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The maternal predisposition to the syndrome of pre-eclampsia Alasaly, Kadria A. 2004

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The maternal predisposition to the syndrome of pre-eclampsia By Dr. Kadria A. Alasaly M.D. Mousl University, Medical College of Physician and Surgeons A THESIS SUBMITTED IN PARTIAL FULLFILMENT OF THE REQUIRMENT FOR THE D E G R E E OF MASTER O F SCIENCE IN THE FACULTY OF GRADUATE STUDIES EXPERIMENTAL MEDICINE PROGRAM DEPARTMENT O F MEDICINE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 2004 © Kadria A. Alasaly, 2004 Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. k f l o g ; / ) ALflsfiLy Afc[ N a m e of Author (please print) Date (dd/mm/yyyy) " i t l e o f T h e s i s : -TX» V*G kem«( pW/</Wfb^ t; D e 9 r e e : Master et - < c , W , Year: JUolf-Departmentof M^cfrc^ f frp-rl^t^ Mg^,;u ( The University of British Columbia Vancouver, BC Canada Abstract Objectives Pre-ec lampsia , w h i c h is characterized b y maternal hypertension, proteinuria, hypoperfus ion o f end organs and a systemic maternal innate inf lammatory response, is a leading cause o f maternal morta l i ty and morb id i ty wor ld -wide . W h e n o f early-onset, pre-eclampsia is associated w i t h fetal intrauterine growth restrict ion ( I U G R ) . I U G R can occur i n isola t ion, so-cal led normotensive I U G R . W h a t is poor ly understood is that some w o m e n develop the maternal syndrome o f pre-eclampsia whi l s t others have o n l y the fetal syndrome (normotensive I U G R ) , despite the fact that the in i t ia t ing event i n bo th is be l ieved to be reduced uteroplacental perfusion. In this thesis I have asked two questions: Firs t , cou ld the maternal innate inf lammatory response o f pre-eclampsia predict an increased l i fe t ime r i sk for developing the systemic inf lammatory response syndrome ( S I R S ) , as it is w i t h later atherosclerosis (another disease o f inf lammat ion)? M y hypothesis was the maternal syndrome o f pre-eclampsia is a form o f the sys temic in f lammatory response syndrome. Second, c o u l d it be that the maternal syndrome o f pre-eclampsia is tr iggered b y a react ivat ion o f chronic infections c o m m o n i n the communi ty , m a i n l y infect ion w i t h Chlamydophila pneumoniae (Chipneumoniae) or cytomegalovirus ( C M V ) infections? T h i s is especia l ly pertinent as these infectious agents have been impl ica ted i n the development o f atherosclerosis. W e wanted to determine whether or not the dichotomous response o f pre-eclampsia and normotensive I U G R cou ld be expla ined b y react ivat ion o f chronic infect ion w i t h Chi pneumoniae or C M V dur ing pre-ec lampsia , but not normotensive I U G R . M y hypothesis was, c o u l d reactivated infectious trigger exp la in the differential maternal response to the shared placental pathology o f pre-eclampsia and ii normotensive growth restriction? This would provide a link between pre-eclampsia and its attendant lifelong risk of atherosclerosis. Study designs SIRS. Cases were selected from women admitted to the intensive care unit (ICU) of St. Paul's Hospital with the diagnosis of SIRS. Controls were women without SIRS, admitted to general medical/surgical wards with the same primary diagnosis and same underlying problem (eg: pneumonia, bowel resection) but unremarkable in-hospital course (NO SIRS). The controls were matched for both age (+5 years) and ethnicity. Chi pneumoniae and C M V . Seroprevalence and levels of an t i -CMV and Chi pneumoniae IgG were compared in a nested case-control study. We compared between women with early-onset pre-eclampsia (<34 weeks'; n=9), late-onset pre-eclampsia (>34+0 weeks'; n=29); normotensive I U G R (birthweight <3 r d centile; n=33), and matched normal pregnancy (n=l 13, up to 2 per case). Results SIRS. Most women in the I C U were too critically i l l to be interviewed. Therefore, this study was deemed not to be feasible and was abandoned because o f the low frequency of female admissions to the St Paul's I C U and, insufficient obstetric information. Chi pneumoniae and C M V . There was a significant difference in both an t i -CMV and Chi pneumoniae E B antibodies between groups (Kruskal-Wallis p<0.05). Women with early-onset pre-eclampsia had higher an t i -CMV levels (median: 79 [95% confidence interval 47, 164]) than women with late-onset pre-eclampsia (26 [22, 82], p<0.05), normotensive I U G R (40 [31, 72], p<0.05), and normal pregnancy (49 [45, 70], p<0.05). Women with i i i normotensive I U G R had s ignif icant ly lower mti-Chlpneumoniae antibodies (0.10 [0.08, 0.38]) than d i d normal pregnancy controls (0.21 [0.20, 0.28], p<0.05). Conclusions S I R S . W e were unable to test the hypothesis as the study, as designed, was not feasible. Chi pneumoniae and C M V . W o m e n w i t h early-onset pre-eclampsia had higher levels o f I g G against Chi pneumoniae and a n t i - C M V anti-bodies i n their serum than i n late-onset pre-eclampsia, normotensive I U G R , and normal pregnancy. Th i s m a y provide a pa thophys io logica l l i n k between pre-eclampsia and the k n o w n increased r isk for subsequent atherosclerosis. C o u l d a reactivated infectious trigger expla in the differential maternal response to the shared placental pathology o f pre-eclampsia and normotensive intrauterine g rowth restriction? In addi t ion, cou ld the association between the maternal I g G response to Chi pneumoniae and C M V provide a l i n k between pre-eclampsia (especial ly o f ear ly onset) and its attendant l i f e long r isk o f atherosclerosis. Ear ly-onset pre-eclampsia m a y t ruly be the ' toxaemia' o f pregnancy. iv T A B L E OF CONTENTS A B S T R A C T i i T A B L E O F C O N T E N T S v L I S T O F F I G U R E S ix L I S T O F T A B L E S ix A C K N O W L E D G E M E N T x C H A P T E R 1: G E N E R A L I N T R O D U C T I O N 1 Maternal predisposition to the syndrome of pre-eclampsia Background 1 Pre-eclampsia 2 1.1. Clinical description 2 1.2. Pathophysiology of pre-eclampsia 4 2 Systemic inflammatory response syndrome 8 (SIRS) and pre- eclampsia. 3 Could subclinical infection be the differentiating trigger between pre- eclampsia and normotensive IUGR? 11 3.1 Human evidence for an infectious trigger in the pathogenesis of pre-eclampsia 11 3.2 Infection in atherosclerosis 13 3.3 Pre-eclampsia and atherosclerosis 1 4 General purpose for research project 1 V C H A P T E R 2: Systemic Inflammatory Response Syndrome (SIRS) 19 Background 20 2.1 Terminology and epidemiology of the systemic inflammatory response (SIRS) 20 2.2 Pathogenesis of sepsis 24 2.3 Pre-eclampsia and the systemic inflammatory response syndrome compared 27 2.3.1 Clinical 29 2.3.2Endothelial dysfunction and inflammatory changes 30 2.3.3Neutrophil activation 30 2.4 Hypothesis and Objectives 2.4.1 Hypothesis.. 32 2.4.2 Objectives 32 2.5 Methods 32 2.5.1 Cases and controls 33 2.6 Data collection 33 2.7 Results 34 2.7.1 Limitations 34 2.8 Discussion 35 VI C H A P T E R 3: A nested case-control seroepidemiological study o f Poss ib le triggers o f the maternal syndrome o f pre-eclampsia 36 3.1 Preamble 37 3.1.1 Background 37 3.2 Methods 39 3.2.1 Research design 39 3.2.2 Identification of cases and controls 39 3.2.2.1 Cases and controls 40 3.2.2.2 Definitions 40 1. Pre-eclampsia 40 2. Normotensive IUGR 41 3. Normal pregnancies 41 3.2.3 Serum bank 41 3.2.4 Data Collection 43 3.2.4.1 Maternal Variables 43 3.2.4.2 Maternal Outcomes 43 3.2.4.3 Fetal Variables 44 3.2.4.4 Fetal Outcomes 44 3.2.5 Laboratory techniques 47 3.2.6 Methods for cytomegalovirus using unlinked anonymous seroprevalence 47 3.2.6.1 Sample selection 47 3.2.6.2 The assay 48 3.2.6.3 Procedure 49 3.2.6.4 Quality control 49 3.2.7 Chi pneumoniae 52 3.2.8 Data analysis 53 3.3 Results.. 53 3.3.1 Analyses 54 3.4 Discussion 57 vii C H A P T E R 4: Conclusion, general approach, future directions for the research 64 4.1 General approach 65 4.2 Summary of the results 65 4.3 Conclusions 66 4.4 Future directions 67 Bibliography 69-90 v i i i Tables and Figures: Figures: Figure 1.1 3 Figure 1.2 7 Figure 1.3 10 Figure 1.4 14 Figure 1.5 17 Figure 2.1 22 Figure 2.2 26 Figure 2.3 26 Figure 2.4 27 Figure 3.1...: 45 Figure 3.2 46 Figure 3.3 46 Tables: Table 1.1 6 Table 2.1 23 Table 2.2 28 Table 3.1 42 Table 3.2 51 Table 3.3 55 Table 3.4.. .....56 Table 3.5 56 ix ACNOWLEDGEMENTS I w o u l d l i ke to take this opportunity to thank m y f a m i l y for he lp ing me through the frustrating times and I thank them for their support and encouragement. A special thanks to m y son O m a r for his support, enthusiasm and inspira t ion. I w o u l d l i k e to thank D r . Peter v o n Dade l szen and D r . K e i t h W a l i e y for supervis ing this thesis and their outstanding support. T h e i r attitude and efforts he lped me to successfully complete m y thesis. Specia l thanks are due to each member o f m y committee: D r s . Delbert R . D o r s h e i d and J i m Russe l l (Cr i t i ca l Care M e d i c i n e ) , and D r . L a u r a M a g e e (Obstetric and Genera l Internal M e d i c i n e ) . A s w e l l I w o u l d l i k e to thank m y e x a m i n i n g committee, D r . M i k e A l l a r d and D r . A n d y Sandford. M o r e o v e r , I w o u l d l i k e to thank D r . N o r m a n W o n g , Di rec tor o f Exper imenta l M e d i c i n e . F u n d i n g for this project was from an Establ ishment Grant f rom the B C Research Institute for C h i l d r e n ' s and W o m e n ' s Hea l th to D r v o n Dade lszen , as w e l l as internal funding f rom the U B C C D C . I a m grateful for the col labora t ion o f m y colleagues at B C W o m e n ' s and the U B C C D C for the publ i shed study o f Chlamydophila pneumoniae and cytomegalovirus i n pre-eclampsia. In particular, I am grateful for the help o f M s Shel ley Soanes, and the Hea l th Records Department at B C W o m e n ' s for their help i n co l la t ing the c l i n i c a l data for the w o m e n studied i n the infect ion study. T h e comple t ion o f this thesis w o u l d not have been poss ible wi thout the contr ibut ion o f these people and thus they are s incerely acknowledged here. X Chapter 1 General introduction: Maternal predisposition to the syndrome of pre-eclampsia Inflammation in itself is not to be considered as a disease ... and in a disease, where it can alter the diseased mode of action, it likewise leads to a cure; but where it cannot accomplish that salutary purpose, it does mischief. John Hunter; Treatise on the blood, Inflammation and Gunshot Wound, London, 1794 1 Background 1. Pre-eclampsia 1.1 Clinical description Pre-ec lampsia is a disorder specific to pregnancy. Pre-eclampsia/eclampsia is the m a i n cause o f maternal mor ta l i ty and is associated w i t h a f ive-fold increase i n perinatal mor ta l i ty i n deve lop ing countries (1). It remains one o f the two commonest causes o f maternal mor ta l i ty i n the developed w o r l d (2-7). Pre-eclampsia is estimated to affect 7% to 10% o f a l l pregnancies i n the U n i t e d States (8). Despite be ing one o f the leading causes o f maternal death and a major contributor o f maternal and perinatal morb id i ty and its importance for the pub l i c health, the aet iology o f pre-eclampsia has not yet been c lear ly established. A c c o r d i n g to the International Socie ty for the Study o f Hyper tens ion i n Pregnancy, pre-ec lampsia is present when a prev ious ly healthy w o m a n develops hypertension combined w i t h proteinuria after the 2 0 t h week o f gestation (9). In the U S A , pre-eclampsia is defined b y the Na t iona l H i g h B l o o d Pressure Educa t ion Program ( N H P B E P ) criteria, as a pregnancy-specif ic increase i n b l o o d pressure (diastolic b l o o d pressure ( d B P ) > 9 0 m m H g , or sys to l ic b l o o d pressure ( s B P ) > 1 4 0 m m H g ) associated w i t h >0.3g proteinuria/d (or protein: creatinine ratio >30mg pro te in /mmol creatinine on random sampl ing (10)). Pre-ec lampsia resolves after de l ive ry (11). There are two syndromes i n pre-eclampsia (Figure 1.1): the first is maternal, characterized c l i n i c a l l y b y hypertension and proteinuria and, pa tho log ica l ly b y endothelial ce l l act ivat ion (12) and systemic inf lammat ion (13). The second is fetal, manifested most c o m m o n l y , b y intrauterine growth restriction ( I U G R ) (14; 15). 2 Trophoblast Immune homeostasis Maternal inflammation Endothelial changes Kidney Uterus Periphery Proteinuria Fetal growth restriction Hypertension Figure 1.1: The fetal trophoblast induces an inflammatory response in the mother, to a state of imbalance with endothelial damage, the extent of which determines the clinical manifestations of pre-eclampsia. Modified from Dietl J (16). T h e maternal c l i n i c a l signs and symptoms defines pre-eclampsia, g iven that incomplete placental implanta t ion is also a feature o f normotensive I U G R (17). I U G R can occur i n i so la t ion ( 'normotensive I U G R ' ) , w h i c h is the fetal syndrome o f pre-eclampsia i n i so la t ion (18) and shares the same under ly ing placental pathology (17), but lacks the maternal response i n terms o f either hypertension and proteinuria or systemic in f lammat ion (19). T h e or igins o f pre-eclampsia and normotensive I U G R l ie i n a mismatch between fetoplacental demands and the maternal vascular supply ('uteroplacental mismatch ' (20)), but result i n d ichotomous c l i n i c a l syndromes (21). Pre-ec lampsia is associated wi th an increased incidence o f I U G R . Infants w h o survive I U G R have an increased r isk o f both phys ica l and mental handicap, and an increased r isk o f mor ta l i ty i n adult l i fe f rom cardiovascular (22) and endocrine diseases (23;24). 3 The pathology o f pre-eclampsia is expressed at three levels (25). T h e p r imary pathology is not yet k n o w n but is at least associated w i t h the presence o f trophoblast. Gesta t ional t rophoblact ic disease, such as hydat id i form mole , is an abnormal i ty i n w h i c h the pregnancy contains ve ry li t t le or no fetal tissue w i t h the products o f concept ion be ing almost exc lus ive ly hyperplast ic trophoblast. It is associated w i t h an increased r isk o f pre-eclampsia . The secondary pa thology o f pre-eclampsia is the maternal adaptation to the impa i red endovascular trophoblast i nvas ion and it includes the def ining signs o f pre-eclampsia: hypertension and proteinuria. U n d e r certain circumstances, the peripheral disturbances o f pre-eclampsia can become so severe that they themselves initiate new or tertiary pathology. The most significant manifestat ion o f tertiary pathology are the convuls ions o f ec lampsia , cerebral haemorrhage, renal failure and the H E L L P (haemolysis , elevated l ive r enzymes, l o w platelets) syndrome (26). 