UMEÅ UNIVERSITY MEDICAL DISSERTATIONS NEW SERIES NO. 1680 ISSN 0346-6612 ISBN: 978-91-7601-149-2

From the Department of Surgical and Perioperative Sciences Anesthesiology and Intensive Care Medicine

Umeå University, Sweden

Severe sepsis Epidemiology and sex-related

differences in inflammatory markers.

Sofie Jacobson

Fakultetsopponent: Professor Else Tönnesen

Dept. of Clinical Medicine - Anaesthesiology Århus, Danmark

Umeå 2014

Cover illustration: "First line of defence" Anders Jacobsson

Copyright © 2014 Sofie Jacobson ISBN: 978-91-7601-149-2 NEW SERIES NO. 1680

ISSN 0346-6612 Layout and printed by: Print & Media

Umeå, Sweden 2014

             

To my family

A goal is a dream with a deadline. ~ Napolean Hill

       

Contents

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CONTENTS

ABSTRACT......................................................................................................................... iv

Svensk sammanfattning .........................................................................................................v

Abbreviations ..................................................................................................................... viii

ORIGINAL PAPERS............................................................................................................x

PROLOGUE ........................................................................................................................ xi

INTRODUCTION..................................................................................................................1 Epidemiology ....................................................................................................................2 The innate immune system, a very brief overview............................................................3 Inflammation .....................................................................................................................5 Cytokines...........................................................................................................................6

Interleukins (IL)............................................................................................................6 Tumor necrosis factor (TNF)........................................................................................7 Interferons (IFN)...........................................................................................................8 Chemokines ..................................................................................................................9

Complement ....................................................................................................................10 Coagulation .....................................................................................................................11 Recombinant Tissue Factor Pathway Inhibitor (rTFPI) ..................................................13 Antithrombin (AT) ..........................................................................................................13 Activated Protein C .........................................................................................................13 Endothelium ....................................................................................................................14 Leptin ..............................................................................................................................15

The protein..................................................................................................................16 Factors affecting leptin production or clearance.........................................................16 The Leptin receptor (LepR, previously denoted Ob-R)..............................................17 Action of leptin...........................................................................................................18 Effects of Adiposity....................................................................................................18 Leptin, inflammation and immunity ...........................................................................19 Leptin and sepsis studies in animals ...........................................................................20 Leptin and sepsis in humans .......................................................................................22

Adiponectin .....................................................................................................................22 Adiponectin Receptors................................................................................................23 Actions of adiponectin................................................................................................24 Adiponectin and inflammation ...................................................................................25 Adiponectin and sepsis studies in animals..................................................................26 Adiponectin and sepsis in humans..............................................................................27

Mannose-binding lectin (MBL).......................................................................................27 Mode of action............................................................................................................28 Genetic determinants and inter individual variations in MBL levels..........................28 Factors effecting MBL-levels .....................................................................................30 MBL and disease ........................................................................................................31 MBL and sepsis in animal studies ..............................................................................32 MBL and sepsis in humans.........................................................................................32

Contents

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AIMS OF THE THESIS ......................................................................................................34

METHODS ..........................................................................................................................35 Study design ....................................................................................................................35 Material, study subjects ...................................................................................................35 Sepsis definition, main inclusion criteria.........................................................................36 TISS scoring....................................................................................................................37 APACHE II scoring.........................................................................................................37 Sequential Organ Failure Assessment (SOFA) Score .....................................................39 Data collection.................................................................................................................40 Northern Sweden Medical Research Bio-bank,...............................................................40 Nested case-referent studies, Papers III and IV...............................................................41

Clinical examinations and biochemical analysis from the MONICA and VIP cohort.....................................................................................................................................44 Blood sampling...........................................................................................................44 Enzyme-linked Immunoassay (ELIZA) and Radioimmunoassay (RIA) ....................44

Statistics ..........................................................................................................................45 Ethical aspects .................................................................................................................46

RESULTS ............................................................................................................................47 Main finding Paper I........................................................................................................48 Main findings Paper II:....................................................................................................52 Paper III and IV...............................................................................................................55 Main findings Paper III ...................................................................................................59 Main findings Paper IV ...................................................................................................62

DISCUSSION ......................................................................................................................65 Aspects of epidemiology and study design .....................................................................65 Findings from a small single center study .......................................................................66 Comorbidities and outcome.............................................................................................67 Predictors of in-hospital death.........................................................................................67 Gender differences ..........................................................................................................68 SOFA score and thrombocytes ........................................................................................69 Gender versus sex, and adiposity ....................................................................................70 Body mass index, diabetes and choletsterol. ...................................................................71 Leptin and adiponectin as predictors of sepsis and in-hospital mortality ........................71 Mannose-binding lectin ...................................................................................................73 Methodological considerations........................................................................................74 Future considerations ......................................................................................................75

CONCLUSIONS..................................................................................................................76

ACKNOWLEDGEMENTS .................................................................................................77

REFERENCES.....................................................................................................................79

Abstract

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ABSTRACT

Background. Sepsis is a syndrome associated with high mortality rates, substantial morbidity and high costs of care. The incidents of sepsis is reported to be high and controversy exists whether gender affect severity or outcome. Little is known about factors determining susceptibility for developing the syndrome and severity of the syndrome once developed. Early detection and adequate antibiotic administration are the mainstay of treatment and means to identify patients with particular high risk of adverse outcome are desirable. There are data to suggest that the course of sepsis and outcome from the syndrome may be influenced by inherited differences in the immunological response among humans

Aims: Paper I: Assess incidence and outcome for ICU-treated sepsis patients in this region; Paper II: Assess if there are gender differences related to characteristics, aspects of treatment or outcome in sepsis in this region. Paper III: Assess the association of baseline levels of leptin and adiponectin and future sepsis event, and association of these adipokines in the cute phase and sepsis severity and outcome. Paper IV: Assess association of baseline levels of mannose-binding lectin (MBL) and future sepsis event, and MBL levels in the acute phase in relation to sepsis severity and outcome.

Results. Paper I: Overall ICU mortality rate was 25%, while the ICU mortality for patients with septic shock was 58% in this retrospective single university hospital cohort analysis. Cardiovascular disease and diabetes were the most prevalent comorbidities among patients who died during hospital stay. Paper II: No gender-related differences in mortality or length of stay was found in this prospective single center observational study. Differences in aspects of treatment were related to differences in site of infection. Men had more often infections in skin and skin-structures, whereas women more often had abdominal infections. Early organ dysfunction assessed as SOFA score at admission was a stronger predictor for hospital mortality for women than for men. The discrepancy was related to the SOFA coagulation-sub score. Paper III: In this nested case-referent study hyperleptinemia at baseline predicted a first-ever sepsis event, even after adjustment for BMI and other cardiovascular risk factors. Hyperleptinemia in the acute sepsis phase was associated with reduced risk of in-hospital death in men, but associated with increased risk of in-hospital death in women. Paper IV: In the same matched cohort as in Paper III high baseline levels of MBL predicted a first ever sepsis event. High MBL levels in the acute phase or an increase from baseline to the acute phase associate with increased in-hospital death in women but not in men. Low MBL levels was not identified as a risk for acute sepsis or in-hospital death.

Conclusions. Mortality from severe sepsis is high, equally affecting men and women. There are differences in patient characteristics and inflammatory markers, which associate with in-hospital mortality differentially in men and women. Aspects of gender should be mandatory, and genetic analysis are desired in future sepsis research.

Keywords: Severe sepsis, Leptin, Adiponectin, Mannose-binding lectin, sex.

Svensk sammanfattningt

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Svensk sammanfattning

Bakgrund

Sepsis är ett syndrom som orsakas av invaderande mikroorganismer, vanligtvis sjukdomsalstrande bakterier. Invasionen av mikroorganismer leder till en inflam-matorisk reaktion genom aktivering av kroppens immunförsvar. Det primära, inne-boende immunförsvaret, aktiveras först och utgörs bland annat av vita blodkroppar, vävnadsmakrofager och celler som bekläder blodkärlens insida, s.k. endotelceller. Till det primära immunförsvaret hör även små proteiner, s.k. cytokiner, chemokiner och opsoniner, som kan verka både inflammationsbefrämjande och inflammations-hämmande. Vid aktivering frisätts dessa proteiner från vita blodkroppar, makro-fager och endotelceller med syfte att dirigera olika aktörer i immunförsvaret till rätt plats för att oskadliggöra inkräktaren. Inflammationen kan vara lokalt begränsad men kan på grund av överdriven aktivering av immunförsvaret bli mer omfattande och påverka hela kroppen. Man säger då att inflammationen är systemisk och eftersom den orsakats av en infektion så definieras tillståndet som sepsis. Vid sepsis aktiveras också olika kaskadsystem, bla koagulation- och komplement- och fibrinolytiska systemet, som interagerar sinsemellan. Endotelceller och glatt muskulatur i blodkärlen påverkas också vilket leder till perifer vasodilatation och kapillärläckage, vilket kännetecknar den kliniska bilden vid sepsis. Sepsis kan ha olika svårighetsgrad och dess allvarligaste form, septisk chock, är förenat med hög dödlighet, även i västvärlden. Dödligheten har i internationella studier jämförts med dödligheten i hjärt-kärlsjukdom. Sepsis är även förenat med betydande sjuklighet och stort lidande för den drabbade, samt stora kostnader för samhället. Tillståndet kan drabba människor i alla åldrar och det finns uppgifter om att förekomsten ökar i omfattning globalt. Det har tidigare varit något oklart hur vanligt det är med intensivvårdskrävande sepsis i Sverige och om dödligheten är lika hög som i andra länder.

Tidigt insatt adekvat antibiotika anses ännu vara grundpelaren i behandlingen. Andra strategier att behandla tillståndet har hittills inte visat bättre resultat vad gäller överlevnad eller sjuklighet. Faktorer som påverkar utgången är bland annat vilken mikroorganism som orsakar infektionen, underliggande sjukdom hos patienten och hur snabbt man påbörjar adekvat behandling. Senare års forskning har visat att nedärvda immunologiska egenskaper kan ha betydelse för förloppet vid septisk sjukdom. Nivåer och funktion hos olika inflammatoriska och immunologiska ämnen styrs genetiskt. Det finns data som talar för att polymorfism (genförändringar) för vissa inflammatoriska ämnen korrelerar till ökad benägenhet att drabbas av infektioner samt en ökad sjuklighet och dödlighet vid vissa infektiösa tillstånd.

Att kunna förebygga sepsis och tidigt kunna upptäcka syndromet eller de som har störst risk att drabbas eller har stor risk att dö av sepsis är ett angeläget forskningsområde.

Svensk sammanfattningt

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Syftet med de två första arbetena som ligger till grund för denna avhandling har varit att kartlägga förekomsten och dödlighet av intensivvårdskrävande sepsis på ett universitetssjukhus i norra Sverige och att undersöka vårdtider och resursförbrukning för dessa patienter samt att utröna om det finns könsrelaterade skillnader med avseende på karakteristik, behandling och utfall i form av dödlighet och vårdtider. Sammanlagt undersöktes journaluppgifter från 208 patienter under åren 2000-2005. Andelen intensivvårdspatienter som hade eller utvecklade sepsis första dygnet var omkring 5 % vilket är färre än vad som rapporterats internationellt. Dödligheten i sepsis var hög men i nivå med internationella siffror under samma tidsperiod, i genomsnitt ca 25 %, och vid septisk chock omkring 50 %. Resursförbrukningen avspeglade både vårdtider och sepsis svårighetsgrad. I den här studien förbrukade yngre människor och de som dog mest resurser. Det fanns skillnader mellan könen i orsaken till sepsis där män oftare hade infektion utgående från hud och hudstrukturer medan kvinnor oftare hade infektion utgående från bukorganen. Detta påverkade vissa aspekter av vården men det gick inte att påvisa någon skillnad i dödlighet eller vårdtid mellan män och kvinnor. Tidigt i förloppet fanns det även skillnader i antalet blodplättar samt nivåer av CRP (en inflammationsmarkör) mellan män och kvinnor som associerade till sjukhusdödlighet hos patienterna. Huruvida det berodde på olikheter i typ av infektionsfokus eller om det representerar skillnader i inflammationssvar hos män och kvinnor kunde denna studie inte ge svar på.

Syftet med tredje och fjärde arbetet var att undersöka om nivåerna av inflammatoriska markörer i blod hos friska individer kan förutsäga utveckling av sepsis och om nivåerna av inflammatoriska markörer i blod i samband med det akuta insjuknandet kan förutsäga sjukdomsförlopp och risk för död. Genom samkörning av databaser från intensivvårdsavdelning vid Norrlands universitetssjukhus med uppgifter i den medicinska biobanken hittades 152 patienter, vilka före sepsisinsjuknandet hade lämnat blodprover i samband med hälsokontroller och donerat dessa till medicinska biobanken. Sepsis-patienterna matchades med två kontrollpersoner som inte drabbats av sepsis och som också donerat blod till biobanken. Utöver dessa prover fanns för 128 patienter även prover från det akuta insjuknandet sparade.

Ämnen som undersöktes var så kallade adipokiner (cytokiner producerade av fettväv), leptin och adiponektin samt mannos-bindande lectin (MBL). MBL är ett ämne som normalt finns i blodet och som fungerar som opsonin, d.v.s det fäster på invaderade mikroorganismer och underlättar för immunförsvaret att inaktivera mikroorganismer. Cirka 10 % av den nordeuropeiska befolkningen beräknas sakna eller ha mycket låga nivåer av MBL. Brist på MBL anses vara kopplat till ökad benägenhet för vissa infektiösa tillstånd. Koncentrationen av leptin och adiponektin är starkt sammankopplad med fettmassa och kvinnor har normalt högre nivåer än män. Leptin och adiponectin är också starkt associerade till metabola sjukdomar som fetma och diabetes samt till hjärtkärlsjukdom.

Svensk sammanfattningt

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Analys av blodproverna visade att höga nivåer av leptin i normaltillstånd associerar till insjuknande i intensivvårdskrävande sepsis och möjligen är det sambandet starkare för män. Detta samband var oberoende av riskfaktorer för hjärt-kärlsjukdom som body mass index (BMI), hypertoni, diabetes, totalkolesterol och rökning. Motsvarande samband sågs inte för adiponectin. I det akuta skedet hade skillnaden i koncentration av leptin och adiponectin mellan män och kvinnor utjämnats, framför allt på grund av att männen ökat sina nivåer. Kopplingen till fettmassa mätt som BMI var också upphävd. Höga nivåer av leptin i det akuta skedet var förenat med minskad risk för män men ökad risk för kvinnor att avlida under sjukhusvistelsen. Motsvarande samband kunde inte påvisas för adiponectin. Studien om mannos-bindande lectin kunde inte påvisa att låga nivåer av MBL i normaltillståndet medförde någon ökad risk att drabbas av intensivvårdande sepsis. Däremot visades att höga nivåer vid akutskedet eller en ökning från basalt tillstånd till det akuta skedet var förenat med ökad dödlighet under sjukhusvistelsen för kvinnor men inte för män.

Sammanfattningsvis, i jämförelse med tidigare studier från andra delar av världen så är dödligheten i intensivvårdskrävande sepsis likartat hög även i norra Sverige. Det gäller både för män och kvinnor även om tillståndet jämförelsevis är mindre vanligt i norra Sverige. Resursförbrukningen är associerad med sjukdomens svårighetsgrad och dödlighet. Det finns könsrelaterade skillnader i aspekter av det inflammatoriska svaret, där leptin och MBL förefaller ha olika betydelse för dödlighet i sepsis hos män och kvinnor. Dessa skillnader är värda vidare undersökning för att i framtiden kunna anpassa förebyggande åtgärder och behandling för män och kvinnor för att kunna förhindra sepsisutveckling och död. För detta krävs större patientmaterial och att man analyserar män och kvinnor var för sig i den statistiska bearbetningen. Genetiska analyser är önskvärda men ställer särskilda krav på etiska överväganden.

Abbreviations

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Abbreviations

APN Adiponectin APM Adiponectin molecule AR Adiponectin receptor AMK Alveolar macrophage killing AMP Adenosine monophosphate APACHE Acute Physiology and Chronic Health Evaluation, version II and III APC Activated Protein C AT Antithrombin BMI Body Mass Index BP Blood Pressure C with suffix Complement CC, CX Chemokine CI Confidence interval CHD Congestive heart disease CLR C-type lectin receptors CSF Colony-stimulating factor COPD Chronic Obstructive Pulmonary Disease CPL Cecal ligation and punction CRF Chronic renal failure C1q Complement factor 1q CRP C-reactive protein CV Confidence interval CVD Cardiovascular disease DIC Disseminated intravascular coagulation DNA Deoxyribonucleic acid DM Diabetes mellitus db diabetes trait (denotes leptin receptor gene deficiency) ELIZA Enzyme-linked Immunoassay ER Endoplasmic reticulum c-GMP cyclic- Guanosine monophosphate ICD International Classification of Diseases ICU Intensive Care Unit IDM Insulin Treated Diabetes Mellitus ICAM Intracellular adhesion molecule IFN Interferon IFG Impaired fasting glucose Ig Immunoglobulins IGF Insulin growth factor IGT Impaired glucose tolerance IL Interleukin IQR Interquartil range KO Knock out (gene deficiency) LOS Length of stay LEP Leptin LEPR Leptin receptor

Abbreviations

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LPS Lipopolysaccharide MBL Mannose-binding lectin MIF Migration inhibiting factor MONICA Monitoring of Trends and Determinants in Cardiovascular Disease NIDM Non-Insulin Treated Diabetes Mellitus NK Natural Killer cells NLR NOD-like receptor NOD Denotes an intracellular receptor protein NO Nitric oxide NSMC The Northern Sweden Maternity Cohort NSMRB The Northern Sweden Medical Research Bank NYHA New York Heart Association Ob Obesity ob/ob Obestity trait (denotes leptin gene deficiency) OB-R Leptin receptor OR Odds ratio OVX Ovaryectomized PAMP Pathogen Associated Molecular Pattern PMN Polymorph nuclear neutrophil PRR Pathogen Recognizing Receptor RCT Double blind randomized controlled trial RIA Radioimmunoassay RIG Retinoic acid-inducible gene RLR Retinoic acid-inducible gene-like receptor SAPS Simplified Acute Physiology Score SIR Swedish intensive care unit registry SIRS Systemic inflammatory response syndrome SNP Single nucleotide polymorphism SOB-R Soluble form of the leptin receptor SOFA Sequential Organ Failure Assessment TC Thrombocytes TF Tissue factor TISS Therapeutic intervention scoring system TNF-α Tumor Necrosis Factor-α TLR Toll-like receptor TGF Transforming growth factor USD US dollar VCAM Vascular cell adhesion molecule VIP Västerbotten Intervention Program WHC Waist-to-hip circumference WHR Waist-to-hip ratio

Original papers

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ORIGINAL PAPERS

This thesis is based on the following publications and manuscript, which are referred to in the text by their Roman numerals: I Jacobsson S, Johansson G, Winsö O.

Primary sepsis in a university hospital in northern Sweden: A retrospective study.

Acta Anaesthesiol Scand 2004; 48: 960—967 II Sofie Jacobsson, Eva Liedgren, Göran Johansson, Martin Ferm,

Ola Winsö. Sequential Organ Failure Assessment (SOFA) scores differ between genders in a sepsis cohort: Cause or effect? Upsala Journal of Medical Sciences. 2012; 117: 415–425

III Sofie Jacobsson, Peter Larsson, Göran Johansson, Margareta Nordberg,

Göran Wadell, Göran Hallmans, Ola Winsö, Stefan Söderberg. Leptin independently predicts development of future sepsis and determines survival in the acute phase. Submitted for publication

IV Sofie Jacobsson, Anna-Maja Åberg, Göran Johansson,

Margareta Norberg, Göran Wadell, Göran Hallmans, Ola Winsö, Stefan Söderberg. Levels of mannose-binding lectin (MBL) predicts sepsis and associates with sepsis-related in-hospital mortality differentially in men and women. Manuscript

The original papers have been reprinted with kind permission from the publishers.

Prolog

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PROLOGUE

Why this thesis?

In January 2000 a young woman was admitted to our intensive care department. She was referred from another hospital where she had collapsed in a fulminant septic shock. Immediate resuscitation was started at the referring hospital, cultures were taken and she received antibiotics. Due to respiratory failure she was intubated before transport. At admission to our hospital she already had multiple organ failure. Despite efforts to restore adequate organ perfusion with infusion of large amounts of fluids and infusion of inotropic and vasoactive drugs, her blood pressure was extremely low. Her heart was dilated and the contractility of the myocardium was reduced. Initially she produced small amounts of urine but soon she became anuric. She had fulminant disseminated intravasal coagulation with petechial bleedings, gangrenous toes and fingers and her platelet count was very low. In the 48 hours to come she gained 30 kg in body weight (60% weight gain) due to fluid accumulation from the efforts to restore circulation with fluids. Despite continuous renal replacement therapy she was edematous beyond recognition. She was difficult to ventilate due to edema formation, which caused low pulmonary compliance and high oxygen demand. Active resuscitation was ongoing during the first 96 hours, and the outcome was uncertain.

On the night of the fifth day, I was "on call" and I witnessed the beginning of the end of the acute inflammatory phase with its extreme vasoplegia and capillary leakage. Suddenly, we were able to reduce the fluid overload by ultra-filtration through continuous renal replacement therapy. The reduction of edema improved the dynamics of ventilation, which allowed adjustment to more normal ventilator settings, and also improved oxygenation. As her circulatory status stabilized, we began tapering down the inotropic and vasoactive support. What happened after that night is another long story but the young woman survived and was eventually discharged from ICU. However, her ICU stay has stayed in my memory.

In 2001 Angus and co-workers published a large epidemiological study on severe sepsis in the United States (1). They found an incidence of 3.0 per 1000 population of whom 51.1% received intensive care. Mortality was 28.6% or 215000 deaths annually, which were as many deaths as from acute myocardial infarction. The costs of care were on average 29900 USD per case for those who received intensive care. The same year, results from a large multicenter double blind randomized controlled trial (RCT) showed that treatment with a new drug significantly reduced mortality in patients with severe sepsis (2). The new drug was very expensive and was heavily marketed in the light of the study from Angus. However, the drug was associated with side effects and contraindications. For instance, the young woman described above would not have received the drug, because she did not have the right treatment indications. Still, marketing of the drug was aggressive.

Prolog

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As an intensive care clinician I asked myself: Are the results from these studies consistent with my own reality, i.e. are they transferable to the patients I meet in my hospital? How many patients with sepsis are admitted to our intensive care department and what are the outcomes for these patients?

These questions were the start of a longer journey now partly presented in this thesis.

Introduction

1

"It is more important to know what sort of person has a disease

than to know what sort of disease a person has"

Hippocrates

INTRODUCTION

Sepsis [σηψις] is the original Greek word for the “decomposition of animal or vegetable organic matter in the presence of bacteria”. It is first met in Homer’s poems, where Sepsis is a derivative of the verb form sepo [σηπω], which means “I rot”. The term sepsis is also found in the Corpus Hippocraticum. Thus, the word “sepsis” has persisted for 2,700 years (3). While the meaning of the word is more or less unchanged, the understanding of the syndrome has evolved.

