A Complement to T Cell Immunity

The human immune system consists of the innate and the adaptive immunity that together protect the body from pathogens. To complete this task, the immune system must be able to recognize and destroy the dangerous foreign structures but also not to react to host structures or innocuous foreign structures, such as proteins of food or the commensal microbes residing in the gut. The innate immunity includes the phagocytes, such as the macrophages and neutrophils, and multiple molecular defensive systems, most importantly the complement system. The innate immunity reacts quickly to pathogens but its functions remain unchanged with repeated encounters with the intruder. The adaptive immunity is slower in its response, but it is more specific and it has memory; upon repeated exposure to a given pathogen, the adaptive immunity is activated more rapidly. Immunological tolerance, the unresponsiveness to self antigens, is a feature of the adaptive immunity. The adaptive immunity includes T and B lymphocytes and the antibodies produced by the B lymphocytes. In spite of their interdependency, the innate and adaptive immune systems have often been studied separately. This thesis focuses on their interface by investigating the role of the innate complement system in the regulation of the adaptive immunity and of the T lymphocyte function in particular. We followed the immune response and the establishment of oral tolerance in a C3 deficient mouse model, where the function of the complement system is blocked. We also studied vaccination responses and mucosal immune homeostasis in C3 deficient human subjects. The mice were immunized with ovalbumin in Complete Freund s adjuvant. In order to induce oral tolerance, some of the mice were given ovalbumin to the gastrointestinal tract prior to the immunizations. The ensuing immune response was monitored by assessing the lymphocyte fractions by flow cytometry and by stimulating splenocytes with ovalbumin and monoclonal antibodies in vitro, and measuring the proliferative response with a radioactive thymidine incorporation assay. The expression of cytokines and transcription factors in isolated cells and tissue samples was analyzed with quantitative real-time PCR. Serum antibody levels were determined by ELISA. We isolated leukocytes from peripheral blood samples collected from the patients and healthy control subjects and analyzed the lymphocyte population with flow cytometry. Serum antibodies specific for intestinal commensal microbes and the vaccine antigens tetanus toxoid and diphtheria toxoid were measured with ELISA. Serum samples were also analyzed for the presence of a set of key cytokines. The results indicate that complement plays a crucial role in the regulation of the functional differentiation of the T helper lymphocytes central to the adaptive immunity. Immunization with ovalbumin produced a weaker T cell proliferative response in the C3 deficient mouse model compared to the wild-type controls. The response of the T lymphocytes was also qualitative different, since the development of a TH1 response was particularly impaired in the absence of a functional complement system, whereas the TH2 response showed no difference between the mouse strains. This was also reflected on the B lymphocyte response: The IgG2a and IgG3 response to the immunization was reduced in the C3 deficient mice but the IgE response was normal. In addition to the general attenuation of the adaptive immunity, the C3 deficiency resulted in a disturbance of the intestinal immune tolerance in both mice and men. The administration of a foreign protein into the gastrointestinal tract of the C3 deficient mice failed to prevent the systemic immune response to the subsequent immunization with the same protein, i.e. the establishment of oral tolerance failed. The C3 deficient human subjects had more mucosally homing activated T lymphocytes in the peripheral blood and higher levels of serum IgG specific for intestinal commensal microbes. A further sign of the deficient immune tolerance in the C3 deficient human system was the lack of IgG4 response to the vaccine antigens. IgG3 antibodies specific for vaccine antigens were present at higher concentrations in the patient sera and the levels of the inflammatory cytokines IL-12 and IL-21 were also elevated. In contrast to the mouse, the profile of serum cytokines and antibody subclasses in the C3 deficient human subjects pointed at a pronounced TH1 response. The work presented in this thesis defines the complement system as a versatile regulator of the adaptive immunity and helper T lymphocytes. The normal functional differentiation of the T lymphocytes requires signals from the complement system and the establishment of immune tolerance both in the mucosal and systemic immune systems is particularly dependent on complement. The results present novel information on the interplay of the innate and adaptive immune systems and will probably affect the treatment strategies for food