Stimulated Cytokine Response Research Articles

Dysfunctional host cellular immune responses are associated with mortality in melioidosis

ABSTRACT Melioidosis is a tropical infection caused by the intracellular pathogen Burkholderia pseudomallei, an underreported and emerging global threat. As melioidosis-associated mortality is frequently high despite antibiotics, novel management strategies are critically needed. Therefore, we sought to determine whether functional changes in the host innate and adaptive immune responses are induced during acute melioidosis and are associated with outcome. Using a unique whole blood stimulation assay developed for use in resource-limited settings, we examined induced cellular functional and phenotypic changes in a cohort of patients with bacteremic melioidosis prospectively enrolled within 24 h of positive blood culture and followed for 28 days. Compared to healthy controls, melioidosis survivors generated an IL-17 response mediated by Th17 cells and terminally-differentiated effector memory CD8+ T cells (P < .05, both), persisting to 28 days after enrolment. Furthermore, melioidosis survivors developed polyfunctional cytokine production in CD8+ T cells (P < .01). Conversely, a reduction in CCR6+ CD4+ T cells was associated with higher mortality, even after adjustments for severity of illness (P = 0.004). Acute melioidosis was also associated with a profound acute impairment in monocyte function as stimulated cytokine responses were reduced in classical, intermediate and non-classical monocytes. Impaired monocyte cytokine function improved by 28-days after enrolment. These data suggest that IL-17 mediated cellular responses may be contributors to host defense during acute melioidosis, and that innate immune function may be impaired. These insights could provide novel targets for the development of therapies and vaccine targets in this frequently lethal disease.

Complement signaling as a T-cell checkpoint in the ovarian cancer (OC) tumor microenvironment (TME)

Abstract T cell checkpoint pathways are barriers to anti-tumor immunity and critical therapeutic targets. We use ascites fluid supernatants (ASC) from patients with newly diagnosed OC as an authentic component of the TME. We previously showed that complement activation is increased in ASC versus paired serum and induces a suppressor phenotype in normal neutrophils characterized by inhibition of critical proliferative, signaling and metabolic pathways in normal human T cells. ASC by itself dramatically inhibited CD3/CD28-stimulated T cell cytokine production (IL-2, IFN-γ, and IL-10) and glucose consumption, but not proliferation. We hypothesized that complement activation in the TME delivers an inhibitory stimulus to T cells that suppresses stimulated cytokine responses required for activation and expansion. Peptide C3 inhibitors (compstatins) restored CD3/CD28-stimulated T cell cytokine responses and glucose consumption. Inhibition of Factor B, a component of the alternative pathway (AP), also rescued cytokine responses, while inhibition of C3aR, C5aR, and C7 had no significant effect. C3b/iC3b deposition on T cells was increased with ASC exposure and reduced by C3 inhibition. Addition of recombinant complement receptor-1 (CR-1) as a decoy of C3b binding to membrane-bound CR-1 fully abrogated the suppressive effect of ASC, while recombinant CD46 had variable effects among different ASC samples. Our results show a novel AP- and CR1-dependent checkpoint of T cell activation in the TME.

Improved Innate Immune Function in Patients with Chronic Lymphocytic Leukemia Treated with Targeted Therapy in Clinical Trials.

Patients with chronic lymphocytic leukemia (CLL) have increased risk of severe infections. Although adaptive immune dysfunction is well described, clinical tools for identifying patients at risk are lacking, warranting investigation of additional immune components. In contrast to chemotherapy, targeted agents could spare or even improve innate immune function. Therefore, we investigated innate immune phenotypes and function in patients with CLL before and during targeted treatment. Baseline and consecutive blood samples were collected from patients with CLL treated with acalabrutinib (n = 17) or ibrutinib+venetoclax (n = 18) in clinical trials. Innate immune function was assessed by TruCulture, a whole-blood ligand-stimulation assay quantifying cytokine release in response to standardized stimuli. Innate immune phenotypes were characterized by flow cytometry. As a proxy for infections, we mapped antimicrobial use before and during treatment. At baseline, patients with CLL displayed impaired stimulated cytokine responses to the endotoxin lipopolysaccharide (LPS) along with deactivated monocytes, enrichment of myeloid-derived suppressor cells and metamyelocytes, and elevated (unstimulated) proinflammatory cytokines. Two/three cycles of acalabrutinib or ibrutinib normalized LPS-stimulated responses, in parallel with decreased duration of infections. Innate immune profiles and elevated proinflammatory cytokines further normalized during longer-term acalabrutinib or ibrutinib+venetoclax, paralleled by decreased infection frequency. Innate immune impairment and infection susceptibility in patients with CLL were restored in parallel during targeted therapy. Thus, targeted treatment may reduce the risk of infections in CLL, as currently under investigation in the PreVent-ACaLL phase 2 trial of acalabrutinib+venetoclax for high-risk CLL (NCT03868722).