1.2 Pathophysiology of pre-eclampsia The mechanisms responsible for the pathogenesis o f pre-eclampsia are unclear. Hyper tens ion associated w i t h pre-eclampsia develops dur ing pregnancy and resolves after de l ivery , imp l i ca t i ng the placenta as a central culpri t i n the disease (27). It has been suggested that release o f factors f rom the placenta i n response to i schaemia results i n endothelial dysfunct ion o f the maternal c i rcula t ion (12;28;29). The vascular endothel ium has many important functions, i nc lud ing control o f smooth musc le tone through release o f vasoconstr ictor and vasodi la tory substances, and regulat ion o f anticoagulat ion, antiplatelet, and f ibr inolys is functions v i a release o f different soluble factors. Ev idence o f endothelial dysfunct ion as an early event i n pre-eclampsia suggests that it is a possible cause (12). A d d i t i o n a l l y , i n w o m e n who develop pre-eclampsia, preexis t ing maternal factors such as chronic hypertension, diabetes, and hyper l ip idaemia m a y predispose the maternal 4 endothel ium to further damage. Increased sensit ivi ty to pressor agents and act ivat ion o f the coagulat ion cascade occur early i n the course o f pre-eclampsia (12). M a n y markers o f endothelial dysfunct ion have been reported i n w o m e n who develop pre-eclampsia , suggesting that pre-eclampsia is an endothelial ce l l disorder. C i r cu l a t i ng levels o f f ibronect in, p lasma th rombomodu l in and v o n W i l l e b r a n d factor are s ignif icant ly elevated i n w o m e n w i t h pre-ec lampsia (30). Platelets also appear to p lay an important role i n the e t io logy o f pre-eclampsia . Enhanced platelet activation, and increased levels o f platelet endothelial ce l l adhesion molecule-1 ( P E C A M - 1 ) , intercellular ce l l adhesion molecule-1 ( I C A M - 1 ) , and vascular ce l l adhesion molecule-1 ( V C A M - 1 ) also occur i n w o m e n w h o develop pre-ec lampsia (12;28;29). M a n y markers o f endothelial dysfunction m a y funct ion as predictors o f the syndrome i n w o m e n who develop pre-eclampsia as many are s igni f icant ly elevated weeks before observ ing o f c l i n i c a l manifestations. Studies over the past decade have provided a better understanding o f the potential mechanisms responsible for the pathogenesis o f pre-eclampsia. The in i t ia t ing event i n pre-ec lampsia has been postulated to be reduced uteroplacental perfusion. T h i s results i n abnormal cytotrophoblast invas ion o f spiral arterioles leading to placental i schaemia . Th i s i schaemia is w i d e l y ci ted as a k e y factor (29;31-33) and, w i t h the placenta b e c o m i n g increas ingly i schaemic as gestation progress, it is thought to lead to widespread act ivat ion/dysfunct ion (Figurel .2) o f the maternal vascular endothel ium (34). It is unclear what triggers this endothelial disturbance i n pre-eclampsia, but neutrophil act ivat ion can result i n release o f a specif ic elastase, capable o f media t ing vascular damage b y des t roying the integri ty o f endothelial cel ls (35;36). Recent f ind ing are more suggestive o f an inappropriate maternal in f lammatory response w i t h i n the framework o f placentation. The innate immune system appears to be p r i m a r i l y 5 i n v o l v e d . Na tura l k i l l e r cel ls cou ld be regulatory cells pushing maternal i m m u n e response toward a T h 2 prof i le , beneficial for fetal surv iva l , or toward a T h l type o f i m m u n e response, w h i c h acts i n synergy (37). The exaggeration o f this maternal intravascular in f lammatory react ion to the i nvad ing trophoblast leads to the manifestation o f pre-ec lampsia i n the mother and feto-placental unit (16). The most popular mode l for the pathogenesis o f the maternal syndrome o f pre-eclampsia describes a process b y w h i c h the injured placenta communicates w i t h the maternal endothel ium, causing a syndrome o f systemic endothelial ce l l dysfunct ion (Table 1.1) and end organ hypoperfusion (12) w h i c h leads to mul t iorgan dysfunct ion. Inadequate de l ivery o f b l o o d to the pregnant uterus can cause a consistent rise i n b l o o d pressure and h y p o x i a cou ld initiate the placental injury (38). L i k e w i s e , the renal morpho log ic changes present i n w o m e n w i t h pre-eclampsia are not found i n any other fo rm o f hypertension. Furthermore, other pathophysiological changes, act ivat ion o f coagulat ion cascade, increased sensi t ivi ty to pressors, reduced p lasma vo lume , and abnormali t ies o f renal p r o x i m a l tubular function, a l l antedate increased b l o o d pressure (12). Table 1.1: Biochemical and morphological evidence supporting the concept of endothelial cell injury in pre-eclampsia Modified From Bolte era/. (39). Biochemical Morphological Factor VIII R : A g f Glomerular endotheliosis Fibronectin f Ultrastructural changes in placental Bed and uterine boundary vessels P A I - l / P A I - 2 ratio | Subendocardial necrosis Disturbance of tPA/PAI-1 balance Disturbance of P G I 2 / T X A 2 balance Endothelin | Thrombomodulin f Growth factor activity | PAI: plasminogen activator-inhibitor; PGI2: prostacyclin; R:Ag: related antigen; tPA: tissue plasminogen activator; TXA 2 : thromboxane 6 cytotrophoblast invasion immunological factors -IUGR poor placentation acute atherosis r j uteroplacental mismatch thrombophilia multiple pregnancy fetal macrosomia (eg DbM) PBLs cytokines PGs lipid peroxides placental debris J t L INTERVILLOUS SOUP endothelial cell activation T_ | maternal syndrome Figure 1.2: A model for the pathogenesis of pre-eclampsia. In this model of pre-eclampsia, the maternal syndrome develops from a number of alternative pathways, leading to uteroplacental mismatch, whereby the fetoplacental demands outstrip the maternal circulatory supply. In response to the mismatch, and probably caused, in part, by recurrent ischaemia-reperfusion injury within the intervillous (maternal blood) space of the placenta and accelerated placental apoptosis, a soup of endothelium-damaging substrates is released, with resulting endothelial cell activation and consequent development of the maternal syndrome of pre-eclampsia. Some elements of the soup namely, activated peripheral blood leukocytes can cause direct end-organ damage. There is cross talk between elements of the soup (not illustrated). Activated protein C could modify both the inflammatory and coagulation consequences of the endothelial cell activation. PBLs , peripheral blood leukocytes; P G s , eicosanoids; R O S , reactive oxygen species. Modified From von Dadelszen etal. (20). Per ipheral b l o o d neutrophils m a y represent a l i n k between the syncytiotrophoblast o f the in terv i l lous space and the systemic vascular endothel ium (40;41), either b y s t imulat ing the endothel ium i n response to syncytiotrophoblast fragments or i n response to pr ior endothelial c e l l act ivat ion b y syncytiotrophoblast fragments themselves. There is evidence o f neutrophi l act ivat ion i n pre-eclampsia superimposed on the neutrophi l ia (42-44) and alterations i n neutrophi l function w h i c h i m p l y that normal pregnancy is a pro inf lammatory state (36;45-48). 7 A s stated, incomplete placental implantat ion is also a feature o f normotensive I U G R (17). W h a t is it that causes one w o m a n to develop the maternal syndrome o f pre-eclampsia; another has a pregnancy compl ica ted sole ly b y I U G R ? In part, this m a y be determined b y a differential neutrophil-mediated maternal inf lammatory response w h i c h characterises both no rma l pregnancy (42;43;45;47) and pregnancy compl ica ted b y normotensive I U G R (19), but is accentuated i n pre-eclampsia (49-51). Neu t roph i l act ivat ion m a y be i n v o l v e d i n the development o f acute atherosis (52), as it is i n atherogenesis (53;54). There is increasing evidence to support the hypothesis that elements o f the syncytiotrophoblast , w h i c h lines the intervi l lous space o f the placenta, m a y be a trigger o f the maternal response modulated b y the endothel ium. W h e n neutrophils are activated, they synthesize and release proteases, such as a specific neutrophil elastase, capable o f media t ing vascular damage b y destroying the integrity o f endothelial cells , the vascular basement membrane, and the subendothelial matr ix (35). T o x i c oxygen species and leukotrienes are also released and can produce membrane l i p i d peroxides, lys is o f endothel ial cel ls , and increased vascular permeabi l i ty and reactivi ty (35;55). B o t h hepatocellular necrosis and A R D S are characterized b y neutrophil infi l trat ion, and are be l ieved to be related to neutrophil-mediated damage (56). 2. Systemic inflammatory response syndrome (SIRS) and pre-eclampsia S I R S is c l i n i c a l l y defined as: tachycardia, tachypnoea, hyper thermia and leukocytos is . It is the beg inn ing o f the inf lammatory cont inuum that includes sepsis. It represents an acute alteration f rom baseline homeostasis i n the absence o f other k n o w n causes. There is strong ep idemio log ic evidence that S I R S , sepsis, severe sepsis, and septic shock represent a h ierarchica l con t inuum o f an inf lammatory response to infect ion (57). A conf i rmed infectious 8 process is required for d iagnosing sepsis. I f S I R S becomes excessive, it means the injury is o v e r w h e l m i n g or is cont inuing because o f inadequate therapy or lack o f control o f injured organs or systems and the patient m a y die. Thus, S I R S is s ick, mul t ip le organ dysfunct ion syndrome ( M O D S ) is sicker, mul t ip le organ failure ( M O F ) is sickest. A systemic in f lammat ion , w i t h the release o f mul t ip le cytokines , plays an important role i n the pa thophys io logy o f S I R S (Figure 1.3). S I R S is associated w i t h most I C U morb id i ty (58). S I R S shares m a n y c l i n i c a l and laboratory characteristics w i t h pre-eclampsia, i nc lud ing a hyperdynamic state, leukocytos is , and organ dysfunct ion (this compar ison is described i n greater detail i n Chapter T w o ) . Pre-ec lampsia m a y persist, often deteriorating transiently, f o l l o w i n g the de l ive ry o f the placenta, m u c h as S I R S develops f o l l o w i n g the resolution o f trauma, burns, sepsis or other in f lammatory events. In both condit ions there is a systemic response w i t h in f lammatory mediator release, endothelial ce l l dysfunction, end organ hypoperfus ion and neutrophi l act ivat ion. T w o decades ago, hypertension-induced cerebral haemorrhage was the most c o m m o n cause o f maternal death associated w i t h pre-eclampsia. Howeve r , since that t ime, w o m e n most c o m m o n l y d ied f rom pre-eclampsia/eclampsia (40 .5% o f maternal deaths ascribed to hypertensive diseases o f pregnancy) either f rom hepatocellular necrosis or the acute respiratory distress syndrome ( A R D S ) (6). Thus the c l i n i c a l picture o f pre-eclampsia is reminiscent o f S I R S as it progress to evolve into M O D S . 9 Toxic stimulus Figure 1.3: Early Biochemical Events in Sepsis. An initial toxic stimulus (e.g., endotoxin) triggers the production of proinflammatory monokines (e.g., tumor necrosis factor and interleukin-1). These cytokines, in turn, result in neutrophil-endothelial-cell adhesion, activation of clotting, and generation of numerous secondary inflammatory mediators, including other cytokines, prostaglandins, leukotrienes, and proteases. Antiinflammatory compounds, such as interleukin-6 and interleukin-10, that may serve as negative feedback to the inflammatory process, are also released. Modified from Wheeler A et a/(59). A question to be asked is whether a w o m a n who develops pre-eclampsia is at increased r isk for other condit ions mediated b y the innate immune system, due to intr insic variations i n her leukocyte function. Such another condi t ion w o u l d be S I R S , w h i c h is most prevalent dur ing the 6 t h and 7 t h decades o f l i fe (60;61). W e have postulated that a h is tory o f pre-ec lampsia w i l l predispose w o m e n to the later development o f S I R S , as has already been established for atherosclerotic disease. The hypothesis is that pre-eclampsia is associated w i t h increased r isk o f systemic inf lammat ion later i n l i fe , as it is possible that there are innate variat ions i n neutrophi l (or other inf lammatory mediator) function that determine whether or 10 not a w o m a n is more l i k e l y to develop pre-eclampsia that w o u l d predispose that w o m a n to S I R S f o l l o w i n g sufficient pro- inf lammatory provocat ion (details i n Chapter 2). 3. Could subclinical infection be the differentiating trigger between pre-eclampsia and normotensive IUGR? Because o f the associat ion between pre-eclampsia and subsequently increased r i sk for atherosclerosis (section 3.3), w e have postulated that w o m e n m a y be more l i k e l y to develop the maternal syndrome o f pre-eclampsia i n the presence o f subc l in ica l infect ion w i t h either Chlamydophila (Chlamydia) pneumoniae (Chipneumoinae) or cytomegalovi rus ( C M V ) , than w o m e n whose pregnancies are either uncomplicated or compl ica ted b y I U G R i n isola t ion (21). Chi pneumoniae and C M V have been l i nked to atherogenesis (section 3.2). B o t h Chi pneumoniae and C M V are especia l ly credible potential pathogens g iven chronic carrier status i n the general popula t ion and the susceptibi l i ty o f pregnant w o m e n to disease reactivation due to reduced cel l -mediated i m m u n i t y i n pregnancy(37). Per iodontal , gastrointestinal and genitourinary tract disease are also prevalent, and the presence o f c l i n i c a l infec t ion delays the resolut ion o f the disease i n w o m e n w i t h severe H E L L P (62). W h a t makes this hypothesis par t icular ly interesting is the potential to intervene w i t h agents that are safe i n pregnancy. Perhaps pre-eclampsia is t ruly the ' toxaemia' o f pregnancy. 3.1 Human evidence for an infectious trigger in the pathogenesis of pre-eclampsia Infection w i t h Chi pneumoniae is suspected to contribute to the pathogenesis o f human atherosclerosis. E p i d e m i o l o g i c a l studies have raised the question o f whether bacterial infec t ion especia l ly w i t h Chi pneumoniae m a y contribute to this inf lammatory process (63;64). His topa thologic studies show a higher prevalence o f Chi pneumoniae i n diseased versus normal coronary arteries (65). O h k u c h i et al found that C M V infect ion i n pregnancy 11 was cer tainly a m i m i c k e r o f pre-eclampsia, i f not a precipitant o f pre-eclampsia (a p laus ible alternative explanation) (66). Case reports have l inked pre-eclampsia to chronic gastrointestinal infestation w i t h parasites such as Schistosomiasis japonica (67) and Strongyloides stercoralis (68), and case-control studies have found associations between ur inary and/or lower genital tract infect ion w i t h Ureaplasma urealyticum and Gardnerella vaginalis (69;70). In a nested, case-control study, us ing mul t ip le logis t ic regression, any "urinary tract infect ion" (bacteria not specified) i n pregnancy increased the r isk for the development o f pre-eclampsia b y 2.5 fo ld (95% confidence interval (CI) 1.3 - 5.0), par t icular ly among pr imigravidae ( O R 5.5 ( 9 5 % C I 2.9 -9.7)) (71). In a more recently described cohort, the presence o f periodontal disease at <26 weeks ' gestation increased the r isk for subsequent pre-eclampsia ( O R 2.3 (95% C I 1 . 0 - 5.4)(72). Per iodonta l disease, w h i c h causes chronic inf lammat ion , has been l inked , i n turn, w i t h atherosclerosis (73). C o u l d the maternal syndrome have been both potentiated and sustained b y a product o f the infectious process, such as G r a m negative-derived endotoxin? Cy tok ines are powerfu l mediators o f the in f lammat ion and sepsis that accompany endotoxin toxaemia. Consistent w i t h a potential role o f cy tokine act ivation i n pre-eclampsia is a study demonstrat ing that an intravenous infus ion o f a very l o w dose o f the endotoxin l ipopolysacchar ide ( L P S ) resulted i n significant and long-term increases i n b l o o d pressure and ur inary a l b u m i n excret ion and significant platelet aggregation i n conscious pregnant rats (74). Decreased renal and hepatic b l o o d f low w i t h pre-eclampsia- l ike his to logical changes fo l lowed exposure to L P S i n pregnant rabbits (75). 