Sepsis is a syndrome which results from the body’s response to an infecting microorganism. It is a drama set within the vasculature. The major actors’ are the immune competent cells, which upon activation cause the release of cytokines and activation of different cascade systems that ultimately, affect the endothelium. This course of events results in what is known as inflammation. In sepsis, the inflammatory process is systemic; it affects every organ system in the body, even remote from the original infection site. If unrecognized and left untreated, there is a considerable risk of progression to the most severe form, septic shock.

Despite enormous advances in the understanding of disease processes and technical progress since the days of Homer, mortality rate in septic shock is still high and morbidity attributable to sepsis is increasing (4-8). Considerable efforts have been made to inhibit or enhance the function of specific factors that are known to play an important part in the development of sepsis, so far with disappointing results (9-12). Some reduction in mortality rates has been reported in studies that focus on early recognition of the syndrome, treatment with antibiotics and standardized organ support (13-15). Emerging data indicates that the course of sepsis and outcome from the syndrome may be influenced by inherited differences in the immunological response among humans.

The main subjects of this thesis are sex-related differences in epidemiology, leptin, adiponectin and mannose-binding lectin (MBL) and the outcome from sepsis. Before those topics are addressed the reader is offered an opportuninty to be guided through some of the epidemiological aspects of sepsis, followed by a glimpse into the innate immune system. After some introduction to the pathophysiology of inflammation, a short review of pertinent cytokines and the coagulation system precedes an exposé over treatment trials. The introduction ends with a presentation of the main actors of this thesis; the adipokines leptin and adiponectin, and the mannose-binding lectin MBL. The material and methods used to study these subjects are then described, followed by the main results of the

Introduction

2

studies. The results are discussed, conclusions are made, and finally future implications are proposed.

Epidemiology

Since Angus and co-workers in 2001 published their study on the incidence, costs and outcome of severe sepsis in the United States, several reports from different parts of the world have been published (1). The results vary with reported incidence ranging from 0.38 to 2.4/1000 population, with hospital mortality rates ranging from 28 to 55% (16-21).

Differences in study design and inclusion criteria are one of the reasons for differences in study results (22). Another contributing explanation is variation in the frequency of co-morbidities in the population, which may affect the severity of sepsis and sepsis outcome (23, 24). Also, there are differences in the prevalence of multi drug-resistant bacteria in different parts of the world, with the Scandinavian countries having among the lowest prevalence (25, 26). Further, cultural and socioeconomic disparities and differences in health care accessibility may influence the propensity to seek medical advice and thus affect the timing of adequate care, which can affect the outcome from an infection (27-29). Ethnicity and gender are not only considered to influence the socioeconomic status, but also the immune system (30-33). Theoretically, inherited immunological differences, genetic polymorphism which affect various factors in the innate immune system, may be responsible for some of the discrepancies in incidence and outcome from sepsis in different parts of the world. As yet there are insufficient data to suggest that this is the case, but the understanding of the influence of individual genetic variations on the immune system and its impact on the course of an infection is beginning to evolve.

Regardless of differences in reported incidence and mortality rates it is clear that sepsis is a common condition in the intensive care setting and it is associated with great suffering, high mortality rates and high costs of care (34). A number of studies and randomized trials have been conducted with the aim to reduce mortality from severe sepsis (2, 35-38). Despite promising results in initial experimental studies or clinical trials, follow-up studies have not convincingly demonstrated reduced mortality (9-12, 39, 40). These discouraging results have contributed to the notion that mortality from sepsis is difficult to affect. However, when planning for randomized trials, mortality data from previous studies are used to estimate the number of patients needed to prove an effect, ie. power calculation. If mortality data from other populations or from other settings than the intended study population are used, there is a risk for miss calculation of the number needed to treat to show an effect (41-43). If mortality in fact is reduced due to uncontrolled factors or if certain patients are excluded (different case mix) it may lead to underpowered studies that are unable to show an existing effect. This fact has gained new actuality through a large epidemiological study from Australia and

Introduction

3

New Zeeland that report an increase of the proportion of patients admitted to the ICU with sepsis, from 7.2% to 11.1% during 2000 to 2012 (44). However, the most encouraging finding was a steady decline in hospital mortality from 35.0 to 18.4 % during the study period. The reduced mortality was in line with improvement occurring also in non-septic patients. This study also underlines the influence of co-morbidity on unfavourable outcome from sepsis. In younger patients without co-morbidities the mortality was less than 5%. However, data from the Swedish Intensive Care Unit Registry (SIR, www.icuregswe.org) during the period 2008-2013 does not show a corresponding decrease in mortality, neither in the general ICU population, nor in patients with sepsis. While 30 day mortality rate in the general ICU-population were approximately 15 %, the corresponding figure for patients with severe sepsis and septic shock was approximately 30%, and even higher if septic shock alone was considered.

The innate immune system, a very brief overview

The immune system is our defense against pathogenic microorganisms such as parasites, viruses, fungi and bacteria’s. It includes unspecific defense mechanisms, such as the skin, mucus membranes and cilia which prevent pathogens from entering the bloodstream. In addition to these mechanical barriers, unspecific chemical factors such as fatty acids secreted on the skin, antibacterial peptides, and the pH of various body fluids are toxic to some microorganisms and constitute the unspecific, very first line of defense (45-48). These barriers are frequently penetrated or violated in the intensive care setting, through cannulation of veins or arteries, insertion of tubes into the airway, ventricle and other orifices. In addition, routine therapeutic measures as administration of ulcer prophylaxis and antibiotics set the primary defense mechanism out of play. Luckily humans, as well as other species, have additional defense mechanisms.

The more specific immune system can be divided into the innate and the adaptive immune systems, both containing humoral and cellular components. The innate immune system is usually called the first line of defense and it come into action if the mechanical barriers are penetrated by intruders. It is a phylogenetically old and well conserved system with vertebrates employing similar defense components as arthropods, plants and sea urchins, which implies a defense mechanism worth conserving through evolution (49, 50).

Microorganisms display on their surfaces structures that collectively are called Pathogen Associated Molecular Pattern = PAMP (51, 52). These are often carbohydrate-rich molecules (lipopolysaccharides, mannose) or nuclei acids or peptidoglycans arranged in repetitive patterns that members of the innate immune system recognize as non-self, thus constituting a threat to homeostasis. Circulating pattern-recognizing molecules like mannose-binding lectin (MBL), immune globulins (IgM, IgG), C-reactive protein (CRP) and complement factors adhere to microorganisms thru binding to PAMPs (53-55).

Introduction

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Cells of the innate immune system, commonly called leucocytes, are formed in the bone marrow and after differentiation the majority migrate to different tissues, while approximately 10 % are found in the blood steam. They are sub-grouped in neutrophils, eosinophils, basophils, mastocytes, monocytes, called macrophages when residing in tissues, and natural killer cells (NK-cells) (Fig 1) (56). They have slightly different functions but have in common surface structures, receptors, capable of recognizing PAMPs. These receptors are commonly called Pathogen Recognizing Receptors (PRRs) and to date several families of PRR are known, the most widely studied being the Toll-like receptors (TLRs) (57-60). Toll-like receptors are trans-membrane structures as is C-type lectin receptors (CLRs) while Retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), NOD-like receptors (NLRs) and the cyclic GMP-AMP synthase, a DNA sensor, are cytoplasmic proteins (61-63). Other non-immune competent cells such as epithelial cells, endothelial cells, and fibroblasts also display PRRs (52).

Figure 1. White blood cells.

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The binding of PAMPs to PRRs initiates complex signaling pathways including the release of various cytokines, chemokines, vasoactive amines, adhesion molecules, eicosanoids and products of proteolytic cascades.

Leucocytes, endothelial cells and fibroblasts do not only display PRRs on their surface, but also cytokine and chemokine receptors. Cytokines and chemokines are pleotropic small peptides that modify vascular endothelial permeability, induce the production of acute-phase proteins and mediate immune cell recruitment in a way that ensures that the appropriate leucocytes are recruited for the mission to finally destroy the intruder, through phagocytosis or lysis (64, 65).

Highly simplified, the recognition of PAMPs, subsequent binding to PRRs and the resultant release of cytokines, chemokines and other pro-inflammatory mediators are the chain of events that initiate the inflammatory response.

What follows is an explosion of events where additional cells and cascade systems are activated, among others kinin-kallikrein-, complement-, eicosanoid-, and the coagulation system including thrombocytes. The adaptive immune system is also activated including B- and T lymphocytes, and antibody formation, but this part of the immune system will not be further reviewed in this thesis.

The interplay between cells and mediators in and between various cascade systems is very complex and incompletely understood. There are numerous mechanisms of amplification and inhibition, feed-back- and feed-forward-loops and “cross-talk” between cells of the immune system, both tissue bound and in circulation, and also with endothelial and epithelial cells, fibroblasts and adipocytes, which are mediated by cytokines, chemokines and their receptors. The exact function of a particular mediator is influenced by the producing cell type, the responding cell type and the phase of the immune response (66-68).

Inflammation

Inflammation is derived from the Latin word inflammatio which means fire, or inflammo I ignite, set alight. Inflammation is the body’s response to injury, irrespective of cause. It involves vascular, neurological, humoral and cellular responses at the site of injury. Traumatic injuries, burns, surgical trauma and infections have in common a disruption of tissue integrity and exposure of molecules that initiate the response previously described. The four cardinal symptoms of inflammation are described in ancient literature as rubor (redness), calor (heat), dolor (pain) tumor (swelling) and the fifth symptom which may be a more modern insertion, function laesa (loss of function) (69, 70). The intensity and extent of the inflammatory response depends on the severity of the injury and the reactive capability of the host. Inflammation can be confined to the redness, warmth, tenderness and swelling around a superficial cut in the skin but can also involve the whole body, affecting every bodily function, a state referred to as Systemic Inflammatory Response Syndrome (SIRS), a syndrome commonly seen in patients cared for in intensive care units (71, 72). To assess whether a patient is

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suffering from SIRS due to an infection or due to other causes is not always easy in clinical practice, but nonetheless crucial for the outcome of the patient. Early antibiotic treatment is still the mainstay of treatment for patients with sepsis (73, 74). In order to avoid superfluous antibiotic use for patients not in need of such treatment and the risk of subsequent antibiotic resistance, many efforts have been made to find markers for early detection of sepsis to guide the distinction from SIRS of other causes. Hitherto, no such marker is available but an immense aggregate of knowledge into the physiological events governing the inflammatory response has emerged since ancient days. The numbers of known inflammatory markers, cytokines, cheomokines, and their receptors has literally exploded the last two decades (75, 76).

To cover the multitude of cytokines, receptors and related substances is not possible in this thesis. The following is a brief introduction to some of the key players in inflammation and sepsis development.

Cytokines

The most studied cytokines up to date belong to the group of interleukins (IL), tumor necrosis factor (TNF) and interferons (IFN), all central to the stimulation of innate immunity and initiation of inflammation. There are numerous other peptides classified as cytokines, such as macrophage migration inhibitory factor (MIF), transforming growth factors (TGF), insulin growth factors (IGF), and Colony-stimulating factors (CSF), among others, that will not be discussed here (77) .

Cytokines can be classified based on the nature of the immune response, with individual cytokines performing specific roles dependent upon cell type from which they are secreted and location at where they act. They can further be sub classed based on identification of common structural motifs and genetic information. Even so, with rapidly increasing discoveries of new substances there is confusion in terms of nomenclature (66, 78).

Interleukins (IL)

The name interleukin was suggested in 1979 and can be translated in the meaning “communication between leucocytes” Inter-between, and –leukin (leucocyte) (79, 80). Many of these proteins are produced by, and act on leukocytes but they can also be secreted by other tissues. They exert complex immune-modulatory functions, including cell proliferation, maturation, migration and adhesion (81-83).

Based on crystallographic data of distinct structural motifs, the known ILs can be divided into four major groups, the IL1-like cytokines, the class I helical cytokines (IL4-like, γ-chain and IL6/12-like), the class II helical cytokines (IL10-like and IL28-like) and the IL17-like cytokines. In addition, there are ILs that have not, yet, been classified in any of the above groups. In total, the human IL gene family contains 55 known IL and IL-related genes (66).

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In the context of sepsis, interleukins are commonly classified after biological function as either pro- or anti-inflammatory. Pro-inflammatory interleukins include IL-1 and Il-6 while IL-10 and IL-12 are described as anti-inflammatory. The most widely studied of the interleukins is the IL-1 family, now including 11 members: IL-1α, IL-1β, the IL-1 receptor antagonist (IL-1Ra), IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36Ra IL-37 and IL-1Hy2, some of which have anti-inflammatory properties. Most thoroughly studied are IL-1α, IL1-β and the receptor antagonist IL-1Ra, all considered central in innate inflammatory and immune responses (84). The expression of IL-1α is constitutive in many cell types, while IL-1β expression is induced in response to microbial molecules or PAMPs, although it can also stimulate its own expression in an autocrine fashion. Single nucleoitid polymorphism in the interleukin family are known, of which several have been associated with different disease conditions (84).

It has been shown that infection and sepsis are associated with altered IL-gene expression, with decreased expression of IL-2, IL-7, IL-23, INF-γ and TNF-α and greater expression of IL-10 and IL-27 compared to controls (85). However, there are inconclusive data regarding the association between single nucleotide polymorphism (SNP) in the IL 1-gene and susceptibility for sepsis (86). Clinical trials with specific antibodies blocking the IL-1 receptor have not been successful in sepsis patients (87-89) but therapy with antibodies blocking IL-1R1 are in clinical use for patients with rheumatoid arthritis (90).

Tumor necrosis factor (TNF)

When discovered in 1975, TNF was initially described as an endotoxin induced-glycoprotein able to induce necrosis in certain tumors, hence the name (91). It is now considered as one of the most important cytokines in the regulation of inflammation and immunity. TNF induces cytokine production, activation or expression of adhesion molecules and growth stimulation, and is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. These pro-inflammatory activities are in part mediated through activation of the transcription factor NF-κB (92). Most ligands are synthesized as membrane-bound proteins, but limited proteolysis can generate soluble forms.

TNF-α is the prototypic member of the TNF superfamily of type II transmembrane proteins that to date includes at least 29 receptors and 19 associated ligands (92). Tumor necrosis factor is a 26 KDa protein expressed by activated monocytes/macrophages, activated NK and T cells, but also by non-immune cells such as endothelial cells and fibroblasts. After cleavage, a 17 kDa soluble TNFα (sTNFα) ectodomain is formed and it is trimers of sTNFα that activate TNF receptors (93).

Chronic and acute inflammatory states such as rheumatoid arthritis and severe sepsis are associated with excessive or inappropriate TNF-expression, and

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mutations in TNF ligands and receptors have been described in rare hereditary disease states, including a condition called TNF-R1-associated periodic fever syndrome (TRAPS, TNF-R1)(94, 95).

The human TNF-gene was cloned 1985 and recombinant anti-TNF is now an established therapy in patients with certain autoimmune disorders (96, 97). However, the therapeutic efficacy of anti-TNF is not sufficiently proven for it to be considered as a standard treatment of sepsis in humans (36, 39, 40, 98-103).

Interferons (IFN)

Interferon was first discovered in 1957 during an in-vitro study on the interference of heat-inactivated influenza A virus on growth of live viruses.It was described as a factor able to interfere with virus replication. The name interferon was suggested by the discoverers (104, 105).

In addition to the antiviral properties, IFNs regulate cell growth and are important regulators of the innate and adaptive immune response (106-110). They have been classified into three major types based on the type of receptor through which they signal (111).

Interferon type I binds to a specific cell surface receptor complex known as the IFN-α receptor (IFNαR) that consists of IFNαR1 and IFNαR2 chains. The type I interferons present in humans are IFN-α (of which there are 13 forms), IFN-β, IFN-ε, IFN-κ and IFN-ω (one form each).

Interferon type II binds to IFNγR that consists of IFNγR1 and IFNγR2 chains. In humans there is only one known form of typ II interferon, designated IFN-γ .

Interferon type III signals through a receptor complex consisting of two chains. The first chain,IL-10Rβ, is expressed by most cells and this chain also partly forms the receptors for IL-10, IL-22 and IL-26. The second chain is an IL-λ specific IFN-λR1 expressed on selective cell types (63, 112, 113)

Type II interferon is induced by cytokines such as IL-12, and its expression is restricted to immune cells such as T cells and NK cells, whereas Type I and Type III interferon exhibit several common features and appear to be co-produced by virtually all cell types upon receptor recognition of viral components, especially nucleic acids (114, 115).

There are a number of genetic deficiencies related to INF-mediated immunity which renders the carriers predisposed to mycobacterial or viral infections (111).

In 1980 the first human fibroblast and leukocyte IFN cDNAs were cloned (116-118). Treatment with recombinant INF-γ for what is called anergy of monocytes or immunoparalysis has been studied in the intensive care setting, including patients with sepsis (119-123). Although promising in smaller trials in selected patients, it is currently not considered as a standard therapy in sepsis treatment. To date, the primary clinical use of interferon is the type 1 INFs α and β in the treatment of hepatitis C and in multiple sclerosis, respectively (124, 125)

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Chemokines

The name is derived from the Greek word for movement (-chinos), in the meaning of an ability to chemically attract other cells in the immune system (126, 127). They are also named chemo-attractants and have the ability to promote migration of endothelial and epithelial cells as well as leucocytes. They are secreted by several cell types, not only immune competent cells (128). Chemokines in humans are currently a family of 48 known members of small, approximately 8 to 14 kDa proteins.

Their 3-dimensional shape is determined by four cysteine residues in conserved locations in the N-terminal. In humans there are four groups of chemokines, classified as CXC, CC, CX3C or C depending on the spacing of their first two cysteine residues in the N-terminal. They are suffixed with an L designating ligand, and a number of the ligand. Their receptors, of which currently 19 are characterized, are composed of seven-transmembrane G-protein-coupled receptors (GPCRs) (64). The receptors are also divided in four families, analogous to the binding ligand. Thus, the nomenclature for chemokines is, e.g.: CCL1 for the ligand 1 of the CC-family of chemokines, and CCR1 for its respective receptor (78, 129) .

Of greater clinical interest is the classification based on function. Chemokines and their receptors can be classified as homeostatic, inflammatory, or both (78). Homeostatic chemokines are constitutively expressed and include CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12 and CXCL13. They are important for many physiological processes under normal conditions, such as directing cell migration of lymphocytes and leucocyte for surveillance and maintenance purposes, and in wound healing and angiogenesis.

The expression of inflammatory chemokines is induced by inflammatory stimuli at the local site. For instance interleukin-1, tumor necrosis factor and interferon-γ can induce various inflammatory chemokines, including CCL2, CCL3, CCL4, CCL5, CCL25, CCL27, CXCL9, and CXCL10, which attract and activate cells from both the innate and adaptive immune system (130).

Overexpression of various chemokines has been associated with chronic disease states such as rheumatoid arthritis, SLE, psoriasis, chronic obstructive pulmonary disease, arteriosclerosis, multiple sclerosis and cancer (131, 132). There are also reports on genetic mutations with impaired chemokine function, causing impairment of pathogen clearance (133, 134) or affecting tumor progression (135).

Several phase 1 and 2 trials with blocking agents have been conducted in different settings including arteriosclerosis, HIV, RA, cancer and pulmonary disease, far from all with encouraging results. However, a CCR5 targeting drug for treatment of patients with HIV and a CXCR4 targeting drug for stem cell mobilization in bone marrow transplantation have been approved by the Food and Drug Administration (FDA) in USA. Still, the success awaits for chemokine-therapy in sepsis (78).

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Complement

In 1896 a heat-labile component of serum was discovered, that could “complement” the antibacterial properties of antibodies. The component was named complement by the discoverer Bordet (136). Today the complement system comprises more than 30 proteins, circulating as inactive precursors in plasma or bound to cell membranes. They represent a vital component of innate immunity and a link to adaptive, anti-body mediated immunity. Their inflammatory actions include opsonisation of microorganisms, chemotaxis of leucocytes and lysis of bacteria and other cells, as well as augmentation of anti-body response and augmentation of immune memory. Their anti-inflammatory action involves “to clean up the mess afterwards” by clearance of immune complex and apoptotic cells (137).

The nomenclature of complement reflects the order of discovery which does not always reflect the sequence of action. Initially the letter C together with a number designated the first discovered inactive complements, whereas activated factors were suffixed with a small letter (a, b, c etc). The discovery of new factors and new pathways required additional labels. As for other cytokines, the nomenclature is not intuitively comprehensible (138, 139).

When triggered by a stimuli, the normally inactive complement proteins become activated through a well co-ordinated sequential enzyme cascade. There are three or perhaps four (138) different pathways of activation; the classical, alternative and lectin. The initiation of each pathway differ but all converge to complement protein C3 and are followed by a common cascade (C5-9). The classical pathway requires interaction between complement factor C1q and anti-bodies attached to pathogens. Through binding with other complement factors, a complex called C3 convertase is formed, which all pathways have in common and that finally culminate in the main effects of the complement system thru formation of C4a/C3a/C5a (anaphylatoxin), C3b, C4 (oposonin) and C5b-C9 (the membrane attack complex, MAC).

The Lectin pathway is analogous to the classic pathway, but instead of a Cq1-antibody complex, the activation is initiated by Mannose Binding Lectin (MBL) in complex vid MBL-associated serine proteases (MASPs) and this complex binding to pathogen associated structures (PAMS).

The alternative pathway includes a pre requisite for an amplification loop and is initiated quite differently by a constitutively low-level, spontaneous hydrolysis of C3 to the C3b analog. Through serial complex-bindings and cleavage, an alternative-pathway C3-convertas is activated which in turn converts C3 to C3a and C3b. Thus converted C3b can by serial bindings form a complex able to activate the alternative pathway-C3convertase, thus forming an amplification loop (140).

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Studies on septic shock in humans have shown increased levels of C3a and C5 which are considered important mediators of increased vascular permeability, hypotension and activation of coagulation. In some of the studies elevated levels of C3a were associated with increased mortality (141-143), or with higher APACHE II score (144), whereas others have not found such associations (145, 146). Levels of C5a were also increased in the study from Furebring, but contrary to findings in animal studies the C5a-recptor on human granulocytes showed reduced expression, a finding that question the potential use of inhibitors of these receptors in the treatment of human septic shock. Although there are case reports on successful treatment with C5a-inhibitors in patients with Escherichia coli-induced hemolytic uremic syndrome (HUS) (147), the only approved indication for C5a-inhibitors is paroxysmal nocturnal hemoglobinuria (PNH). Substitution with C1-inhibitor (C1-INH) has been studied in septic shock with no detrimental effect (148) but currently its use is only approved for hereditary angioedema (HAE). Thus, therapeutics interfering with the complement system are still confined to a small set of conditions (149).

Coagulation

Coagulation includes both cellular and humoral components. Thrombocytes (TC) are the smallest blood cells, derived from megakaryocytes in the bone marrow. They have no nucleus and their lifespan is short, approximately 10 days. Normally they are disc shaped but activated they transform and become more spherical with long dendritic extensions facilitating adhesion. Younger thrombocytes have better functionality than older TC´s. They display different receptors on their surface and contain granule in their cytoplasm with factors primarily enhancing coagulation and adhesion (150).

