f rom B E A S - 2 B cel ls even though signif icant v i ra l repl icat ion was measured (Figures 3.1 a and b). The cause o f this discrepancy remains unclear; it may be that in some cases elevated control cytokine/chemokine secretions may have masked observable differences in R V infected cel ls . These elevated contro l levels may be attributable to changes in ce l l phys io logy w i th passage number, or poss ib ly some other confound ing factor. However , it seems cur ious that this discrepancy cou ld on ly be observed in R V 1 4 experiments. Du r i ng tandem experiments (where both R V 1 4 and R V 1 A designated cel ls were plated s imultaneously) R V 1 A infected cel ls showed clear increases in IL-6 and IL-8 secretion relative to contro l wh i le R V 1 4 d id not, even though contro l levels for A 5 4 9 cel ls were s imi lar between R V serotypes. It may be that the v i ra l dose for R V 1 4 106 may be near some threshold for e l i c i t ing a cytokine/chemokine response w h i c h cou ld be inf luenced by other factors such as ce l l age. O r perhaps R V repl icat ion and IL-6/IL-8 responses are contro l led by different mechanisms wh i ch may or may not be related. Fo r R V 1 A a consistent trend was observed for both serotypes and ce l l l ines result ing in s ignif icant v i ra l repl icat ion and IL-6/IL-8 release in a l l cases. Regardless o f this observed discrepancy, it is clear that R V infect ion o f the a i rway cel ls is capable o f induc ing v i ra l repl icat ion and the secretion o f pro-inf lammatory cytokines and chemokines for both intercel lular adhesion molecule-1 ( I C A M - 1 ) and low density l ipoprote in receptor ( L D L R ) u t i l i z ing R V serotypes. The treatment o f B E A S - 2 B cel ls w i th Ech inacea extracts also y ie lded interesting results (Chapter 4). Nei ther the aqueous polysaccharide-rich ( E l ) nor the a lcohol ic a lkamide-rich (E2) Ech inacea extracts had any observable effect on R V 1 4 or R V 1 A repl icat ion. A l t hough the presence o f c i chor ic ac id in Ech inacea has been shown to have some v i ruc ida l effects on viruses such as herpes s implex virus-1 (B inns et a l . , 2002) , the lack of impact on R V repl icat ion further supports the not ion that Ech inacea exerts most o f its effect through the host immune response. N o effect o f Ech inacea on IL-6 secretion was observed in uninfected B E A S - 2 B cel ls . Th is result is surpris ing cons ider ing that many studies have reported st imulatory effects o f Ech inacea on var ious immune cel ls and a i rway epithel ial cel ls (Brush et a l . , 2006 ; Brousseau & M i l l e r , 2005 ; Cur r ie r & M i l l e r , 2000 ; G o e l et a l . , 2005 ; M o z z a r o n i et a l . , 2005 ; Sasagawa et a l . , 2006; Sharma et a l . , 2006). It may be that the extracts used were not concentrated enough to produce such st imulatory effects, or some o f the active constituents had degraded dur ing storage. However , there was a clear effect o f Ech inacea when administered to R V infected ce l ls , a l though the results were marked ly different between R V serotypes. Fo r R V 1 4 , E l d id not have any s ignif icant effect on R V infected ce l ls , wh i l e E 2 treatment st imulated further IL-6 secretion f rom R V 1 4 infected cel ls . However , both E l and E 2 treatment o f R V 1 A infected cel ls complete ly inhib i ted IL-6 secretion. If Ech inacea confers its health benefit by inh ib i t ing p ro -in f lammatory cytokine secretion (and therefore co ld symptoms) then in this case a health benefit may be observed dur ing R V 1 A infect ion, but not for R V 1 4 , where E 2 cou ld arguably exaggerate the in f lammatory response. Bear ing this in m i n d , the evaluat ion of 107 Ech inacea treatment o f R V infected cel ls should consider not on ly the active constituent prof i le o f the herb extracts but also the R V serotype responsible for infect ion. The strength o f m y research is the characterization o f R V infect ion for two different receptor-uti l iz ing serotypes in two dist inct a i rway epithel ia l ce l l models over the course