allergies and inflammatory bowel diseases.Ihmisen immuunijärjestelmä jakautuu luontaiseen ja hankittuun immuniteettiin, jotka yhdessä suojelevat kehoa taudinaiheuttajilta. Onnistuakseen tässä tehtävässä immuunijärjestelmän on kyettävä tunnistamaan ja tuhoamaan vaaralliset vieraat rakenteet ja oltava reagoimatta kehon omiin rakenteisiin ja harmittomiin vieraisiin rakenteisiin, kuten ruuan proteiineihin ja suoliston normaaliflooran mikrobeihin. Luontaiseen immuniteettiin kuuluvat fagosyytit, kuten makrofagit ja neutrofiilit, sekä erilaiset puolustusmolekyylit, tärkeimpänä näistä komplementtijärjestelmä. Luontainen immuniteetti reagoi nopeasti taudinaiheuttajiin, mutta sen toiminta ei kehity toistuvien kohtaamisten myötä. Hankittu immuniteetti reagoi hitaammin, mutta sen tunnistuskyky on tarkempi ja siihen liittyy immunologinen muisti; kun elimistö kohtaa saman taudinaiheuttajan uudelleen, aktivaatio tapahtuu nopeammin. Myös toleranssi, eli reagoimattomuus omiin rakenteisiin, on hankitun immuniteetin ominaisuus. Hankittuun immuniteettiin kuuluvat T- ja B-lymfosyytit, sekä jälkimmäisten tuottamat vasta-aineet. Luontainen ja hankittu immuniteetti ovat riippuvaisia toisistaan, mutta niitä on usein tutkittu erillään. Tässä väitöskirjassa paneudutaan niiden rajapintaan selvittämällä luontaiseen immuniteettiin kuuluvan komplementtijärjestelmän toiminnan vaikutuksia hankitun immuniteetin säätelyyn, sekä erityisesti T-lymfosyyttien toimintaan. Tutkimus on toteutettu seuraamalla immunisaatiovastetta ja oraalisen toleranssin kehittymistä hiirikannassa, jonka komplementtijärjestelmä ei toimi C3-tekijän puutteen vuoksi. Lisäksi tutkimme rokotusvasteita ja kartoitimme suoliston immuunijärjestelmän tasapainoa C3-puutteisilla potilailla. Koejärjestelyssä hiiret immunisoitiin ovalbumiinilla ja adjuvanttina käytettiin Complete Freund s adjuvanttia. Oraalisen toleranssin synnyttämiseksi osalle hiiristä annosteltiin ovalbumiinia mahasuolikanavaan ennen immunisaatiota. Kehittynyttä immuunivastetta tutkittiin analysoimalla lymfosyyttipopulaatioiden koostumusta virtaussytometrialla ja stimuloimalla lymfosyyttejä soluviljelmässä ovalbumiinilla ja monoklonaalisilla vasta-aineilla, seuraten jakautumisvastetta radioaktiivisen tymidiinin sitoutumiseen perustuvalla koejärjestelyllä. Sytokiinien ja transkriptiotekijöiden ilmentymistä soluissa ja kudosnäytteissä tutkittiin reaaliaikaisella PCR:llä. Seerumin vasta-aineiden määrittämiseen käytettiin ELISA- menetelmää. Potilaiden ja terveiden verrokkien verinäytteistä eristimme valkosolut ja analysoimme lymfosyyttipopulaatioiden koostumusta virtaussytometrialla. Suoliston normaaliflooraa, tetanustoksoidia ja difteriatoksoidia vastaan kehittyneitä seerumin vasta-aineita tutkimme ELISA-menetelmällä. Seeruminäytteistä määritettiin myös immuunivasteessa keskeisten sytokiinien pitoisuudet. Tutkimuksen tulokset osoittavat, että komplementti vaikuttaa hankitun immuniteetin keskeisten solujen, auttaja T-lymfosyyttien, toiminnalliseen erilaistumiseen ratkaisevasti. C3-puutteisessa hiirikannassa immunisaatio ovalbumiinilla tuotti heikomman T-lymfosyyttien jakautumisvasteen kuin villityypin hiirissä. T- lymfosyyttien vaste immunisaatioon oli myös laadullisesti erilainen, sillä TH1-tyyppisten solujen kehitys oli erityisesti heikentynyttä komplementin puuttuessa, mutta TH2 vaste oli normaali. Tämä heijastui myös B-lymfosyyttien tuottamiin vasta- aineisiin: IgG2a ja IgG3 vaste immunisaatioon oli heikentynyt C3-puutteisissa hiirissä, mutta IgE vaste oli normaali. Hankitun immuniteetin yleisen heikentymisen lisäksi C3-puutos aiheutti sekä hiirissä että ihmisissä suoliston immunologisen toleranssin häiriön. C3-puutteisille hiirille ei kehittynyt oraalista toleranssia, eli mahasuolikanavaan annosteltu vieras proteiini ei kyennyt estämään immuunivastetta myöhemmässä immunisaatiossa. C3-puutteisilla potilailla puolestaan oli veressään enemmän suolistoon matkalla olevia aktivoituneita T-lymfosyyttejä, sekä enemmän suoliston normaaliflooraan kohdistuvia IgG-luokan vasta-aineita. Toleranssin häiriöön viittasi myös C3-puutteisten potilaiden puuttuva IgG4-vaste rokotuksille. Rokoteproteiineja vastaan tuotettuja IgG3 vasta-aineita potilaiden seerumissa oli merkitsevästi enemmän kuin terveillä verrokeilla. Potilaiden seerumissa oli myös enemmän tulehduksellisia IL-12 ja IL-21 sytokiineja. Ihmisellä C3-puutos vaikuttaisi siis johtaneen TH1-vasteen voimistumiseen, toisin kuin hiirellä. Väitöskirjani tulokset osoittavat, että komplementtijärjestelmä säätelee hankitun immuniteetin ja etenkin auttaja-T-lymfosyyttien toimintaa laaja-alaisesti. T- lymfosyyttien normaali toiminnallinen erilaistuminen tarvitsee komplementin aktivaation tuottamia viestejä ja etenkin toleranssin kehittyminen sekä suoliston alueella että immuunijärjestelmässä laajemmin häiriintyy komplementin puuttuessa. Löydökset tuovat uutta tietoa luontaisen ja hankitun immuniteetin yhteistoiminnasta ja asettavat etenkin allergioiden ja tulehduksellisten suolistosairauksien hoitomenetelmät uuteen valoon