Historical perspectives and the nomenclature of coagulation factors are reviewed by Wright, Giangrande and Shapiro (151-153). Analogous with the complement system, coagulation factors circulate in the blood as precursors in an inactive form which through coordinated sequential enzyme-induced cleavage become activated, a process called the coagulation cascade. The coagulation can be activated in two different ways, the intrinsic, and extrinsic pathway which converge by the activation of coagulation factor X to factor Xa. Factor Xa then initiates the common pathway. The intrinsic pathway starts with the activation of factor XII to XIIa and ends with the formation of a complex of activated factors, VIIIa, IXa, Xa and calcium whereas the extrinsic pathway is initiated by the exposure of tissue factor (TF) to factor VII which becomes activated and subsequently forms a complex of TF, factor VIIa, Xa, and calcium. The common pathway is initiated when factor X is activated (Xa) and then through a series of complex-formations and enzyme induced-cleavage, thrombin is formed, the master regulator of the coagulation (154). Thrombin cleaves fibrinogen which first form soluble fibrin monomers that subsequently polymerize and form a fibrin clot at the

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site of the injury. The clot then becomes reinforced through activation of factor XIII by thrombin. Activated factor XIII also activates thrombocytes that further enhances the clot formation and activates factor V and VIII which in a positive feed-back loop further activates the coagulation cascade. This step serves to repair the initial damage and also encapsulate potential microorganisms. To avoid total obstruction of blood flow through the blood vessels, the pro-coagulant phase is followed by an early fibrinolysis due to increased expression of plasminogen-activators (t-PA and u-PA). This anti-coagulant phase is rapidly inhibited by release of plasminogen activator inhibitor-1, the anti-fibrinolytic phase, to prevent re-bleeding. Thus, there are several possible steps of interference and amplification in the coagulation system. In sepsis the main activation of coagulation is through the extrinsic pathway, starting with the expression or exposure of TF. Until recently, the common notion has been that bacterial products (endotoxin, cytokines or PAMPS) activate monocytes, endothelial cells, and also thrombocytes, to express tissue factor (155). However, this notion is under debate and data indicates that it is only the monocytes that constitutively express TF, which is normally encrypted but becomes unmasked upon stimulation (especially by IL-6). Thrombocytes might be a part of this process (156). Another possibility is a microbe-specific activation of coagulation occurring via the FXII-dependent contact driven (intrinsic) pathway, upon binding of FXII to negatively charged structures, abundantly expressed on bacterial surfaces (157). Nevertheless, the inflammatory and pro-coagulant host responses to infection are intricately linked. Coagulation abnormalities are common in sepsis, which in its most dramatic forms leads to disseminated intravascular coagulation (DIC) that subsequently may lead to multiple organ failure (158).

Thrombocytopenia is associated with severity of illness in critical care and sepsis patients and seems to be related to unfavourable outcome (159-162). Not only low platelet counts but a drop (160) and, in a unselected intensive care population, inability to increase platelet count during ICU-stay were associated with poor outcome (163). One study on surgical intensive care patients that presented data stratified on gender did not find gender associated with thrombocytopenia (164).

Several studies have been conducted with the aim to find a remedy for the perturbation of coagulation associated with severe sepsis and septic shock. Most studies concern inhibitory factors targeting thrombin, its actions or its formation, or inhibition of tissue factor TF. The largest studies include the natural anticoagulants antithrombin (AT), Protein C (PC or recombinant activated rhAPC) and tissue factor pathway inhibitor (TFP-I1). The following is a brief review over clinical trials in this area which all have in common promising initial effects in animal models or smaller human studies.

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Recombinant Tissue Factor Pathway Inhibitor (rTFPI)

A randomized, double-blind, placebo-controlled, multicenter, phase 3 clinical trial conducted 2000 to 2001 included 1754 adult patients with severe sepsis and PKINR>1.2. Patients were randomized to receive an infusion of rTFPI (tifacogin) during 96 hour or placebo. They did not find any effect on all-cause mortality in the treatment group but administration of rTFPI was associated with an increased risk of bleeding, irrespective of baseline PKINR (37). A later randomized double-blind placebo-controlled three- armed multicenter trial, including 2,138 adult patients with severe community acquired pneumonia, compared infusion during 96 hours of rTFPI in two different dosages with placebo. They were also unable to prove an effect on 28-days mortality despite favourable effects on coagulation indices. The frequency of adverse events did not differ between groups (9).

Antithrombin (AT)

In animal studies (165-167) and smaller phase II placebo controlled randomized trials in patients with severe sepsis (35, 168-170), treatment with AT had shown promising results in terms of improved indices of coagulation and organ dysfunction and reduced 30-days mortality. A meta-analysis of the results from the human studies estimated a near 23% reduction in 30-days mortality (35). These studies were followed by a randomized, double-blind, placebo-controlled, multicenter, phase 3 clinical trial during 1999 to 2000 including 2314 adult patients with severe sepsis. Two equal groups were randomized to receive AT (30 000IU in total) or placebo during 4 days. They were unable to find an effect on 28-day mortality from treatment with AT in the studied dosage and there were an increased risk of hemorrhage in patients simultaneously treated with heparin (10).

Activated Protein C

In 2001 recombinant human activated protein C or drotrecogin alfa (activated) DrotAA) was approved by FDA for the treatment of severe sepsis. This approval was based on results from one randomized, double-blind, placebo-controlled, multicenter, phase 3 clinical trial. Patients with sepsis were randomly assigned to treatment with an infusion of DrotAA during 96 hours or placebo. The study was designed to include 2280 patients but was stopped early because of efficacy. At that point 1690 patients had been included (840 in the placebo group and 850 in the DrotAA group). They found beneficial effects on coagulation with signs of reduced thrombotic activity (reduction of D-dimers) and reduction of inflammatory markers as IL-6. They also found a significant reduction in mortality, 24.7% in the treatment group compared to 30.8% in the placebo group. The incidence of serious bleeding was relatively low but higher in the DrotAA group than in the placebo group (3.5 percent vs. 2.0 percent, P=0.06) (2).

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As mentioned in the prolog the results from this study was used to heavily market the drug (Xigris®). However, subgroup analyses indicated that it was the patients with more severe forms of sepsis (APCHE II >24 or more than one sepsis related organ dysfunction) that benefitted most from treatment with DrotAA. This prompted the FDA to restrict the use of the drug to “adult patients with high risk of death” until further data could confirm or discard an effect in less severely ill patient and in children. Further trials including less severely ill adult patients and children were initiated. Both studies were stopped early, this time due to futility (37, 171). Further, data from the trial on adults could not confirm beneficial effects in high risk patients. In 2007 the European Medicines Agency requested an additional randomized placebo controlled trial with the motivation that there were sufficient doubts regarding the efficacy and safety in the use of DrotAA to warrant a new investigation. From March 2008 to August 2011 patients were enrolled at 208 sites in Europe, North and South America, Australia, New Zealand and India.

The inclusion criteria were septic shock, strictly defined, with evidence of hypoperfusion. They recruited 1697 patient out of 27816 potential patients and were able to assess the primary outcome, mortality 28 days after randomization, in 1680 patients. Over 70% of patients were recruited at European sites, and approximately 14% from North America and 14% from other countries. The conclusion was that DrotAA did not significantly reduce mortality at 28 (26.4% vs 24.2%) or 90 days (34.1 vs 32.7), regardless of severity of disease or Protein C concentration at inclusion. Serious adverse events were rare and did not differ between groups (12). Xigris® is no longer registered as a drug for treatment in sepsis.

Despite initial studies showing positive effects on sepsis associated coagulation disturbances and encouraging results regarding outcome, none of the drugs above are currently considered as standard treatment in sepsis. Even if it have been performed, none of the studies have reported results stratified for sex.

Endothelium

The endothelium is the innermost layer of the vascular system and constitutes a vast surface area in contact with the blood and its components. Instead of representing a passive barrier endothelial cells are indeed highly adaptive and interactive, and crucial for maintaining normal hemostasis (172). The understanding of its importance in the pathophysiology of inflammation and innate immunity has steadily increased during the last decades (173). Besides its importance for the coagulation system, as part of the immune system, endothelial function includes directing and enhancing the migration of blood cells into the tissues by expression of adhesion molecules and production of chemo-attractants. The endothelium is also important for the regulation of blood pressure, the microcirculation and vasopermeability (174). Under normal conditions the endothelium inhibit coagulation through different anti-thrombotic mechanisms

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such as expression of antithrombotic molecules and inhibition of thrombocyte aggregation through production of prostacyclin and nitric oxide which also are involved in vasoregulation. (175, 176). Under normal conditions endothelial adhesion molecules are not expressed and the endothelial surface serves to reduce friction, maintain optimal rheological conditions to ensure adequate microcirculation. When activated due to trauma or inflammatory stimuli (IL1α, IL1β, TNF-α, IL-6, activated complement factors) the endothelium may transform to a pro-coagulant state with loss of anti-thrombotic molecules. Whether endothelial cells themselves express tissue factor or not is under debate (156), but TF is involved in the endothelial transition to radically increase the production of inflammatory mediators, chemo-attractants, vasoactive substances and expression of adhesion molecules (177, 178). This results in increased leukocyte adhesion and transmigration, increased capillary permeability, a shift in the hemostatic balance towards the procoagulant side and an alteration in vasomotor tone. All of these alterations serve to direct the first line of defense to the site of the injury to incapacitate the intruder (158). In the process of bacteria killing, proteolytic enzymes, peroxidases, reactive oxygen species and other cytotoxic substances are released from activated neutrophils and macrophages which cause damage to endothelial cells with subsequent exposure of TF expressed by cells underlying endothelial cells. Exposed TF can thus contribute to a perpetuation of the inflammatory process if not inhibitory mechanisms modulate the process. In certain conditions associated with properties of both the invading microorganism and the host, this activated state may become overactive and encompass sites distant to the injury. This distant interference can affect organ functions and lead to multiple organ failure (172-174, 179, 180). This was what happened to the woman in the prolog.

Leptin

Having been hypothetically postulated in 1953 and its existence supported by animal experiments (parabiosis) in 1959, it was not until 1994 that the gene encoding this factor was cloned (181-183).

A spontaneous mutation occurred in laboratory mice in 1949 that rendered a cohort of mice severely obese (184). The trait was inherited in an autosomal recessive fashion and the mutation was localized to chromosome 6. The gene was designated obese (ob). In 1966 a similar syndrome was identified in mice with another mutation, located on chromosome 4 and designated diabetes (db)(185). Mice homozygote for the obese-gene (ob/ob) or the diabetes gen (db/db) display similar symptoms of overeating, early onset obesity, insulin resistance or diabetes, infertility and reduced locomotion (186). In 1994 the obese gene was cloned and it was shown that it encoded a protein, that (ob/ob) mice are devoid of (183). When exogenously administered the protein was found to correct the hyperfagia and obesity in (ob/ob) mice. Since it made the animal thinner, the name leptin was

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proposed, derived from the Greek root leptόs, meaning thin (187). The (db/db) mice, who have high circulating levels of leptin, did not respond to exogenous leptin treatment and later it was found that this was related to a defect receptor function (188). The discoveries concerning the (ob/ob) and (db/db) and the use of these mice in laboratory experiments has since been the mainstay for research in the area of satiety, metabolism, obesity and diabetes (189). In recent years the interplay between metabolism, obesity and inflammation has become increasingly clear and adipocyte derived cytokines, such as leptin and adiponectin are thought to represent links between metabolism and inflammation.

The protein

Leptin is a protein of 167 amino acids with extensive homology among vertebrates which suggests a highly conserved function. After splicing of a signal sequence it is a 146 amino acid, 16 kD protein normally circulating in blood (183, 187). Threading analysis and its crystal structure reveals a three dimensional four-helical bundle structure strikingly similar to cytokines belonging to the long-chain helical cytokine family (IL-6, IL11, IL12, LIF, G-CSF, CNTF and oncostatin), even though the primary amino acid sequence lack homology with these or other proteins (190).

Factors affecting leptin production or clearance

Leptin is mainly expressed by white adipose tissue and its serum concentration is directly correlated with fat mass and adipocyte size. For a given fat mass or BMI, women have higher circulating levels of free leptin than men (191-193). Leptin decreases with age independent of BMI, with a more pronounced decrease in women (194).The levels of leptin are influenced by sex- hormones as estrogens and testosterone (195, 196). Ethnical influence on leptin levels are reported with higher levels in South Asians and Asian Indians than in Caucasians or Europids (197, 198). Insulin, cortisol, growth hormone (199, 200), and leptin itself (201, 202) affect leptin levels. Several other factors influence leptin production as well. For instance, glucose and lipid infusion induce leptin expression in skeletal muscle in rodents (203) and the beta-agonist isoprenalin is shown to inhibit leptin production in cultured human adipocytes which could be reversed by propranolol (204). Interesting, since both glucose and lipid infusions as well as infusions with beta-agonists is common in the treatment of ICU-patients, but data on humans in this respect is hard to find. Alcohol and smoking also affect leptin levels (205, 206). Cytokines such as tumor necrosis factor (TNF), leukemia inhibitory factor (LIF), and interleukin 1 (IL-1) are known to stimulate leptin synthesis and will be discussed later (207, 208)

There is a normal physiological circadian variation in the secretion of leptin with the highest concentration in the middle of the night, opposite to what is seen

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in cortisol (194, 209). The circadian variation in leptin secretion is abolished in sepsis (210).

Other tissues found to express leptin are the bone marrow, ovary, mammary gland, stomach, lymphoid tissue and skeletal muscle. However, these organs express leptin at much lower levels than white adipose tissue (183, 201, 203, 211-214).

Leptin is mainly cleared by the kidneys and elevated levels are seen in end stage renal disease (215-217). The liver does not seem to be involved in the clearance of leptin since there is no net flux over the splanchnic bed (218). However, higher levels of leptin are reported in patients with liver cirrhosis. This has led to the notion that other cells than hepatocytes may be able to express leptin after stimulation or trauma. Data from animal experiments support this theory (219-221).

The Leptin receptor (LepR, previously denoted Ob-R)

The leptin receptor LepR was first isolated from mouse choroid plexus by expression-cloning and was identified as a member of the cytokine-1 family of receptors (222). Soon it was confirmed that LepR had signaling capabilities of interleukin 6-type cytokine receptors (223). Today there are six known sub types or isoforms of leptin receptors which all have the same extracellular ligand-binding domain at the amino-terminus but differ at the intracellular carboxyl terminus (188, 224). The receptors can be divided in three classes: secreted (or soluble), short, and long form. The single soluble isoform, LepRe (sOBR), has only extracellular domains whereas the other five isoforms (LepRa, LepRb, LepRc, LepRd, and LepRf) also have common trans-membrane domains. However, only LepRb (the long from previously OB-RL) contains intracellular motifs that enables the activation of the signal transducers that ultimately results in transcription of DNA (Janus-kinase JAK-STAT) and other signal transduction pathways (223, 225-228).

The soluble form LepRe is mainly secreted by the liver into the blood stream and by binding to leptin it regulates the concentration of the free, biologically active form (229).

The short forms (ie m-RNA for the receptor protein) (LepRa, LepRc, LepRd, and LepRf) (OB-RS), are detected in the choroid plexus, the lungs and kidneys and in many other tissues but to a lesser extent. The short form receptors have no known signaling capability. It has been speculated that they may be involved in transporting leptin from the blood into the CSF, where it by diffusion can enter the brain, thus exerting its actions. Another theoretical function concerns clearance of leptin (230, 231).

In contrary, m-RNA for the long form LepRb (Ob-RL) is less abundant and even though detectable in most tissues it is present in much lower levels, except for in the hypothalamus and certain nuclei of the midbrain and brain stem, areas considered vital for regulation of appetite and weigh control (225). Studies in

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genetically modified mice have demonstrated that the mediated effect of leptin through LepRb in the hypothalamus and midbrain alone is sufficient to regulate body weight, feeding, energy expenditure, and glucose metabolism, without the contribution from peripheral actions of leptin (232-235).

Action of leptin

Centrally and peripherally mediated actions of leptin include fertility and reproduction (236, 237), hematopoiesis (238), angiogenesis (239, 240), wound healing (241-243), endothelial interactions (244) cartilage and bone formation (245, 246), immunity and inflammation (247-249).

However, the main function of leptin is regulation of body weight, satiety, energy expenditure, and glucose metabolism. This is accomplished through a highly intricate circuit. White adipose tissue produces and secretes leptin into the bloodstream, which represents the nutritional status, and through binding to LepRb expressed by neurons primarily in hypothalamus, the energy thermostat, leptin affects behavior and metabolic events in order to maintain weight and metabolic homeostasis.

As evident from the global epidemic of obesity, this tight regulatory function is frequently set out of play in the modern society, and affect nearly 35% of the adult population in most developed countries (WHO statics 2013) (250). Even if an imbalance between energy-intake and energy expenditure has an impact on body weight, the fact that obese individuals have high leptin levels indicate a defect or dysfunction in the leptin-LepR signaling system. The underlying mechanisms that lead to a dys-regulation in the axis of adipose tissue-leptin-hypothalamus are not completely understood, but the term leptin-resistance is used to describe the phenomenon. Several mechanisms have been proposed to explain the condition, including defective leptin transport across the blood–brain barrier, attenuation of leptin signaling, deficiency and variations of LEP and LEPR genes, endoplasmatic reticulum (ER) stress, inflammation, excessive bioavailability of heavy metals, and other mechanisms, nicely reviewed in the following references (202, 230, 251-253).

Effects of Adiposity

White adipose tissue (WAT) contains not only mature adipocytes but also fibroblasts, endothelial cells, preadipocytes, and in particular macrophages residing in the stromavascular part of WAT. Adipocytes account for only about half of the total WAT cell content in obese individuals. Adipocytes produce and release not only leptin but a variety of pro-inflammatory and anti-inflammatory factors, including other adipokines (protein that is secreted from and synthesized by adipocytes); adiponectin, resistin, and visfatin, as well as chemokines and cytokines (254) (Fig 2).

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Figure 2. Adipose tissue and inflammation.

Adipocytes function as a reversible excess energy storage, a depot of excess nutrition. When overloaded, hypertrophy and hyperplasia of adipocytes follow, which result in cellular stress that subsequently initiate oxidative stress and inflammatory responses in adipose tissue with secretion of cytokines and chemokines and migration of macrophages. Adipocyte hyperplasia with its accompanying increase in number of infiltrating macrophages leads to increased local and systemic levels of various pro-inflammatory cytokines, including TNF-α, IL-6, IL-1b, CC-chemokineligand 2, monocyt chemoattractant protein-1 (MCP-1), and acute phase reactants as C-reactive protein, thus perpetuating the loop of chronic low grade inflammation. This is also considered the prerequisite for development of insulin resistance, diabetes and cardiovascular disease (248).

Leptin, inflammation and immunity

The role of leptin in regulation of inflammation and immunity has started to unravel during the last two decades. As previously mentioned the structure and function of leptin resembles that of other pro-inflammatory cytokines and its receptor, LepR, has structural similarities with members of the class I cytokine receptor superfamily (190, 255). The LepRb is expressed by various immune cells from both the innate and adaptive immune system, such as neutrophils, monocytes, macrophages, subpopulations of T cells and B cells, mast cells, dendritic cells (DC), and natural killer (NK) cells (223, 225, 238, 256). Through binding to its receptor in macrophages and monocytes leptin improves phagocytosis by regulat-ing oxidative stress. Leptin also induces eicosanoid and nitric oxide synthesis, acts as a chemo-attractant and increases the secretion of cytokines, such as IL-1RA, IL-

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1, IL-6, TNF-α (27), among other actions (257-260) . Leptin, like IL-6, promote acute phase production and release from human hepatocytes (261)

Leptin protects T lymphocytes from apoptosis, regulates T-cell proliferation and activation, and induces a shift towards an increase in pro-inflammatory T1 helper (Th1) cells over anti-inflammatory T-helper 2 (Th2) cells (262, 263). Thus, leptin has many different functions in inflammation and immunity, not all of which are listed above. Furthermore, the interactions between leptin and inflammation are bidirectional. Certain inflammatory and infectious stimuli, such as IL-1, lipopolysaccharide (LPS), and TNF-α can increase leptin levels, which correlate with the level of inflammation. Thus, pro-inflammatory cytokines increase the synthesis and release of leptin, which in turn helps to perpetuate the loop of inflammation (207, 264). This constitutes in part a pathophysiological explanation for chronic inflammation associated with obesity, which in turn leads to metabolic disturbances, insulin resistance, hyperglycemia, arteriosclerosis and cardiovascular disorders.

Leptin and sepsis studies in animals

The ob/ob and db/db mice is frequently used in animal experiments concerning leptin not only in association to metabolism but also in studies on the association between leptin and immune function, infections and sepsis. Mice with the ob/ob trait and starved wild life mice are said to represent chronic and acute leptin deficiency, respectively, while db/db mice and fed wild life mice often are used as control animals. Experiments on these mice models have shown that leptin is important in many aspects of immune response both innate and adaptive.

Leptin deficient mice have an increased susceptibility to different infectious agents with reduced bacterial clearance and reduced chemotactic capacity of monocytes, lymphocytes or leucocytes. This is shown in systemic infection with Listeria Monocytogenes (265) and in pulmonary infection caused by Mycobacterium tuberculosis which also was associated with reduced capacity to form granuloma and impaired IFN-γ production (266).

Reduced numbers of lung neutrophil polymorph nuclear leukocytes (PMNs), lower levels of cytokines (IL-6, MIP-2 and leukotriene), and impaired alveolar macrophage bacterial killing (AMK) was seen in starved wildlife mice with pneumonia caused by Streptococcus penumoniae, compared with fed littermates (267). Contrary to starved wildlife mice, ob/ob mice with streptococcal pneumonia had increased levels of cytokines (TNF-α, MIP-2, prostaglandin E2) and PMN counts, but they too had a defective alveolar macrophage phagocytosis and PMN killing of S. pneumonia and greater lethality compared to wildlife mice (268). Klebsiella pneumonia was associated with increased mortality in ob/ob (leptin-deficient) mice compared to pneumonia in wild-type mice, with reduced bacterial clearance and defective alveolar macrophage phagocytosis. There were no

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difference in cytokine levels (TNF-α, IL12 and MIP2) but leukotriene levels were reduced in ob/ob mice.

In the above experiments exogenously administered leptin reduced bacteremia, restored bacterial clearance, both systemically and in the lungs. In the lung models AM phagocytosis and PMN-killing capacity and levels of leukotriene were restored which improved survival. Thus, leptin-deficient mice display impaired host defense in bacterial pneumonia that may be due to a defect in alveolar macrophage phagocytosis and leukotriene synthesis (269).

Lipopolysaccride (LPS) is used to induce endoxemic/inflammatory response mediated by IL-1β and TNF-α in animal and human models. Leptin secretion is increased within hours of LPS administration in fasted animals, which is thought to be mediated by IL-1β and TNF-α (264, 270). In a study on LPS-, or bacterial-, or virus induced lung injury, leptin expression was induced in injured human and murine lungs and leptin was effective in directing alveolar airspace neutrophilia independent of other cytokines. Thus, leptin may exert a direct chemotactic effect to recruit lung neutrophils (271).