Monitoring functional immune responses with a cytokine release assay: ISS flight hardware design and experimental protocol for whole blood cultures executed under microgravity conditions.

Introduction: Long-term space missions trigger a prolonged neuroendocrine stress response leading to immune system dysregulation evidenced by susceptibility to infections, viral reactivation, and skin irritations. However, due to existing technical constraints, real-time functional immune assessments are not currently available to crew inflight. The in vitro cytokine release assay (CRA) has been effectively employed to study the stimulated cytokine response of immune cells in whole blood albeit limited to pre- and post-flight sessions. A novel two-valve reaction tube (RT) has been developed to enable the execution of the CRA on the International Space Station (ISS). Methods: In a comprehensive test campaign, we assessed the suitability of three materials (silicone, C-Flex, and PVC) for the RT design in terms of biochemical compatibility, chemical stability, and final data quality analysis. Furthermore, we thoroughly examined additional quality criteria such as safety, handling, and the frozen storage of antigens within the RTs. The validation of the proposed crew procedure was conducted during a parabolic flight campaign. Results: The selected material and procedure proved to be both feasible and secure yielding consistent and dependable data outcomes. This new hardware allows for the stimulation of blood samples on board the ISS, with subsequent analysis still conducted on the ground. Discussion: The resultant data promises to offer a more accurate understanding of the stress-induced neuroendocrine modulation of immunity during space travel providing valuable insights for the scientific community. Furthermore, the versatile nature of the RT suggests its potential utility as a testing platform for various other assays or sample types.

Design and fabrication of a vaccine candidate based on rOmpA from Klebsiella pneumoniae encapsulated in silk fibroin-sodium alginate nanoparticles against pneumonia infection

The present study describes the design and fabrication of a novel vaccine candidate based on the outer membrane protein A (rOmpA) from Klebsiella pneumoniae (K. pneumoniae) encapsulated in silk fibroin-sodium alginate nanoparticles (SF-SANPs) against K. pneumoniae-mediated pneumonia. The physicochemical properties, toxicity, release profile, and in vivo potency of SF-SANPs encapsulated with rOmpA were evaluated. The spherical nano vaccine was created with an average particle size of 160 nm and an encapsulation efficiency of 80 %. Antigen release from SF-SANPs was 40 % after 22 days release assay. The SF-SANPs showed a zeta potential of −24.8 mV and had no toxic effect on the L929 cells in vitro. It was found that SF-SANPs in the vaccine formulation promoted systemic and mucosal antibodies and also stimulated cytokine responses, inducing both humoral (Th2) and cellular (Th1) immune responses, with a Th1-polarized response. The vaccine candidate was effective in protecting the mice lung against experimental pneumonia and reducing inflammation. These findings suggest that the rOmpA-based vaccine encapsulated in SF-SANPs could be a promising strategy for preventing pneumonia caused by K. pneumoniae.

Depressive symptoms and other negative psychological states relate to ex vivo inflammatory responses differently for men and women: Cross-sectional and longitudinal evidence

An array of negative psychological states – including depressive symptoms, perceived stress, rumination, and negative affect – have been linked to immune function and inflammatory responses. Herein we show evidence of gender-dependent associations between ex vivo lipopolysaccharide (LPS)-stimulated cytokine responses and such psychological states, in both cross-sectional and longitudinal analyses from three annual waves (N = 162 at baseline, 67.3% female). In cross-sectional analyses (at baseline), gender moderated the associations of depressive symptoms (previously reported), perceived stress (B = -0.043, 95%CI [-0.080, -0.015]), rumination (B = -0.500, [-1.015, -0.232]), negative affect (B = -0.020, [-0.020, -0.005]), and positive affect (B = 0.024, [0.008, 0.047]) with LPS-stimulated cytokine responses. In each analysis, negative psychological states were positively associated with LPS-stimulated cytokine responses among men but negatively among women (with associations for positive affect in the opposite direction). In longitudinal analyses (across three annual measurements), similar associations were seen for depressive symptoms (B = -0.024, [-0.059, -0.004]), perceived stress (B = -0.045, [-0.069, -0.024]), and rumination (B = -0.381, [-0.622, -0.120]). These results indicate that gender is a critical factor in associations between a broad array of negative psychological states and inflammatory responses and identify one pathway by which gender may influence psychosomatic health.