12 3.2 Infection in atherosclerosis Increasingly, there is evidence that chronic infect ion m a y be an important co-factor i n in i t ia t ing atherogenesis (54). The ep idemiolog ica l associations between atherosclerotic cardiovascular disease and infect ion w i t h C M V and, part icular ly, Chi pneumoniae (54;76-78) (Figure 1.4) i m p l y an infectious element to atherogenesis . C M V and Chi pneumoniae have been ident i f ied w i t h i n atheromatous plaques (54;79;80). Potential mechanisms inc lude direct loca l effects o n endothel ium (on vascular smooth muscle cel ls and/or o n macrophages w i t h i n the atherosclerotic lesion) , or ampl i f ica t ion o f the systemic inf lammatory response. C M V has a seroprevalence o f 60-85%, can persist i n the human b o d y for several years and can cause recurrent infect ion, also speci f ica l ly i n pregnancy (81). C M V is associated w i t h the accelerated atherosclerosis compl ica t ing cardiac transplantation (82) and direct endothel ial damage i n systemic sclerosis (83). An t ibod ie s to Chi pneumoniae have a popula t ion prevalence o f 5 0 % b y the age o f 20, r i s ing to 5 5 % among w o m e n at 40 (84). These ep idemio log ica l data support the premise that chronic infect ion cou ld l i n k pre-eclampsia w i t h later atherosclerosis, especia l ly g iven the increased suscept ibi l i ty to chronic infect ion due to reduced T h l responses (cel l-mediated immuni ty ) i n pregnancy (37). Recent ly , increased seroprevalence o f mti-Chlpneumoniae I g G (not I g A or I g M ) has been described i n pre-eclampsia pregnancy when compared w i t h normal pregnancy (85). These changes were specif ic to Chi pneumoniae and not to either Chi trachomatis or Chi psittici. That this associat ion between chronic infect ion and atherosclerosis exists is supported by the fact that macrol ides , effective against Chi pneumoniae, m a y reduce the r i sk o f recurrent coronary events (86;87), and that prophylact ic post-transplant ganc ic lov i r , act ive against cytomegalovirus , reduces the r isk o f accelerated post-transplant atherosclerosis (88). 13 Protease Induction 7 LDL.. oxidation Autoimmunity to Hsp60 Plaque Foam ceils Inflammatory responso - cytokines - CAMS -CRP Changes: in 1 lipid metabolism - triglycerides .. -HDL Atherosclerosis C H O HspSO - heart -schoc* cotoin SO CAM' «tetlulaf &dhcnon molecules LCI - tow eternity lipoprotein CRP = C-reaCTtve protein HZil i J-> density lipoprotein • , CHD . * coronary heart disease Figure 1.4: Pathogenic mechanisms by which Chlamydia pneumoniae infection could affect the development of atherosclerosis and coronary heart disease. Modified from Leinonen M etal(79). A compat ib le loca l immune balance has been shown to be o f considerable importance for successful pregnancy. The fetoplacental unit is k n o w n to secrete a wide range o f cytokines and other immunomodu la to ry substances important for this immune balance (89). T h l responses are thought to be detrimental to pregnancy whereas T h 2 responses are benef ic ia l . There is a compensatory upregulat ion o f T h 2 i m m u n i t y i n normal pregnancy (37). T h e percentage o f T h l and T h 2 , and the ratios o f T h l :Th2 correlate w i t h cy tokine ( I F N - a and I L -4) secretion l eve l . The type 1 cytokine I F N - a and the type 2 cytokine I L - 4 , as w e l l as the immunosuppress ive cytokine I L - 1 0 , have been reported to be present l o c a l l y i n the uterus dur ing human pregnancy (90-92). Up-regula t ion o f T h l responses and down-regula t ion o f T h 2 responses occur i n the early phases o f pre-eclampsia, thereby inh ib i t ing placentation. The prof i le o f secreted cytokines shifts i n favour o f T h l ac t iv i ty (extremely h i g h I F N - y and l o w I L - 6 and I L - 1 0 secretion). Changes i n N K and T lymphocyte subsets fo l l owed w i t h T h l cy tokine I F N - y over-act ivi ty , and T h l / T h 2 imbalance cou ld affect loca l immunoregula tory mechanisms i n th i rd trimester o f w o m e n w i t h pre-eclampsia. 14 T h e sensit ized monocytes and granulocytes o f a generalized T h 2 response, i n later pregnancy, w o u l d be the target o f the reactivated infect ion and that w o u l d trigger the excessive T h 2 upregulat ion and systemic in f lammat ion characteristic o f pre-eclampsia (13;93), especia l ly early-onset pre-eclampsia (13), w h i c h differs i n degree f rom that found i n normotensive I U G R (21). Recent ly , Cunn ingham et al assessed maternal i m m u n e status i n patients w i t h the H E L L P syndrome (62). The results o f their study showed a depression o f both T and B c e l l potential and impaired monocyte hand l ing o f intracel lular pathogens (up to 3 3 % , 1 1 % and 17% o f control values, respectively) , and that the r isk o f opportunist ic infections m a y therefore be increased i n the patient w i t h the H E L L P syndrome because o f the general ized immunosuppress ion and profound decrease i n monocyte phagocyt ic and bacter ic idal act ivi ty . 3.3 Pre-eclampsia and atherosclerosis T h e development o f pre-eclampsia predicts later cardiovascular morb id i t y and morta l i ty through atherosclerosis (94-98), and one o f the pathognomonic f indings i n the pre-eclampsia placenta is that o f acute atherosis (99). The arterial les ion observed i n pre-eclampsia w h i c h is characterized b y focal endothelial disruption, f ib r ino id necrosis o f the arterial w a l l , the inf i l t ra t ion o f per ivascular spaces b y mononuclear cells and endovascular accumula t ion o f l ip id - l aden macrophages. A c u t e atherosis shares w i t h atherosclerosis (Figure 1.5) s imi la r pathology, pathogenesis (wh ich includes in f lammat ion (13; 100)), c l i n i c a l settings i n w h i c h it occurs (endothelial ce l l damage (12;54)), and an association w i t h adverse long-term cardiovascular outcomes i n the mother (94). B o t h acute atherosis and atherosclerosis occur i n the presence o f hyper l ip idaemia (Figure 1.5) (especial ly the presence o f o x i d i z e d l ipoproteins and increased p lasma cholesterol (101-104)) and invo lve a role for monocytes/macrophages (105-109). C a n these s imilar i t ies provide us w i t h a clue to the pathogenesis o f pre-eclampsia? 15 There is an ep idemio log ica l l i n k between atherosclerosis and chronic infect ion and it is poss ible that such a l i n k exists between pre-eclampsia and chronic infect ion. C o u l d an infectious trigger exp la in the differential maternal response to the shared placental pa thology o f pre-eclampsia and normotensive intrauterine growth restriction? W e bel ieve that infect ion m a y be important i n the pathogenesis o f pre-eclampsia, both i n terms o f its in i t ia t ion (by increas ing the r isk o f acute uteroplacental atherosis) and/or its potentiation (by ampl i fy ing the maternal systemic inf lammatory response). 16 intimal deposition of lipoprotein • native / modified LP phospholipids cholesterol apoprotein B triglycerides lipid peroxides •albumin foam cell formation anionic phospholipids binding of Factors IXa, Xa activated protein C HYPERUProAEMIA lipids, lipoproteins, cholesterol, lipid peroxides prostacyclin basal lamina extracellular matrix 'secretory* phenotype Figure 1.5: Atherosclerosis and acute atherosis sis. (a) Atherosclerosis; (b) acute atherosis. Modified from Simionescu (1992). LP: lipoprotein; S T B M : syncytiotrophoblast microvillous membrane. 17 4. General purpose for research project W e attempted to investigate the relationship between a w o m a n ' s i m m u n e system and vu lnerab i l i ty to develop pre-eclampsia. Firs t , w e attempted to investigate whether or not a personal his tory o f pre-eclampsia increases the r isk for later S I R S . Th i s l i nked the postulated in f lammat ion resul t ing from defective or excessive innate i m m u n i t y that m a y occur i n both syndromes. T h i s in i t i a l study indicated that to test this hypothesis a very h igh number w o u l d be required, and is described i n Chapter T w o . Second, us ing a nested case-control design, w e have tested the hypothesis that infect ion m a y be important i n the pathogenesis o f pre-eclampsia, both i n terms o f its in i t ia t ion (by increasing the r i sk o f acute uteroplacental atherosis) and/or its potentiation (by ampl i fy ing the maternal systemic inf lammatory response) (21). T h e coro l la ry to that is that w o m e n whose pregnancies are affected sole ly b y normotensive I U G R w o u l d be less l i k e l y to have a pregnancy associated w i t h reactivation o f a chronic infect ion. T h i s project w i l l be described i n Chapter Three. 18 Chapter 2 Systemic Inflammatory Response Syndrome (SIRS) "The study ofproblems which fall on the borderline between diverse specialties ...affords unique opportunities to study the pathophysiology of disease ". -Soma Weis, 1941 19 Background: Pre-ec lampsia is a pregnancy-specif ic condi t ion, it is the p r inc ipa l hypertensive disorder o f human pregnancy (110). Death from pre-eclampsia most c o m m o n l y fo l lows the onset o f neutrophil-mediated compl ica t ions , hepatocellular necrosis and the acute respiratory distress syndrome ( A R D S ) (111). Hepatocel lu lar necrosis and A R D S are both features o f established systemic inf lammatory response syndrome ( S I R S ) (112). Pre-ec lampsia m a y persist, often deteriorating transiently, f o l l o w i n g the de l ive ry o f the placenta, m u c h as S I R S persists f o l l o w i n g the resolut ion o f trauma, burns or sepsis. In both condi t ions there is a systemic response w i t h inf lammatory mediator release, endothelial ce l l dysfunct ion, a hyperdynamic state, end organ hypoperfusion, and neutrophi l act ivat ion, resul t ing i n the c l i n i c a l syndromes. B o t h pre-eclampsia and S I R S result f rom differential responses to in i t ia t ing factors shared w i t h other c l i n i ca l outcomes. Pre-ec lampsia results f rom incomplete placentation, w h i c h also underlies the development o f intrauterine growth restrict ion i n i so la t ion (the fetal syndrome o f pre-ec lampsia wi thout the associated maternal syndrome). S i m i l a r l y , sepsis, t rauma and burns also occur i n patients w h o have unremarkable recoveries rather than deve lop ing S I R S . Is pre-ec lampsia a fo rm o f S I R S ? 2.1 Terminology and epidemiology of the systemic inflammatory response (SIRS) There is n o w general agreement that sepsis and the systemic inf lammatory response ( S I R S ) are accompanied b y the inab i l i ty to regulate the inf lammatory response. The systemic response to infect ion has been termed sepsis (57; 113). H u m a n sepsis is associated w i t h a general ized act ivat ion and systemic expression o f the host's in f lammatory pathways. T h i s 20 act ivat ion occurs v i a s t imulat ion o f the host immune effector cel ls that subsequently synthesize and release potent mediators o f ce l l in f lammat ion (56). Thus , it has been referred to as the systemic inf lammatory response syndrome (114). S I R S is the systemic inf lammatory response to variety o f severe c l i n i ca l insults (115), and it is be l ieved to be the f inal c o m m o n pathogenic l i n k between a number o f disorders (113). Sepsis has been defined as the presence o f var ious pathogenic organisms and their toxins i n the b l o o d or tissues (116). T h e causes o f the perturbations o f S I R S are s t i l l unknown . Despi te more than 2 0 years o f extensive research, sepsis and S I R S remain the ch i e f cause o f death i n the intensive care unit, w i t h mor ta l i ty rates between 30-70% (117). The incidence o f sepsis has increased 139% over the past ten years despite progression i n management and therapy for sepsis (118). T h e existence o f resistant microorganisms m a y contribute to the increased incidence o f sepsis as w e l l as increased numbers o f immuno-incompetent patients. Sepsis is an increas ingly c o m m o n cause o f morb id i ty and mortal i ty , par t icular ly i n elderly, immunocompromised , and c r i t i ca l ly i l l patients. It has been estimated to occur i n 1 % o f a l l hospi ta l ized patients, affecting more than 40,000 people i n Canada every year. There are approximate ly 751,000 cases (3.0 cases per 1,000 popula t ion and 2.26 cases per 100 hospi tal discharges) each year i n the Un i t ed States (119). The incidence o f sepsis and septic shock has not been clear because i n the past there was no un i fo rm term for def in i t ion o f sepsis and septic shock. T h i s also affects the statistical and ep idemio log ica l issues i n this matter (120). T o address some o f the above issues, the A m e r i c a n C o l l e g e o f Chest Physic ians and the Socie ty o f the C r i t i c a l Cafe M e d i c i n e proposed new defini t ions for sepsis ( T a b l e 2.1). T h e y recognized that sepsis was the systemic inf lammatory response to a documented infect ion, but acknowledged that this response also m a y be observed i n a number o f other c l i n i c a l condi t ions. T h e y conc luded that sepsis was a 21 con t inuum o f injury response, ranging from sepsis to septic shock, u l t imate ly leading to the mul t i -organ dysfunct ion syndrome ( M O D S ) (121). The end result o f the host response to infect ion m a y be the development o f diffuse endovascular injury, microvascula r thrombosis , organ i schemia , mul t iorgan dysfunction, and death (122) (Figure 2.1). Inflammatory T h r o m b o t i c Fibrinolytic R e s p o n s e R e s p o n s e R e s p o n s e to Infection to Infection to Infection Figure 2.1: The systemic inflammatory, procoagulant, and fibrinolytic host responses to infection: The inflammatory and procoagulant host responses to infection are intricately linked. Infectious agents and inflammatory cytokines such as tumour necrosis factor (alpha) (TNF-a) and interleukin-1 activate coagulation by stimulating the release of tissue factor from monocytes and the endothelium. The presentation of tissue factor leads to the formation of thrombin and a fibrin clot. Inflammatory cytokines and thrombin can both impair the endogenous fibrinolytic potential by stimulating the release of plasminogen-activator inhibitor 1 (PAI-1) from platelets and the endothelium. PAI-1 is a potent inhibitor of tissue plasminogen activator, the endogenous pathway for lysing a fibrin clot. In addition, the procoagulant thrombin is capable of stimulating multiple inflammatory pathways and further suppressing the endogenous fibrinolytic system by activating thrombin-activatable fibrinolysis inhibitor (TAFI). Endothelial injury results in decreased thrombomodulin levels. The end result of the host response to infection may be the development of diffuse endovascular injury, microvascular thrombosis, organ ischemia, multiorgan dysfunction, and death. Modified from Bernard et al. (122). 22 Table 2.1: Definition of SIRS, sepsis, septic shock, and M O D S . Modified from Balk A (120) A C C P / S C C M Consensus Conference Definitions of Sepsis, Severe Sepsis, and Septic Shock Systemic Inflammatory Response Syndrome (SIRS). The systemic inf lammatory response to a w i d e variety o f severe c l i n i ca l insults, manifested b y two or more o f the f o l l o w i n g condi t ions: 1. Temperature > 38 ° C or < 36 ° C 2. Heart rate > 90 beats/min 3. Respi ra tory rate > 20 breaths/min or P a C 0 2 < 33 m m H g 4. W h i t e b l o o d ce l l count > 12,000/mm3, < 4000 /mm3, > 1 0 % immature (band) forms. Sepsis. The systemic inf lammatory response to a documented infect ion. In associat ion w i t h infect ion, manifestations o f sepsis are the same as those prev ious ly defined for S I R S . It should be determined whether they are a direct systemic response to the presence o f an infectious process and represent an acute alteration f rom baseline i n the absence o f other k n o w n causes for such abnormalit ies. The c l in i ca l manifestations w o u l d inc lude two or more o f the f o l l o w i n g condit ions as a result o f a documented infect ion: 1. Temperature > 3 8 ° C or < 3 6 ° C 2. Heart rate > 90 beats/min 3. Respi ra tory rate > 20 breaths/min or PaC02 <32 m m H g 4. W h i t e b l o o d ce l l count > 12,000/ml, > 4000 /ml , or > 10% immature (band) forms. Severe Sepsis/SIRS. Sepsis ( S I R S ) associated w i t h organ dysfunction, hypoperfusion, or hypotension. Hypoper fus ion and perfusion abnormalit ies m a y include, but are not l imi t ed to, lact ic acidosis , o l igur ia , or an acute alteration i n mental status. Sepsis (SIRS)-Induced Hypotension. A systol ic b l o o d pressure <90 m m H g or a reduct ion o f > 40 m m H g f rom baseline i n the absence o f other causes for hypotension. Septic Shock/SIRS Shock. A subset o f severe sepsis ( S I R S ) and defined as sepsis (S IRS) - induced hypotens ion despite adequate f lu id resuscitation a long w i t h the presence o f perfusion abnormali t ies that m a y include, but are not l imi t ed to, lact ic acidosis , o l igur ia , or an acute alteration i n mental status. Patients rece iv ing inotropic or vasopressor agents m a y not longer be hypotensive b y the t ime they manifest hypoperfus ion abnormali t ies or organ dysfunction, yet they w o u l d s t i l l be considered to have septic ( S I R S ) shock. Multiple Organ Dysfunction Syndrome (MODS). Presences o f altered organ function i n an acutely i l l patient such that homeostasis cannot be maintained wi thout intervention. *From B o n e R C , B a l k R A , Cer ra F B , et al: A m e r i c a n Co l l ege o f Chest Phys ic ians / Socie ty o f C r i t i c a l Care M e d i c i n e Consensus Conference: Def in i t ions for sepsis and organ failure and guidel ines for the use o f innovat ive therapies i n sepsis. Chest 101: 1644-1655, 1992. 