Experiments have shown that leptin deficient ob/ob mice are hypersensitive to the lethal effects of LPS and TNF-α, while hyperleptinemic db/db mice are resistant (272-274). In an experiment with hyperleptinemic db/db mice, devoid of the long-form leptin receptor, and db/db mice deficient of membrane-bound receptors (only expressing the soluble receptor form), endotoxic shock was induced through intra-peritoneal LPS administration (275). High doses of LPS (100µg) caused 50% mortality in mice with membrane bound receptors devoid of the long form, but no mortality in mice only expressing the soluble form of receptor. Both had an increase in leptin, corticosterone and TNF, and a drop in rectal temperature from lower doses of LPS (10 µg, lethal effect on ob/ob mice). Pretreatment of mice only expressing the soluble form of receptor with leptin blunted the increase in corticosterone and the drop in rectal temperature but had no effect on TNF-α. These findings indicate that LPS resistance is due to absence of membrane-bound receptors and the increased circulating levels of leptin rather than the absence of leptin signaling. The authors hypothesized that an excess of free leptin may cross-react with other cytokine receptors and in the absence of long form receptors modulate the anti-inflammatory response. They further hypothesized that leptin administration might be beneficial in reducing the low-grade inflammatory response associated with obesity or diabetes (275). As of 2014 no such treatment is in clinical practice but further studies have shown that the long-form leptin receptor is involved in the up-regulation of endothelial adhesion molecules, which leads to endothelial dysfunction and increased morbidity and mortality in sepsis models (276).

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Leptin and sepsis in humans

Bornstein et al found threefold higher circulating leptin levels in survivors of sepsis than in non-survivors. They also found the diurnal variation in leptin secretion to be abolished in sepsis patients (210). In a subsequent experimental study, the same group found no significant change in plasma leptin levels from baseline values after administration of endotoxin in humans (277). Further research from the same group revealed a strong negative correlation between mean 24 h plasma IL-6 and leptin which suggested that IL-6 was not the principal stimulus of leptin hyper-secretion in patients with sepsis (278).

Others have also reported higher leptin levels in sepsis patients and a lack of correlation between leptin levels and BMI. One of the studies reported higher leptin levels in sepsis survivors, while data on outcome were not presented in the other report (279, 280). In patients with peritonitis, insufficient elevation in serum leptin was associated with an increased risk of death (281). In a subsequent study the same group investigated the impact of leptin-gene and leptin-receptor polymorphisms on the outcome of patients with secondary peritonitis. Their conclusion was that the poor outcome in patients with -2548 and 223-polymorphism was related to lower levels of serum leptin, in line with the findings in their previous study (282). Further studies have found increased leptin levels in sepsis patients, some with correlation between leptin levels and severity of sepsis, but no reports on outcome (276, 283, 284). Higher levels in non-survivors than in survivors have also been reported, although with non-significant difference. In the same study increases in leptin levels at the onset of sepsis correlated with insulin and insulin resistance and leptin levels decreased during ICU-stay (285).

There are human sepsis studies were no significant changes in serum leptin concentration have been found and no association to outcome (286-288). In a study on patients with SIRS or at risk for MODS compared to controls, serum leptin levels were higher in SIRS patients but there were no association with severity or mortality. However, the majority of patients had SIRS of non-infectious causes (289).

Leptin levels in patients with respiratory failure and intensive insulin therapy, showed low leptin levels, but mortality was not reported (290). Thus, there are previous studies that have found an association between sepsis and increased levels of leptin and also survival-benefits associated with high levels of leptin in the acute phase of sepsis, while others have not. A relatively limited number of patients are included in the majority of these studies, and inclusion criteria and timing of blood sampling varies, making comparison difficult. What all of the studies have in common are that they have not reported their results stratified for sex.

Adiponectin

Adiponectin is a 244 amino acid, 30 kDa protein, first described by Scherer et al in 1955 and thought to be exclusively synthesized by adipocytes. It is the most

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abundant adipokine accounting for up to 0.01% of the total plasma proteins (291). Later discoveries have found expression of adiponectin in tissues as bone marrow, osteoblasts, fetal tissue, myoctes, cardiomyocytes, epithelial cells and salivary glands, but the contribution to circulating adiponectin from these tissues is considered negligible (292).

Adiponectin is structurally related to complement factor C1q and is a member of a growing family of C1q- tumor necrosis factor-α-related proteins (CTRPs), all of which contain a C-terminal globular C1q-like domain of approximately 135 amino acids, highly conserved during evolution.

The crystal structure of adiponectin globular domain is very similar to the structure of tumor necrosis factor (TNF)-α (293). There are about 25 proteins belonging to the C1q/TNF-α superfamily (294). Except for the globular domain adiponectin displays additionally three distinct domains; a signal peptide at the N terminus, a short variable region, and a collagenous domain (294). The collagene-like domain allows oligomerization of the protein. The homotrimer (LMW) is the basic circulating form but oligomerization to hexameres (MWM) and multimeres (HMW) occur and are the most abundant forms found in circulation. Full length monomeric forms and a proteolytic fragment that corresponds to the globular domain of the protein are also found but in much lower concentrations, about 1% of total adiponectin. The higher oligomers seem to have greater activity in endothelial and hepatic cells than the other forms which exert their effect in other tissues, such as skeletal muscle (295).

Adiponectin Receptors

There are two known receptors, AdipoR1 (AR1), AdipoR2 (AR2) and a third putative receptor, T-cadherin. AdipoR1 and AdipoR2 are commonly expressed, structurally related and also highly conserved between species. AdipoR1 is highly expressed in heart and skeletal muscle, whereas AR2 is predominantly expressed in the liver. Both are widely expressed in hypothalamus, pituitary gland and the brain stem, suggesting a function of metabolic regulation and energy expenditure (296). Both AR1 and AR2 are seven-transmembrane domain receptors, but in contrast to other G-coupled protein receptors, they have the carboxy terminus extracellularly and the amino terminus intracellularly (297). AdipoR1 is a high-affinity receptor for globular adiponectin and a low-affinity receptor for full-length adiponectin, whereas AdipoR2 has an intermediate affinity for both the full-length and globular species. The AR1 andAR2 receptors mediate increased activity of adenosine monophosphate (AMP) kinase and peroxisome proliferator-activated receptor-a (PPARa) ligand activities, as well as fatty-acid oxidation and glucose uptake. The regulation of AdipoR1 and AdipoR2 expression differs in responses to different stimuli (296).

Mice deficient of AdipoR1 become obese, glucose-intolerant and have de-creased energy expenditure. In contrast, AdipoR2 deficient mice are lean and

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resistant to high-fat-diet induced obesity and show increased energy expenditure (298).

The third putative receptor T-cadherin is a unique molecule that lacks the trans-membrane and cytoplasmic domains, and is anchored to the surface membrane through glycosyl-phosphatidylinositol (GPI) (299). Cadherin is commonly expressed in humans in smooth muscle and endothelial cells, most highly in central parts of the circulation, in the heart, the aortic, carotid, iliac and renal arteries (300). This receptor binds exclusively the hexameric and HMW forms of adiponectin but not the globular or trimeric forms (299). Even though T-cadherin lacks the intracellular domain needed for signal transduction, it can participate in intracellular signaling cascades by competing with AdipoR1 and AdipoR2 receptors for adiponectin binding (301).

Actions of adiponectin

As with leptin, substantial knowledge about pathophysiological actions of adiponectin has been gained from studies on mice. Mice devoid of adiponectin develop symptoms characteristic of the metabolic syndrome such as insulin resistance, glucose intolerance, hyperglycemia and hypertension (302-304). Similar relationships between adiponectin and metabolic or cardiovascular disease apply also to humans. In humans, serum levels of adiponectin are diminished in obese individuals and correlate negatively with the degree of obesity and most important to the degree of visceral fat accumulation (305, 306). Plasma levels are diminished in diabetes type 2 and are inversely correlated with insulin resistance in type 2 diabetes (307, 308). Low adiponectin levels are associated with hypertension, coronary artery disease and myocardial infarction in men, and proceed the development of insulin resistance in both sexes (309-312).

Adiponectin levels in relation to age seem to be dependent on population studied. Both race, sex and the age category affect the results. In both the Africans and Japanese, adiponectin increased with age, but in Caucasian populations there are divergent results (313-317). Both increases and unchanged levels are reported for Caucasian women, and increases in men over 65-70 years of age but not in younger men. The effect on ageing may or may not be a result of a decrease in renal function with increasing age (313, 314, 318). Differences in study design may very well contribute to some of the disagreements in study results.

There are only minor diurnal variations in serum adiponectin levels with a nadir during early morning (319). Adiponectin levels are higher in women from puberty and onward due to the inhibitory effect of androgens on the synthesis of adiponectin. Women have both higher proportions and absolute amounts of HMW forms than men (320-322).

Ethnic differences are reported with South Asians and Chinese men and women having lower levels than Caucasian and Candian Aboriginals, also adjusted for age and waist circumference (197)

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Factors known to down regulate production of adiponectin in different tissues include TNF-α, insulin, prolactin, growth hormone, glucocorticoids, and β-agonism (323-328). Plasma levels of adiponectin in cigarette smokers are presumed to be lower than in non-smokers and increases after smoking cessation (329, 330).

Moderate alcohol consumption (40g ethanol/day) improved insulin sensitivity in relatively insulin-resistant middle-aged men, an effect that the authors ascribed to alcohol-induced increases in adiponectin (331).

Adiponectin is increased in renal dysfunction and both HMW adiponectin and AR1 och AR2 have been found to be up regulated in end-stage renal disease and not related to insulin resistance or anthropometric measures (332, 333).

Adiponectin and inflammation

The notion of adiponectin as the prototype of anti-inflammatory adipokines has been modified since high levels of adiponectin are reported in a number of inflammatory conditions. There are indications that adiponectin differs in effect in immunological and inflammatory diseases compared to in metabolic and cardiovascular diseases. Conflicting results from in-vitro studies on different tissues or cells support the concept that adiponectin may have different effects depending on biological context (334). Also, adiponectin circulates in different forms, low molecular weight (LMW) and higher molecular weight (HMW) as well as proteolytic globular fragments which have been shown to activate different signaling pathways (335). Thus, the effects of adiponectin appear to depend on its molecular structure and on the particular cells or tissues studied and their state of activation.

Adiponectin induces the production of the anti-inflammatory mediators IL-10 and IL-1Ra in primary human monocytes, monocyte-derived macrophages, and dendritic cells. In addition, adiponectin significantly impair the production of the pro-inflammatory cytokine IFN-γ in human macrophages. Further, adiponectin-treated macrophages exhibit a reduced phagocytic and allo-stimulatory capacity (336). In porcine macrophages activated with lipopolysaccharide, adiponectin increased IL-10 expression and suppressed both TNF-α and IL6 production (337). In a series of experiments with human monocytes, it has been shown that adiponectin in physiological concentrations directly and dose dependently activates NF-κB, an essential transcription factor for pro inflammatory proteins (334). This effect is only exerted by high molecular weight (HMW) adiopnectin. The same group showed that adiponectin does inhibit inflammatory stimuli by LPS, but not by TNF α, and only after an initial activation of NF-κB by adiponectin (pre incubation). They also found that globular adiponectin is a direct and potent activator of NF-κB at high concentrations. Despite strong resemblance between globular adiponectin and TNF-α, the activation of NF-κB is through a different pathway than that of TNF-α. The native adiponectin has a moderate effect as NF-κB activator.

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Different Adiponectin molecule isoforms do share common effects on monocytic cells but also induce isoform-specific responses. This was shown in a study of the effect of recombinant LMW versus HMW adiponectin on apoptosis and inflammation. Only LMW-adiponectin displayed anti-inflammatory properties. Both LMW- and HMW induced apoptosis, activation of adenosine monophosphate-activated protein kinase (AMPK), and reduced macrophage scavenger receptor (MSR)A-mRNA expression. However, HMW-adiponectin induced the secretion of interleukin (IL)-6 in human monocytes and THP-1 cells but did not suppress lipopolysaccharide (LPS)-induced IL-6 secretion. In contrast, LMW-adiponectin reduced LPS-mediated IL-6 release and stimulated IL-10 secretion (335).

The activation of NF-κB by HMW and globular isoforms of adiponectin in human monocytes shown by Haugen et al. (334) are consistent with the activating effect of globular adiponectin on NF-κB in vascular endothelial cells, which in turn induces expression of pro-inflammatory and adhesion molecules (338). However, there are reports of adiponectin mediated inhibition of endothelial NF-κB signaling through a cAMP-dependent pathway (339). Differences in study results that may be related to differences in adiponectin isoforms and other experimental conditions, such as cell lines.

Adiponectin and sepsis studies in animals

Several studies using sepsis induced lung injury models, where sepsis is induced by cecal ligation and puncture (CLP), have shown ameliorating effects of exogenously administered adiponectin, with a reduction of pro-inflammatory cytokines and increased survival (340, 341).

Adiponectin knockout mice (APN-KO) had reduced survival and a more prominent pro inflammatory cytokine profile compared to controls in a CLP-model. Pretreatment with a substance that increases the plasma adiponectin concentration improved survival in wildlife but not in KO mice (342).

Adiponectin also modulates the expression of coagulation and endothelial adhesion molecules (VCAM-1 and ICAM-1, E-selectin) during experimental sepsis in male mice. Male APN-KO mice exhibit increased macrophage and neutrophil infiltration in various organs, and vascular leakage and end-organ dysfunction in the liver and kidney, but not in the lungs (343, 344).

Studies on gonadal-intact and ovariectomized female rats in lipopolysaccharide (LPS)-induced sepsis models reveal different patterns of adiponectin and adeponectin receptor expression in gonadal intact versus ovariectomized rats. Further, serum TNF-α levels and hypothalamic and hepatic IL-6 and TNF-α mRNA were higher in gonadal intact than ovariectomised rats. The response pattern also differed from studies on male rodents. This could imply that estrogen affects the modulatory effect of adiponectin and AdipoR on inflammation under septic conditions, at least in rodents (345).

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Adiponectin and sepsis in humans

Adiponectin studies in humans with sepsis are few. Some are experimental and/or include a relatively small set of subjects. The aims of the studies differ as does the timing of blood sampling. Only a few report data on mortality. Not surprising, there are conflicting results regarding adiponectin and sepsis.

Unaltered levels of total adiponectin and low and high molecular weight adiponectin, and also a suppression of whole blood mRNA for adiponectin receptors, was found in 20 healthy men and women (10/10) after LPS injection (346). No change in adiponectin was found in serial blood sampling during 24 hours from 23 (19 men/4 women) after endotoxin injection (347).

In a clinical study no difference in adiponectin levels between healthy controls and critically ill patients was found, regardless of if they had sepsis or not. However, they found that low adiponectin levels at ICU-admission was an independent predictor of short and long term survival (348).

In a study including patients receiving treatment with activated protein C, non-treated patients had higher adipoectin levels compared to healthy controls and patients treated with APC. However there were no differences in adiponectin levels between survivors and non-survivors (349). Vassiliadi also found increased levels of adiponectin in sepsis patients compared to controls, but no correlation to severity of sepsis or outcome (350). Two studies reported lower adiponectin levels in sepsis patients compared to controls, but no data on outcome (290, 351, 352)

None of the studies have reported data stratified for sex.

Mannose-binding lectin (MBL)

Mannose-binding lectin (MBL) is a 248 amino acid, 24kDa large glycoprotein, exhibiting four domains. First a short cysteine rich N-terminal region essential for effective oligomerization, the second major collagen-like domain representing the long tail of the molecule, a short α-helical neck region, and finally the large C-terminal C-type carbon hydrate recognition domain representing the prominent globular head, also termed the lectin domain (Fig 3). The name "lectin" is derived from the Latin word legere, in the meaning "to select". Mannose-binding lectin was first isolated from the rabbit-liver cytosol in 1978 and subsequently mannose-binding protein was isolated from human serum in 1983(353). The human gene encoding MBL was localized to chromosome 10 in 1989 (354, 355).

Mannose- binding lectin is mainly synthesized in the liver and secreted into the blood stream in oligomeric forms. The main portion of MBL in serum consists of trimers and tetramers of three and four triple helices of functional MBL subunits, but both higher (pentamers and hexamers) and lower oligomeric forms are found (356, 357).

Mannose-binding lectin belongs to the family of collectins which share common structural features. They are structurally and functionally related to the

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first component of the classical complement pathway, C1q, and their mission in the innate immune system is to opsonize microorganisms, to activate phagocytosis and complement activation. It is the lectin domain that binds to carbohydrates on microorganisms or apoptotic cells, while the collagenous regions are ligands for the collectin receptor on phagocytes and also mediate the activation of the classical complement pathway. Other members in the collectin family include lung surfactant proteins SP-A and SP-D. Collectins represents an ancient mechanism of host defense common with plants and invertebrates (358-360).

Mode of action

MBL binds to the surface of a wide range of microorganisms, functioning either as a direct opsonin, or through activation of the complement system, thereby enhancing phagocytosis of microorganisms by macrophages and neutrophils. Activation of the complement system can occur through the classical, the alternative or the lectin pathway. Mannose-bindning lectin circulates in the blood in complex with a C1s-like serine protease, designated MBL-associated serine protease (MASP). The activation of the complement system by MBL is mediated by MASP-2 and is called the lectin pathway (361). Additional MAPS has been discovered, MASP-1, MASP-2 and MASP-3 and a non-enzymatic protein, MAp19. They are structurally similar to C1r and C1s. MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4bC2b (362).

As previously described activation of complement factor C3 finally results in release of chemoattractants, deposition of membrane-attack-complex (MAC) on target cells and lysis to clear microorganisms and endotoxin (140)

Genetic determinants and inter individual variations in MBL levels

The protein-encoding region of the MBL gene (MBL2) consists of four exons interrupted by three introns. Exon 1 encodes the signal peptide and a part of the collagen structure, exon 2 encodes the larger part of the collagen structure of the triple helix, exon 3 encodes the neck region and exon 4 the carbon hydrate binding site (355) (Fig 3).

The promoter sequence of the MBL2 gene contains several consensus elements, indicating that MBL is an acute phase reactant as transcription is enhanced by IL-6, dexamethasone and heat shock protein but inhibited by IL-1 (354, 355, 363). Further, the MBL2 gene contains an extra alternative exon, denoted exon 0, which also may initiate transcription of the MBL2 gene (364). The main production of MBL originates from the liver and is mainly transcribed from exon 1 but approximately 10% may be the result of transcription from exon 0. Further, evidence of MBL2 transcription in the small intestine and testes are at hand, showing transcripts initiated from both exon 1 and 0. The extra hepatic production may represent a part of a local defense mechanism (365).

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Figure 3. MBL transcription with artistic freedom.

In humans a number of single base substitutions in exon 1 are known to inde-pendently reduce the level of functional MBL in serum. The single base substitu-tions are located at codon 52, 54 and 57 and are designated allele D, B and C. The normal (wild type) allele is designated A (366-368). All three alleles affect the level and function of MBL in serum, decreasing the level of functional MBL as much as 90%, even in heterozygotes. The variant proteins are more unstable and degrade to lower oligomeric forms which possess lower capacity of ligand binding and complement activation. As the effect on serum MBL is rather similar in the variant allele forms B, C and D they are often pooled together and given the common designation O (369). The instability of the variant proteins are thought to contribute to shorter half-life in the circulation and may lead not only to reduced function, but also to a reduced concentration of variant MBL in the circulation (370).

In addition, polymorphic sites in the promoter region are also known to affect the serum concentration of MBL, independent of the structural variant alleles (B, C and D). These polymorphic sites are located at positions −550, −221, and +4 in the promoter region and are designated H/L, X/Y and P/Q.

The following common haplotypes have been identified: HYPA, LYPA, LYQA, LXPA in individuals carrying the A allele (normal chromosomal background), and HYPD, LYPB and LYQC on chromosomes carrying structural variant alleles (D, B and C respectively). These seven common (classical) MBL2 haplotypes are associated with high, intermediate and low MBL serum concentrations, respectively (368, 371). Additionally at least 80 polymorphic sites have been detected, of yet unknown clinical relevance (372).

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In functional promoter analysis it has been found that the three truncated haplotypes HY, LY and LX correlate with high, medium and low promoter activity in agreement with the serum concentrations (364).

In epidemiological and statistical contexts the most common way of presenting data is to pool the structural alleles B–D to one O allele and only show the most significant promoter allele in position −221 (X/Y) which is only found on a normal A haplotype background (YA or XA) (369, 373). Epidemiological studies show various frequencies of the structural alleles and promoter polymorphisms in different ethnic groups, explaining some of the variations in MBL-serum levels in different populations (374). MBL deficiency is common, as frequent as in 10-20% of the population, depending on studied population and definition used. However, even though the structural alleles and polymorphisms in the promoter region may be the main determinants for functional MBL-levels, individuals with identical genotypes for all known MBL variants may still differ substantially in MBL levels (375, 376). Thus, the genetics governing structure, function and serum levels of MBL in humans is complex and still not fully understood, and even less is known about the clinical relevance of different polymorphisms and haplotypes.

Factors effecting MBL-levels

The constitutional MBL-level is considered to be very stable over time (years), do not display any circadian variation, and is not effected by physical exercise, or menstrual cycle (377, 378). Whether there are variations in relation to age and sex is a matter of studied cohort, but the common perception is that age and gender does not significantly affect MBL-levels, among adults. However, serum MBL levels are lowest at birth, approximately one third of the maternal levels, then increases rapidly to its highest levels during the first month of life after which there is a decline in MBL-levels until adult levels are reached at approximately 12 year of age (379). Levels stay fairly constant until midlife. Whether or when MBL-levels decline further appear to vary depending on ethnicity and age of the study population. Declining levels from the forth decade are reported from a Chinese population, from the sixth decade in a mainly Caucasian population in Australia, and in a Japanese population mean levels were fairly constant from the third decade of life (380-382). However, frequency distribution in the Japanese study changed from a bimodal pattern with peaks of MBL-levels at both very low and high values, to a distribution with lower frequency of high levels after the age of 30. In a cohort of Danish blood donors 18-64 years of age, MBL-levels were independent of age (378).

Stratification on sex in studies of MBL polymorphism or MBL-levels are scarse and when assessed the majority report no differences between men and women in serum MBL levels or in distribution of mbl2 haplotypes (378, 380, 381). However, in a Korean cohort of 129 individuals mean serum levels of MBL were higher in women compared to men (383)

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31

Studies on weight reduction after bariatric surgery in obese subjects have reported both increased and unchanged MBL-levels. Both studies included a limited number of patients. (384, 385) Weight reduction due to a low caloric diet have no influence on MBL-levels and there is no evidence that MBL is produced in adipose tissue (386)

There are studies on the associations between MBL2 polymorphism and outcomes from various conditions in smokers versus non-smokers, but if smoking per se affects MBL-levels is hardly explicitly investigated (387-389). However, in a study of periodontitis, they found significantly higher MBL-levels among heavy smokers than in non-smokers, a finding that needs to be confirmed in larger cohorts (390). Serum concentrations of MBL are significantly elevated in patients with type-1 diabetes and even more so in patients with vascular complications (391-394). However, a large and well conducted study on the association between MBL2 polymorphisms and diabetes type-1 found no association between MBL2 gene and type-1 diabetes or diabetic nephropathy. Thus, high MBL levels in diabetic patients may rather be a consequence of diabetes micro vascular angiopathia or a marker of a yet unknown factor, than the cause of diabetic complications (395).