Can we treat post cardiac arrest shock by removing cytokines from circulation with high cut-off veno-venous hemodialysis?

Non peer reviewe

Association of Sequential Organ Failure Assessment (SOFA) components with mortality

Background Sequential Organ Failure Assessment (SOFA) is a practical method to describe and quantify the presence and severity of organ system dysfunctions and failures. Some proposals suggest that SOFA could be employed as an endpoint in trials. To justify this, all SOFA component scores should reflect organ dysfunctions of comparable severity. We aimed to investigate whether the associations of different SOFA components with in-hospital mortality are comparable. Methods We performed a study based on nationwide register data on adult patients admitted to 26 Finnish intensive care units (ICUs) during 2012-2015. We determined the SOFA score as the maximum score in the first 24 hours after ICU admission. We defined organ failure (OF) as an organ-specific SOFA score of three or higher. We evaluated the association of different SOFA component scores with mortality. Results Our study population comprised 63,756 ICU patients. Overall hospital mortality was 10.7%. In-hospital mortality was 22.5% for patients with respiratory failure, 34.8% for those with coagulation failure, 40.1% for those with hepatic failure, 14.9% for those with cardiovascular failure, 26.9% for those with neurologic failure and 34.6% for the patients with renal failure. Among patients with comparable total SOFA scores, the risk of death was lower in patients with cardiovascular OF compared with patients with other OFs. Conclusions All SOFA components are associated with mortality, but their weights are not comparable. High scores of other organ systems mean a higher risk of death than high cardiovascular scores. The scoring of cardiovascular dysfunction needs to be updated.Peer reviewe

Intensive care-treated cardiac arrest : a retrospective study on the impact of extended age on mortality, neurological outcome, received treatments and healthcare-associated costs