23 Based on the hypothesis that excessive levels o f in f lammat ion dur ing infect ion can mediate the organ dysfunct ion, the pathogenesis o f septic shock, and lethal i ty o f sepsis and septic shock, different therapies have been proposed (117). V a r i o u s ant i - inf lammatory agents have been used i n human c l i n i c a l trials back to 1963. S o m e o f those ant i - inf lammatory agents are: a h i g h doses cort icosteroid (123), and mediator-specif ic non-g lucocor t i co id anti-inf lammatory agents, interleukin-1 receptor antagonist ( I L - l r a ) (124), soluble tumour necrosis fac tor-a ( T N F - a ) receptors (125), a n t i - T N F - a antibodies (126), platelet-activating factor ( P A F ) antagonists (127;128), and arachidonic ac id metabolites (prostaglandin E l and thromboxane) (129; 130). Unfortunately, the his tory o f c l i n i c a l trials i n sepsis has conf i rmed that even extremely successful results form animal models o f sepsis cannot necessari ly be translated into the c l i n i c a l settings i n humans and, have been disappoint ing and fai led to show signif icant benef ic ia l effect o n outcome unt i l now. It seems i n the pathogenesis o f systemic inf lammatory response and septic shock, other important mechanisms are i n v o l v e d . 2.2 Pathogenesis of sepsis T h e mechanisms o f sepsis and septic shock start w i t h infect ion and/or endotoxin product ion (131) (Figure 2.2). E n d o t o x i n or products f rom bacterial cel ls activate the host defence system. D u r i n g the onset o f sepsis, the inf lammatory system becomes hyperact ive, i n v o l v i n g both ce l lu lar and humora l defence mechanisms (Figure 2.3) (117) H u m o r a l factors p lay an important role i n ini t ia t ing a systemic in f lammatory response and i n determining outcome (132). The pr imary effector organ for ce l l injury i n S I R S is the activated immunocy te , i nc lud ing neutrophils, monocytes , macrophages, and lymphocytes . In septic patients w i t h l ive r failure, Rosenb loom et al. (133) demonstrated that a l l c i rcu la t ing 24 leukocytes , i n c l u d i n g neutrophils, lymphocytes and monocytes , are activated and that the levels o f T N F - a , I L - 6 , I L - 1 , IL -8 are increased (TNF-oc and I L - 6 are increased i n severe pre-eclampsia) . Simul taneously , robust product ion o f acute phase proteins, such as C-react ive protein and humora l defence mechanisms such as the complement system are activated (117), resul t ing i n product ion o f pro- inf lammatory mediators, i nc lud ing C 5 a . It is poss ible that po lymorph i sms i n the genes encoding these mediators o f in f lammat ion c o u l d va ry between those patients w h o develop either S I R S and/or pre-eclampsia, and those w h o do not. T h e c i rcu la t ing i m m u n e effector ce l l act ivat ion was proport ional to mean c i rcu la t ing I L - 6 levels . These cel ls can induce organ system dysfunct ion across different vascular beds and at sites remote f rom the in i t ia t ing s t imul i . Endothel ia l and epithelial cel ls , as w e l l as neutrophils , macrophages and lymphocytes , produce powerful pro- inf lammatory mediators. In part icular, the neutrophi l is a central ce l lu lar defence type i n the media t ion o f the inf lammatory response. Its recruitment, act ivat ion, and cy to toxic capabi l i ty are essential aspects o f the body's ab i l i ty to ward o f f infect ion (56). C la s s i ca l ly , leukocyte act ivat ion is characterized b y loss o f the const i tut ively expressed ce l l surface protein L-se lec t in (as i n pre-eclampsia) . T h i s is necessary for the in i t i a l weak ce l l adhesion, f o l l owed b y an increasing d i sp lay o f both C D 1 l b and C D 1 8 , the P2 integrins essential for f i rm adhesion o f c i rcu la t ing neutrophils to endothelial cel ls , and C D 3 5 , a complement receptor (132). 25 increasing Inflammation High risk of infection Prophylactic antibiotics, immune stimulants In fec t ion SIRS plus infection Antibiotics, antitoxins Coagulation inhibitors, antioxidants Septic shock Antiinflammatory cytokines, inflammatory cytokine antagonists Figure 2.2: Scheme for preventing and treating sepsis. Before sepsis develops, augmentation of immunologic defense mechanisms offers the best opportunity for protection. However, if host defense mechanisms are impaired, brief administration of agents that inhibit proximal mediators of inflammation may help block multiple-organ failure. SIRS denotes systemic inflammatory response syndrome. Adapted from Bernard et a/.(134) Epithelial ceils Prtwnflaftti cytokines * > ehemokines . ROS. i jducw En.-.-* cnkm Excessive inflammatory response E E « c HYPERREACTIVE j IMMUNE RESPONSE! Vascular pormeabilityf Br a ?11 . f DiC Tachypnea Fever Leukocytosis Ta>:'w.".:"i i Peripheral resistance^ EtJema Tissue damage Organ failure Lsukocytoperaa Shutdown of phagocyte SuscopSPiltty toft* HYPOREACTIVE IMMUNE RESPONSE, IMMUNE PARALYSIS Dynamic time-course of the inflammatory response during sepsis Figure 2.3: Excessive inflammatory mediator production during sepsis. Various stimuli can cause activation of different cell types and serum proteins, as well as the coagulation and complement systems, leading to excessive production of pro-inflammatory cytokines and chemokines and upregulation of adhesion molecules on endothelial cells and polymorphonuclear leukocytes (PMNs). Monocytes, PMNs and other phagocytes release large amounts of granular enzymes and generate R O S in response to the original stimulus in the early (hyperreactive) phase of sepsis. As result of excessive pro-inflammatory mediator production, vascular permeability increases, tissue damage and organ failure occur and crucial innate immune functions become defective, resulting in increased susceptibility toward infection in the later (hyporeactive) phase of the immune response, often along with immune paralysis.DIC, disseminated intravascular coagulopathy. Modified from Riedemann N et al. (117). 26 A d h e s i o n and transmigration o f leukocytes across the endothelial ce l l layer is a mul t i step process i n v o l v i n g sequential interaction o f specific adhesion molecules on the endothelial surface w i t h counter receptors on the leukocyte surface. The first step is r o l l i n g o f the leukocyte o n the endothelial surface, the second step is act ivat ion o f the neutrophi l , w h i c h is achieved b y t r iggering molecules such as selectins, platelet act ivat ing factor, C 5 a , chemokines and other inf lammatory mediators. The third step is f i rm adhesion. The f inal step is t ransmigrat ion (Figure 2.4). C e l l - C e l l I n t e r a c t i o n s d u r i n g I n f l a m m a t i o n L y m e n o f P o s t - c a p i l l a r y V e n u l e mediated .<-' : r o l l i n g f ree (. H ) t r a n s -f i r m e n d o t h e l i a l a d h e s i o n m i g r a t i o n .Vasculap- EndotheHujn \ l g l f t Tissue Space^ r t C t t o n Figure.2.4: Sequential activation of normal circulating PMNs and their adherence to the vascular endothelium. The first step is constituted by the activation of a constitutively expressed L-selectin leading to the initial weak cell adhesion with endothelial cellular adhesion modelules (ICAM-1) in the post-capillary venule. Secondarily, there is an activation of the b2 integrins, including CD11b/CD18, leading to a firm adhesion. Activated PMNs can now release their toxic reactive species into the micro-environment between the two cells, causing highly localized damage and microbe killing. Finally, if chemokines have established a concentration gradient, subsequent transendothelial migration of PMNs into the interstitium occurs. Modified from Adrie etal. (132). 2.3 Pre-eclampsia and the systemic inflammatory response syndrome compared A s stated i n the Genera l Introduction (Chapter 1), S I R S shares many c l i n i ca l and laboratory characteristics w i t h pre-eclampsia, i nc lud ing a hyperdynamic state, leukocytosis 27 and organ dysfunct ion. W h i l e discussed br ief ly i n the Introduction to this thesis, I w i l l n o w describe the s imilar i t ies i n greater detail ( T a b l e 2.2). Table 2. 2: Pre-eclampsia and S IRS share clinical and laboratory features. Clinical Finding Laboratory Finding Acute respiratory distress syndrome Hepatic necrosis Disseminated intravascular coagulation Microvascular thrombosis and haemolysis Acute renal failure Cardiomopathy Hyperdynamic state End organ hypoperfusion t vascular permeability Differential response to initiating agent (placenta/ trauma/infection) Persistence following removal of initiating agent Endothelial function and inflammation Tplasma T N F - a tplasma IL-6 TPAI-l:PAI-2 Tthromboxane: prostacyclin ratio T endothelin t caerulopasmin t a / - Antitrypsin Complement Activition •I plasma transferrin Hypoalbuminaemia i activated protein C Neutrophil activation Neutrophilia and delayed neutrophil apoptosis t plasma neutrophil elastase t neutrophil CD1 lb expression T basal intracellular ionized calcium t oxidative stress Table 2.2: Pre-eclampsia and systemic inflammatory response syndrome share clinical and laboratory features, [up arrow]: increased compared with normal; TNF: tumour necrosis factor; IL: interleukin; PAI: plasminogen activator inhibitor; [down arrow]: decreased compared with normal. Modified from von Dadelszen et al (20). A t first glance, the statement that pre-eclampsia resembles S I R S is counter-intuit ive, as S I R S is considered to be 'characterized' b y hypotension, and pfe-eclampsia 'characterized' b y hypertension. H o w e v e r , hypotension is not an obl igatory feature o f S I R S , but o f severe sepsis, septic shock or the mul t ip le organ dysfunct ion syndrome ( M O D S ) ; 9 6 % o f patients 28 w h o fu l f i l the diagnost ic cri teria for S I R S do not require posi t ive inotrope support (57). A l s o , pre-eclampsia can occur i n the absence o f hypertension, 2 1 % o f w o m e n h a v i n g no documented hypertension pr ior to their first eclamptic seizure (135). A d d i t i o n a l l y , pre-ec lampsia can also be compl ica ted b y M O D S , a hypotensive disorder. In the Col labora t ive E c l a m p s i a T r i a l , m a n y o f the 456 w o m e n who either d ied or suffered serious morb id i t y related to ec lampsia had one or more findings consistent w i t h M O D S : pu lmonary oedema (40 women) , renal failure (125 women) , l ive r failure (43 women) , and coagulopathy (213 women) (136). 2.3.1 C l i n i c a l W h a t remains clear is the s imi la r i ty between the two c l i n i ca l entities. F o r example, S I R S can be characterized b y hepatic failure and A R D S , w h i c h , as was stated, are both neutrophi l -mediated and the most c o m m o n causes for maternal morta l i ty i n w o m e n w i t h pre-eclampsia (3-5). B o t h disorders persist f o l l o w i n g the remova l o f the in i t ia t ing agent, w h i c h is poor placentat ion (pre-eclampsia) or gram negative sepsis ( S I R S ) , but do not occur un i fo rmly i n the presence o f that respective in i t ia t ing agent. Disordered act ivat ion o f the c lot t ing cascade occurs i n both syndromes, result ing i n disseminated intravascular coagula t ion and microvascu la r thrombosis and haemolysis i n pre-eclampsia (111) and S I R S (56; 112; 133; 137). R e n a l failure can compl ica te both syndromes (111;112; 137), a l though proteinuria is not seen characteris t ical ly i n S I R S . In addit ion, cardiomyopathy m a y complicate both pre-eclampsia (138) and S I R S (139). Increased vascular permeabil i ty , most c lear ly presenting as oedema, is prominent i n both condit ions (111;112;137), al though it m a y be dif f icul t to differentiate from the phys io log i ca l oedema o f pregnancy i n pre-eclampsia, increased vascular permeabi l i ty is related to altered endothelial ce l l function (140). 29 2.3.2 Endothelial dysfunction and inflammatory changes A s i n pre-eclampsia, S I R S is characterized b y systemic endothelial dysfunct ion, w h i c h m a y persist l o n g after resolut ion o f the inc i t ing event. Examples o f this dysfunct ion are the elevated concentration o f factor VHI- re l a t ed antigen (141), p lasminogen activator inhibi tor-1 (56;112; 137), and endothel in (142), as w e l l as an increased thromboxane: p ros tacyc l in ratio, a l l o f w h i c h also occur i n pre-eclampsia (143-145). Endothe l ia l ce l l act ivat ion and dysfunct ion are felt to underl ie the coagulopathy characteristic o f both syndromes. M a n y c l i n i c a l manifestations o f pre-eclampsia and S I R S are related to end-organ hypoperfusion, secondary to endothelial ce l l - induced vasoconstr ic t ion and microvasc luar coagulat ion (12;56). That pre-eclampsia is a form o f systemic in f lammat ion is supported b y the changes i n the acute phase reactants, caeruloplasmin (146), a 1-antitrypsin (147), complement (148), transferrin (149;150) and a lbumin (151) concentrations that paral lel changes noted i n S I R S (56;112;137;152-154). The p lasma concentrations o f the inf lammatory cytokines , I L - 6 and T N F - a , are increased i n both pre-eclampsia (155-157) and S I R S (56;112;137). TNF-cc i n v o k i n g a poss ible role for peripheral b l o o d monocytes i n the development o f both c l i n i c a l syndromes. A s stated, i n pre-eclampsia, it has been postulated that peripheral b l o o d neutrophils m a y prov ide an important l i n k between the poo r ly perfused in tervi l lous space o f the pre-eclampsia placenta and the endothel ium (145). 2.3.3 Neutrophil activation The current m o d e l for the development o f organ dysfunct ion i n patients w i t h S I R S includes a central ro le for activated neutrophils (Table 2.2). In S I R S , it is felt that s t imul i , 30 such as bacterial endotoxin, injured tissue and ischaemia, result i n disseminated act ivat ion o f the innate i m m u n e system. S I R S and its sequelae, result f rom the act ion o f host-derived mediators o n host tissues. Measures o f neutrophil act ivat ion present i n both pre-eclampsia and S I R S inc lude increased plasma concentration o f neutrophi l elastase (36;56), increased oxida t ive stress (158-161), increased surface expression o f C D 1 l b (49;50;162), increased concentrat ion o f basal intracel lular free-ionized ca l c ium (163;164), and, i n v i t ro , increased chemoattractant-induced neutrophil superoxide product ion (165; 166). N e u t r o p h i l i a is a prominent component o f normal pregnancy, pre-eclampsia and S I R S . In normal pregnancy spontaneous neutrophil apoptosis is s ignif icant ly retarded w i t h respect to nonpregnant values, that explains the neutrophi l ia o f normal pregnancy (19). The delay i n spontaneous neutrophil apoptosis is s ignif icant ly greater i n neutrophils f rom w o m e n w i t h pre-eclampsia than i n neutrophils f rom w o m e n w i t h normal pregnancies, and i n normotensive I U G R (19). Pre-ec lampsia is associated w i t h both a relative neutrophi l ia w h e n compared w i t h normal pregnancy and greater delays i n spontaneous neutrophil apoptosis than i n either normal pregnancy or normotensive I U G R (19). D e l a y e d spontaneous neutrophi l apoptosis m a y exp la in the neutrophi l ia o f both pre-eclampsia (19) and S I R S (162). T h i s delay i n neutrophi l apoptosis m a y expla in , i n part, the persistence o f the c l i n i ca l syndrome, be it either pre-eclampsia (111) or S I R S (112; 137), f o l l o w i n g e l imina t ion o f the in i t ia t ing agent, be that a placental s t imulus i n pre-eclampsia or either an infectious or traumatic insul t i n S I R S . Therefore, i n both pre-eclampsia and S I R S , there is a transient, usua l ly loca l i zed , i nc i t ing s t imulus, leading to a differential systemic response w i t h inf lammatory mediator release, endothelial ce l l dysfunct ion and neutrophil act ivat ion, resul t ing i n the phenotypic organ dysfunct ion o f the c l i n i c a l syndrome. A s trauma, burns and bacteria are to neutrophils i n S I R S , so m a y placental debris be to neutrophils i n pre-eclampsia. H o w e v e r , the c l i n i c a l and 31 laboratory pictures o f both pre-eclampsia and S I R S are further compl ica ted b y the int r ins ic in te r - indiv idual differences i n neutrophil response to the same in i t ia t ing pathology. 