MBL and disease

A large number of studies have examined the relationships between different disease states, and MBL levels and/or MBL genotypes (396-399). Since MBL is involved in the innate immune response, early reports showing an association between low MBL levels and recurrent infections, infectious diseases and autoimmune disorders have gained special attention in this respect. The common notion is that MBL-low level states correlate to susceptibility to different infectious diseases and worse outcome, but there are alternative views (400-406). No evidence for significant differences in infectious disease or mortality in MBL-deficient individuals versus controls was found in a large Danish population based study were they had genotyped 9,245 individuals. The follow up period was 24 years for hospitalization due to infection and other serious common diseases, and the follow up period for mortality was 8 years. They concluded that MBL deficiency is not a major risk factor for morbidity or death in the adult Caucasian population (407)

As previously stated, MBL exhibit similarities with other acute phase reactants on a transcriptional level (363). In immunologically mature individuals MBL is thought to play a part in the early stages of an infection, before the generation of the specific humoral or cellular responses (408). However, in response to surgical trauma and infection the change in MBL-levels is far more unpredictable than of other classical acute phase reactants. Compared to C-reactive protein (CRP), both slower and less obvious increases are seen, and even decreases in MBL-

Introduction

32

concentration. Whether these variations are governed by the genetic polymorphisms or relates to MBL-function is not clear (376, 408-411).

MBL and sepsis in animal studies

Partly because mice have two functional MBL genes, it was not until 2002 the first report of experiments with MBL-gene deficient mice (MBL-A KO) was published (412). These mice are apparently healthy, contrary to their relative’s ob/ob, db/db and APN-KO mice (413).

In a septic peritonitis model (CLP) they found MBL-A null mice to have better survival compared to wild-type mice and significantly decreased TNF-α and IL-6 levels in the blood and peritoneal cavity (412). In other models of Influenza A virus and Stafylococcus aureus infections, MBL-A KO mice have displayed a less severe inflammatory response, with less pronounced cytokine and chemokine production and less severe illness compared to wild life mice (414, 415). There are also studies indicating an increased susceptibility in MBL-deficient mice to infection with certain pathogens, such as o S. aureus, Pseudomonas aeruginosa, Herpes simplex virus-2, and Candida albicans. Additionally, there is evidence of a pro coagulant effect of MBL (416).

MBL and sepsis in humans

There are variable results from studies on susceptibility, severity or outcome from sepsis in relation to MBL 2 polymorphism and/or serum levels of MBL.

Garred et al found a similar frequency of MBL genotypes between patients with SIRS and healthy control subjects (417). However, MBL variant alleles were significantly more common in patients with a confirmed infection compared to those without an infection. Further, patients with sepsis carried variant alleles in higher frequency, compared to the patients without sepsis and patients carrying MBL variant alleles had a higher risk of developing severe sepsis and septic shock. They also found a linear trend in susceptibility to sepsis, severe sepsis, and development of septic shock, from the highest expressing MBL genotypes to genotypes encoding MBL deficiency, compared with that for patients with SIRS. MBL-concentration showed the same pattern with decreasing concentration corresponding to greater disease severity. Finally, variant alleles associated with and increased risk of fatal outcome (417)

In a study from Australia the median MBL-levels for the whole study group remained constant during the study period, regardless of genotype or sepsis severity. Analysis of individual patients response over time revealed that 31.3% of the patients had a positive acute-phase response, 27.3% had a negative response, while 41.4% did not display any significant change in MBL-levels during hospital stay (409).

In another study from the same group, MBL functional deficiency (C4b deposition) in adult patients with blood stream infection and pneumonia were

Introduction

33

compared with healthy blood donors. Median MBL function, but not median MBL-levels were lower in sepsis patients than in controls. No difference in frequency of MBL levels defined as deficiency or low MBL-producing genotypes were seen between cases and controls. Function (deposition of C4b) or MBL-levels did not differ between patients with sepsis or septic shock. No significant association between MBL- genotypes, MBL-levels or MBL-functional deficiency and mortality was found even though MBL-functional deficiency was associated with a high SOFA score day 3 (418).

Studies with higher frequency of MBL2-gene polymorphism in patients with severe sepsis and septic shock compared with normal healthy adults have been reported, but without significant difference in MBL-2 genotype or haplotype frequency between survivors and non survivors. There was a strong relationship between MBL-2 haplotype and plasma MBL concentration also in this study, and the mortality rate was higher among those with MBL levels <1000 mg/L compared to patients with levels >1000 mg/L with a p-value of 0.05. In this study individual plasma levels were variable and increased between days 1 and 7 (419).

By reanalysis of previously conducted studies, Eisen et al found MBL<0.5 mikrog/L to be a reliable predictor of low producing states. This study confirmed that MBL levels < 0.5 mikrog/L was associated with increased risk of death due to infection, even in the intensive care setting, especially if streptoccous pneumoniae was the infecting agent (420).

The frequency of genetic polymorphism and MBL-concentration in patients with severe sepsis and septic shock was compared to blood donors in a Korean population. Homozygosity at codons 54 (A/A) and -550 (H/H) was associated with severity, but not the outcome of sepsis, whereas low MBL levels (<1.3 microg/L) were a risk factor for death in patients with septic shock. They concluded that the genotype and serum level for MBL2 may have different clinical implications (421).

In one Spanish case-control study MBL2 genotypes did not associate with either community acquired pneumonia or invasive pneumococcal disease (422), but in another Spanish study assessing genotypic variants impact on mortality in patients with pneumococcal sepsis MBL2 polymorphism (AO/OO) was independently associated with 90-day mortality (423).

No wonder that the authors of the latest meta-analysis on the subject states that “MBL structure variants might be associated with susceptibility to sepsis but further studies with a large sample size should be conducted to confirm these finding” (424).

None of the studies have reported results stratified for sex.

Aims of the thesis

34

AIMS OF THE THESIS

To assess incidence, length of stay, cost of care, and outcomes for ICU-treated sepsis patients in a selected tertiary hospital.

To assess if there are gender differences related to characteristics,

treatment or outcome in sepsis patients requiring intensive care. To assess whether leptin, adiponectin and mannose-binding lectin

(MBL) in a pre-septic state associate with future development of ICU-requiring sepsis. Further to assess their associations with sepsis severity and outcome.

To assess if levels of leptin, adeponectin and MBL in the acute

phase of sepsis, or the change from a pre-sepsis state to the acute phase associate with in-hospital mortality.

Methods

35

“Insanity: doing the same thing over and over again and expecting different results.” Albert Einstein

METHODS

Study design

Paper I was a retrospective observational study in a single tertiary care hospital. Paper II was a prospective observational study. Papers III and IV were case-referent studies, nested within the framework of two population based health surveys, where prospectively collected blood samples were analyzed based on retrospectively identified ICU sepsis cases (Table 1).

Table 1. Summary of papers. Aim Study design Included patients Study period Main study parameters

Paper IFrequence, outcome, costs of care

Retrospectiv, observational study

81 2000-2002TISS, APACHE II, ICU-LOS, ICU- and one-month mortality

Paper II

Gender differences in characteristics, outcome and treatment

Prospective, observational study

127 2003-2005

TISS, APACHE II, SOFA, ICU- and hospital LOS, ICU-, hospital- and long term mortality, aspects of treatment

Paper III

Leptin and adiponectin as predictors of sepsis, sepsis severity and outcome

Nested case- referent study

152 patients /304 referents

1988-2008Sex-related differences in leptin, adiponectin, sepsis severity and association with hospital mortality

PaperIVMBL as predictor of sepsis, sepsis severity and outcome

Nested case- referent study

152 patients /304 referents

1988-2008Sex-related differences in MBL, sepsis severity and association with hospital mortality

Material, study subjects

All patients in the four studies were cared for in the Intensive Care Unit at the University Hospital of Umeå, and were identified for the studies based on demonstrating sepsis criteria within 24 hours of admission. The same basal inclusion criteria were used for all patients included in Papers I-IV.

Data on age, sex, referral pattern, admission category, co-morbidities, and microbiological agents was collected. Source of infection was evaluated from X-ray examinations, laboratory results from virological and microbiological analyses and cultures, and from documentation from the daily infection consultant. APACHE II and SOFA-scores were calculated on the basis of routine chemical analyses and documents of parameters of vital sign monitoring. Severity of sepsis was evaluated based on these data. ICU and in-hospital mortality were primary end points in all four studies and length of stay in ICU and hospital were secondary endpoints in Papers I and II.

Methods

36

Additional data collection included TISS scoring in Paper I and II, data on aspects of treatment in Paper II, chemical analyses of leptin and adiponectin in Paper III and analyses of Mannos Binding Lectin (MBL) in Paper IV.

Sepsis definition, main inclusion criteria

The studies included only adult sepsis patients (18 years of age or older). If sepsis syndrome was identified after 24 hours, then the patients were not eligible for inclusion in the study. Other simultaneous treatments or co-morbidities were allowed.

The criteria used in this study were consistent with those presented in 1992 (71), with specification of definitions of organ dysfunction and hypoperfusion in accordance to limits in the SOFA score (425).

Infection was defined as the presence of pus in a normally sterile body fluid; perforated viscus; radiographic evidence of pneumonia in association with the production of purulent sputum; or conditions associated with a high risk of infection (e.g. ascending cholangitis, necrotizing fasciomyositis, but not pancreatitis), or other confirmed or highly suspected infectious conditions.

Systemic inflammatory response syndrome (SIRS) was present if at least two of the following criteria were full filled:

Temperature dysregulation (>38°C or <36°C)

Tachycardia (>90 beats per minute)

Tachypnea (>20 breaths per minute or arterial PCO2<4.3 kPa)

Altered white blood cell count (>12x109/L or <4x109/L or > 10% of immature forms).

Sepsis was defined as the presence of systemic inflammatory response syndrome (SIRS) caused by an infection (or strong suspicion of infection).

Severe sepsis was defined as sepsis associated with signs of hypoperfusion, organ dysfunction or sepsis induced hypotension.

Hypoperfusion abnormalities included P-lactate >2 mmol/l, oliguria less than 0.5 ml/kg/h for more than one hour despite adequate fluid administration, and acute alteration of mental status.

Organ dysfunction was present if thrombocyte count was less than 100 x109/L, P-bilirubin more than 30 µmol/L, P-creatinine higher than 170 µmol/L and base deficit > 5 or pH<7.30 of no other causes.

Sepsis-induced hypotension was defined by the presence of a systolic blood pressure of less than 90 mmHg or its reduction by 40 mm Hg or more from baseline or a mean arterial pressure of less than 70 mm Hg for at least 1 hour despite adequate fluid resuscitation, adequate intravascular volume status or the use

Methods

37

of vasopressors in the absence of other causes for hypotension (eg, bleeding, overt sedation or presence of epidural)

Septic shock was defined as sepsis-induced hypotension resistant to fluid resuscitation.

TISS scoring

The Therapeutic Intervention Scoring System includes multiple aspects of intensive care treatment and nursing activities, which has been validated as a tool to quantify need for ICU resources and personnel workload for individual patients. The TISS assigns values ranging from 1 to 4 for a number of medical and surgical interventions to measure the intensity of care during a 24-hour period. The items fall into four groups; active treatment, ICU monitoring-personnel intensive, ICU-monitoring- technology intensive, and standard care. It has been used as a proxy for cost of care, in terms of use of personnel and resources other than medications and disposables. Several revisions have been made in order to simplify the use of the score (426-429). Though initially intended to classify severity of illness, other scoring systems have gained a wider spread in that respect, APACHE being almost contemporary in its origin.

APACHE II scoring

The Acute Physiology, Age, Chronic Health Evaluation II (APACHE II) is a scoring system developed to quantify severity of critical illness with the aim of predicting survival (430). The score has been well validated as a marker of severity of illness, though not as a prognostic tool for individual patients. It is used in this thesis since it was the best widely available tool for quantifying severity of illness in the intensive care setting, when the studies of this thesis were conceived. One criticism of APACHE II is that it is highly detailed and demanding to manage. As a research tool, it has been commonly used for many years, though the original database is now quite outdated in regard to critical care mortality. It has been revised (431, 432), but currently another severity of illness assessment tool is more commonly used in Scandinavian ICUs with a more modern validating database, the Simplified Acute Physiology Score (SAPS) (433-435). Since SAPS came into wider use after the work of this thesis began, APACHE II is used for assessment of severity of illness in this thesis. It assesses several physiological aspects on a scale from 0 (normal) to 4 (most deteriorated values), with the exception of level of consciousness which is scored from 0 (lucid) to 12 (no response, out of consciousness). It is the most divergent values of each parameter, during the first 24 hours after admission to the ICU that is included in the score. In addition to physiological parameters, co-morbidities are also evaluated and depending on if the admission is due to elective or acute surgical reasons or not the presence of a chronic illness is given a score of 2 or 5 points. Age is also included in the score (Table 2).

Methods

38

Table 2. APACHE score APACHE score 0 1 2 3 4

Temp 36.0-38.4 34.0-35.9 32.0-33.9 30.0-31.9 <30.0 (°C) or 38.5-38.9 or 39.0-40.9 or >40.9

MAP 70-109 50-69 130-159 <50(mm Hg) or 110-129 or >159

Heart rate 70-109 55-69 40-54 <40(bpm) or 110-139 or 140-179 or >179

Respiratory rate 12-24 10-11 6-9 35-49 <6(bpm) or 25-34

FiO2/PaO2 (kPa) <2.1 2.10.3.99 4.00-4.99 5.0

a-pH 7.33-7.49 7.50-7.59 7.25-7.32 7.15-7.24 <7.15or 7.60-7.69 or >7.70

S-Na 130-149 150-154 120-129 111-119 <111(mmol/L) or 155-159 or 160-179 or >180

S-K 3.5-5.4 3.0-3.4 2.5-2.9 6.0-6.9 <2.5(mmol/L) or 5.5-5.9 or 7.0

S-Creatinine 50-129 130-179 <50 300-599 >600(µmol/L) or 180-299

Renal failure 50-129 130-179 <50 300-599 >600Creatinine or 180-299

Hb 90-139 140-149 61-89 60(g/L) or 150-179 or 180

B-LPK 3.0-14.9 15-19.9 1.0-2.9 <1.0

(109/L) or 20-39.9 or score

RLS RLS 1 0RLS 2 2RLS 3 5RLS 4 7RLS 5 9RLS 6 10RLS 7 11RLS 8 12

Cronic deseaseelective surgery 2

emergency surgery or no surgery 5

AGE 44 045-54 255-64 365-74 5

75 6 Chronic diseases are defined as: Cardiovascular: (NYHA IV). Pulmonary: (PCO2 > PO2; PO2 <8 kPa; PHT 40 mmHg; ventilator dependent). Hepatic: (verified cirrosis; portal hypertension; hepatic encephalopathy; bleeding oesophageal varices). Renal: (chronic dialysis). Immune system: (corticosteroid treatment corresponding to metyl prednisolon ≥15 mg/kg for 5 days or more; chemo- or radiation therapy witin 3 months; Immunosuppressive conditions as leukemia, lymphoma, AIDS or disseminated malignancy).

Methods

39

APACHE also includes a list of admission diagnosis (the reason for admission to ICU), which is coupled to a coefficient that introduced in an equation together with the score gives an estimate of risk of death in 28 days after admission to the ICU. Only the scoring of parameters and the admission diagnosis are used in this thesis. The admission diagnosis according to APACHE II or III was used as search criteria in Papers I, III and IV (Table 4).

Sequential Organ Failure Assessment (SOFA) Score

The initial name of the score was Sepsis-related Organ Failure Assessment and the score was intended as a tool to describe and quantify the degree of organ dysfunction/failure over time in groups of, or individual patients. It was not designed to predict outcome but to describe the evolution of disease processes in critically ill patients, as objectively as possible (425). It has since been shown that there is a good correlation between higher scores and ICU related mortality, and it is applicable to all categories of ICU patients (425, 436). The SOFA score contains an assessment of six organ systems; respiration, coagulation, hepatic function, circulation, central nervous system (level of consciousness) and renal function. Scoring should be performed on a daily basis, with a summation of the most divergent values during the preceding 24 hour period. The score for each organ system range from 0 (normal) to 4 (greatest divergence from normal). Conventionally organ failure is defined as a score of 3 or more for each organ system and a score of (1)-2 is defined as organ dysfunction (437) (Table 3).

Table 3. Sequential Organ Failure Assessment (SOFA) scores. SOFA score 0 1 2 3 4

Respiration

PaO2/FiO2 (kPa) >53 <53 <40 <27 <13

ventilator support ventilator supportCoagulation

Platelets 109/L >150 <150 <100 <50 <20

LiverBilirubin (µmol/L) <20 20-32 33-101 102-204 >204

Cardiovascular No MAP <70 dopamine 5 or dopamine >5 or dopamine >15 orHypotension hyptension dobutamine (any) norepinephrine 0.1 norepinephrine >0.1

or epinephrine>0.1 orLevosimedan any dose

CNSGlascow Coma Score 15 13-14 10-12 6-9 <6RLS 1 2 3 4-5 6-8

RenalCreatinine (µmol/L) <110 110-170 170-299 299-440 >440urine output (mL/d) and >500 and >500 and >500 or<500 or<200

Cardiovascular support administered for at least 1 h (doses given are in µg/kg/min). Oxygen delivery via nasal sprongs: 1L O2:FiO2 =0.22; 2L O2:FiO2= 0.24; 3L O2:FiO2=0.26; 4L O2:FiO2=0.28.

Methods

40

Data collection

Demographics were entered in the ICU journal system, and included age, sex, referral pathways, admission category (medical, surgical- elective or acute, or obstetrical), co-morbidities, and length of stay in ICU and discharge pathways from ICU. Data on vital signs, and for Paper II, pharmaceutical and surgical or technical treatments were retrieved from the ICU-records. Pharmaceutical treatment included administration of antibiotics, anti-viral and anti-fungal drugs, immunoglobulin, corticosteroids, anticoagulants, blood and blood-products, fluids grouped as crystalloids, colloids and nutritional solutions, vasoactive drugs, sedation and enteral nutrition. Technical support included endotracheal intubation, ventilator support and renal replacement therapy. Surgical procedures included removal of obstruction (urinary, biliary or gastrointestinal), surgical drainage and debridement. The use of advance hemodynamic monitoring was also retrieved from the ICU journal. Both the ICU journal system and the hospital journal system provided results from routine chemical analyses, radiographic examinations, microbiological and serological results. The hospital record also provided information on hospital length of stay and hospital mortality. For the earliest subjects (Papers III and IV), data were collected from paper journals (starting in 1988), since digitalized patient journals were first introduced in the ICU of the cohort center in 1999, and at various dates for the hospital journal depending on affiliate clinic. Long-term mortality data were retrieved from a national database.

Northern Sweden Medical Research Bio-bank,

Papers III and IV included blood samples and data retrieved from a research bio bank. The Northern Sweden Medical Research Bio-bank (NSMRB), comprises blood samples donated from participants of population based health surveys, the Northern Sweden Health and Disease Study (NSHDS) and the Northern Sweden Maternity Cohort (NSMC), which are well described elsewhere (438-441). Following is a brief description of the separate surveys. The Northern Sweden Health and Disease Study (NSHDS) consists of 3 sub studies: the Västerbotten Intervention Project (VIP), the Northern Sweden part of the World Health Organization (WHO) study for Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA) including the counties of Västerbotten and Norrbotten, and the Mammary Screening Project (for the county of Västerbotten). All participants were asked to donate blood to the Northern Sweden Medical Research Bank (NSMRB) for research purposes. NSMC includes all women in the study area who were screened for rubella immunity during pregnancy. Remaining samples from rubella screening as well as from routine clinical serological and viral analyses have been stored at -20°C since 1975 at the Department of Virology, Umeå University. Until the 31st of December 2009, VIP consists of 85600 unique individuals with 104800 blood samples, MONICA of 10300 individuals with

Methods

41

19000 blood samples, Mammary Screening Project of 28700 individuals with 54300 blood samples and the Maternity cohort of 91000 individuals with 125000 blood samples.

Nested case-referent studies, Papers III and IV

In addition to being 18 years or older and developing sepsis within 24 hours from admittance to the ICU, in Papers III and IV, patients should have donated blood samples to NSMRB before the onset of sepsis. The cases were retrieved from the retro- and prospective studies, Paper I and Paper II. In order to improve the power of the study cases were also retrieved from a search of records of patients admitted to the ICU at Umeå University Hospital, from the 1st of January 1988 to the 31st of December 1999, and from the 1st of January 2006 to the 31st of October 2008 (Fig4). The search criteria used was sepsis related APACHE II and APACHE III admission diagnosis (depending on the year searched) and ICD 9 and ICD 10 diagnosis where primary sepsis could be expected (Table 4).

Personal identification data were cross-linked to the Northern Sweden Medical Research Bio-bank (NSMRB). By co-analyzing patients with sepsis syndrome with registries of the population based studies in NSMRB, 152 unique individuals were identified who prior to their sepsis event had donated blood samples to the medical bio-bank (NSMRB) (Fig 4).

For every patient with pre-sepsis collected blood sample, two referents from the same sub cohort were identified and matched for age, gender, smoking habits, time-point of blood sampling, and being alive at the date of the case admission to ICU. From cross-linkage with the bio bank at the Department of Virology, sera from the acute phase was found. Altogether 128 out of 152 patients had also retrievable samples collected at the day of ICU admission.

Met

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42

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IV).

Methods

43

Table 4. Search criteria of the data base according to APACHE III admission diagnosis and according to the ICD 10 classification.

Major disease classification in the APACHE III Prognostic System

Nonoperative:

Respiratory

Parasitic pneumonia

Aspiration pneumonia

Bacterial/viral pneumonia

Chronic Obstructive Pulmonary Disease

Other respiratory diseases

Gastrointestinal (GI)

GI perforation/obstruction

GI inflammatory disease

Other GI disease

Neurologic

Neurologic infection

Sepsis

Sepsis other than urinary tract

Sepsis of urinary tract origin

Haematological

Other haematological disease

Renal diseases

Other renal disease

Postoperative

Respiratory

Respiratory infection

Other respiratory disease

Gastrointestinal (GI)

GI perforation/rupture

GI inflammatory disease

GI obstruction

GI cholecystitis/cholangitis

Other GI disease

Neurologic

Other Neurologic disease

Diagnosis according to The 10th international disease classification (ICD 10)

All A and B diagnosis

J: 00-06, 10-18, 20-22, 36, 39, 98

G: 00-03

K: 65

L: 00-08

M: 00-03, 86 Corresponding diagnosis for the ICD 9 was used for patients admitted before 1990 and APACHE II for patients admitted before 2000

Methods

44

Clinical examinations and biochemical analyses from the MONICA and VIP cohort.

Data on weight, length, smoking habits, systolic and diastolic blood pressure, fasting glucose levels and post load glucose levels, total cholesterol were retrieved from the baseline examinations in the MONICA and VIP registries. In these examinations participants were classified as smokers or non-smokers. Blood pres-sure was measured in the recumbent position; adjustments were made for sitting posture blood pressure measurements. Hypertension was defined as systolic blood pressure >140 mmHg and/or diastolic blood pressure >90 mmHg and/or on anti-hypertensive medication. The presence of diabetes was based on self-reported data and/or on fasting glucose levels ≥7 mmol/L and/or post-load glucose levels ≥11 mmol/L (≥12.2 mmol/l in the VIP-cohort based on capillary plasma). Total serum cholesterol was analyzed using a bench-top analyzer or by an enzymatic method. Data regarding smoking habits, hypertension, and diabetes were not available in the maternity cohort.

Blood sampling

Baseline plasma sampling was obtained in the morning after a minimum of four hours fasting. After fractionation into plasma, buffy coat, and erythrocytes, plasma was stored at –80°C at the Northern Sweden Medical Research Bio-bank). In the Maternity cohort, blood samples were drawn from pregnant women according to routine procedures for serological testing. Sera were collected and stored at –20°C after screening for Rubella. In the acute phase, blood samples were drawn from patients at admittance to the ICU in accordance to routine serological testing. After routine analysis, sera was stored at –20°C at the Department of Virology, Umeå University.