o f a typica l infect ion. I have demonstrated that R V infect ion results in signif icant v i ra l repl icat ion and pro-inf lammatory cytokine/chemokine st imulat ion through both the I C A M -1 and L D L R pathways; however, the specif ic trends observed are dependent on both ce l l mode l and R V serotype choice. M o s t pr ior R V studies o f this nature have been carried out by choos ing one specif ic ce l l type and one R V serotype and sampl ing on l y at l imi ted t ime intervals. Moreove r , in compar ing R V studies, many researchers treat R V serotype and ce l l choice as largely redundant factors, but m y f indings suggest that such broad comparisons may be o f questionable va l id i ty . I have also supported the f indings o f Sharma et al. (2006), where IL-6 secretion f rom airway epithel ia l ce l ls st imulated by Ech inacea treatment was different between R V infected and uninfected ce l l s , suggesting the invo lvement o f largely unknown but complex b iochemica l pathways. Overall Significance and Future Studies M y G r o w t h Cu r ve exper iments strengthen the g row ing consensus that R V infect ion causes an increase in pro-inf lammatory cytokine secretion that can be observed beyond peak v i ra l repl icat ion. I demonstrated this effect under control led condit ions for two different a i rway epithel ia l ce l l models and for two different receptor-uti l iz ing R V serotypes. These results offer a more comprehensive exper imental examinat ion than prev ious ly demonstrated and show that R V repl icat ion and st imulat ion o f ce l l pro -in f lammatory cytokine/chemokine secretion occurs for both the I C A M - 1 and L D L R pathways. Future studies should consider u t i l i z ing other a i rway epithel ia l ce l l models and pr imary cultures, as we l l as addit ional R V serotypes. The U V experiments supported previous f indings in B E A S - 2 B cel ls that genetical ly intact v irus is necessary to induce a pro-inf lammatory cytok ine response for both R V 1 4 and R V 1 A (Gr iego et al . , 2000 ; Papadopoulos et al . , 2001) . Furthermore, my results demonstrated that quantitative real -t ime polymerase chain reaction ( qRT-PCR ) is capable o f dif ferent iat ing between U V treated and untreated R V 1 4 , an issue wh i ch is in dispute in the literature ( M a et a l . , 1994; 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 IL-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 metabol ized and b io log i ca l l y avai lable in the b loodstream, may be o f lesser benefit than preparations that cou ld be appl ied direct ly to the affected tissues (e.g. nasal sprays). Overa l l , the development o f an effect ive cure for the c o m m o n co ld wou ld universa l ly benefit a l l people, imp rov i ng the wel l-being o f the healthy, and potent ia l ly saving the l ives o f the vulnerable. Conclusions Descr ibed be low are the general conc lus ions that can be drawn f rom this thesis in relation to the objectives and hypotheses proposed in Chapter 1. Chapter 2 (In Vitro Characterization of Rhinovirus Infection in Airway Epithelial Cells - Growth Curves): 1. A s hypothesized, R V infect ion d id not cause any observable ce l l death or C P E in B E A S - 2 B or A 5 4 9 cel ls support ing evidence that R V is not cytotoxic to a irway epithel ia l cel ls . 2. A s hypothesized, R V replicated at relat ively l ow levels in a i rway epithel ia l cel ls (0 to 0.1 infect ious virus particles per cel l ) when compared to permiss ive H I cel ls (1-100 infect ious virus particles per cel l ) . Furthermore, R V 1 4 fa i led to replicate in the alveolar A 5 4 9 cel ls . 3. S igni f icant R V repl icat ion was observed between D l and D 2 post-infection for al l ce l l l ines ( H I , B E A S - 2 B and A 5 4 9 ) . N o signif icant repl icat ion was detected f rom D 3 onward. The R V repl icat ion trends observed supported the hypothesis that v i ra l repl icat ion w o u l d peak post-infection and gradual ly decl ine to control levels over t ime. 4. R V infect ion st imulated pro-inf lammatory IL-6 and IL-8 secretion f rom a i rway epithel ia l ce l l l ines. M a x i m u m cytokine/chemokine levels were typ ica l l y measured between D 2 and D 7 and, once st imulated, usual ly remained elevated (relative to control ) over the course of infect ion. IL-6 and IL-8 levels d id not typ ica l l y decl ine by D 7 as was or ig ina l l y hypothesized. 