2.4.1 Hypothesis A s it is be l i eved that the maternal syndrome o f pre-eclampsia is a fo rm o f the S I R S ; w e have postulated that a his tory o f pre-eclampsia w i l l predispose w o m e n to the later development o f S I R S , as has already been established for atherosclerotic disease (see f o l l o w i n g chapters) (95;96). 2.4.2 Objectives T h e p r imary objective o f this prospective study was to determine whether pre-eclampsia is associated w i t h increased r isk o f S I R S later i n l i fe , as it is possible that there are innate variat ions i n neutrophil function that determine whether or not a w o m a n is more l i k e l y to develop pre-eclampsia that w o u l d predispose that w o m a n to S I R S f o l l o w i n g sufficient pro-inf lammatory provocat ion . The second objective was to determine whether specif ic po lymorph i sms i n gene cod ing are more prevalent i n patients w i t h SIRS/pre-ec lampsia . Pro- inf lammatory cytokines , i n c l u d i n g T N F - a , I L - 6 and I L - 1 , and the endotoxin receptor ( C D - 1 4 ) , are be l i eved to p lay a central ro le i n the in i t ia t ion and ampl i f ica t ion o f both S I R S and pre-eclampsia (36;56;167). P o l y m o r p h i s m s i n the genes cod ing for these and other inf lammatory mediators have been associated w i t h a poor outcome (168). Es tab l i sh ing association between these po lymorph i sms and the c l i n i c a l syndrome requires demonstration o f increased leve l o f the mediators that they code for. 2.5 Methods T h i s was a prospect ive case-control study. 32 2.5.1 Cases and controls Cases were w o m e n admitted to the intensive care unit ( I C U ) o f St. P a u l ' s Hosp i t a l w i t h the diagnosis o f S I R S . The w o m e n w i t h S I R S were identif ied b y a Research Assoc ia te and the student. A l l e l ig ib le subjects (or their p r o x y dec is ion maker where the w o m a n herse l f was obtunded) were approached to obtain informed consent. Cont ro ls were w o m e n without S I R S , admitted to general medica l / surg ica l wards w i t h the same p r imary diagnosis and same under ly ing p rob lem (eg: pneumonia , b o w e l resection) but unremarkable in-hospi ta l course (NO S I R S ) . T h e controls were matched for both age (+5 years) and ethnici ty. Informed consent was obtained from the patients b y the student. 2.6 Data collection Eth ics approval was obtained f rom the U n i v e r s i t y o f B r i t i s h C o l u m b i a . A detailed obstetric history was taken that inc luded: numbers o f pregnancies, year(s) o f pregnancy (ies), gestational age(s) at del ivery, b i r th weight(s), gender(s), and his tory o f ' t oxaemia ' o f pregnancy (hypertension, proteinuria), o r miscarr iage. T h e obstetric h is tory was taken either f rom the patient, the fami ly , or f rom the med ica l chart b y the student. In the absence o f an adequate obstetric his tory be ing obtained from either the patient or her next -of -k in , the patient 's general practi t ioner was contacted b y telephone i n an attempt to rev iew the w o m a n ' s obstetric history. 33 2.7 Results In a per iod o f s ix months, w e recruited o n l y s ix cases and two controls inc lud ing : Cases: 39yo pneumonia , respiratory distress 54yo divert icular abscess, b o w e l infect ion 75yo perforated duodenal u lcer and col i t i s 22yo pneumonia and respiratory distress 66yo pneumonia , diabetic ketoacidosis , urosepsis 70yo left lower lobe pneumonia and thoractomy Controls: 72yo pneumonia and pu lmonary oedema 58yo pneumonia N u m b e r o f S I R S w o m e n patients admitted to I C U at our study per iod was 25 patients. 2.7.1 Limitations Most women in the ICU were too critically ill to be interviewed, or when able to answer questions, were too confused to be able to remember details of their obstetric history. T w o o f the w o m e n were immigrants f rom the developing w o r l d without clear diagnoses, despite histories suggestive o f pre-eclampsia; both came f rom environments i n w h i c h the costs o f accessing m e d i c a l care were prohibi t ive . W h e n contacted, none o f these w o m e n ' s general practit ioners had med ica l records dat ing back to their obstetric history. Therefore, this study was deemed to be not feasible and was abandoned because o f the l o w frequency o f female admissions to the St Pau l ' s I C U , the insufficient obstetric in format ion avai lable i n the I C U setting, the poor qual i ty data, and the fact that it was not 34 poss ible to go back to general practit ioner charts as most w o m e n had del ivered outside B r i t i s h C o l u m b i a . 2.8 Discussion Clinical and Research Challenges T h i s study was abandoned i n response to the reali ty o f obta in ing accurate obstetric histories i n the intensive care unit setting. These w o m e n were either too u n w e l l , too confused, or came f rom an environment i n w h i c h accurate obstetric diagnoses were not made, to be able to p rov ide accurate data. In addit ion, their next-of-kin, generally husbands or chi ldren , were not aware o f speci f ic diagnoses that were made. These points argue for the population-based approach used i n Scand inav ia and Scot land, i n w h i c h maternal-infant databases can be l inked w i t h general hospi tal databases to provide useful l o n g term data (95;96). Therefore, w e were not able to test the hypothesis that the maternal syndrome o f pre-ec lampsia is a fo rm o f systemic inf lammat ion , us ing this experimental design. T h i s was a useful experience for a scientist-in-training, as I ident i f ied some o f the challenges i n v o l v e d i n data co l lec t ion , par t icular ly prospective data co l l ec t ion rather than chart r ev iew. E s p e c i a l l y important was the p lan to launch a p i lo t study in i t i a l l y to determine the feasibi l i ty o f the study design, and our dec is ion to abandon this study once it was c lear ly not feasible. Instead, the dec i s ion was made to pursue a related l ine o f enquiry, that be ing the relat ionship between chronic infect ion and pre-eclamspia. That study w i l l be the topic o f the next chapter o f this thesis. 35 CHAPTER 3 A nested case-control seroepidemiological study of possible triggers of the maternal syndrome of pre-eclampsia 36 3.1. Preamble: A s stated i n the Introduction to this thesis (Chapter One) , it is plausible that the maternal syndrome o f pre-eclampsia m a y be triggered b y reactivated chronic infect ion o f those agents l i nked to the development o f later atherosclerosis (21). These agents are C M V and Chi pneumoniae. W e used three different experimental methods i n this study. First , we l i n k e d two databases to ident ify cases and controls. Second, w e undertook C M V serology. T h i r d , w e performed Chi pneumoniae serology. 3.1.1 B a c k g r o u n d The evidence for a causal l i n k between maternal infect ion and pre-eclampsia is incomplete at best. Th i s body o f evidence is summarized i n Chapter One . O f the p laus ible infectious triggers o f the maternal syndrome o f pre-eclampsia, we feel that C M V and Chi pneumoniae are o f part icular interest because o f their putative role(s) i n atherogenesis. C M V - r e l a t e d chronic v i l l i t i s ( typified b y an inf lammatory infiltrate dominated b y maternal leukocytes w i t h i n fetal chor ionic v i l l i ) is associated w i t h both pre-eclampsia (169), normotensive I U G R (170), and st i l lbir th (21). Placental C M V D N A can be identif ied i n the setting o f chronic v i l l i t i s and intrauterine fetal death, without immunohi s tochemica l evidence o f infec t ion (171). In addi t ion, w o m e n suffering f rom recurrent spontaneous abort ion have a marked ly impai red lymphocy te proliferat ive response to C M V (172). Recurrent spontaneous abort ion is probably part o f the spectrum o f disease that includes pre-eclampsia (18). In pregnant w o m a n , diagnosis o f C M V infect ion is , most o f the t ime, performed indi rec t ly b y detecting C M V antibodies. Detect ion o f o f p r imary infect ion is based on the observat ion o f a seroconvert ion or the detection o f both I g G and I g M w h i c h is rarely observed, and the presence o f I g M antibody is sometime very hard to interpret (173). 37 A c c u m u l a t i n g evidence suggests that infect ion b y Chi pneumoniae m a y be i n v o l v e d i n the endothelial in jury that marks atherosclerosis. Chi pneumoniae m a y contribute to atherosclerosis b y b r ing ing monocytes to areas o f arterial damage to form foam cel ls , leading to plaque, and thrombosis (174). S i m i l a r l y , past, persistent, or recurrent infect ion w i t h Chi pneumoniae m a y contribute to abnormal vascular function, atherosis, and the abnormal placental perfusion that marks the first stage o f pre-eclampsia. The I g G seroprevalence to C pneumoniae was more c o m m o n among w o m e n w i t h pre-eclampsia than among w o m e n w i t h unaffected term pregnancies (85). There is no association to pre-eclampsia for I g G seroprevalence to C trachomatis or Cpsittaci suggests a specif ic associat ion between pre-ec lampsia and the part icular species o f Chlamydia that has been associated w i t h atherosclerosis. A n associat ion to pre-eclampsia- was not found for I g A or I g M seroprevalence to C pneumoniae. B o t h o f these f indings suggest that it is past, persistent, or chronic act ive infect ion and not acute infect ion or reinfect ion w i t h Cpneumoniae that is associated w i t h pre-eclampsia. A c u t e infect ion or reinfect ion w o u l d cause an elevation i n I g M titers (85). In animal models , the induc t ion o f atherosclerosis has required persistent or chronic active infect ion, rather than a single past exposure to C pneumoniae. Repeated respiratory infect ion w i t h C pneumoniae, and not a s ingle, acute infect ion, is needed to promote the growth o f atherosclerotic plaque i n rabbits. A n t i b i o t i c treatment can prevent or reduce atherosclerotic changes i n animals be ing reinfected w i t h Cpneumoniae (175). 38 3.2. Methods 3.2.1 Research design The study was a nested case-control study o f prospect ively col lected serum (P rov inc i a l maternal rube l la status screening programme). T h e primary outcome was the leve l o f maternal i m m u n o g l o b u l i n G (IgG) antibodies (persistent / reactivation o f infection) against C M V and Chi pneumoniae i n maternal serum. The nested case-control study is an efficient ep idemio log ica l des ign whereby a case-control approach is employed w i t h i n an established cohort. It is a solu t ion to the p rob lem o f the smal l sample sizes; it starts w i t h a group o f people w i t h the disease, the cases, and compar ing them to a second group without the disease, the controls. In this case, both groups were s imi la r w i t h the respect o f the t ime o f the del ivery . 3.2.2 Identification of cases and controls Subjects were identif ied from the maternal database at the Chi ldren ' s and W o m e n ' s Hea l th Centre o f B r i t i s h C o l u m b i a ( C W H C B C ) . Diagnoses were conf i rmed by hand search o f patient files, us ing the criteria established b y the Na t iona l H i g h B l o o d Pressure Educa t ion P rog ram ( N H P B E P ) i n the U S A (2000) (see Sect ion 3.4). There was no direct patient contact. Research data were un l inked f rom patient ident i fy ing data b y anonymous u n l i n k e d seroprevalence methods, this method hav ing been used w i t h s imi la r sampl ing techniques i n B r i t i s h C o l u m b i a i n recent years (176) and hav ing proven acceptable under the laws o f the Prov ince . E th ics approval was granted b y both the U n i v e r s i t y o f B r i t i s h C o l u m b i a and the C h i l d r e n ' s and W o m e n ' s Hea l th Centre o f B C Eth ics Commit tees . T h e cases and controls were identif ied from the I C D 9 codes i n Hea l th Records , B C W o m e n ' s Hosp i t a l , and each chart was manua l ly rev iewed to determine match ing for 39 select ion cri teria, the relevant data abstracted, and identifiers forwarded to U B C C D C . D u e to c o d i n g delays (12 months) and the t ime between rube l la serology (usual ly 12-16 weeks ' gestation) and del ivery , o n l y a three month per iod was avai lable for study. A t the U B C C D C , the serum was identif ied from the banked rubel la serum, and a l l analyses were performed u n l i n k e d to ident i fy ing maternal data. Labora tory staff personnel were b l inded to pregnancy outcome diagnoses. 3.2.2.1 Cases and controls T h e cases were w o m e n w i t h early-onset pre-eclampsia, late-onset pre-eclampsia , and normotensive ( I U G R ) , and up to two matched controls per case, (i) w o m e n w i t h early-onset pre-eclampsia (meeting the N H B P E P cri teria (Sect ion 2.4, below))(177) w i t h c l i n i c a l onset pr ior to 34 weeks ' gestation, ( i i ) w o m e n w i t h late-onset pre-eclampsia (same criteria), w i t h the disease onset at >34+0 weeks ' gestation, and ( i i i ) w o m e n w i t h normotensive I U G R (defined as a b i r th weight < 3 r d centile for gestational age and gender). The controls were w o m e n w i t h no rma l pregnancy outcomes matched for maternal age ( ± 5 y ) and pari ty (0, 1, >2). T h e d i v i s i o n o f pre-eclampsia samples into two groups is designed to discr iminate between those w o m e n whose disease is more homogeneous and c l i n i c a l l y significant (early-onset pre-eclampsia) f rom those whose disease m a y be a phys io log ica l variant at term i n p r i m i g r a v i d pregnancies (late-onset pre-eclampsia)( l 78). 3.2.2.2 Definitions 1. Pre-eclampsia: was defined b y the N H B P E P criteria, namely: a pregnancy-specif ic increase i n b l o o d pressure ( d B P > 9 0 m m H g , or s B P > 1 4 0 m m H g ) associated w i t h >0.3g proteinuria/d or >2+ dips t ick proteinuria. T w o or more episodes o f d ips t ick proteinuria ' + ' was accepted i n the presence o f other evidence o f pre-eclampsia (e.g. hyperur icaemia) . 40 2. Normotensive IUGR: was defined as a bir th weight < 3 r d centile for gestational age and gender, f o l l o w i n g pregnancies compl ica ted b y neither hypertension nor proteinuria. 3. Normal pregnancies: were defined as pregnancies associated w i t h a n o r m a l l y g r o w n fetus, de l ive r ing at term (37-42 weeks), without evidence o f either pre-eclampsia or gestational diabetes. These w o m e n had no his tory o f major significant med ica l problems that predated the pregnancy (e.g. systemic lupus erythematosis, chronic hypertension, and renal disease). T h e M e d i c a l Records cod ing o f cases and controls is described o n (Table 3.1). M a t c h e d controls were age-matched (wi th in + 5 years) and pari ty-matched (0, 1, >2). C o n t r o l patients were w o m e n w i t h normal pregnancy who had undergone rube l la serology testing close to the t ime o f their appropriate case. Th i s was designed to reduce confounding due to both the pregnant state and seasonal variat ion i n infectious diseases. O n l y s ingleton pregnancies were examined, and cases related to fetal hydrops o r gestational trophoblast diseases were excluded. 3.2.3 Serum bank Cases and controls serum were identif ied from the Un ive r s i t y o f B r i t i s h C o l u m b i a Centre for Disease C o n t r o l ( U B C C D C ) serum bank o f rubel la serology. T h i s contains 9 0 % o f a l l rube l la tests performed i n the Prov ince o f B r i t i s h C o l u m b i a , a l l samples be ing kept for 24 months. 