Enzyme-linked Immunoassay (ELIZA) and Radioimmunoassay (RIA)

In Paper III, leptin and adiponectin were analyzed with a double-antibody radioimmunoassay (RIA) method (Millipore Corporation, Billerica, MA, USA). The assay’s detectable level is 0.5 ng/mL. The total coefficient of variation (CV) for leptin was 4.7% at both low (2–4 ng/mL) and high (10–15 ng/mL) levels. For adiponectin, the total CV was 15.2% at low (2–4 µg/mL) levels and 8.8% at high (26–54 µg/mL) levels. This method has shown high stability after various storage temperatures, repeated freeze and thaw cycles, and no interference with free hemoglobin or triglycerides up to 10 µg/L, and equivalent results from serum and plasma (heparin or EDTA) specimens (442).

In Paper IV, mannose-binding lectin (MBL) was analysed with an ELISA technique using a commercially available kit (MBL Oligomer ELISA Kit 029, BioPorto Diagnostics, Gentofte, Denmark), with pre-coated wells and ready-to use calibrators and working solutions. Analysis was performed in accordance with the instructions from the manufacturer. The absorbance was read on a spectro-

Methods

45

photometer (Labsystems Multiskan MS, Triad Scientific Inc., USA). Assay range was 5-4000 ng/mL, with limit of detection 0.02 ng/ml. Since the standard curve included a zero standard, readings corresponding to concentrations below 5ng/ml were possible. The assay measures oligomerized functional MBL, in serum and plasma. ELISA assays have shown high stability after various storage tempera-tures, repeated freeze and thaw cycles, and equivalent results from serum and plasma speciment (plain, clotted, or EDTA) (443).

Statistics

In the original papers, data are presented as numerical values or percentages for categorical variables. Continuous data are presented as mean with standard deviation, or median and first and third quartiles, or interquartile range, according to distribution. For statistical comparisons between groups, depending on sample size, Pearson chi-square tests or Fisher’s exact test were used for categorical variables and for continuous variables Student t-test or Mann–Whitney U test according to proof of normality. Mortality rates are presented as proportions with 95% confidence intervals (CI), except in original paper I were mortality is presented as proportion only.

A p-value of less than 0.05 was considered significant. No correlation for multiple testing has been used. The statistical analysis was performed with the SPSS software package (version 11.0 (Paper I), version 19 (Papers II-III); SPSS Inc., Chicago, IL paper, and version 22.0 IBM (Paper IV)

In Papers II-IV non-conditional logistic regression analysis was used to assess the risk for in-hospital death expressed as odds ratios (OR) with 95% CI. Initial univariable logistic regression was performed to estimate individual co-variables association with in-hospital death. Interaction analysis between sex and SOFA score (Paper II), and sex and leptin (Paper III) and sex and MBL (Paper IV) was performed with hospital death as the dependent variable. Independent co-variables significantly associated with in-hospital death were introduced in a multivariate backward stepwise logistic regression adjusted for age and gender (Paper II) and in forward and backward stepwise logistic regression, stratified for sex (Paper III and IV).

In Papers III-IV many of the main variables showed a skewed distribution and were loge-transformed (natural logarithm) prior to analysis to attain approximate normal distribution and as continuous variables these are presented as geometric means with 95% CI. Pearson correlation was used for test of associations, and partial correlation coefficients were calculated, adjusted for sex and BMI. Paired sample t-test was used for intra-individual comparison of leptin and adiponectin at baseline compared to the acute phase.

In Paper IV, the main variable (MBL) exhibited skewed distribution in a tri-modal fashion, which was not possible to transform into a normal distribution. As a continuous variable, MBL is expressed as median and interquartile range. In

Methods

46

comparison between groups, MBL is expressed as a continuous variable or categorical when appropriate. For comparison between groups, non-parametric tests, Mann–Whitney U- and Kruskal Wallis test, were used for MBL as a continuous variable and Fisher’s exact test was used for the comparison of proportions. Spearman´s rho was used for test of correlations. Wilcoxon signed ranks test was used for intra-individual comparison of MBL at baseline compared to the acute phase.

In Papers III and IV, univariate and multivariate conditional logistic regression was used to estimate the risk for future sepsis which was expressed as odds ratios (OR) with 95% CI.

In Paper III partial and bivariate correlation analysis to assess correlations between adipokines and markers of cardiovascular disease and their confounding effect preceded the logistic regression analysis. Analyses were made for the whole cohort and stratified for sex. In Paper III, leptin and adiponectin were tested both as continuous (loge-transformed) and categorical (quartiles) variables. Cut-offs for quartiles were based on the sex-specific distribution amongst referents (risk for sepsis) or amongst cases (risk for death).

In Paper IV, different cut offs were used based on deficiency states reported in the literature. The detection limit of the assay was also used as cut off to assess the risk for future sepsis.

Ethical aspects

All four studies were vetted and approved by the Regional Review Board for Ethical Human Research in Umeå. The data handling procedure was approved by the National Computer Data Inspection Board. Informed consent was obtained from all participants for the prospectively collected baseline specimens. Informed consent was not obtained for the samples from the acute phase because the majority of patients were not alive at the time for chemical analysis. Consent was assumed on basis of former positive approach, samples were already collected and individual results are not possible to identify. No genetic analyses were made. It was concluded that no further harm or complications could be inflicted on the patients or their next of kin. The main questions in Papers III and IV could not be addressed otherwise. New knowledge about factors associated with mortality in this category of patients is needed, and informed consent is impossible to gain in many categories of critically ill patients. This affects the possibility to conduct clinical studies, or at least the information that can be gained, from studies on intensive care patients. It is important to have an accurate assessment of what can potentially be of harm for patients, and what can benefit others. These are principles used based on the obtained approval for the study by The Regional Review Board for Ethical Human Research at Umeå University, Sweden.

Results

47

“It's not that I'm so smart , it's just that I stay with problems longer .”

Albert Einstein

RESULTS

A summary of patient characteristics and outcome of all patients included in the work of this thesis are displayed in table 5 and 6.

Table 5. Patient characteristics Female Male P

Number, n n=167 (47.9) n=182 (52.1)

n(%) n(%)

Referral pattern Admission from the community 31(18.6) 22(12.1) 0.10

ICU transfer from within hospital 111(66.5) 133(73.1) 0.12

Transfer from other institution 25(15.0) 27(14.8) 1.00

Community acquired 107(64.1) 124(68.1) 0.43

Nosocomial 60(35.9) 58(31.9) 0.43

Patient category Medical 80(47.9) 108(59.3) 0.04

Surgical emergency 59(35.32) 54(29.7) 0.30

Surgical elective 12(7.2) 20(11.0) 0.27

Obstetrical 16(9.6) 0 <0.001

Comorbidities Congestive heart failure 4(2.4) 13(7.1) 0.05

Chronic lung disease 5(3.0) 6(3.3) 1.00

Chronic liver disease 3(1.8) 4(2.2) 1.00

Chronic renal insuff. 4(2.4) 8(4.4) 0.38

Diabetes 25(15.0) 36(19.8) 0.26

Insulin treated 14(8.4) 16(8.8) 1.00

Non-insulin treated 11(6.6) 20(11.0) 0.19

Cancer

Hematological 10(6.0) 11(6.0) 1.00

Localized 20(12.0) 23(12.6) 0.87

Metastatic 10(6.0) 11(6.0) 1.00

Immunosuppresants

Chronic steroids 15(9.0) 18(9.9) 0.86

Chemotherapy 15(9.0) 13(7.1) 0.56

Radiotherapy 6(3.6) 3(1.6) 0.32

Other immunosupression 14(8.4) 15(8.2) 1.00

Number of co-morbidities

0 81(48.5) 63(34.6) 0.009

1 39(23.4) 59(32.4) 0.07

2 22(13.2) 37(20.3) 0.09

>3 25(15.0) 23(12.6) 0.54

Primary infection site Pneumonia 28(16.8) 31(17.0) 1.00

Abdominopelvic 59(35.3) 41(22.5) 0.009

Urinary tract 17(10.2) 31(17.0) 0.09

Other 61(36.5) 72(39.6) 0.58

Unknown 3(1.8) 8(4.4) 0.22

Microorganism Gram positive bacteria 74(44.3) 78(42.9) 0.83

Gram negative bacetria 42(25.1) 57(31.3) 0.24

Fungi 15(9.0) 15(8.3) 0.85

Other 10(6.0) 12(6.6) 1.00

Polymicrobial 14(8.4) 22(12.1) 0.29 Other microorganism include tuberculosis avium, malaria falsiparum, Pneumocystis carinii, Nephropathia epidemica benigna (NEB), endemic hemorrhagic fever caused by a Puumala virus from the Hanta group. Epstein Barr virus (EBV). Cytomegal Virus (CMV). One man and one woman was judged to have two foci of infection. Fisher`s exact test was used to compare male and female.

Results

48

Table 6. Summary of included patients outcome. Women Men

Mean 95% CI Mean 95% CI P

Number of patients (N=349) n=167 n=182

Age 54.4 (51.9 - 57.0) 59.5 57.4-61.7 0.002

Scoring

APACHE II-score 19 (17.8 - 20.0) 19.6 (18.5-20.7) (18.5-20.7) 0.42

SOFA day 0 6.8 (6.1 - 7.4) 7.4 (6.8-8.0) (6.8-8.0) 0.13

Length of stay (days) Median 25-75 percentile Median 25-75 percentile

ICU LOS (days) 4.2 (1.6 - 9.8) 3.7 (1.5-11.4) 0.72

Hospital LOS (days) 19 (11.0 - 35.0) 17 (9.8-34.2) 0.57

Outcome n(%) 95% CI n(%) 95% CI

ICU mortality (%) 29(17.4) (11.6 - 23.2) 33(18.1) (12.5 - 23.8) 0.89

Hospital mortality (%) 32(19.2) (13.1 - 25.2) 44(24.2) (17.9 - 30.5) 0.30

Severe sepsis, septic shock n=127 n=145 0.44

ICU mortality (%) 29 (22.8) (15.4 - 30.2) 33 (22.8) (16.0 - 29.9) 1.00

Hospital mortality (%) 32 (25.2) (17.5 - 32.8) 42 (29.0) (21.6 - 36.7) 0.50

Septic shock n=46 (27.5) n=38 (20.9) 0.17

ICU mortality (%) 20 (43.5) (28.6 - 58.4) 16 (42.1) (25.7 - 58.6) 1.00

Hospital mortality (%) 20 (43.5) (28.6 - 58.4) 17 (44.7) (28.2 - 61.3) 1.00

Main findings Paper I

The aim of the first study was to investigate the frequency of sepsis and associated mortality, length of stay and costs of care. Main findings in Paper I are summarized in table 7, figure 5, 6 and 7.

Figure 5. ICU-lenght of stay (days) in relation to outcome. Non-survivors had significant shorter ICU-LOS than survivors, P=0.02 using Mann-Whitney U-test. Abbreviations: ICU, intensive care unit; LOS, length of stay

Results

49

0

50

100

150

200

250

300

350

400

0-40 41-64 65-84

TISS total

Age (years)

A*

0

5

10

15

20

25

30

0-40 41-64 65-84

APACHE

Age (years)

B

0

2

4

6

8

10

12

0-40 41-64 65-84

LOS (days)

Age (years)

C*

0

5

10

15

20

25

30

35

40

0-40 41-64 65-84

Mortality (%)

Age (years)

D

Figure 6. Total TISS (panel A), APACHE score (panel B), Length of stay (panel C) and mortality (panel D) in relation to age group (0-40, n=15; 41-64, n=39; 65-84, n=27).

Disease severity expressed as APACHE II and ICU-mortality did not differ between age groups. Patients younger than 40 years of age had longer ICU-stay and correspondingly higher total TISS score than older patients (* P=0.03 for both ICU-LOS and TISS using Mann-Whitney U-test). Data are presented as median. Abbreviations: APACHE, acute physiology age and chronic health evaluation, TISS, therapeutic intervention scoring system; ICU, Intensive care unit; LOS, length of stay.

Results

50

Figure 7. TISS per day in relation to outcome. The use of resourses per day was higher in non-survivors than in survivors, P=0.001, using Mann-Whitney U-test.

Of 2125 patient admissions during the study period, 81 (3.8%) patients met the inclusions criteria, of which 47 (58%) were men. The median age was 55 years (range 18-84 years). The main finding was a high mortality rate, nearly 58% (95% CI, 37-78), for patients in septic shock and an overall mortality rate of near 25% (95% CI, 15-34) (Table 7). The length of stay in the ICU was related to outcome and to age. Non-survivors had shorter ICU-LOS than survivors, (P=0.02) (fig 5). Patient 40 years or younger had longer ICU-LOS than older patients (P=0.03). The use of resources expressed as TISS per patient was related to length of stay and to outcome. Patients 40 years or younger had higher total TISS score than patients older than 40 years (P=0.03) (fig 6). While there was no difference between survivors and non-survivors in total TISS per patient, TISS/day was higher in non-survivors than in survivors, (P=0.001) (fig 7).

Secondary findings were that nearly a third of the patients had diabetes mellitus (32.1%), and that the majority of deaths were related to cardiovascular events and most deaths (80%) occurred early, within 5 days. Data not presented in the original paper and not illustrated are that there were no differences between sexes in age (P=0.11), severity of disease expressed as APACHE (P=0.84), TISS (P=0.23), TISS/day (P=0.37), sepsis, sever sepsis or septic shock (Pearson chi P=0.41, liner by liner 0.46), and there were no differences in ICU (P=0.80) or 28 day mortality (P=0.46), or in length of stay (P=0.27).

Results

51

Table 7. Outcome Paper I. Sepsis Severe sepsis Septic shock Total

n=20 n=35 n=26 n=81Median (IQR) Median (IQR) Median (IQR) Median (IQR)

Age (years) 63.5 (23.0) 55.3 (26.0) 52.0 (27.0) 55.0 (24.0)

APACHEII score 21.0 (9.5) 20.0 (9.0) 29.0 (12.0) 22 (10.5)

TISS 76 score 83 (244) 156 (590) 226 (366) 139 (338)

TISS76/day 34.8 (11.6) 36.2 (10.8) 43.9(29.4) 38.0 (12.6)

ICU_LOS (days) 2.4 (7.5) 4.9 (13.2) 5.0 (10.6) 4.2 (10.8)

Outcome %(95% CI) %(95% CI) %(95% CI) %(95% CI)

ICU-mortality 0 14.3 (2.1-26.5) 57.7 (37.3-78.0) 24.7 (15.1-34.3)

28-days mortality 4.7 (-0.5-15.5) 20.0 (6.1-33.9) 61.5 (41.5-81.6) 29.6 (19.5-39-8) APACHE = acute physiology age and chronic health evaluation, TISS = Therapeutic Intervention Scoring System; ICU, Intensive Care Unit; LOS, Length of stay.

Table 8. Summary of gender-related differences in patient characteristics and treatment, (Paper II).

Women Men P

n (%) n (%)50 (40) 77 (60)

Patient category

Medical 28 (56) 59 (76) 0.02 Surgical_emergency 16 (32) 9 (12) <0.01

Site of infection

Abdominal 17 (34) 8 (10) <0.01 Skin or skin structures 0 (0) 9 (12) 0.01

CRPmax Median (IQR) Median (IQR) All 260 (110) 242 (171) 0.25 Survivors 272 (116) 225 (170) 0.03 Non-survivors 231 (159) 296 (192) 0.14

Treatment n (%) n (%) Surgical proceures 25 (50) 24 (31) 0.04 Red blood cells¤ 36 (72) 41 (53) 0.04 Cefuroxime# 11 (22) 16 (21) 0.02 Ampicillin# 5 (10) 10 (13) 0.04

Data are presented as numbers (%) or median (IQR, inter quartile range). For categorical data Fisher´s exact test was used and for CRP max, Mann Whitney U-test was used. ¤ denotes number of patients, # denotes number of doses administered. Surgical procedures include debridement, removal of obstruction, and drainage

Results

52

0

2

4

6

8

10

12

14

16

18

NO YES NO YES

Hospital dead

Female Male

SOFA-max at admission C

**

Main findings Paper II:

The aim of the study was to assess gender differences in characteristics, outcome and treatment modalities in patients with severe sepsis. Main findings in Paper II are summarized in table 8, figure 8 and 9.

0

2

4

6

8

10

12

14

16

NO YES NO YES

Hospital dead

Female Male

SOFA score at admission A

*

0

2

4

6

8

10

12

14

16

NO YES NO YES

Hospital dead

Female Male

SOFA score day1 B

*

*

Figure 8 A-C. SOFA score in relation to outcome stratified for sex.

Mann-Whitney U-test was used to compare survivors and non-survivors among men and women. SOFA score was significantly higher in non-survivors than survivors among women (P=0.001 at admission and P=0.008 day 1, panel A-B) but not among men (P=0.2 at admission, panel A). Contrary, day 1 non-surviving men had lower SOFA score than surviving men (P=0.04, panel B). The maximum SOFA score was higher among non-survivors in both sexes (women, P=0.001 and men P=0.02, panel C).

Of 2271 admitted patients, 127 (5.6%) full filed the inclusion criteria for severe

sepsis or septic shock, of which 77 (60.6%) were men. There were differences be-tween sexes in terms of admission category and site of primary infection (Table 8). Women were more often admitted due to emergency surgical reasons with the ab-domen as primary infection site, while men were more often admitted due to medi-cal reason and the primary infection site was more often the skin and skin struc-

Results

53

tures. There was no difference between sexes in severity of disease expressed as APACHE II- or SOFA score, but early SOFA score was a stronger predictor for hospital mortality in women than in men (Fig 8). This difference was mainly related to the coagulation sub-score. CRP levels differed between genders in relation to hospital mortality. At admission surviving women had higher CRP than surviving men (P=0.02). Among non survivors men had higher CRP than women, however not significant (p=0.07). Surviving women had higher CRP than non-surviving women (p=0.04), contrary in men where non-surviving men had higher CRP than surviving men (p=0.04).

The same pattern was seen in maximum CRP, surviving women had higher CRP than surviving men (P=0.03). Among non survivors men had higher CRP than women, (p=0.05). Surviving women had higher CRP than non-surviving women (p=0.02), contrary in men where non-surviving men had higher CRP than surviving men, however not significant (p=0.08) Fig 9, panel a and b. There was no difference between men and women in ICU- or long term mortality during a 2-year follow up (Fig 10). The ICU-mortality was lower but in the same range as in Paper I, 20% in women and 22% in men, and one-and two-year mortality was 42% in women and 46% in men. There was no difference between sexes in length of stay in ICU or hospital, but a tendency for longer hospital stay in women. Differences in treatment were related to differences in reason for admission and primary infection site, where women more often were subjected to surgical procedures and more often received transfusion with red blood cells than men. Choice of antibiotics also reflected differences in site of infection. The use of resources expressed as TISS score did not differ between men and women.

0

50

100

150

200

250

300

350

400

Surviv

ors

Non-s

urvivo

rs

Surviv

ors

Non-s

urvivo

rs

Female Male

CRP at admission A

**

0

50

100

150

200

250

300

350

400

Surviv

ors

Non-s

urvivo

rs

Surviv

ors

Non-s

urvivo

rs

Female Male

CRP max B

*

Figure 9 A-B. CRP at admission and CRP-max in relation to outcome stratified for sex. Mann-Whitney U-test was used to compare survivors and non-survivors among men and women (see text).

Results

54

Figure 10. Two years survival, men (green line) and women (blue line).

Factors associated to unfavourable outcome included medical admission type, chronic steroid treatment and early SOFA score (Table 9). Notable secondary findings included that the number of days platelet transfusion was needed associated with worse outcome. No other treatment modality was associated with hospital death.

Table 9. Multivariate stepwise backward logistic regression analysis, hospital death as dependent variable.

Adjusted OR 95% CI P

Medical admission type 4.07 1.20-13.87 0.02

Chronic corticosteroid treatment 14.60 1.70-125.6 0.02

SOFA score at admission 1.17 1.02-1.35 0.02

Platelet transfusion 1.66 1.14-2.43 <0.01

Multivariate logistic regression analysis, risk of in-hospital death expressed as OR (95% CI), adjusted for age and sex.

Results

55

Paper III and IV

Papers III and IV include the same study cohort of 152 patients and their 304 matched referents, where 63 % of both cases and referents were women (matched), and the mean age was 51 years at baseline (matched). Cases were more obese than their referents at baseline, (BMI 27.1 vs. 26.1, P<0.05).

Mannose-binding lectin did not correlate with leptin (P=1.0), adiponectin (P=0.4), BMI (P=0.4) or total cholesterol (P=0.9) at baseline. Mannose-binding lectin was analyzed separately.

The acute event was classified as sepsis in 26%, as severe sepsis in 52% and as septic shock in 22%, with no differences between men and women. The ICU mortality was 17.8% and the hospital mortality was 21.0%.

Women were younger when developing sepsis (P=0.02), and the period between the baseline survey and the sepsis event was longer for men than for women (P=0.02). Severity of sepsis, APACHE II- and SOFA scores, length of stay, ICU- and in-hospital mortality did not differ between men and women.

The main results are displayed in tables for adiponectin, leptin and MBL combined (Table 10 and 11).

Res

ults

56

Tab

le 1

0. P

aper

III

and

IV

. A

dipo

kine

s an

d M

anno

se-b

indi

ng le

ctin

at b

asel

ine

A

llM

en

Wo

me

nR

efer

ents

Cas

esO

R95

% C

IR

efer

ents

Cas

esO

R95

% C

IR

efer

ents

Cas

esO

R95

% C

IL

ep

tin

(b

ase

lin

e)

All

Uni

varia

teQ

1_3

229

922.

041.

30–3

.20

8631

2.39

1.18

–4.8

614

361

1.83

1.02

–3.2

7Q

475

5828

2447

34M

odel

11.

891.

14–3

.13

2.60

1.20

–5.6

11.

380.

70–2

.74

Mod

el 2

1.77

1.04

–3.0

01.

850.

78–4

.39

1.35

0.67

–2.7

3S

ep

sis

Uni

varia

teQ

1_3

5523

1.62

0.66

–3.9

626

101.

820.

48–6

.89

2913

1.46

0.43

–4.9

2Q

423

158

615

9M

odel

11.

750.

65–4

.69

2.93

0.54

–15.

81.

110.

27–4

.52

Mod

el 2

1.89

0.67

–5.3

43.

550.

50–2

5.5

1.44

0.31

–6.5

5S

eve

re s

ep

sis U

niva

riate

Q1_

311

748

1.84

1.01

–3.3

447

182.

090.

82–5

.31

7030

1.68

0.77

–3.6

5Q

441

3017

1324

17M

odel

11.

390.

69–2

.80

1.92

0.69

–5.3

50.

960.

36–2

.58

Mod

el 2

1.42

0.64

–3.1

21.

530.

37–6

.29

1.00

0.36

–2.8

1S

ep

tic

sho

ckU

niva

riate

Q1_

357

213.

741.

29–1

0.9

133

7.20

0.81

–64.

244

182.

860.

81–1

0.1

Q4

1113

35

88

Mod

el 1

4.40

1.31

–14.

87.

950.

82–7

7.5

3.30

0.76

–14.

4M

odel

23.

891.

06–1

4.3

0.45

0.00

–141

.93.

480.

76–1

6.0

Ho

spit

al

de

ath U

niva

riate

Q1_

354

185.