5. Cons ider ing that R V repl icat ion was no longer detectable after D 2 , m y results support the hypothesis that it is the pro-inf lammatory cytokines/chemokines that are responsible for co ld symptoms wh i ch typ ica l l y last for at least one week. 110 6. A s hypothesized, the bronchia l B E A S - 2 B cel ls were more susceptible to R V infect ion than the alveolar A 5 4 9 cel ls . Th is may suggest that bronchia l cel ls der ived f rom higher in the a i rway ep i the l ium are more susceptible to infect ion than alveolar cel ls . 7. In contrast to what was hypothesized, R V 1 A produced more pronounced effects than R V 1 4 in terms o f v i ra l repl icat ion and IL-6/IL-8 secretion in the a i rway epithel ia l cel ls suggesting substantial differences between R V serotypes. Chapter 3 (The Effects Ultraviolet Inactivated Rhinovirus on Airway Epithelial Cells): 8. Contrary to what was hypothes ized, U V treatment o f R V 1 4 and R V 1 A complete ly inhibi ted v i ra l repl icat ion and IL-6 secretion in B E A S - 2 B ce l ls , ind icat ing that genet ical ly intact ( infectious) v irus was necessary to stimulate this response. Chapter 4 (The Effects of Echinacea Extracts on Rhinovirus Infected and Uninfected Airway Epithelial Cells): 9. A s hypothesized, Ech inacea treatment had no effect on R V repl icat ion suggesting that Ech inacea does not affect the R V repl icat ion cyc le . 10. In contrast to what was hypothes ized, Ech inacea d id not stimulate IL-6 secretion f rom uninfected B E A S - 2 B ce l l s ; however, as hypothesized Ech inacea d id affect IL-6 secretion in R V infected cel ls suggesting complex interactions between Ech inacea extracts and R V infected cel ls . 11. U n l i k e the or ig ina l hypothesis, different effects were observed for R V 1 4 and R V 1 A ; therefore, Ech inacea may not confer benefit against a l l R V s . 12. A s hypothesized, the two distinct Ech inacea extracts produced different IL-6 results indicat ing that the effects o f Ech inacea may depend on their chemica l prof i les . Understanding the pathogenici ty o f R V infect ion is cruc ia l in treating the c o m m o n co ld and further e luc idat ing its role in respiratory disease. Furthermore, the evaluat ion o f co ld treatments, such as Ech inacea , helps the publ ic and health practit ioners make more 111 in formed treatment decis ions. M y in vitro characterization of R V infect ion strengthens the current sc ient i f ic knowledge o f this v i ra l in fect ion, and such studies offer a backbone f r om wh i ch further in vivo studies and c l in i ca l trials may be developed. 112 REFERENCES Binns, S., J . Hudson, S. Merali, and J . Arnason 2002. 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Rossmann 2004. Structural and v i ro log ica l studies o f the stages o f v irus repl icat ion that are affected by ant irhinovirus compounds. Journal o f V i r o l o g y . 78:11061-11069. Zhu, Z. , W. Tang, A. Ray, Y. Wu, O. Einarsson, M . L . Landry, and J . M . Gwaltney, Jr. 1996. Rh inov i rus st imulat ion o f interleukin-6 in vivo and in vitro. The Journal o f C l i n i c a l invest igat ion. 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 © © © 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 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) Sample i 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 A549 Cells. R V stock solutions used in a l l experiments were harvested f rom lysed H I cel ls wh i ch were c lar i f ied by centr i fugat ion at 10,000 x g in order to remove ce l lu lar debris and produce concentrated R V conta in ing stocks suspended culture med ium. However , these R V stocks were not pur i f ied f rom other smal l ce l lu lar components (e.g. soluble proteins, organelles) wh i ch cou