41 Table 3.1: The field name and definitions as described by Health Records at CWHCBC. Field Name Definition chart jocatn Chart Location. Records Processing Dept. will need this info to pull charts women with pre-eclampsia (ICD9-CM pre-eclampsia codes used: *642.4, *642.5, *642.6, *642.7) M# Mother's unit# Term_dig Terminal digit of mother's unit#, required for record pulling Disch Mother's discharge date Age Mother's age Dob Mother's date of birth Name Mother's name Phn PHN# (PHN# = 10000000000 or 00000000000 means the PHN# was unavailable Deliv_date Delivery date Deliv_hr Baby's delivery hour. Delivery date/hour used to find control patients Deliv_type C /S , Vag Nullip Y, N Parity # previous deliveries G a gestational age at time of delivery. G A of clinical onset of condition is not captured in Med2020 or B C R C P , therefore G A at delivery was used. Smoker_code Current, Former, Never, Blank = not documented on chart and can be interpreted as "never". This field is abstracted from the Antenatal Record. 64271 Present "Yes" means that the ICD10 code "642.71= Pre-eclampsia or eclampsia superimposed on pre-existing hypertension" was present. "No" means that the code was not present. This field is present only for the pre-eclampsia <34 weeks and >=34 weeks data. M# Mother's unit# repeated so print-out of baby data can be matched with maternal data. B_serv Baby's patient service. NB = Newborn S B = Stillborn B# Baby's unit# b-dob Baby's date of birth B_discharge Newborn's discharge date from B C W H . Baby may have been transferred to B C C H or another hosp, and that discharge date has not been provided. Sex M=Male, F=Female Wt Newborn weight at delivery. 3%ile_wt 3 r a percentile birthweight vs G A vs Gender. This field is only present on the Normotensive w IUGR data. Excluded cases: - congenital infection due to toxoplasmosis, rubella, cytomegalovirus - any chromosomal abnormality involved - hydatidiform mole - fetal hydrops or gestational trophoblast disease - multiple pregnancies 42 3.2.4 Data Collection The data extracted retrospectively from the w o m e n ' s charts and, i f required, the charts o f infants admitted to a nursery i n the C W H C B C , were entered into an A c c e s s ™ database, i n c l u d i n g maternal variables and outcomes (see Sect ion 3.2.4.2), newborn variables and outcomes (see Sec t ion 3.2.4.4), and B C C D C laboratory results. The laboratory data entered f rom the day o f admiss ion , for the day o f de l ivery unt i l the day o f discharge for the purpose o f the analyses. 3.2.4.1 Maternal Variables D a t a o n the f o l l o w i n g maternal variables were col lected b y chart r ev iew: maternal age, gestational age o n admiss ion , pregnancy weight , pre-pregnancy b o d y mass index ( B M I ) ( K g / m 2 ) , weight gain dur ing pregnancy , b l o o d pressure ( s B P and d B P ) , total leukocyte count, mean arterial pressure, proteinuria (24h urine, d ips t ick proteinuria), creatinine, ur inary output, b i l i r u b i n , u r i c acid , aspartate aminotransferase ( A S T ) , lactate dehydrogenase ( L D H ) , b i l i r u b i n , a lbumin , platelet count, mean platelet v o l u m e ( M P V ) (Figure 3.1). 3.2.4.2 Maternal Outcomes W e developed, b y D e l p h i c consensus, a combined adverse maternal outcome. T h e D e l p h i Consensus technique is consensus method used to determine the extent o f agreement o n an issue. The technique involves ask ing a panel o f experts to take part i n a series o f rounds to identify, c lar i fy , refine and finally to gain consensus on the part icular issue (179). E a c h D e l p h i round compr ised a questionnaire, an analysis, and a feedback report. It is concerned w i t h de r iv ing quantitative estimates through quali tat ive approaches. T h e consensus process begins w i t h a predetermined objective i n m i n d , and consensus is reached w h e n objections to that predetermined pos i t ion have been quieted. In this case, the D e l p h i c consensus inc luded 43 15 internal medic ine , c r i t ica l medic ine , maternal-fetal medic ine , and paediatric intensive care med ic ine specialists f rom Canada, the U K , the U S A , Aus t r a l i a and N e w Zealand . The maternal adverse outcome consis t ing of: mortal i ty , hepatic failure, hepatic haematoma/rupture, G l a s g o w coma scale ( G C S ) <13, stroke, seizures, cor t ica l bl indness, renal d ia lys is , renal transplantation, posi t ive inotropic support, infus ion o f 3rd antihypertensive, myoca rd i a l infarct ion, >50% 0 2 > 1 hour, intubation and transfusion o f > 1 0 U i n total o f any combina t ion o f b l o o d or b lood products (Figure 3.2). T h i s combined adverse outcome has received general support at scientif ic meetings since its der ivat ion b y our research group, i n col laborat ion w i t h the same Canadian , B r i t i s h , A m e r i c a n , and Aus t ra las ian experts. 3.2.4.3 Fetal Variables W h e r e avai lable, w e entered the f o l l o w i n g ultrasound data into the database: amniot ic f lu id index ( A F I ) as percentile for gestational age and the ultrasound estimated fetal weight ( E F W ) as percenti le for gestational age into the database. T h e data were present for most cases and for o n l y a mino r i t y o f controls, as routine ultrasound evaluat ion o f ' n o r m a l ' pregnancies is not associated w i t h improved pregnancy outcomes (Cochrane rev iew) . S i m i l a r l y , placental pathology was available for o n l y a mino r i t y o f controls, as routine placental evaluat ion is not recommended i n the absence o f c l i n i c a l indicat ions ( A m C o l l e g e o f Pathologists) . 3.2.4.4 Fetal Outcomes W e also developed a combined adverse prenatal outcome, us ing the same D e l p h i c consensus. Included i n the definit ions were: s t i l lbir th , neonatal death, perinatal mortal i ty , intraventricular haemorrhage ( I V H , grade 3 or 4) , retinopathy o f prematuri ty ( R O P ) , cys t ic 44 periventr icular l eukomalac ia ( c P V L ) , bronchopulmonary dysplasia ( B P D ) and necrot is ing enterocolit is ( N E C ) (Figure 3.3). Patient ID [MRNJ] location f"~ Lastneme Fret Name PHN P | StudylD P ! DQA ' Mother G T P SA TA Pas? obstetric Httiw . POYIOUC Pfe-ecl«<rei zl •^pregnancy diabetes, j zi HSA | A' ; Personal Histafjr, I Smoking j zl Obesity zi . DVT/PE | New Partner j zi Cocaine j zi zi • Renal rjseawt zi Ofhef J : IHD/oth«he«tdwas8exceotMVP Fomtjy history of Preedampsia rJUOR K©«rt dt«a« "3 DVT/PE j j Hvoertension Othm I " 3 "3 Prepregnancywerghi j height i GestasePET/nlUGfl Gejl age @ tetrrration j j Present pregnancy Weight] ~Z3 height lieett | height 6nchet)( -7J Weeks Days I Record. " l < i f ^ Treatment wth. Si«(ddi fori«sl kjruj mat Anffln-pet-tawtves Steroids for H E L L P n^dtome MgS04 Other j ' mi &;i Labs : Form 40 Uiine output fml/H) j 174 Total Bilirubin (uM MRN: ^ Test data | 21-Feb-OO Gestational age (WksVdays) WBC Icefc/rrl) BP Sy5tofc(mmHg) BP Diastolic (mmHgJj TT? AST (u/L) SaDEpJejRA | LDH (u/L) D of Secures Afaman (oA| UttcacidluM) I 605 Platelets (exp 9/L| Creatinine (uM i Unnary albumin cyD Dip stick protein S3 MFVIJU (sVU Study ID Ul Postpartum days AH Xile for GA (0-1ODT |unknown _-J EFW Xte tat GA (0-10021 (unknot Diastolic How I EXCEPTIONS: I Record; I<1 • 1 f 1 > 1>H>*I of M7 Figure 3.1: Maternal variables and laboratory information. 45 Figure 3.2: Maternal outcome Neonatal Outcome Form HwiomiDJMBNjj ] StudylO Date 14-Feb-Q Location T , E O P E T t j j LOPETItf : ntUGB* f Control toKROPETH 1 nlUGFffl f 1 norvtnegrancji 8 Moths'! MM | Fotm lor jangjetortotbaby^jj Baby's DOB f ~ Sender [Fafiute""jJ weight {gn») | Ratinopathy • CysfePVL ROS 8ioncr>o pumori-Sy d .^3i3Cl2 j u f ^ c w ^ J .[ NEC Admission to neonatal fCU It of days Date 'tii'-bd$ d/c (I aprJcabie) Uve8«th Jye* 3 jYes.DQD i Yes cause SfilBtrth no Neona»d death f no "3 Perinatei rnoit*j (20/40-2M) (no BW Xie tat gender /GA § 3 i r "3 Ge^afionalageaf the time of de&vwy {forpierfiaturityl I Record: n| • if 1 • i " M of 14 Figure 3.3: Fetal outcome 46 3.2.5 Laboratory techniques A nested case-control study o f serum from a population-based bank was performed. Seroprevalence and levels o f a n t i - C M V and Chi pneumoniae I g G were compared between groups. Genera l ly , these samples were taken at 12-20 weeks o f pregnancy as part o f routine c l i n i c a l care. Per forming rubel la and syphi l i s serology (+ H I V and/or hepatitis B ) , a long w i t h b l o o d group t yp ing and a l lo immuniza t ion antibody screening, is routine at this t ime. Samples are saved so that serology can be rev iewed at a later stage should c l i n i c a l suspic ion o f congenital infect ion arise dur ing the index pregnancy or w i t h i n one o f the expected data o f de l ivery . T h e specimens, frozen at - 2 0 ° C were thawed. A n aliquot o f each specimen was separated f rom the o r ig ina l serum and de l inked from ident i fying data after the baseline demographic data were recorded. Case-control pairs or trios were assayed i n batches to m i n i m i s e inter-assay var iab i l i ty . 3.2.6 Methods for cytomegalovirus using unlinked anonymous seroprevalence 3.2.6.1 Sample selection Stored specimens col lected dur ing 2000-2001 for cases and controls were used. A n al iquot o f each specimen was separated from the or ig ina l sera and the f o l l o w i n g were recorded: A g e at t ime the b l o o d was drawn C i t y or t o w n o f residence N e w study specimen number (not l i nked to previous specimen). N o unique ident i fy ing data were recorded for study purposes. 47 A n t i - C M V I g G serology was performed on a V I D A S analyser ( B i o M e r i e u x , St Laurent, Quebec) , accord ing to manufacturer 's instructions. It is an easy, rapid , and reproducible test. It was evaluated i n a mult icentre study to differentiate between p r imary C M V infections and past infections or reactivations (173). In immunocompetent patients, and especia l ly i n pregnant w o m e n (considered immunocompetent patients), diagnosis o f C M V infect ion is , most o f the t ime, performed indi rec t ly b y detecting C M V antibodies and not the antigens. 3.2.6.2 T h e assay T h e assay combines an enzyme immunoassay method w i t h a f inal fluorescent detection ( E L F A ) . Resul ts were classif ied as be ing negative (<4mU/ml) , equivoca l ( 4 - 6 m U / m l ) , and pos i t ive (>6mU/ml ) . The lower threshold for detection was 4 m U / m l , and the upper range for the scale is 4 0 0 m U / m l . F o r data entry, values < 4 m U / m l were assigned the value '3' , and values >400 were assigned the value '401' . A l l reagents were from B i o M e r i e u x . V I D A S C M V I g G is an automated assay for the V I D A S system, w h i c h enables a n t i - C M V I g G i n human serum to be measured quantitatively. A l l reaction steps are performed b y the V I D A S instrument. V I D A S ki ts are stored at 2 - 8 ° C . A k i t contains: disposable so l id phase receptacle ( S P R s ) , reagent strips, a posi t ive control serum, a negative control serum and one standard, w h i c h is also k n o w n as a 'ca l ibrator ' . S P R s are l i ke pipette-tip disposable devices, coated on the ins ide surface w i t h inactivated virus , and serve both as a combina t ion pipette tips and react ion vessels dur ing the assay. A l l o f the other reagents (mouse monoc lona l ant i-human I g G antibodies label led w i t h a lkal ine phosphatase, wash ing buffers, and substrate) are avai lable i n a 10-wel l foi l-sealed strip. 48 Before beg inn ing an assay, the k i t must be brought to r o o m temperature at least 30 minutes pr ior to use. Cal ibrator , controls and samples were m i x e d thoroughly w i t h a vortex to obta in a better result reproducibi l i ty . 3.2.6.3 Procedure (see Table 3.2) W e entered patient and assay data o n the V I D A S terminal to create a w o r k list. T h e assay code for V I D A S C M V G is C M V G . W e m i x e d the sera thoroughly w i t h a vortex, then pipetted lOOpl o f each into the sample w e l l o f the reagent strips i n the order shown i n the w o r k list . The C M V G S P R and C M V G reagent strip were p laced i n the posi t ions indicated b y the V I D A S w o r k list and, w e fo l l owed the procedure as directed i n the V I D A S User 's M a n u a l . T h e assay was completed i n approximately 40 minutes. Once the E L F A assay was p h y s i c a l l y completed b y the V I D A S , the a n t i - C M V I g G were mathemat ica l ly calculated b y the V I D A S , and expressed i n U / m l . The calcula t ion involves compar ing the fluorescence o f each control and patient sample to the current cal ibrat ion curve, and their ant ibody levels mathemat ica l ly determined i n relat ion to the cal ibrat ion curve w h i c h is stored i n the V I D A S ' s memory . 3.2.6.4 Quality control E v e r y new lot number when used for the first t ime, and every 14 days thereafter, had a cal ibra t ion curve made by running the C M V G calibrator assayed i n duplicate ("according to the M a n u a l M e n u " ) . Cal ibrators are sera w i t h a very accurately k n o w n ant ibody leve l . The V I D A S determines the amount o f fluorescence for each calibrator, compares it to the expected fluorescence for that antibody leve l , and makes a mathematical ca lcula t ion to construct a curve. T h e curve is a graph w i t h concentration plotted against fluorescence. I f the 49 calibrators are repeatedly out o f acceptable range, the curve is i n v a l i d and the k i t must be discarded. Pos i t ive and negative controls are inc luded i n each V I D A S C M V I g G ki t . Cont ro l s are s i m i l a r to calibrators, i n that they are sera w i t h a k n o w n ant ibody l eve l . These controls were performed once da i ly , at the beg inn ing o f the day, to ver i fy the accuracy o f the curve, the integri ty o f the k i t component, and the proper operation o f the V I D A S instrument, pr ior to ana lyz ing patient specimens. I f the control value deviated from the expected values, a new curve must be made b y rerunning the calibrator, and then the controls must be rerun. I f after repeated attempts, one or both o f the controls remains out o f range, then the k i t must be discarded, and the process begun again w i t h a new kit . 50 Table 3.2 CMV: Kit Composition (a), Description of the C M V G Reagent Strip (b), and Thresholds and Interpretation of Results (c). a: Kit Composition (60 tests): 60 CMVG strips Ready-to-use 60 CMVG SPRs 2x30 Ready-to-use. SPRs coated with CMV antigen (AD169). Each SPR is Clearly marked. Only remove the required number of SPRs. Made sure the pouch is well closed after opening Positive CMVG control 1 x 1.5ml (liquid) Human serum containing anti-CMVG IgG + 1 g/l of sodium azide. Titre in arbitrary unit/ml: range given on vial label. Negative CMVG control 1 x 1.5 ml (liquid) Human serum not containing anti-CMVG IgG + 1 g/l of sodium azide. CMVG calibrator 1 x2 ml (liquid) Human serum containing anti-CMV IgG + 1 g/l of sodium azide. Titre in arbitrary unit/ml given on vial label. b: Description of the CMVG Reagent Strip: Wells Reagent 1 Sample well. 2 Serum diluent: Phosphate buffer( 10 mmol/l) pH 7.2 - Tween + protein and chemical stabilizers + 1 g/l of sodium azide (300ul) 3 Pre-washing solution : Phosphate (10mmol/l) pH 7.2 - Tween + protein and chemical stabilizers + 1 g/l of sodium azide (600LI|) 4 - 5 - 7 - 8 Washing solution : TRIS (50 mmol/l) pH 7.4 + 1 g/l of sodium azide (600nl) 6 Conjugate: Alkaline phosphatase labelled monocional anti-human IgG antibodies(mouse) + 1 g/l of sodium azide ( 400LJ) 9 Empty well. 10 Cuvette with substrate: 4 methyl-umbelliferyl-phosphate + 1 g/l of sodium azide ( 300 L J ) . c: Thresholds and Interpretation of results: Value (U/ml) Interpretation <4 Negative From >4 to <6 Equivocal >6 Positive For all assays with results between 4 and 6, samples were assayed a second time to better determine true seroprevalence and levels. 