131.

64–1

6.1

225

6.61

1.37

–31.

832

133.

680.

68–1

9.9

Q4

1014

48

66

Mod

el 1

5.18

1.57

–17.

06.

411.

32–3

1.1

5.02

0.63

–39.

9M

odel

24.

721.

32–1

6.9

5.01

0.82

–30.

62.

780.

33–2

3.4

Con

diti

onal

logi

stic

reg

ress

ion

anal

ysis

. Val

ues

show

n ar

e od

ds r

atio

s (O

R)

wit

h 95

% C

I fo

r le

ptin

, adi

pone

ctin

, and

man

nose

-bin

ding

lect

in a

nd r

isk

for

a fu

ture

sep

sis

even

t. L

epti

n an

d ad

ipon

ecti

n w

ere

cate

gori

zed

into

qua

rtil

es b

ased

on

the

sex-

spec

ific

dis

trib

utio

n am

ongs

t ref

eren

ts. T

he O

Rs

repr

esen

t the

incr

ease

d ri

sk a

ssoc

iate

d w

ith

the

4th

quar

tile

of

lept

in a

nd a

dipo

nect

in, r

espe

ctiv

ely,

ver

sus

the

low

er th

ree

quar

tile

s (r

efer

ence

wit

h O

R 1

.00)

. U

niva

riat

e an

d m

ulti

vari

able

ana

lyse

s, m

odel

1 in

clud

ed

BM

I an

d m

odel

2 in

clud

ed B

MI,

hyp

erte

nsio

n, r

educ

ed g

luco

se to

lear

ance

(IF

G, I

GT

a/o

DM

), a

nd to

tal c

hole

ster

ol.

MB

L w

as d

icho

tom

ized

bas

ed o

n cu

t-of

fs

repr

esen

ting

MB

L-d

efic

ienc

y ac

cord

ing

to r

epor

ts f

rom

pre

viou

sly

publ

ishe

d st

udie

s. A

ddit

iona

lly

a cu

t off

rep

rese

ntin

g th

e hi

ghes

t ass

ay li

mit

was

use

d. T

he O

Rs

repr

esen

t the

incr

ease

d ri

sk a

ssoc

iate

d w

ith

valu

es a

bove

the

cut-

off

vers

us v

alue

s be

low

(re

fere

nce

wit

h O

R 1

.00)

. Pot

enti

al c

onfo

unde

rs (

BM

I, a

ge, l

epti

n, a

dipo

nect

in

and

tota

l cho

elst

erol

) di

d no

t cor

rela

te w

ith

MB

L a

nd a

re n

ot in

clud

ed in

the

anal

yses

.

Res

ults

57

Tab

le 1

0. P

aper

III

and

IV

. A

dipo

kine

s an

d M

anno

se-b

indi

ng le

ctin

at b

asel

ine

(c

onti

nued

).

Ta

ble

10

(co

nti

nu

ed

).A

llM

en

Wo

me

nR

efer

ents

Cas

esO

R95

% C

IR

efer

ents

Cas

esO

R95

% C

IR

efer

ents

Cas

esO

R95

% C

IA

dip

on

ect

in (

ba

seli

ne

)A

llU

niva

riate

Q1_

322

911

70.

850.

53–1

.38

8646

0.60

0.26

–1.3

814

371

1.03

0.57

–1.8

6Q

475

3328

947

24M

odel

10.

920.

56–1

.49

0.61

0.26

–1.4

11.

180.

64–2

.18

Mod

el 2

0.88

0.53

–1.4

70.

560.

22–1

.43

1.07

0.57

–2.0

1M

an

no

se-b

ind

ing

le

ctin

(b

ase

lin

e)

All

(ng/

ml)

Uni

varia

teM

BL<

100

4124

0.84

0.49

-1.4

314

90.

750.

30-1

.85

2715

0.89

0.45

-1.7

4M

BL>

100

263

128

100

4816

380

Uni

varia

teM

BL<

500

8946

0.95

0.62

-1.4

728

150.

920.

45-1

.86

6131

0.98

0.56

-1.6

8M

BL>

500

215

106

8642

129

64

Uni

varia

teM

BL<

1000

130

581.

220.

81-1

.84

4020

1.00

0.52

-1.9

390

100

1.38

0.82

-2.3

3M

BL>

1000

174

9474

3710

057

Uni

varia

teM

BL<

4000

258

119

1.81

1.01

-3.2

691

401.

790.

82-3

.91

167

791.

840.

75-4

.49

MB

L>40

0046

3323

1723

16

Con

diti

onal

logi

stic

reg

ress

ion

anal

ysis

. Val

ues

show

n ar

e od

ds r

atio

s (O

R)

wit

h 95

% C

I fo

r le

ptin

, adi

pone

ctin

, and

man

nose

-bin

ding

lect

in a

nd r

isk

for

a fu

ture

sep

sis

even

t. L

epti

n an

d ad

ipon

ecti

n w

ere

cate

gori

zed

into

qua

rtil

es b

ased

on

the

sex-

spec

ific

dis

trib

utio

n am

ongs

t ref

eren

ts. T

he O

Rs

repr

esen

t the

incr

ease

d ri

sk a

ssoc

iate

d w

ith

the

4th

quar

tile

of

lept

in a

nd a

dipo

nect

in, r

espe

ctiv

ely,

ver

sus

the

low

er th

ree

quar

tile

s (r

efer

ence

wit

h O

R

1.00

). U

niva

riat

e an

d m

ulti

vari

able

ana

lyse

s, m

odel

1 in

clud

ed B

MI

and

mod

el 2

incl

uded

BM

I, h

yper

tens

ion,

red

uced

glu

cose

tole

aran

ce (

IFG

, IG

T

a/o

DM

), a

nd to

tal c

hole

ster

ol.

MB

L w

as d

icho

tom

ized

bas

ed o

n cu

t-of

fs r

epre

sent

ing

MB

L-d

efic

ienc

y ac

cord

ing

to r

epor

ts f

rom

pre

viou

sly

publ

ishe

d st

udie

s. A

ddit

iona

lly

a cu

t off

re

pres

enti

ng th

e hi

ghes

t ass

ay li

mit

was

use

d. T

he O

Rs

repr

esen

t the

incr

ease

d ri

sk a

ssoc

iate

d w

ith v

alue

s ab

ove

the

cut-

off

vers

us v

alue

s be

low

(r

efer

ence

wit

h O

R 1

.00)

. Pot

enti

al c

onfo

unde

rs (

BM

I, a

ge, l

epti

n, a

dipo

nect

in a

nd to

tal c

hoel

ster

ol)

did

not c

orre

late

wit

h M

BL

and

are

not

incl

uded

in

the

anal

yses

.

Res

ults

58

Tab

le 1

1. P

aper

III

and

IV

. A

dipo

kine

s an

d M

anno

se-b

indi

ng le

ctin

in th

e ac

ute

phas

e A

llM

en

Wo

me

nS

NS

OR

95%

CI

SN

SO

R95

% C

IS

NS

OR

95%

CI

Le

pti

nU

niva

riate

Q1

368

1.00

34

1.00

334

1.00

Q2_

463

211.

500.

60–3

.73

347

0.15

0.03

–0.8

529

143.

981.

18–1

3.46

AP

AC

HE

II s

core

1.47

0.56

–3.8

80.

050.

01–0

.48

4.18

1.17

–15.

00A

dip

on

ect

inU

niva

riate

Q1

3211

1.00

83

1.00

248

1.00

Q2_

467

180.

780.

33–1

.85

298

0.74

0.16

–3.4

338

100.

790.

27–2

.28

AP

AC

HE

II s

core

0.78

0.31

–1.9

30.

430.

08–2

.41

0.86

0.29

–2.6

1M

an

no

se-b

ind

ing

le

ctin

(b

ase

lin

e)

Uni

varia

teA

. 10

00 n

g/m

l58

112.

190.

93-5

.15

207

0.71

0.18

-2.8

838

44.

941.

44-1

6.89

MB

L>10

0041

1716

425

13A

PA

CH

E II

sco

re2.

430.

96-6

.14

0.68

0.14

-3.2

56.

101.

58-2

3.50

Uni

varia

teB

. M

edia

n70

152.

090.

89-4

.94

259

0.50

0.09

-2.7

345

64.

581.

47-1

4.26

at b

asel

ine

2913

112

1811

AP

AC

HE

II s

core

2.41

0.95

-6.0

70.

640.

11-3

.85

5.20

1.53

-17.

62

Uni

varia

teC

. M

edia

n ac

ute

5310

2.07

0.87

-4.9

416

70.

460.

11-1

.84

373

6.64

1.73

-25.

46

ac

ute

phas

e46

1820

426

14A

PA

CH

E II

sco

re2.

430.

95-6

.24

0.38

0.08

-1.8

49.

922.

13-4

6.16

Uni

varia

teD

. In

crea

se62

141.

680.

72-3

.90

208

0.47

0.11

-2.0

642

63.

671.

19-1

1.28

from

bas

elin

e37

1416

321

11A

PA

CH

E II

sco

re1.

740.

71-4

.28

0.45

0.08

-2.3

63.

811.

17-1

2.45

V

alue

s sh

own

are

Odd

s ra

tios

(O

R)

wit

h 95

% C

I fo

r le

ptin

and

adi

pone

ctin

, and

man

nose

-bin

ding

lect

in a

nd r

isk

for

in-h

ospi

tal d

eath

. Lep

tin

and

adip

onec

tin

wer

e ca

tego

rize

d in

to q

uart

iles

bas

ed o

n th

e co

hort

and

sex

-spe

cifi

c di

stri

buti

on a

mon

gst c

ases

bas

elin

e va

lues

. The

OR

s re

pres

ent t

he in

crea

sed

risk

ass

ocia

ted

wit

h th

e hi

gher

th

ree

quar

tile

s vs

. the

low

est (

refe

renc

e w

ith

OR

1.0

0).

Uni

vari

able

and

adj

uste

d fo

r A

PA

CH

E I

I sc

ore.

Adj

ustm

ent

wit

h S

OF

A s

core

gav

e si

mil

ar p

oint

est

imat

es, (

data

no

t sho

wn)

. M

anno

se-b

indi

ng le

ctin

(A

, B a

nd C

) an

d th

e ch

ange

in M

BL

-lev

els

from

bas

elin

e to

the

acut

e ph

ase

(D)

and

risk

for

in-h

ospi

tal d

eath

. MB

L w

as

dich

otom

ized

wit

h th

ree

diff

eren

t cut

off

s. A

. MB

L-l

evel

s ab

ove

or b

elow

100

0ng/

ml,

repr

esen

ting

cut

-off

for

def

icie

ncy

or lo

w-l

evel

sta

tes

as r

epor

ted

in p

revi

ousl

y pu

blis

hed

stud

ies.

B. L

evel

s ab

ove

or b

elow

the

sex-

spec

ific

med

ian

valu

e am

ongs

t cas

es b

asel

ine

valu

es a

s an

inte

r-in

divi

dual

ref

eren

ce f

or p

re-s

epsi

s le

vels

. C. L

evel

s be

low

or

abov

e th

e se

x-sp

ecif

ic m

edia

n of

the

acut

e va

lues

as

an in

ter-

indi

vidu

al r

efer

ence

for

the

acut

e ph

ase.

D. R

epre

sent

s th

e ch

ange

fro

m b

asel

ine

to th

e ac

ute

phas

e w

ith

no c

hang

e (z

ero)

as

cut-

off.

The

OR

s re

pres

ents

the

incr

ease

d ri

sk a

ssoc

iate

d w

ith

valu

es a

bove

the

cut-

off

vers

us v

alue

s be

low

(re

fere

nce

wit

h O

R 1

.00)

. Uni

vari

able

an

d ad

just

ed a

naly

ses

wer

e pe

rfor

med

, mod

el 1

incl

uded

AP

AC

HE

II

scor

e an

d m

odel

2 in

clud

ed S

OF

A s

core

. (S

OF

A s

core

gav

e si

mil

ar p

oint

est

imat

es, o

mit

ted

from

ta

ble.

A

bbre

viat

ions

: MB

L, m

anno

se-b

indi

ng le

ctin

; S, i

n-ho

spit

al s

urvi

vors

; NS

, in-

hosp

ital

non

-sur

vivo

rs; A

PA

CH

E, A

cute

Phy

siol

ogy

Age

Chr

onic

Hea

lth

Eva

luat

ion;

SO

FA

, Seq

uent

ial O

rgan

Fai

lure

Ass

essm

ent.

Q1,

low

est q

uart

ile;

Q2_

Q4,

thre

e hi

ghes

t qua

rtil

es. A

, MB

L c

ut o

f 10

00 n

g/m

; B, M

BL

cut

of

sex-

spec

ific

m

edia

n va

lue

at b

asel

ine;

C, M

BL

cut

of

sex-

spec

ific

med

ian

valu

e in

the

acut

e ph

ase;

D, M

BL

incr

ease

fro

m b

asel

ine

to th

e ac

ute

phas

e

Results

59

Main findings Paper III

The aim of the study was to determine if levels of the adipocyte-derived hormones leptin and adiponectin (adipokines) predict sepsis development and if intra-individual changes in circulating levels from baseline to the acute phase affect outcome.

Hyperleptinemia in the pre-sepsis period (at baseline) predicted future sepsis event, and retained its predictive value after adjustment for BMI, hypertension, diabetes and total cholesterol (OR 1.77, 95% CI 1.04–3.00, P=0.03). The response was graded, with stronger association between hyperleptinemia and the more severe forms of sepsis and also with hospital death. Hyperleptinemia predicted sepsis in both men and women. After stratification for sex the association remained in men after adjustment for BMI, but not in women. Adiponectin did not associate with future sepsis event (Table 10.)

High leptin levels in the acute phase predicted death irrespective of disease severity (APACHE II) in women (OR=4.18, 95% CI 1.17–15.00, P=0.03), whereas high leptin levels in men were associated with a decreased risk of death (OR 0.05, 95% CI 0.01–0.48, P=0.01). Adiponectin in the acute phase did not associate with hospital death (Table 11).

There were sex-related differences in regard to change in adipokine levels from baseline to the acute phase. Men had a larger increase in adipokine levels than women, and in the acute phase men and women had similar levels of both leptin and adiponectin (Fig 11B and Fig 12B). Stratified for hospital death surviving men had a higher increase in leptin than surviving women (P<0.001), but the increase in leptin levels did not differ between men and women among non-survivors (P=0.60)(Fig 11C). Non-surviving women had a larger increase than surviving women P=0.03). Adiponectin increased more in men than in women among non-survivors (fig 12C).

Secondary findings, were that BMI and reduced glucose tolerance associated with increased risk for future sepsis event, and total cholesterol associated with a reduced risk. BMI remained associated with sepsis in women after stratification for sex, but not for men, whereas low cholesterol levels and diabetes associated with sepsis in men but not in women. However, none of these confounders remained significant when introduced in the final model. At baseline BMI correlated to adiponectin (r= -0.24, P<0.001) and leptin (r=0.36, P<0.001), but in the acute phase, the correlation between BMI and adipokines were abolished, (adiponectin, r=-0.05, P=0.4) and (leptin, r=-0.05, P=0.6).

Results

60

C

P =0.001 P =0.6

A

P <0.001 P =0.6

B

P <0.05 P =0.3

Figure 11 A-C. Leptin levels stratified for sex in survivors and non-survivors at baseline (A) and acute phase (B). Panel C shows difference between acute phase and baseline. P-values denotes differences between men and women among survivors and non-survivors respectively.

Results

61

C

P= 0.14 P= 0.04

A

P <0.001 P <0.001

B

P= 0.3 P= 0.8

Figure 12 A-C. Adiponectin levels stratified for sex in survivors and non-survivors at baseline (A) and acute phase (B). Panel C shows difference between acute phase and baseline. P-values denotes differences between men and women among survivors and non-survivors respectively.

Results

62

Main findings Paper IV

The aim of the study was to determine if levels of mannose-binding lectin predict sepsis development and if intra-individual changes in circulating levels from baseline to the acute phase affect outcome.

The main finding was that high baseline levels of MBL predict future sepsis event requiring ICU treatment (OR 1.81, 95% CI 1.01-3.26, P<0.05), but did not associate with in hospital mortality. Contrary, low levels did not predict future sepsis event and did not associate with severity of sepsis or in-hospital mortality (Table 10).

High MBL levels in the acute phase or an increase from baseline to the acute phase predicted hospital death in women (OR=6.10, 95% CI 1.58–23.5, P<0.01) adjusted for disease severity expressed as APACHE II score. High MBL levels did not associate with in-hospital mortality in men (OR 0.68, 95% CI 0.14–3.25, P=0.6) (Table 11).

Distribution of circulating MBL-levels at baseline did not differ between cases and referents (P=0.6), and the distribution of subjects with low (below 100), medium (100-1000) and high (above 1000 ng/ml) levels did not differ. Among referents MBL-levels were higher in men than in women (P=0.03), a difference not seen between men and women developing sepsis (P=0.3) (data not shown).

MBL in the acute phase

Co-morbidities, sources of infection and infecting microorganisms did not differ in those with MBL-levels below 500 ng/ml or above 500 ng/ml (data not shown). Circulating MBL levels were similar in men and women (P=0.8), and levels did not differentiate between sepsis severity (P=0.4), or in-hospital mortality (P=0.4) (data not shown). However, differences were seen in the stratified analysis. Women who died had significantly higher levels than men who died (P=0.02) (Fig 14B). Furthermore, non-surviving women had significantly higher levels than surviving women (P=0.004), whereas circulating MBL levels were similar among in-hospital surviving and non-surviving men (P=0.4).

Circulating MBL levels in the acute phase were significantly lower compared to baseline (P=0.003), with a similar intra-individual pattern in men (P=0.02), and in women (P=0.06). Stratified for outcome, intra-individual levels decreased from baseline to the acute phase in survivors (P=0.001), and this change was more pronounced in women (P=0.003) than in men (P=0.08). Non-survivors did not change significantly from baseline to the acute phase (P=0.7), but levels tended to increase from baseline to the acute phase in non-surviving women (P=0.06) (Fig 14C).

Results

63

Changes in MBL levels between baseline and the acute phase were similar in men and women (P=0.5) as were the proportion showing a 25% decrease from baseline to the acute phase (men 55% and women 53%, P=0.9) (Fig13).

Non‐survivors

Survivors

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Baseline Acute phase

MBL (men)ng/mLA

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Baseline Acute phase

MBL (women)ng/mLB

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Baseline Acute phase

MBL (men)ng/mLC

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Baseline Acute phase

MBL (women)ng/mLD

Figure 13 A-D. Change in MBL-levels from baseline to the acute phase in hospital non-survivors (panel A-B) and in survivors (panel C-D). Men to the left women to the right Abbreviations: MBL, mannose-binding lectin

Results

64

C

P= 0.02P= 0.7

However, levels increased in non-surviving women compared to surviving women (P=0.005), a difference not seen in men (P=0.6). Non-surviving women showed a higher increase in circulating MBL levels from base line to the acute phase than non-surviving men (P=0.02) (Fig 14C).

A

P= 0.7 P= 0.3

B

P= 0.4 P= 0.02

Figure 14 A-C. MBL levels stratified for sex in survivors and non-survivors at baseline (A) and acute phase (B). Panel C shows difference between acute phase and baseline. P-values denotes differences between men and women among survivors and non-survivors respectively.

Discussion

65

“If you can't explain it simply, you don't understand it well enough”

Albert Einstein

DISCUSSION

Aspects of epidemiology and study design

As in other aspects of epidemiology, it is almost impossible to address the subject of sepsis without discussing the importance of sample size and definition. The current definition of sepsis came in to general use after a consensus conference in 1992 (71). Attempts have been made to improve the definition but the basic elements of the definition has stood the test of time(72). However, most clinical and intervention- studies have used more well defined cut-offs for blood chemical- or physiological parameters to define organ dysfunction and organ failure. There has been a drift towards more severe signs of organ dysfunction in order to classify sepsis as severe sepsis or septic shock (2, 44). Ensuring a certain degree of severity is a way to include patients with higher probability of unfavourable outcome. Given that the studied treatment has an effect on mortality, the number of patients needed to treat to show an effect is less, if mortality is high. Thus, fewer patients need to be included which can reduce the duration of the study period and this will minimize the confounding effect of time. The introduction of other therapeutic measures not directly related to sepsis that may have an indirect effect on survival is one such “time” confounder. Another possible time-related confounder is the constant changes in medical routines and, one hope, an increasing knowledge and awareness of factors of importance for the outcome of intensive care patients. Shortage of staff or personnel replacements may also affect the medical care and outcome and these are difficult to control. It is not certain that the effect of staffing is less during a shorter study period, but it can be.

In large epidemiological studies, different coding systems are used to define and classify sepsis patients as suffering from severe sepsis and septic shock. The accuracy of the original registered data is highly dependent not only of the definitions used but also of the interpretation and understanding of the definition (22, 42). In the best of worlds, highly qualified and dedicated personnel should be responsible for the assessment and coding procedure based on well documented pertinent clinical facts. Unfortunately, this is far from the clinical or administrative reality in many hospitals. Still, both multicenter clinical studies and studies based on administrative data have an enormous strength in the possibility of inclusion of large patient populations, enabling more robust statistical calculations than a single center study ever can achieve.

The first study in this thesis was hypothesis-generating and the original idea for the second study was to create a web based registry in order to be able to gather information from a lager set of patients with the best possible data collection. Great

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effort was put into that respect, but due to lack of local resources and the development of a national database (Swedish Intensive Care Registry, SIR) the project was not successful in including patients from other hospitals. Thus, there are a limited number of patients in the second as well as in the first study. However, the prospective approach of the second study identified more patients than in the retrospective study as suffering from sepsis during the first 24 hours in ICU. The term “primary sepsis” was used in the first study to denote that sepsis was the admission diagnosis. This term may be of debatable validity but the intention was to differentiate sepsis as a cause of admission from sepsis arising later during ICU-LOS, which may represent different phases in the immune response (444, 445).

Findings from a small single center study

Some things can be learned from small single center studies. The first two Papers in this thesis reflect the local or regional circumstances and provide an estimate of how many patients per year suffer from sepsis within the first 24 hours in the intensive care unit of the University Hospital of Umeå. About 5% of patients admitted during 2000-2005 suffered from sepsis which is relatively few compared to a frequency of 10-15%, more commonly reported by others. This information does not tell the true incidence of sepsis in Northern Sweden, but one can deduce that the incidence of sepsis requiring intensive care is far less than that reported from Angus (3 of 1000 population of which 51% received intensive care) (1). More than 80% of patients in our studies were from the primary referral area with a population of approximately 120 000, which gives a rough estimation of an incidence less than 0.5 per 1000 population, more in line with reports from Finland and Iceland (19, 20). The discrepancies in incidence might very well be an effect of the different methods used, but other circumstances may have had an impact as well. For instance, a fairly well developed and accessible medical health care system may prevent patient delay in care-seeking when an infection arises. Early treatment can prevent sepsis development, thus reducing the need for intensive care treatment. An easily accessible health care can also prevent deterioration of comorbidities in the general population making them less vulnerable to an infectious insult. There are population-based data from this region indicating a fairly low prevalence of comorbidities, although there has been an increase in BMI, and diabetes prevalence during the last decades (446-448). Low prevalence of comorbidities is also reflected in the studies of this thesis with the exception of diabetes. This is not so surprising since diabetic patients are more prone to infections and thus may develop sepsis, but it has not been shown for certain that diabetic patients have less favourable outcome from sepsis than non-diabetic patients (24, 449).