ld have potential ly affected cytokine/chemokine secretion, independent f rom v irus, when administered in R V inocu la dur ing experiments. Therefore, the effects o f pur i f ied and unpur i f ied R V on IL-6 and IL-8 secretion were compared in order to con f i rm that the use o f unpur i f ied R V stocks in experiments was va l id , and that any observed effects on cytokine and/or chemokine secretion were a result o f v irus on ly (and not other H I residual ce l lu lar components) . Methods: R V 1 4 and R V 1 A stocks (harvested f rom H I cel ls) were al iquoted into sterile tubes (0 .5mL each) and ultracentrifuged under vacuum at 100,000 x g in order to pellet the virus. These virus pellets were then either re-suspended in their or ig ina l culture med ium (unpurif ied) or fresh med ium (purif ied). The pur i f ied and unpur i f ied R V stocks were then used to infect B E A S - 2 B and A 5 4 9 cel ls ( M O I = l ) using standard infect ion procedures (n=6). The effects o f unpurif ied/purif ied R V on v i ra l repl icat ion and IL-6 (for B E A S - 2 B ) or IL-8 (for A 5 4 9 ) secretion after 48 hours were ascertained by plaque assays and E L I S A s . Results: There were no statist ical ly s ignif icant (a=0.05, 1-way A N O V A s and post-hoc Tukey tests) differences found between pur i f ied and unpur i f ied R V inocu la on infect ious virus in B E A S - 2 B or A 5 4 9 cel ls 48 hours post-infection (Figures C I and C2 ) . Furthermore, there were no signif icant differences (a=0.05, 1-way A N O V A s and post-hoc T u k e y tests) found in IL-6 or IL-8 st imulat ion in the B E A S - 2 B and A 5 4 9 cel ls respect ively between pur i f ied and unpur i f ied R V inocu la 48 hours post-infection (Figures C 3 and C4 ) . 118 1 6 Q. 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. RV 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 RV inocula for either RV14 or R V 1 A (n=6). o 4 H > CO o r> 3 j RV14 RV1A RV Serotype Figure C2: Effect of purified and unpurified RV14 and R V 1 A on hours post-infection. See Fig. C2 legend for further details. nfectious rhinovirus in A549 cells 48 119 400 E 300 O) Q. C o I 200 c d> o c o O <o 100 0 mam Pure BEAS-2B/IL-6 05133 Unpure • Bl I RV14 RV1A RV Serotype 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 RV1A (n=6). A549/IL-8 RV14 R V 1 A 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 no difference observed i n : infect ious virus (repl ication), IL-6 secretion f rom B E A S - 2 B cel ls , or IL-8 secretion f rom A 5 4 9 cel ls between unpur i f ied or pur i f ied R V 1 4 and R V 1 A inocula . Therefore, the effects on cytokine/chemokine secretion observed in both ce l l l ines are attributable to the virus and not other H I ce l l proteins and/or remnants der ived f rom R V harvesting protocols. Overa l l , the use o f unpur i f ied (clarif ied) R V stocks as inocu la is exper imenta l ly va l id . 120 APPENDIX D: Cell Counts for Rhinovirus Infected and Uninfected BEAS-2B and A549 Cells. C e l l counts were conducted for uninfected and R V infected A 5 4 9 and B E A S - 2 B cel ls on DO, D 2 , and D 7 post-infection in order to assess whether R V infect ion had any effect on ce l l growth and number, and to con f i rm that ce l l numbers remained relat ively unchanged once cultures had reached conf luence. Th is experiment was carr ied out to con f i rm that changes in ce l l number were not contr ibut ing factors in R V repl icat ion, R V 1 4 R N A , and cytokine/chemokine data. M e t h o d s : B E A S - 2 B and A 5 4 9 cel ls were grown to conf luence under standardized condit ions (see Chapter 2) in 24 we l l plates. Supernatants were then aspirated and inocu la of: R V 1 4 , R V 1 A or med ium alone were added and a l lowed to infect for 1 hour at 35°C. A f te r infect ion, cel ls were washed wi th D M E M and fresh culture med ium was added to the wel ls ( 1 % F B S ) . A t DO, D 2 , and D 7 post-infection cel ls were t ryps in ized, suspended in l m L o f fresh med ium, and counted by hemacytometer (with trypan blue exc lus ion for dead cel ls) w i th a l O u L loading vo lume (n=6). Re su l t s : There were no statistical differences (a=0.05, 2 way A N O V A s , Dunnet ' s Test) found in number o f cel ls between any o f the treatments (Cont ro l , R V 1 4 , or R V 1 A ) at any o f the sampl ing time-points for either the B E A S - 2 B or A 5 4 9 ce l l l ines (Figures D l and D2) . Furthermore, ce l l number remained fa i r ly constant f rom DO to D 7 in R V infected and control cel ls . There were very few dead cel ls present and supernatants contained v i r tua l ly no cel ls . Anecdota l l y , the culture med ium for R V 1 4 and R V 1 A infected cel ls d id appear more acidic than that o f the uninfected B E A S - 2 B and A 5 4 9 cel ls . 121 7 H CD £ 6 CD E o O CD O 4 H Control RV14 RV1A BEAS-2B 0 2 7 Time After Infection (Days) Figure Dl: 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 RV infected cells at any time (n=6). E 3 o O a> O 5 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 rway epithel ia l cel ls w i th R V 1 4 or R V 1 A had no effect on ce l l number when compared to controls and ce l l number remained quite constant for the duration o f the sampl ing per iod for both B E A S - 2 B and A 5 4 9 ce l l l ines. Therefore, changes in ce l l number d id not affect to R V repl icat ion, R V 1 4 R N A , or cytokine/chemokine secretion data in m y exper imental designs. 122 APPENDIX E: HI , BEAS-2B, and A549 Cell Counts at Confluence C e l l counts i n 6-well plates at conf luence for H I , A 5 4 9 , and B E A S - 2 B ce l l l ines were conducted in order determine ca lculat ion o f v i ra l infect ion dose ( M O I = l ) for R V infect ion experiments. M e t h o d s : Ce l l s were grown under standardized condit ions ( 3 m L D M E M or 50:50 D M E M / F 1 2 wi th 5-10% F B S ) at 35°C for unt i l f reshly conf luent in 6-well plates. A f te r 48 hours, supernatants were aspirated, cel ls t ryps in ized, di luted 5:1 in culture med ium and counted by hemacytometer (n=5) wi th trypan blue exc lus ion to stain dead cel ls w i th a loading vo lume o f l O u L . Resu l t s (Figure E l ) : C e l l count at conf luence was s igni f icant ly (a=0.05) higher in H I cel ls by 1.4-fold compared to B E A S - 2 B and A 5 4 9 cel ls but no difference in ce l l number was observed between the a i rway ce l l l ines (1-way A N O V A and post-hoc Tukey test). C e l l counts were (in number o f cel ls/wel l ) : H l = 1 . 9 x 10 6 ±3.3 x 10 4 , B E A S - 2 B = 1 . 3 x 10 6± 8.2 x 10 4 and A549=1.4 x 10 6 ± 8.5 x 10 4 . A l l ce l l counts were at an order o f magnitude o f 10 6 cel ls per we l l . R V infect ion doses o f 1 infect ious v i ra l particle per ce l l ( M O I = l ) in al l R V infect ion experiments were based on these ce l l counts. BEAS-2B Cell Type A549 Figure E l : Cell counts for uninfected HI , 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 106 order of magnitude. 123 APPENDIX F: Rhinovirus Stability The stabi l i ty o f R V 1 4 and R V 1 A under typ ica l experimental condit ions was ascertained, in order to determine whether infect ious virus assayed at g iven G row th Curve sampl ing times (D0-D7) represented da i ly secretions or accumulated R V secretions up to sampl ing day. M e t h o d s : R V 1 4 and R V 1 A stocks di luted w i th culture med ium ( D M E M ) and 1 % F B S to an in i t ia l starting concentrat ion o f 10 4 " 5 p fu/mL and al iquoted into sterile 6-well trays ( 3 m L of R V solut ion per wel l ) in order to mock typica l G r o w t h Curve condit ions (n=4). Samples were taken da i ly , f rozen at -80°C, and assayed for infect ious virus (pfu/mL). Resu l t s : Fo r both R V 1 4 (Figure F I ) and R V 1 A (Figure F2) infect ious virus decreased s igni f icant ly (a=0.05, 1-way A N O V A s and post-hoc T u k e y tests) f rom day to day unt i l it was no longer detectable on D 4 for R V 1 4 , and somewhere between D 4 and D 7 for R V 1 A . However , the in i t ia l concentration o f R V 1 A (DO) was higher than for R V 1 4 probably accounting for t ime difference in degradation. Ove ra l l , infect ious virus decreased by 1-2 orders o f magnitude dai ly for both R V 1 4 