51 3.2.7 Chi pneumoniae T h e enzyme- l inked immunosorbent assays ( E L I S A s ) were developed i n the U B C C D C research laboratory to detect I g G antibodies against Chi pneumoniae A R 3 9 elementary b o d y ( E B ) us ing an E L I S A method prev ious ly published(180). In brief, E L I S A plates (Corn ing , N Y , U S A ) were coated w i t h l O O u L / w e l l o f E B ( 0 . l u g / w e l l or l x l 0 5 I F U / w e l l ) , at 4 ° C overnight. The E B s were di luted i n 0.1 M N a H C 0 3 (S igma , St L o u i s , M O ) , at p H 9.0. The plates were washed once w i t h phosphate buffered saline ( P B S ) - T w e e n 20 (Sigma) , 150uX 3 % bovine serum a lbumin ( B S A , S i g m a , Miss i s sauga , O N ) i n P B S was added to each w e l l , and the plates incubated for 1.5hr at 3 7 ° C . Plates were washed w i t h P B S - T w e e n (Sigma) . Serum samples were added (diluted 1:200 i n 0 .5% B S A / P B S ) 1 0 0 p L to each w e l l , and incubated overnight at 4 ° C . T h e plates were washed three t imes. 100uX o f horseradish peroxidase-conjugated goat ant i-human I g G (1:2000 d i lu t ion ; Pha rmaGen , Oca la , F L ) i n 0 .5% B S A / P B S was added to each w e l l . Plates were incubated for 2 hour at 3 7 ° C and then washed three times. 100uX o f 2 ,2-azino-bis(3-ethylbenzthiazol ine-6-sulphonic acid) ( A B T S ; A m e r s h a m Biosc iences , A m e r s h a m , B u c k s , U K ) substrate was added to each w e l l , and a l lowed to develop i n the dark for 6 0 m i n , and then the plates read at O D 4 0 5 . The substrate was prepared b y adding 1ml A B T S stock to 2 0 m l 0 . 0 1 M citrate buffer and 20uX 3 0 % hydrogen peroxide. A B T S stock was prepared b y adding 160mg A B T S to 10ml H 2 0 , stored i n 5 0 0 - 1 0 0 0 p L aliquots at - 2 0 ° C . F o r Chi pneumoniae, seroposi t ivi ty was defined as an OD450 reading >0.2 arbitrary units at a 1:200 d i lu t ion o f serum. Seroposi t ive means that there is a significant l eve l o f ant ibody detected i n the serum, above an arbitrary cut -of f determined b y non-specif ic b i n d i n g i n k n o w n negative controls. I f the serum contains a h igh level o f antibody, it w i l l g ive higher O D values. A l t h o u g h i n our 52 study w e considered any posi t ive O D value as seropositive. W e also recorded the absolute amount o f ant ibody (up to the upper l i m i t o f 400) for our analysis. 3.2.8 Data analysis Data were entered into a database and analysed us ing P r i s m 3.0 (GraphPad , San D i e g o , C A ) software, us ing % 2 , K r u s k a l - W a l l i s , and M a n n - W h i t n e y U tests, as appropriate. P<0.05 was considered statistically significant. Base l ine maternal and pregnancy outcome data are presented parametr ical ly , as those data were no rma l ly distributed (Table 3.3). N o statistical analyses were made between groups i n this table. T h e p r imary outcome o f interest was the leve l o f a n t i - C M V and anti-Chlpneumoniae I g G . T h e secondary outcome was the seroprevalence o f a n t i - C M V and/or anti-Chl pneumoniae I g G . F o r seroprevalence comparisons, the % 2 was used to test the hypothesis that the seroprevalence o f a n t i - C M V and/or anti-Chl pneumoniae I g G w o u l d be greater i n w o m e n w i t h pre-eclampsia , especial ly pre-eclampsia o f early onset. F o r the comparisons o f levels o f a n t i - C M V and anti-Chl pneumoniae I g G , the K r u s k a l -W a l l i s test was used as a non-parametric analysis o f variance between mul t ip l e groups. W h e r e the K r u s k a l - W a l l i s test found statistically significant differences w i t h i n a compar ison, i n d i v i d u a l groups were compared us ing the M a n n - W h i t n e y U test to identify the group(s) under ly ing the s ignif icant K r u s k a l - W a l l i s result. 3.3 Results F r o m the Hea l th Records Department, B C Women ' s Hosp i t a l and Hea l th Centre, w e ident i f ied 18, 55, and 50 cases o f early-onset pre-eclampsia, late-onset pre-eclampsia , and normotensive I U G R , respectively. 246 controls were identif ied, up to two per case. The 53 U B C C D C had adequate stored samples and matched controls for serology for 9 (13 controls) , 29 (47 controls) , and 33 (53 controls) cases o f early-onset pre-eclampsia, late-onset pre-eclampsia , and normotensive I U G R , respectively. T h e three groups o f control w o m e n d i d not differ f rom each other i n terms o f their pregnancy details or their antibodies, so were amalgamated into a s ingle group for analyses. Ma te rna l and perinatal characteristics and outcomes o f the matched cases and their controls are detailed i n (Table 3.3). W o m e n w i t h pre-eclampsia were un i fo rmly hypertensive, and more often suffered from either transaminitis and/or thrombocytopenia than d i d other groups. One w o m e n w i t h early-onset pre-eclampsia had trace proteinuria but suffered f rom the H E L L P syndrome, and, therefore, fu l f i l led our def ini t ion. 3.3.1 Analyses 238 (78%) and 119 (39%) o f the total popula t ion (n=304) were seroposit ive for C M V and Chi pneumoniae, respectively. The seroprevalence results for the matched cases and controls are s h o w n i n (Table 3.4). There was no significant difference between groups (% p>0.13). There were significant differences i n both a n t i - C M V I g G and anti-CA/pneumoniae E B antibodies across the groups (Table 3.5), w i t h the early-onset pre-eclampsia group s h o w i n g increased antibodies compared w i t h the other three groups ( K r u s k a l - W a l l i s test, p=0.03). W o m e n w i t h early-onset pre-eclampsia had higher a n t i - C M V titres than w o m e n w i t h late-onset pre-eclampsia ( M a n n - W h i t n e y U test, p=0.012), normotensive I U G R (p=0.023), and norma l pregnancy controls (p=0.028). W h i l e there was a trend for w o m e n w i t h early-onset pre-eclampsia to have greater mti-Chlpneumoniae titres than w o m e n w i t h normotensive I U G R ( M a n n - W h i t n e y U test, p=0.092), normotensive w o m e n del ivered o f an I U G R infant had s igni f icant ly l o w e r mti-Chlpneumoniae antibodies than d i d normal pregnancy controls ( M a n n - W h i t n e y U test, p=0.010). A l t h o u g h the median value for late-onset pre-eclampsia 54 cases was the same as that for normotensive I U G R (0.103), this group was not significantly different from normal controls (Mann-Whitney U p=0.133). Table 3.3: Maternal and perinatal demographics and outcomes (mean (SD) or N [%]). j Variable Normal EO PET LO PET nlUGR | pregnancy 1 (n=9) (n=29) (n=33) (n=113) Maternal outcomes 1 Maternal age (y) 30.8 (5.4) 28.8 (8.1) 30.9 (6.1) 30.6 (4.9) Nulliparous 94 [83] 8 [89] 22 [76] 27 [82] Obesity* 25 [22] 2 [22] 9 [20] 6 [20] j Significant past history 5 [4] 0[0] 2 [7] 0[0] (Gestational age at delivery (wk) 39.0 (2.4) 30.9 (2.7) 38.5 (1.8) 37.3 (4.7) 1 Combined adverse maternal outcome 0[0] 1 [11] 0[0] 0[0] Highest mean arterial pressure (mmHg) 97.7 (8.5) 124.8(14.3) 125.7(10.8) 97.4 (9.9) i Proteinuria (>'+')11 14(12.4) 8 (88.9) 28 (100) 2(6.1) (n=28) Highest AST (IU/L) 25.0 (7.7) 75.0 (84.8) 69.7(158.2) -(n=5) (n=8) (n=25) 5 Lowest platelet count (x109/L) 191.9 (56.4) 123.8 (51.9) 176.2 (63.7) 202.1 (56.9) j (n=54) (n=8) (n=29) (n=16) j Highest uric acid (uM) 319.8 (21.6) 374.4 (93.4) 369.6 (80.3) 315.0 (93.0) j (n=5) (n=9) (n=23) (n=3) | Perinatal outcomes jBirthweight (g) 3342 (571) 1614(319) 3270 (524) 2281 (467) IUGR" 0[0] 1 [11-1] 0[0] 33 [100] i Perinatal l o s s n 0[0] 0[0] 0[0] 0[0] Combined adverse perinatal outcome * 0[0] 1 [11-1] 0[0] 1 [3.0] EO: early-onset (<34+0 weeks' gestation); LO: late-onset (>34+0 weeks' gestation); nlUGR: normotensive intrauterine growth restriction; PET: pre-eclampsia. Matched normal pregnancy controls were analysed as individual results. *body mass index >25 (3 missing values for n lUGR); Significant past history: previous pre-eclampsia, recurrent miscarriage, thromboembolic disease, hypertension, renal disease, or heart disease; Combined adverse maternal outcome: maternal death; hepatic failure, hepatic haematoma or rupture; Glasgow coma scale <13, stroke, >2 seizures, or cortical blindness (transient or permanent); positive inotrope support, myocardial infarction, or infusion of any 3 r d antihypertensive; dialysis, or renal transplantation; requirement of >50% 0 2 fo r>1h, or intubation; and/or transfusion of >10U of blood products (in total), ^proteinuria considered significant if >'+', despite risks of false positives; one early-onset pre-eclampsia case had only trace proteinuria, but had hypertension and hyperuricaemia. **IUGR: intrauterine growth restriction: birthweight <3 r d percentile for gender and gestational age. ^Perinatal loss: stillbirth >20 weeks' gestation and neonatal death in the first 28d of postnatal life. ^Combined adverse perinatal outcome: bronchopulmonary dysplasia; grade III or IV intraventricular haemorrhage; cystic periventricular leukomalacia; retinopathy of prematurity, stage 3 or 4; necrotising enterocolitis. 55 Table 3.4: Chlamydophila pneumoniae and CMV seroprevalence in women with normal pregnancies, early- and late-onset pre-eclampsia, and normotensive intrauterine growth restriction (n (%)). Normal EO PET (n=9) LO PET (n=29) nlUGR (n=33) pregnancy (n=113) CMV seropositive* 92 (81) 9 (100) 21 (72) 26 (79) Chi pneumoniae EB seropositive* 60 (53) 6(67) 11 (38) 10 (30) CMV & Chi pneumoniae seropositive for both* 46 (36) 6(67) 8 (28) 10 (30) * x2p>0.05; EO: early-onset (<34+0 weeks' gestation); LO: late-onset (>34+0 weeks' gestation); nlUGR: normotensive intrauterine growth restriction; PET: pre-eclampsia. Matched normal pregnancy controls were analysed as individual results. Table 3.5: Levels of IgG against Chlamydophila pneumoniae and CMV differ between women with normal pregnancies, early- and late-onset pre-eclampsia, and normotensive intrauterine growth restriction (median [interquartile range]). Normal EO PET LO PET nlUGR pregnancy (n=9) (n=29) (n=33) (n=71) CMV* Titre 49 79 26 40 [27, 65] [49, 179] [3, 68] [13, 66] Chi pneumoniae** EB titre 0.207 0.354 0.103 0.103 [0.105,0.359] [0.067, 0.659] [0.046, 0.315] [0.031, 0.216] Kruskal Wallis *p=0.024, **p=0.038. EO: early-onset (<34+0 weeks' gestation); LO: late-onset (>34+0 weeks' gestation); nlUGR: normotensive intrauterine growth restriction; PET: pre-eclampsia. Matched normal pregnancy controls were analysed as paired results when appropriate 56 3.4. Discussion Pre-ec lampsia is a dangerous disease o f human pregnancy, w h i c h affects both the mother and her baby. Pre-ec lampsia is a disease o f a major obstetric importance throughout the w o r l d . Nonetheless it remains enigmatic. The disorder takes several months dur ing pregnancy to develop, remits spontaneously immediate ly , or w i t h i n a few weeks after, de l ivery . It is a complex disorder that cannot be attributed to any one single cause. O u r p re l iminary study is the first to associate both C M V and Chi pneumoniae serological status w i t h early onset pre-eclampsia. It adds support to the evidence for both a causal l i n k between maternal infect ion and pre-eclampsia and for a l i n k between pre-eclampsia and later atherosclerosis (21), and recently reported sole ly for Chi pneumoniae i n pre-eclampsia compared w i t h normal pregnancy (85). W e also noted that a lower level o f antibodies to Chi pneumoniae is associated w i t h normotensive I U G R . T h i s latter point m a y help to exp la in the conundrum o f w h y w o m e n w i t h the same intrauterine pathology, namely uteroplacental mi sma tch (20), develop a dichotomous maternal response, the first be ing the systemic disorder o f pre-eclampsia , the second be ing the fetal syndrome o f pre-ec lampsia i n i so la t ion (21). Pre-ec lampsia and atherosclerosis share many c o m m o n pa thophys io logica l features and r i sk factors. O n e c o m m o n feature is endothelial dysfunction, w h i c h m a y derive f rom inf lammat ion . In atherosclerosis, injury- induced mononuclear ce l l accumula t ion , migra t ion and prol i ferat ion o f smooth muscles , and formation o f fibrous tissue u l t imate ly lead to plaque format ion and vessel obstruction have suggested an inf lammatory o r ig in to the altered endothelial dysfunct ion seen i n atherosclerosis. In pre-eclampsia, too, heightened intravascular in f lammat ion has been suggested as the o r ig in o f the abnormal endothelial funct ion (93). 57 A c c u m u l a t i n g evidence suggests that infect ion b y Chi pneumoniae, a c o m m o n pathogen m a y be i n v o l v e d i n the endothelial injury that marks atherosclerosis. Chi pneumoniae is prevalent i n atherosclerotic lesions but generally absent i n normal coronary arteries (181). It induces atherosclerotic lesions i n m ice and rabbits (175; 182), par t icular ly i n associat ion w i t h hypercholesterolaemia. The role o f infect ion as a possible trigger to the p rob lem is consistent w i t h an important an imal mode l (pregnant rat) o f pre-eclampsia (74). Faas et al. developed this m o d e l for pre-eclampsia i n w h i c h a single very l o w dose endotoxin infus ion induced a pregnancy-specif ic pre-eclampsia- l ike state, based on M c K a y et al. 's (183) observation that pre-eclampsia resembles the Shwar tzman reaction. These changes were prevented b y the administrat ion o f either l o w dose aspir in or superoxide dismutase. Cer ta in ly , subc l in ica l endotoxaemia has been detected i n those w i t h the systemic inf lammatory response syndrome ( S I R S ) (184), w i t h w h i c h pre-eclampsia shares m a n y characteristics (13). W h i l e M i t t e n d o r f et al. (71) have postulated that the use o f antibiotic p rophylax is might reduce the r i sk o f pre-ec lampsia i n pr imigrav idae w i t h a his tory o f ur inary tract infect ion, g iven the f indings o f Faas et al., a s ingle infectious event m a y be enough to trigger the in f lammatory response o f pre-eclampsia . Chi pneumoniae is an intracel lular pathogen that m a y reside w i t h i n macrophages o f atheromatous arteries i n a chronic , persistent state w i t h l o w metabol ic act ivi ty . A b o u t 7 0 % o f persons w i t h acute myocard ia l infarct ion show a seroresponse to c h l a m y d i a l l ipopolysacchar ide epitope and elevated titres against Chi pneumoniae i n sera f rom such patients point to an exacerbation i n a chronic infect ion as does a change i n the nature o f i m m u n e complexes containing ch lamydia l L P S . The presence o f antibodies to Chi pneumoniae proteins i n i m m u n e complexes suggests an intimate association o f the pathogen w i t h the vascular system (185). 