BackgroundCardiac arrest (CA) is a leading cause of death worldwide. As population ages, the need for research focusing on CA in elderly increases. This study investigated treatment intensity, 12-month neurological outcome, mortality and healthcare-associated costs for patients aged over 75 years treated for CA in an intensive care unit (ICU) of a tertiary hospital.MethodsThis single-centre retrospective study included adult CA patients treated in a Finnish tertiary hospital's ICU between 2005 and 2013. We stratified the study population into two age groups: 75 years. We compared interventions defined by the median daily therapeutic scoring system (TISS-76) between the age groups to find differences in treatment intensity. We calculated cost-effectiveness by dividing the total one-year healthcare-associated costs of all patients by the number of survivors with a favourable neurological outcome. Favourable outcome was defined as a cerebral performance category (CPC) of 1-2 at 12 months after cardiac arrest. Logistic regression analysis was used to identify independent associations between age group, mortality and neurological outcome.ResultsThis study included a total of 1,285 patients, of which 212 (16%) were >= 75 years of age. Treatment intensity was lower for the elderly compared to the younger group, with median TISS scores of 116 and 147, respectively (pPeer reviewe

Early hyperoxemia is not associated with cardiac arrest outcome

Aim: Studies suggest that hyperoxemia increases short-term mortality after cardiopulmonary resuscitation (CPR), but the effect of hyperoxemia on long-term outcomes is unclear. We determined the prevalence of early hyperoxemia after CPR and its association with long-term neurological outcome and mortality. Methods: We analysed data from adult cardiac arrest patients treated after CPR in tertiary ICUs during 2005-2013. We retrieved data from the resuscitation and the first arterial blood sample collected after return of spontaneous circulation (ROSC) (severe hyperoxemia defined as PaO2 > 40 kPa and moderate as PaO2 16-40 kPa). We inspected two outcomes, neurological performance at one year after resuscitation according to the Cerebral Performance Category and one-year mortality. We used logistic regression to test associations between hyperoxemia and the outcome and interaction analyses to test the effect of hyperoxemia exposure on the outcomes in smaller subgroups. Results: Of 1110 patients 11% had severe hyperoxemia, prevalence was 10% for out-of-hospital arrests, 13% for in-hospital arrests and 9% for in-ICU arrests. In total 585(53%) patients had an unfavourable neurological outcome. Compared to normoxemia, severe (Odds ratio [OR] 0.81, 95% confidence interval [CI] 0.50-1.30) and moderate hyperoxemia (OR 0.94 95%CI 0.69-1.27) did not associate with neurological outcome. Additionally, hyperoxemia had no association with mortality. In subgroup analyses there were no significant associations between severe hyperoxemia and outcomes regardless of cardiac arrest location, initial rhythm or time-to-ROSC. Conclusion: We found no association between early post-arrest hyperoxemia and unfavourable outcome, Subgroup analysis found no differential effect depending on arrest location, initial rhythm or time-to-ROSC.Peer reviewe

The Association Between Arterial Oxygen Level and Outcome in Neurocritically Ill Patients is not Affected by Blood Pressure

Background In neurocritically ill patients, one early mechanism behind secondary brain injury is low systemic blood pressure resulting in inadequate cerebral perfusion and consequent hypoxia. Intuitively, higher partial pressures of arterial oxygen (PaO2) could be protective in case of inadequate cerebral circulation related to hemodynamic instability. Study purpose We examined whether the association between PaO2 and mortality is different in patients with low compared to normal and high mean arterial pressure (MAP) in patients after various types of brain injury. Methods We screened the Finnish Intensive Care Consortium database for mechanically ventilated adult (>= 18) brain injury patients treated in several tertiary intensive care units (ICUs) between 2003 and 2013. Admission diagnoses included traumatic brain injury, cardiac arrest, subarachnoid and intracranial hemorrhage, and acute ischemic stroke. The primary exposures of interest were PaO2 (recorded in connection with the lowest measured PaO2/fraction of inspired oxygen ratio) and the lowest MAP, recorded during the first 24 h in the ICU. PaO2 was grouped as follows: hypoxemia ( 18.3 kPa, the highest 10th percentile), and MAP was divided into equally sized tertiles ( 68 mmHg). The primary outcome was 1-year mortality. We tested the association between hyperoxemia, MAP, and mortality with a multivariable logistic regression model, including the PaO2, MAP, and interaction of PaO2*MAP, adjusting for age, admission diagnosis, premorbid physical performance, vasoactive use, intracranial pressure monitoring use, and disease severity. The relationship between predicted 1-year mortality and PaO2 was visualized with locally weighted scatterplot smoothing curves (Loess) for different MAP levels. Results From a total of 8290 patients, 3912 (47%) were dead at 1 year. PaO2 was not an independent predictor of mortality: the odds ratio (OR) for hyperoxemia was 1.16 (95% CI 0.85-1.59) and for hypoxemia 1.24 (95% CI 0.96-1.61) compared to normoxemia. Higher MAP predicted lower mortality: OR for MAP 60-68 mmHg was 0.73 (95% CI 0.64-0.84) and for MAP > 68 mmHg 0.80 (95% CI 0.69-0.92) compared to MAP <60 mmHg. The interaction term PaO2*MAP was nonsignificant. In Loess visualization, the relationship between PaO2 and predicted mortality appeared similar in all MAP tertiles. Conclusions During the first 24 h of ICU treatment in mechanically ventilated brain injured patients, the association between PaO2 and mortality was not different in patients with low compared to normal MAP.Peer reviewe