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Comorbidities and outcome

In addition to this background, the mortality rates observed in Papers I and II were very high, especially for patients in septic shock; approximately 50% of the patients died during hospital stay. This is a mortality rate about five times higher than in the average ICU population, but comparable to previous reports from others. Looking in to more detail of the underlying conditions of the patients who died, only one out of 24 patients had no (zero) known comorbidity, in the first study. There were two major categories of comorbidities. Metabolic and cardiovas-cular disorders, including diabetes, were most prevalent among those who died followed by autoimmune disorders and conditions treated with immunosuppressive therapy. This makes the high mortality rate more understandable, but still disturb-ingly high.

In this context, it is worth noting that the APACHE II score only includes chronic heart failure, NYHA class IV, not taking other cardiovascular risk patients in to account. Thus, there is a high likelihood of underestimation of the severity of disease or risk of death in patients with less severe cardiovascular disease than end stage heart failure. In this respect one can favour the use of the SAPS (III) score, where cardiovascular disease is classified in three separate groups and hypertension is also taken into account. In this thesis this fact is of minor importance since we did not use APACHE score for mortality prediction. However, findings from our first study indicate that not only immunosuppressive states, which already are known to be associated with worse outcome in sepsis, but also cardiovascular or metabolic disorders are strongly associated with unfavourable outcome from sepsis. This finding raises the question of whether it is the increased circulatory demand imposed on the patient due to sepsis that leads to unfavourable outcome, or if there are other mechanisms affecting the outcome that may relate to inflammation and immunity. This is a question that will be addressed later in this discussion.

Predictors of in-hospital death

In the second study, logistic regression analysis was used to assess which factors predict sepsis- related in-hospital death. In this model, age, admission due to medical reasons, hematological disease and chronic steroid treatment were associated with in-hospital death as well as the SOFA and APACHE II score. There was a strong correlation between APACHE II and SOFA score, and since the APACHE II score contains information about both age and immunosuppressive states, only the SOFA score was used in the multivariate analysis. However, the SOFA score does not contain factors related to chronic cardiovascular disease, instead only the acute circulatory status. Thus, in this model underlying cardiovascular disease is not assessed. After adjustment for age, sex, and SOFA score, chronic steroid treatment and admission due to medical reasons remained independently associated with in-hospital death. The wide confidence interval for

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chronic steroid treatment is due to limited numbers of patients, calling for caution when interpreting the odds ratio. However, chronic steroid treatment and hematological diseases are known from studies including larger patient populations to predict unfavourable outcome in sepsis. The fact that admission-type medical by definition contains the group of patients with hematological diseases, and to a lesser extent patients with chronic steroid treatment, complicates the interpretation. One reflection that can be made is the following; how much of the predictive value of the admission type medical is related to the group of patients with hematological disease and to what extend do cardiovascular and metabolic diseases contribute? Unfortunately, this study was not designed to answer this question, and this specific question has arisen now with hindsight. In future studies, this would be a relevant factor to address.

Gender differences

The main objective in the second study was to assess if there are gender related differences in characteristics, aspects of care, and outcome in sepsis patients. There were differences between men and women related to focus of infection, with men more often having infections in the skin and skin structures whereas women more often had abdominal infections. Women were also more often admitted due to emergency surgical reasons. The reason for admission and the focus of infection was strongly correlated and affected some aspects of the treatment of patients, but this did not translate to any significant differences in outcome. In a newly published large European point prevalence study (2014 EPIC II) abdominal infections accounted for about 20% of the infections, and this finding was associated with significantly higher mortality rates than other infections (P<0.001) (450). As in most sepsis studies men accounted for 60% of the patients, but data was not stratified for sex. In our study abdominal infection was associated with borderline significant increased survival (P<0.05).

The term gender, encompassing men and women and their cultural and social roles, was used in the second study. We hypothesized that there could be differences between men and women in the delivery of care that were not related to the medical condition. From other areas in medicine, it has been shown that men and women receive different treatment for the same condition, despite no apparent medical reason for these differences. We examined several treatment modalities common in the critical care setting, as categorical parameters (received/not received) and the use of resources expressed as TISS score. Fluid resuscitation was examined as the time to administration (expedience), volume, and choice of fluids. We were unable to find any differences between men and women that were not medically indicated. Women were more often exposed to surgical procedures, they received more blood transfusions during the first 24 hours of ICU-stay and the choice of antibiotics differed between men and women. All of these differences were related to the higher prevalence of abdominal infection in women. Different

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infection sites are related to different microbiological floras. Whereas gram positive cocci are the dominating agents in skin infections, gram negative rods are the dominating microbes in abdominal infections, where multi-resistant bacteria also are a more common feature. Further, a skin infection is easier to detect and surgical interventions like drainage of abscesses and debridement are (often) easier to execute outside- than inside the body. Thus, a higher mortality from abdominal infections compared to other infection sites, which was found in larger studies, is not surprising. Notably, we did not find an increased mortality related to abdominal infections. Considering anatomical differences between men and women it is not difficult to imagine that women more often have abdominal infections than men. Somewhat more surprising, none of the women had skin-infections. Question arise: Is this an effect of gender-related differences in behavior or other social structures? Do women seek medical care also for small wounds and get treatment in time, while men delay care-seeking until the infection has become severe and they need intensive care? Is this a reflection of work-related differences? These are questions left unanswered from this study.

SOFA score and thrombocytes

In the second study we found a difference between men and women in the association between early SOFA score and hospital mortality. This became apparent in the univariate analysis when SOFA at admission and the maximum SOFA score were associated with in-hospital death, in contrast to SOFA for the first 24 hour period which was not. This was an unexpected finding and provided a reason for further investigation. In further analysis it became apparent that the early SOFA score behaved differently in men and women, and that gender significantly affected the predictive value of SOFA on hospital mortality. A sub-score analysis revealed that the coagulation score was the main contributor to this effect. Further analysis of men and women with platelet counts of 50 x109/L or less showed that women with low platelet counts at admission and Day 1 died in-hospital more often than men, and also more often than women with platelet-counts over 50 x109/L. Low platelet counts were not associated with abdominal infection. At admission, only one patient with abdominal infection had a platelet count below 50 x109/L and five patients day 1. Of patients admitted due to surgical reasons, 2 out of 8 with low platelet counts died in hospital. This makes surgery a less probable cause for the differences in SOFA score. Hematological disease is associated with low platelet count, and with higher mortality rates from sepsis, P=0.001 compared with other patients, in this study. Hematological disease, thrombocytopenia, and mortality in this group of patients, affected men and women equally. Thus, hematological disease is also less likely to account for the difference in the predictive power of the SOFA score between genders. Related to this finding, platelet transfusion independently predicted in-hospital death. Questions arise: is there an intrinsic effect in the thrombocyte that is important to the immune response or is thrombocytopenia a sign of some other factor that can affect the

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immune response? This question seems to be answered from recent studies. Animal experiments indicate that platelets are involved in several aspects of the immune system. Low platelet count is associated with reduced survival in sepsis in animal experiments and affect both macrophage function and cytokine release, among other effects (451-454).

In our study, there was a sex-related difference in the SOFA score in relation to in-hospital death and the association between platelet transfusion and in-hospital death was stronger in women (P=0.02) than in men (P=0.1). Another question arises; are there gender-related differences in the action or function of platelets as players in the immune system? This cannot be answered from analysis of our findings.

Gender versus sex, and adiposity

The terms gender and sex are often used interchangeably. The reason for exchanging “gender” in Paper II to the term “sex” in Papers III-IV is a change of focus, to the biological state of men and women. One apparent anatomical difference between men and women has already been touched upon in relation to site of infection. Another sex-related difference is the amount and distribution of adipose tissue in men and women. The dichotomous separation into a male (android) and female (gynecoid) type of fat accumulation, where men more often have a central- (apple) and women more often a pear-shaped (hips and buttocks) fat accumulation is perhaps an oversimplification, but it is not the shape per se that is important. Of relevance are the metabolic properties of the adipose tissue where excess energy is deposited. In obese individuals the shape-characters become more distinct. The android shape is associated with an excess of visceral fat, whereas the gynecoid shape is associated with subcutaneous and intramuscular fat deposits. The association between obesity and metabolic or cardiovascular disease has since long been recognized. Body mass index has been used as a measure of obesity and high BMI is, in addition to smoking, hypertension and cholesterol associated with cardiovascular disease. High BMI also associates to type II diabetes. However it has become evident that the ratio of waist-to-hip circumference (WHC) or waist-to-hip ratio (WHR) more strongly correlates to metabolic complications and to cardiovascular outcomes than the BMI (455, 456). This relates to the different properties of visceral adipose tissue and subcutaneous adipose tissue. Visceral adipose tissue associates with several markers of inflammation, increased cytokine levels, dys-fibrinolysis, a pro-coagulant profile, dyslipidemia and insulin-resistance. The adipose tissue has been suggested to be the link between inflammation and cardiovascular and metabolic diseases, as insulin resistance and diabetes (457). Proposed mediators in this respect are among others the adipocyte derived cytokines leptin and adiponectin, the pleiotropic peptides presented in the introduction.

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Body mass index, diabetes and choletsterol.

In Paper III, we investigated the association between these adipokines and sepsis adjusted for some of the known markers of cardiovascular risk, BMI, hypertension, diabetes and smoking. Of note was that the women who later developed sepsis were more obese at baseline than their referents, but there was no difference among men. Men who developed sepsis had lower total-cholesterol levels and more often diabetes than their referents, a difference not seen among women. Thus, differences in metabolic profile in relation to future sepsis development existed between genders, but there were no differences in adipokine levels between cases and their referents.

We confirmed an association between obesity, expressed as high BMI and hyperleptinemia in both men and women. As shown by others high BMI correlated with high leptin levels and with low adiponectin levels, and women had higher adipokine levels than men at baseline. Further, leptin associated with high post-load glucose independent of BMI and sex. Adiponectin was not associated with glucose levels.

To our knowledge it has not been previously shown that BMI and abnormal glucose tolerance are associated with sepsis development. Here, high BMI predicted sepsis in women but not in men, and a high post-load glucose level, including diabetes, predicted sepsis in men but not in women.

However, after adjustment for BMI, none of the known markers of cardiovascular disease, smoking, hypertension, high post-load glucose levels or diabetes, remained predictive of sepsis. In contrast, a high total cholesterol level was protective and remained protective in men but not in women after adjustment for BMI. A cholesterol protective effect is in agreement with a recent study showing that low levels of cholesterol are associated with increased risk of post cardiopulmonary bypass sepsis (458) . Cholesterol is thought to have endotoxin neutralizing properties and in-vitro experiments with cultured macrophages have shown that HDL can attenuate LPS-induced TNF-α production, but only if added concomitantly with, but not after, LPS exposure (459). As usual, possible sex-related differences has not been addressed in these studies.

Leptin and adiponectin as predictors of sepsis and in-hospital mortality

Our main focus was on the adipokines, and one of the main findings was that hyperleptinemia but not adiponectin predicted sepsis. Additionally, there were stronger BMI-independent associations between hyperleptinemia and more severe forms of sepsis compared to milder forms of sepsis, and possibly stronger association in men. These findings awakened new questions: Is it leptin itself that makes patients susceptible to sepsis or is leptin a marker for some other yet unknown factor and if so, are there sex-related differences in that respect? High

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leptin levels are associated with several of the known metabolic markers of cardiovascular risk, including BMI, but these markers were taken in to account.

High leptin levels lead to a low-grade pro-inflammatory state where cells in the innate immune system are activated and induced to release cytokines. Does a constant low-grade pro-inflammatory state contribute to increased susceptibility to an infection and to a more severe form of sepsis once developed? If so, the mechanisms are relevant for further study. Our study was not designed to answer this type of questions, but one can speculate. The term leptin resistance is used to describe a situation which is characterized by elevated circulating leptin levels and decreased leptin sensitivity, in terms of satiety and metabolic aspects of leptin, and this leads to, or are caused by obesity. Several explanatory models exist that relate to the metabolic effect of leptin and hypothalamic receptor signaling. These include defective transport of leptin across the blood brain barrier, attenuation of leptin receptor signaling, mutation in the receptor or co-factors important for the signal function, endoplasmic reticulum stress, inflammation, and “other” causes (202). LEP or LEPR gene contains a number of single nucleotide polymorphisms respectively, which can lead to the impaired LEPR expression or defect function of leptin but the association between polymorphism and obesity is relatively rare (183, 460). Whether these mechanisms per se are related to impaired immune response is not clear. However, since leptin can activate immune cells, constant high levels of leptin may lead to some form of “fatigue” or desensitization to leptin, i.e receptor down regulation or attenuated sensitivity to leptin in cells of the innate immune response. An excess of leptin may also cross-react with other class I cytokine receptors (275). If this is the case, and an infection is imposed, the host may have difficult to respond in an appropriate way to avoid detrimental effects of an infection.

The other main finding, that high levels of leptin in the acute phase predicted in-hospital mortality in women whereas high leptin levels in men associated with reduced in-hospital mortality is contradictory. This sex-difference raises questions about mechanisms of this interaction. Men increased their leptin levels from baseline to the acute phase of sepsis, whereas women maintained or slightly decreased their levels, with an equalizing effect on leptin concentration between men and women. Additionally, leptin levels in the acute phase did not correlate to BMI in men or women. The increase in leptin levels in men are understandable, since activation of the innate immune system induces leptin secretion, but why is a further increase in women detrimental?

Does this equilibrium reflect the transition of function of leptin, from a hormone of metabolic regulation to an acute phase cytokine? One can speculate that there might be an ideal leptin level for facilitating a strong survival response, though this remains to be established. It will be necessary to study not just plasma levels of leptin, but also cell responses to leptin when conditioned by sepsis. This was beyond the scope of the studies in this thesis.

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Some aspects of obesity and lipid metabolism was not assessed in this study, since we did not have access to serum levels of triglycerides, or cholesterol specified as low density or high density lipoprotein (HDL and LDL) for the whole cohort. Further, we did not have measures of the above mentioned WHR or WHC, which may be a more accurate, but not absolute, measure of visceral adiposity. One can only speculate whether these factors would have had an impact on the results.

Adiponectin, the most abundant of hormones, also increased in men from baseline to the acute phase, but not in women. The changes in adiponectin or its absolute concentration was not associated with in-hospital mortality. From its presumed anti-inflammatory properties one would have expected a protective effect, but as mentioned in the introduction adiponectin have different effects in different situations and in different milieu, and the net effect may not be easy to predict. As in the other studies these findings generate new questions.

Mannose-binding lectin

In Paper IV, high MBL levels were shown to predict sepsis, and high levels in the acute septic state or an increase from baseline to the acute phase associated with unfavourable outcome in women but not in men. This contradicts earlier reports which have found that genotypes associated with low MBL levels were likely to make patients more susceptible to infections and sepsis development, and that the low MBL level-genotypes associate with more severe forms of sepsis and unfavourable outcome (417). In our study MBL levels decreased from baseline to the acute phase in both men and women, and stratified for in-hospital death, surviving women had a more pronounced decrease than men. Among non-survivors, men and women, MBL levels did not change significantly from baseline to the acute phase. A theoretical explanation for the difference in MBL-kinetics between survivors and non survivors could be related to the opsonizing and phagocytosis facilitating function of MBL. If MBL opsonizes a microorganism and subsequently become phagocytized, MBL would be “cleared” from the circulation or at least “occupied” in such way that the assays measuring MBL cannot detect the molecule and this would be perceived as a decrease in MBL levels. If, for some reason the function to opsonize is defective, or if some other factor has occupied the sites were MBL normally would attach this would represent a defect in innate immune response with reduced capacity of phagocytosis of bacteria. In the latter scenario, MBL levels of circulating free MBL may be detected by the assay and levels perceived as unchanged. The reduced phagocytosis of bacteria may led to unfavourable outcome.

Further, there are data indicating a slower response of MBL than for instance for CRP, to a stimuli. If the response is delayed, with slow MBL synthesis and secretion, if MBL arrives ‘on the scene’ too late, or if it is not the best immune element for fighting a specific bacterial infection, it may be that MBL has accumulated in patients that are not ‘winning’ their immune battle against

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infection. This is speculation about MBL mechanisms and related plasma concentrations. Why there should be differences in MBL levels and responses between men and women is not clear. There may be important interactions with other actors of innate immunity, such as thrombocytes, CRP or adipokines. Thrombocytopenia was more strongly associated with in-hospital death in women than in men, and C-reactive protein was higher in surviving women than in men, and higher in non-surviving men than non-surviving women (unpublished results). This awakens a question that there might be an interaction between them.

Methodological considerations

Concerning Papers I and II, as discussed in the outset, the strength in collecting data from multiple centers is to reduce the possibilities that confounding factors in a single center strongly influence the results. Also, data can be identified and collected over a shorter period, reducing the influence of time and uncontrolled factors on the results, when collecting from multiple centers. In our study the duration of data collection was fairly extended, and data can be influence by local factors including changes in local treatment programs, changes in the intensive care unit personnel, and other factors.

In Paper I, there are clear limitations related to retrospective data collection as far as making generalizations from the findings. This study was ’kick-off’ in the long-term project to build a new and strong sepsis database with the aim to assess sepsis mortality, along with a clear appraisal of the extent and character of our local patient population. We used TISS score to assess utilization of resources and in the first study we used the registered TISS scores and found that patients who died used more resources per day than survivors. This was not seen in the second study were we re-scored the patients. In both studies TISS paralleled the length of ICU stay. Though not a measure of the total cost of care and perhaps of little interest to the patient and the average clinicians, this kind of data can be of use in decision-making concerning ICU- management and planning.

Concerning Paper II, even with prospective data collection, there is always a weakness to generalize from data collected from a single center.

In Papers III and IV the Northern Sweden Medical Research Bank (NSMRB) for research purposes, where blood samples collected in conjunction with health surveys in the framework of Northern Sweden Health and Disease Study (NSHDS) and the Northern Sweden Maternity Cohort (NSMC) was a strong asset for these studies. These health surveys have since the mid-80s used standardized protocols to collect data from population based surveys with overall high participation rates and without signs of major selection bias (441, 461-464). Though this is not quantified, one can suspect that some intensive care patients are less active concerning health maintenance, given the fact that substance abuse is relatively common among the general intensive care unit population. Clearly, though, there are regional and national differences concerning the sociodemographic profile of the intensive care

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unit population (27). The Mammary and Maternity cohorts are not as complete as the VIP and MONICA cohorts, concerning data on cardiovascular risk markers. However, omitting the maternity cohort from the analysis in Paper III did not influence the results. Further, cases and referents were matched within the respective cohort, thus minimizing effects of differences between cohorts.

The sepsis cohort represents an ethnic homogenous population with more than 99% Caucasians, though data on ethnicity from the health surveys is lacking.

A potential weakness in this material is the period of time between collection of the blood sample and the sepsis event and also a long period of time with sample storage, which in the worst case can affect the quality of the sample.

In the logistical regression in Papers II and Paper III we encountered an effect of opposing trends in men and women, in parameters associated with the outcome (in-hospital mortality). The opposing effect resulted in a non-significant association of the respective parameter with the outcome when men and women were analyzed together, and adjustment for sex did not disclose this effect. An interaction analysis could confirm the effect of gender on outcome, and when men and women were analyzed separately the opposing effect was disclosed. Thus, it became clear during the analysis that women and men needed to be analyzed separately. When men and women are analyzed together, no relation of leptin in the acute phase to mortality can be noted. Other statistical challenges for analysis of adipokines and MBL have been the skewness and disproportion. The distribution of MBL was not at all normal, and this called for assessment by category or nonparametric tests

Future considerations

To summarize, future studies have to include larger patient populations, preferably with genetic analysis and assessment of other cytokines, their receptors and signaling function. Also, additional aspects of cardiovascular risk assessment would be valuable. In future studies men and women should be analyzed separately in order to disclose potential sex-related differences.

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CONCLUSIONS

Severe sepsis has high in-hospital mortality.

Severe sepsis frequency is relatively low in our region.

There is no difference in severe sepsis in-hospital mortality between men and women.

Early SOFA score is a stronger predictor of in-hospital mortality for women compared to men, and this probably relates to differences in thrombocyte score.

Need for thrombocyte transfusion independently predict in-hospital mortality in sepsis patients.

High baseline leptin levels predict sepsis events and in-hospital mortality in men.

High leptin levels in the acute phase of sepsis associate with increased in-hospital mortality in women.

High leptin levels in the acute phase of sepsis associate with reduced in-hospital mortality in men.

High levels of mannose-binding lectin predict sepsis event.

High levels of mannose-binding lectin in the acute phase of sepsis associate with increased in-hospital mortality in women, but not in men.

An increase in levels of mannose-binding lectin from baseline to the acute phase of sepsis associate with increased in-hospital mortality in women, but not in men.

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ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to all who in some way have contributed to the completion of this thesis. Special thanks to the following persons:

Professor Ola Winsö, my main supervisor for all the support and enjoyable moments during the long and winding road leading to this thesis.

Professor Stefan Söderberg, my co-supervisor, for valuable insights into the world of adipokines and statistical pitfalls.

Peter Larsson MD, Phd, my co-supervisor, for developing the ideas for using bio bank material in the study of predictive markers of sepsis.

Professor Michael Haney, my colleague over many years, who in my opinion have transformed from an annoying man to a dear friend, knowledgeable and efficient in the academic reality.

Göran Johansson, my coauthor, Excel wizard and music lover for all support and nice tunes, in the assistance with practical and theoretical dilemmas in presenting tables and figures.

Eva Liedgren, Lennart Styrke, Marianne Ekström and Björn Byström, my colleagues at the Intensive Care Unit at University Hospital Umeå, for many laughs and valuable help with data capture and collection.

Anna-Maja Åberg and Margareta Danielsson for valuable help and laboratory assistance, analyzing mannose-binding lectin, leptin and adiponectin.

Åsa Ågren and Hubert Sjödin at The Northern Sweden Medical Research Bank for all the work and assistance in the process of cross-linking database information, matching and identification blood samples.

Professor Göran Hallmans, the Northern Sweden Medical Research Bank and Professor Göran Wadell the Northern Sweden Maternity Cohort, for enabling the study including bio-bank material.

Associate Professor Margareta Norberg, co-author, for valuable aspects on study presentation, Martin Ferm and Marie Eriksson for valuable advise in statistics.

Camilla Brorsson and Maria Sehlin, my comrades during our doctoral studies, for encouraging in-put in the completion process of this thesis.

Colleagues and leadership at Operationscentrum, the Intensive Care Unit, and Department of Neurorehabilitation, University Hospital of Umeå, for keeping up the good clinical work during my absence in the making of this thesis.

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My ever encouraging sister Mia, the Queen of positive sayings and quasi-scintific interpretations, my artistic and caring brother Anders and friends, you know who you are, for in so many different ways helping me maintain sanity and perspective during the tedious process of research.

To my father Torsten and my late mother Marta for all your patience and your love and affection.

To Lisa for permission to tell your story and all who have suffered greately from such small adversaries.

To all not mentioned above who in have contributed to the completion of this thesis.

Grants.

These studies were supported by grants from:

The Faculty of Medicine at Umeå University, the Kempe foundation, the Capio Research foundation, the Swedish Society of Medicine, and through a regional agreement between Umeå University and the Västerbotten County Council (ALF).

References

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