58 H o w might Chi pneumoniae increase the r isk o f pre-eclampsia? Pre-ec lampsia can be considered a two-stage disorder, w i t h the first stage i n v o l v i n g abnormal placental implanta t ion and the second be ing the abnormal maternal adaptation to the pregnancy that results i n systemic dysfunct ion (186). In healthy pregnancies, the maternal spiral arteries undergo extensive remodel l ing , this remodel l ing is incomplete i n pre-eclampsia (187) and, that the vascular change most c o m m o n l y associated w i t h normal pregnancies was phys io log i ca l change and subint imal th ickening o f both segments o f the spira l arteries. In pre-eclampsia , i nvas ion o f the uterine spira l arteries is l imi t ed to the p r o x i m a l decidua, w i t h 3 0 % to 5 0 % o f the spiral arteries o f the placental bed escaping endovascular trophoblast r emode l l i ng (8;31). M y o m e t r i a l segments o f these arteries remain ana tomica l ly intact and undi la ted, and adrenergic nerve supply to the spiral arteries is not affected. The mean external diameters o f the uterine spiral arteries i n w o m e n w i t h pre-eclampsia are less than one h a l f o f the diameters o f s imi la r vessels f rom uncompl ica ted pregnancies (32). In pre-eclampsia med ia l disorganisat ion and hyperplasia i n the myomet r i a l arteries and acute atherosis i n decidual arteries were c o m m o n . The outcome o f abnormal spiral artery remode l l ing , m a y contribute to reduced placental perfusion and, is considered to be the root cause o f pre-eclampsia . Chi pneumoniae m a y also contribute to the second stage o f pre-eclampsia through endothelial dysfunct ion (188). Th i s endothelial pa thophysio logy is shared b y obesity, diabetes, hyper l ip idaemia and hypertension, a l l o f w h i c h are established r i sk factors for coronary heart disease and, when present i n a w o m e n entering pregnancy, greatly enhance their l i k e l i h o o d o f deve lop ing pre-eclampsia (189). S a i k k u et al. (63) reported an association o f I g G ant ibody to Chi pneumoniae and coronary heart disease among young F i n n i s h men and subsequent seroepidemiologic studies 59 suggested an associat ion o f I g G ant ibody to Chi pneumoniae w i t h both coronary and carot id atherosclerosis. These studies fo l lowed b y mount ing evidence f rom pathologica l , an imal experimental , and molecula r studies that supported a possible e t io logic ro le o f infect ion w i t h Chi pneumoniae i n atherosclerosis (65;76). S imi l a r l y , past, persistent, or recurrent infect ion w i t h Chi pneumoniae m a y contribute to abnormal vascular function, atherosis, and the abnormal placental perfusion that marks the first stage o f pre-eclampsia. An t ibod ie s against both C M V and Chi pneumoniae have been correlated w i t h react ivat ion o f chronic infect ion i n a number o f other c l i n i c a l scenarios. Spec i f i ca l ly , for C M V , ant ibody levels against C M V have been correlated w i t h v i r a l load and c l i n i ca l state. D u r i n g the T h l downregula t ion o f normal pregnancy (37), it is recognised that i m m u n e control over ch ron ica l ly carried C M V can be lost (190; 191). It is uncertain whether the same w i l l be noted for Chi pneumoniae as w e l l . W e also tested for the presence o f a maternal i m m u n e response ( IgG) to C M V , w h i c h is found i n 60 -80% o f the populat ion. C M V is thought to be responsible o f a large proport ion o f graft versus host disease i n bone mar row transplantation (192). C M V is a member o f the pherpesvirinae, a subfami ly o f the herpesviridae, and after infect ion the vi rus is general ly not e l iminated, m a y main ta in latency i n many tissues o f the host, and is capable o f infect ing trophoblast (193). V i r u s excret ion after infect ion continues for a l o n g t ime. C M V i s the most frequent cause o f congenital infections (1 -2% children). A fetal infect ion can lead to a preterm labour. The diagnosis can be done on the basis o f ident if icat ion o f specific antibodies, v i rus cu l t iva t ion and P C R from urine, sa l iva , b lood , and cerebrospinal f lu id . A n ant ivi ra l drug ganc ic lov i r can be used for treatment. A n t i - C M V vaccines are under development (194). 60 Several pieces o f evidence suggest that vascular endothel ium m a y be a site o f latent herpetic v i r a l infect ion, and that act ivat ion o f such infect ion might cause or aggravate atherosclerosis. Other viruses (Herpes s implex 1, Herpes s implex 11, C o x s a c k i e B ) have been ident i f ied as potential culpri ts i n atherogenesis, but evidence is less compe l l i ng than for C M V . The mechanisms are reported b y w h i c h infected endothel ium m a y be damaged b y marginated in f lammatory cel ls (195), and be transformed f rom an anticoagulant to a procoagulant tissue (196). T h i s transformation w o u l d promote the adherence o f neutrophils and platelets to the endothel ium. V i r a l infect ion o f the endothel ium induces the expression o f v i r a l g lycoproteins and adhesion molecules , w h i c h promote neutrophil and monocyte adhesion. V i r a l infect ion also induces the procoagulant molecule , tissue factor, i n endothelial cel ls . These enhanced procoagulant effects are associated w i t h the loss o f anticoagulants, i n c l u d i n g th rombomodul in , pros tacycl in and tissue p lasminogen activator (196). These studies support the speculat ion that v i r a l infect ion in vivo promotes vascular injury and thrombosis , w h i c h m a y contribute to disease states such as atherosclerosis and pre-eclampsia. C M V - r e l a t e d chronic v i l l i t i s has been associated w i t h pre-eclampsia (169), I U G R (170), and intrauterine fetal death (171). In addit ion, there is an impai red lymphoprol i fe ra t ive response to C M V i n w o m e n suffering from recurrent spontaneous abort ion (172), w h i c h is probably part o f the spectrum o f disease that includes pre-eclampsia (18). An t ibod ie s against bo th C M V and Chi. pneumoniae have been correlated w i t h react ivat ion o f chronic infect ion i n a number o f other c l i n i c a l scenarios. Spec i f ica l ly , for C M V , antibody levels against C M V have been correlated w i t h v i ra l load and c l i n i ca l state i n condit ions as var ied as human immunode f i c i ency v i rus infect ion and extraterrestrial travel as an astronaut (197; 198). 61 W i t h Chi pneumoniae, serum I g G antibodies have been correlated w i t h atherosclerotic disease act ivi ty . D u r i n g the T h l downregulat ion o f normal pregnancy (37), it is recognised that i m m u n e control over chron ica l ly carried C M V can be lost (190;191). It is uncertain whether the same w i l l be noted for Chi pneumoniae as w e l l . A l t h o u g h based upon a prospective population-based cohort, our study was l im i t ed i n a number o f ways . Firs t , a l l cases were identif ied i n a tertiary referral centre. Second, not a l l o f the cases and controls identif ied w i t h i n this centre had either enough or any serum sample i n the rube l la bank to permit its use i n this study. A s a consequence, not a l l cases were either fu l ly matched (two controls per case) or matched at a l l (controls were ident i f ied for o n l y 9 o f 18 early-onset pre-eclampsia cases), w h i c h reduced the statistical power o f the results o f this p re l imina ry study. F i n a l l y , the sample size for early-onset pre-eclampsia, i n particular, was sma l l , a l though w e used a rigorous case def ini t ion for pre-eclampsia and w e l l established methods for the detection o f the infectious agents. The seroprevalence for both C M V and Chi pneumoniae was s imi la r to the publ i shed experience for w o m e n o f reproductive age (81 ;84). The sample size reduced the power o f the study to determine any true differences i n seroprevalence between groups, al though they d i d appear to be higher i n early-onset pre-eclampsia. W e achieved sufficient power to detect 2 7 % , 4 5 % , and 4 7 % differences i n the seroprevalence for C M V , Chi pneumoniae and both C M V and Chi pneumoniae, respectively, at 8 0 % power. A t 6 0 % power, the difference between groups for seroprevalence became significant for a l l comparisons. These data w o u l d be consistent w i t h those o f He ine and colleagues (85), w h o found increased seroprevalence o f anti-Chl pneumoniae I g G (and not I g M ) i n pre-eclampsia when compared w i t h normal pregnancy and not to Chi trachmatis or Chi psittaci, w h i c h suggest a specif ic associat ion between infect ion w i t h Chi pneumoniae and pre-eclampsia (85). 62 In the presence o f enough other predisposing factors for pre-eclampsia , l eading to uteroplacental mismatch (20), the presence o f a reactivated infect ion w i t h either C M V and/or Chi pneumoniae cou ld precipitate that excessive innate immune response and in f l ammat ion especia l ly characteristic o f early-onset disease (19; 178; 199). Ear ly-onset pre-eclampsia differed from normal pregnancy, w h i l e late-onset pre-ec lampsia d i d not. T h i s supports the conjecture that early-onset pre-eclampsia is a different cond i t ion f rom late-onset disease (178). Early-onset disease carries a far greater burden o f maternal (200) and fetal (20;201) r isks , and differs more f rom the, often gestational age-dependent, phys io log ica l adaptations o f normal pregnancy than does late-onset pre-eclampsia (202), perhaps support ing (19;20;156) the conjecture that late-onset pre-eclampsia m a y be adaptive i n evolut ionary terms (203), proffering surv iva l advantages for a fetus exposed to uteroplacental mismatch (203-205) w h i l e m i n i m a l l y increasing maternal r i sks (200). T h e results here presented suggest that pre-eclampsia cou ld be a mul t i fac tor ia l disease and our f indings cou ld expla in w h y it has been imposs ib le so far to point out a unique disease responsible gene and suggest that other infectious agents cou ld have a s imi l a r inf luence on the aet iology o f this disease. A l s o , the infectious agents inc luded i n this study m a y serve as indicators o f one or more other infectious agents, spreading i n the same manner w h i c h m a y contribute to the development o f pre-eclampsia. G i v e n the f indings o f this p re l iminary study, w h i c h require prospect ive populat ion-based conf i rmat ion o f both serology and direct measurement o f infectious part icle D N A b y reverse transcriptase polymerase chain reaction w i t h i n buffy coat cel ls , it is possible that antibiotics and antivirals m a y develop a role i n pre-eclampsia prophylaxis . Early-onset pre-eclampsia m a y t ruly be the ' toxaemia' o f pregnancy. 63 Chapter 4 Conclusion, general approach, future directions for the research 64 4.1 General approach T h i s thesis examined the role o f immune adaptation to pregnancy i n in f luenc ing the development o f the maternal syndrome o f pre-eclampsia (13;20;93;206). The first approach was a case-control study o f w o m e n admitted to an I C U w i t h the systemic in f lammatory response syndrome ( S I R S ) . T h i s was ul t imately unsuccessful. The second approach was an examinat ion o f the seroepidemiology o f two infectious agents be l ieved to be i n v o l v e d i n atherogenesis, namely cytomegalovirus ( C M V ) and Chlamydophila pnemoniae (Chi pneumoniae) (21). The development o f pre-eclampsia dur ing a w o m a n ' s reproductive career predicts later atherosclerotic morb id i ty and mor ta l i ty (95;96;207). W e undertook these two investigations to test two hypotheses. Firs t , as it is be l ieved that the maternal syndrome o f pre-eclampsia is a fo rm o f the S I R S ; w e postulated that a his tory o f pre-eclampsia w i l l predispose w o m e n to the later development o f S I R S , as has already been established for atherosclerotic disease. T h i s experiment was described i n Chapter T w o . Second, w e postulated that w o m e n m a y be more l i k e l y to develop the maternal syndrome o f pre-eclampsia i n the presence o f subc l in ica l infect ion w i t h either Chi pneumoniae or C M V , than w o m e n whose pregnancies are either uncompl ica ted or compl ica ted b y I U G R i n isola t ion . 4.2 Summary of the results There were insufficient data from the pi lo t study o f w o m e n admitted to the I C U at St Pau l ' s Hosp i t a l to undertake any v a l i d analyses. O v e r a s ix month per iod, I ident if ied, 65 consented, and obtained data f rom o n l y s ix cases and two controls. The data col lected were incomplete at best. Therefore, the pi lo t project was abandoned. W e proceeded to undertake a nested case-control study o f serum from a populat ion-based bank. Seroprevalence and levels o f a n t i - C M V and Chi pneumoniae I g G were compared (non-parametr ical ly) between w o m e n w i t h early-onset pre-eclampsia (<34 weeks' ; n=9), late-onset pre-eclampsia (>34+0 weeks' ; n=29); normotensive I U G R (birthweight < 3 r d centile; n=33), and matched normal pregnancy (n=l 13, up to 2 per case). There was a signif icant difference i n both a n t i - C M V and Chi pneumoniae E B antibodies between groups ( K r u s k a l - W a l l i s p<0.05). W o m e n w i t h early-onset pre-eclampsia had higher a n t i - C M V titres (median: 79 [95% confidence interval 47, 164]) than w o m e n w i t h late-onset pre-eclampsia (26 [22, 82], p<0.05), normotensive I U G R (40 [31, 72], p<0.05), and normal pregnancy (49 [45, 70] , p<0.05). W o m e n w i t h normotensive I U G R had s ignif icant ly lower anti-CWpneumoniae antibodies (0.10 [0.08, 0.38]) than d i d normal pregnancy controls (0.21 [0.20, 0.28], p<0.05). 4.3 Conclusions N o f i rm conclus ions can be drawn from the case-control study undertaken i n the I C U . F r o m the seroepidemiology study w e concluded that the a n t i - C M V and anti-CA/pneumoniae antibodies were higher i n early-onset pre-eclampsia than i n late-onset pre-eclampsia , normotensive I U G R , and normal pregnancy. Th i s m a y prov ide a pa thophys io logica l l i n k between pre-eclampsia and the k n o w n increased r isk for subsequent atherosclerosis (186). T h i s informat ion cou ld lead to nove l therapeutic manoeuvres to attenuate the inf lammatory response o f pre-eclampsia, thereby safeguarding against fetal growth restr ict ion and iatrogenic prematurity. A reduction o f the pro-coagulant environment w o u l d reduce the 66 inc idence o f occ lus ive placental events, thereby protecting both the maternal perfusion o f the in tervi l lous space and, consequently, fetal nourishment. S i m i l a r l y , some fetuses o f w o m e n w i t h pre-eclampsia are frequently del ivered whi ls t s t i l l t h r iv ing in utero because o f the severi ty o f the maternal syndrome. A n y manoeuvre, such as macro l ide / nitrofurantoin ant ibiot ic p rophylax is or short course ant iviral agents, that was able to reduce the rate o f the development o f the maternal syndrome w o u l d have a direct effect on prematuri ty and its consequences. A s stated, pre-eclampsia is the commonest cause o f iatrogenic prematurity. Ear ly-onset pre-eclampsia m a y t ruly be the ' toxaemia' o f pregnancy. 4.4 Future directions F r o m the first, unsuccessful, study, it m a y be possible to answer the questions raised b y the hypothesis us ing a number o f different approaches. First , a l ong term fo l low-up study designed to investigate the inf lammatory sequelae o f hav ing developed pre-eclampsia , i nc lud ing both atherosclerosis (an inf lammatory condit ion) and S I R S . Second, us ing populat ion-based data records, such as exist i n Scandinavia and Scot land, to l i n k maternal obstetric outcomes w i t h later hospital admissions/death cert if icat ion. T h e results o f the second study o f Chi pneumoniae and C M V seroepidemiology require va l ida t ion . Fi rs t w e must perform a prospective sample o f cases and controls to determine i f the seroprevalence is greater i n pre-eclampsia. In particular w e are interested i n early-onset pre-eclampsia , than i n other groups w h i c h control for disease severi ty (late-onset), abnormal placentat ion (normotensive I U G R ) , and pregnancy (normal pregnancy and non-pregnancy). Second, w e need to identify both Chi pneumoniae and/or C M V D N A i n buffy coat (ie lymphocytes and monocytes) cells o f w o m e n f rom the groups out l ined above. T h i s w o u l d 67 reveal that there was t ruly active infect ion at the t ime o f disease diagnosis i n pre-eclampsia. A s opposed to the infect ion hav ing been cleared prev ious ly this cou ld be undertaken us ing molecu la r b i o l o g i c a l techniques such as semi-quantitative reverse transcriptase polymerase cha in reaction. T h i r d , to investigate the expression o f receptors hand l ing these infectious agents, such as T o l l - l i k e receptors ( T L R ) - 2 and -4, and po lymorph i sms i n the genes encod ing those receptors (tlr-2 and tlr-4). Four th , w e require a population-based seroepidemiological to study the Chi pneumoniae and C M V t imed to coinc ide w i t h the triple marker screen (208) used to screen for aneuploidy and open neural defects, l i nked w i t h pregnancy outcomes. Th i s w o u l d ident ify i f the change f rom adaptive to innate i m m u n i t y i n early pregnancy w o u l d permit the ident i f ica t ion o f a group o f w o m e n subsequently at r isk for developing pre-eclampsia, us ing levels o f an t i -CA/ pneumoniae and a n t i - C M V as a screening test. F i f t h us ing rube l la serology as a control for prospective va l ida t ion . F i n a l l y , were these studies to prove successful, then intervention trials cou ld be designed to prevent the onset of, especial ly early-onset, pre-eclampsia. 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