and 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 o 4 to 3 3 1 I 2 o CD I 1 0 a RV1A b Jjjiijl J 1 c { ^ * d S B e 0 1 2 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 med ium, R V 1 4 and R V 1 A stock solutions undergo dai ly degradation o f 1-2 orders o f magnitude. However , R V remains detectable for at least 3 days under these condit ions depending on in i t ia l R V concentrations. Therefore, G r o w t h Curve repl icat ion data f o l l ow ing the in i t ia l peak in infect ious virus (usual ly around D l ) cou ld represent gradual ly degrading virus synthesized on D l , or a combinat ion o f accumulated and newly synthesized R V . Furthermore, this experiment does not take into account the poss ib i l i ty that R V contained w i th in cel ls is protected f rom the degradation observed in the extracel lular environment. Add i t i ona l l y , in culture systems cel ls may secrete factors wh i ch faci l itate the degradation o f R V beyond what can be observed i n culture med ium alone. 125 APPENDIX G: Interleukin-6 and Interleukin-8 Stability The stabi l i ty o f IL-6 and IL-8 under typ ica l exper imental condit ions was assessed in order to determine whether cytokine/chemokine assayed at given time-points (D0-D7) in experiments represented da i ly secretions or accumulated IL-6/IL-8 secretions up to sampl ing day. M e t h o d s : Cy tok ine secretion was stimulated in B E A S - 2 B cel ls by infect ion w i th R V 1 A . A f te r 72 hours supernatants were co l lected, comb ined , centr i fuged at 1000 x g to remove ce l lu lar debris, re-distributed to sterile 6-well plates ( 3 m L per plate), and incubated at 35°C. Samples were col lected immediate ly (DO) and seven days after re-distribution (D7) , stored at -20°C, and assayed by E L I S A (n=3). Re su l t s : Bo th IL-6 (Figure G l ) and IL-8 (Figure G2 ) were stable under the g iven experimental condit ions up to D 7 post-incubation at 35°C (a=0.05, 1-way A N O V A and post-hoc Dunnet ' s test). 300 250 H & 200 S 150 c o o CD - 50 1 100 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 E Q. 600 1 J5 400 1 c o o c o O co 200 H IL-8 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 : Exper imenta l l y measured IL-6 and IL-8 secretions most l i ke l y represent accumulated cytokine/chemokine levels up to the speci f ied day o f sampl ing (unless the cel ls secrete factors w h i c h degrade IL-6 and IL-8). Th is mode l is the typ ica l mode l used for cytokine/chemokine secretion. A l ternat ive ly , one cou ld remove and replace the med ium in cel l cultures da i l y ; however , this is not usual ly done because o f the problems that it may cause. Fo r example , da i l y removal o f supernatant wou ld also remove any secreted virus in the med ium thus interfer ing w i th repl icat ion data samples. Furthermore, the addit ion o f fresh da i ly med ium may disturb ce l ls , and addit ion o f new nutrients may encourage ce l l growth and other ce l lu lar 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 by subtracting values f rom the previous sampl ing day was done; however, for the most part this d id not affect overa l l trends. F igure G 3 shows an example o f such a data modi f i ca t ion for trial 1 IL-6 secretion o f the R V 1 A / B E A S - 2 B G row th Curve (or ig ina l ly F igure 2.3b). The overa l l trend is s imi la r w i th m a x i m u m IL-6 secretion occurr ing on D 5 and D 7 ; however, cons ider ing that these sampl ing times may represent accumulated IL-6 over 2 days the actual da i l y secretion w o u l d l i ke l y be lower (possibly ha l f this value). Th i s cou ld result in a much less pronounced effect on D 5 and D 7 ; however, these values wou ld remain the m a x i m u m increases in IL-6 secretion relative to their controls. > 127 2000 1500 w 1000 co Q o > CD 500 H -500 Control RV Trial 1: BEAS-2B/RV1A i T 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. RV and control IL-6 data from the trial 1 BEAS-2B Growth Curve with RV1A (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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