Ventilation during continuous compressions or at 30:2 compression-to-ventilation ratio results in similar arterial oxygen and carbon dioxide levels in an experimental model of prolonged cardiac arrest

Background: In refractory out-of-hospital cardiac arrest, transportation to hospital with continuous chest compressions (CCC) from a chest compression device and ventilation with 100% oxygen through an advanced airway is common practice. Despite this, many patients are hypoxic and hypercapnic on arrival, possibly related to suboptimal ventilation due to the counterpressure caused by the CCC. We hypothesized that a compression/ventilation ratio of 30:2 would provide better ventilation and gas exchange compared to asynchronous CCC during prolonged experimental cardiopulmonary resuscitation (CPR).Methods: We randomized 30 anaesthetized domestic swine (weight approximately 50 kg) with electrically induced ventricular fibrillation to the CCC or 30:2 group and bag-valve ventilation with a fraction of inspired oxygen (FiO(2)) of 100%. We started CPR after a 5-min no-flow period and continued until 40 min from the induction of ventricular fibrillation. Chest compressions were performed with a Stryker Medical LUCAS (R) 2 mechanical chest compression device. We collected arterial blood gas samples every 5 min during the CPR, measured ventilation distribution during the CPR using electrical impedance tomography (EIT) and analysed post-mortem computed tomography (CT) scans for differences in lung aeration status.Results: The median (interquartile range [IQR]) partial pressure of oxygen (PaO2) at 30 min was 110 (52-117) mmHg for the 30:2 group and 70 (40-171) mmHg for the CCC group. The median (IQR) partial pressure of carbon dioxide (PaCO2) at 30 min was 70 (45-85) mmHg for the 30:2 group and 68 (42-84) mmHg for the CCC group. No statistically significant differences between the groups in PaO2 (p =0.40), PaCO2 (p = 0.79), lactate (p = 0.37), mean arterial pressure (MAP) (p = 0.47) or EtCO2 (p = 0.19) analysed with a linear mixed model were found. We found a deteriorating trend in PaO2, EtCO2 and MAP and rising PaCO2 and lactate levels through the intervention. There were no differences between the groups in the distribution of ventilation in the EIT data or the post-mortem CT findings.Conclusions: The 30:2 and CCC protocols resulted in similar gas exchange and lung pathology in an experimental prolonged mechanical CPR model.Peer reviewe

Serum fibroblast growth factor 21 levels after out of hospital cardiac arrest are associated with neurological outcome

Fibroblast growth factor (FGF) 21 is a marker associated with mitochondrial and cellular stress. Cardiac arrest causes mitochondrial stress, and we tested if FGF 21 would reflect the severity of hypoxia-reperfusion injury after cardiac arrest. We measured serum concentrations of FGF 21 in 112 patients on ICU admission and 24, 48 and 72 h after out-of-hospital cardiac arrest with shockable initial rhythm included in the COMACARE study (NCT02698917). All patients received targeted temperature management for 24 h. We defined 6-month cerebral performance category 1-2 as good and 3-5 as poor neurological outcome. We used samples from 40 non-critically ill emergency room patients as controls. We assessed group differences with the Mann Whitney U test and temporal differences with linear modeling with restricted maximum likelihood estimation. We used multivariate logistic regression to assess the independent predictive value of FGF 21 concentration for neurologic outcome. The median (inter-quartile range, IQR) FGF 21 concentration was 0.25 (0.094-0.91) ng/ml in controls, 0.79 (0.37-1.6) ng/ml in patients at ICU admission (PPeer reviewe