Different PPD-stimulated cytokine responses from patients infected with genetically distinct Mycobacterium tuberculosis complex lineages

ObjectivesThe genetic diversity of Mycobacterium tuberculosis complex (MTBC) influences the immune response of the host, which may affect the immunodiagnostic tests and biomarker assessment studies used for tuberculosis (TB). This study aimed to determine whether the mycobacterial-antigen-stimulated cytokine responses vary with the genotype of the MTBC infecting the patient. MethodsEighty-one patients with confirmed active pulmonary TB were recruited, and MTBC clinical strains were isolated from their sputum for bacterial lineage single-nucleotide polymorphism typing. Whole blood was drawn from the patients to measure the purified protein derivative (PPD)-stimulated cytokine responses (GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, TNF-α, IFN-α, IL-12, eotaxin, IL-13, IL-15, IL-17, MIP1-α, MIP1-β, MCP1, IL1RA, IP10, IL2R, MIG) with the Luminex multiplex immunoassay. ResultsOf the 24 cytokines studied, three were produced differentially in whole blood dependent on the infecting lineage of MTBC. Decreased production of IL-17 was observed in patients infected with modern lineages compared with patients infected with ancestral lineages (P < 0.01), and production of IFN-γ and IL-2 was significantly decreased in patients infected with lineage 4 strains compared with patients infected with lineage 3 strains (P < 0.05). ConclusionMTBC strains belonging to lineage 4 induced a decreased whole-blood PPD-stimulated pro-inflammatory cytokine response.

Prospective association of maternal immune pro-inflammatory responsivity and regulation in pregnancy with length of gestation.

The immune system represents a leading pathway of interest in the pathophysiology of preterm birth. The majority of human clinical studies interrogating this pathway have utilized circulating immune biomarkers; however, these concentrations typically reflect only basal production but not key functional properties of the immune system, particularly variation in the pro-inflammatory response to antigen challenge and the regulation of this response. Thus, in this study, we utilized an ex vivo stimulation protocol that quantifies these processes, and we examined their prospective association with the gestation length and risk of preterm birth. Immune responsiveness and regulation were assessed in 128 pregnant women in mid-gestation using an ex vivo stimulation protocol. Maternal pro-inflammatory responsivity of leukocytes was quantified by assessing the release of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in response to antigen stimulation, and regulation of the pro-inflammatory response was quantified by assessing the suppression of stimulated cytokine response upon co-incubation with increasing dexamethasone concentrations (ie, glucocorticoid receptor resistance; GRR). Higher maternal GRR, indicating impaired regulation of the pro-inflammatory response, was significantly and independently associated with shorter gestational length (β=-0.42, p=.0091) and a 3.0-fold increase in risk for preterm birth (OR=3.01, 95% CI=1.17-7.70, p=.0218). Basal circulating IL-6 and TNF-α were not associated with either outcome. The association of maternal GRR with length of gestation and preterm birth risk suggests that the processes represented by this measure-maternal pro-inflammatory propensity and immune regulation-may provide further mechanistic insight into the pathophysiology of preterm birth.

Associations between spontaneous parental perspective-taking and stimulated cytokine responses in children with asthma.

Cognitive empathy in parents-reflecting the extent to which one considers the perspectives and emotions of others-is hypothesized to contribute to family social environments in ways that affect youths' physical health. Using a novel assessment technique for cognitive empathy, the current study examined associations between spontaneous parental perspective-taking and key inflammatory processes implicated in pediatric asthma. One hundred thirty children (ages 9-17) with physician-diagnosed asthma, along with 1 parent, participated in the current study. Parents completed an interview from which statements of perspective-taking were coded and youths provided blood samples. Youths whose parents demonstrated greater spontaneous perspective-taking during the interview had cells that mounted smaller inflammatory responses to stimulation by nonspecific, asthma-specific, and viral analogue ligands, as well as cells that showed greater sensitivity to the anti-inflammatory effects of glucocorticoids. These results were not accounted for by parental warmth or parent or youth depressive symptoms, nor by covariates of race, age, gender, parental education level, use of asthma medications over the past week, or asthma severity. These findings suggest that parental perspective-taking may have implications for biological processes relevant to childhood asthma. (PsycINFO Database Record

Impaired cytokine responses in patients with cryopyrin-associated periodic syndrome (CAPS).

Cryopyrin-associated periodic syndrome (CAPS) is characterized by dysregulated inflammation with excessive interleukin (IL)-1β activation and secretion. Neonatal-onset multi-system inflammatory disease (NOMID) is the most severe form. We explored cytokine responses in 32 CAPS patients before and after IL-1β blocking therapy. We measured cytokines produced by activated peripheral blood monuclear cells (PBMCs) from treated and untreated CAPS patients after stimulation for 48 h with phytohaemagglutinin (PHA), PHA plus IL-12, lipopolysaccharide (LPS) or LPS plus interferon (IFN)-γ. We measured IL-1β, IL-6, IL-10, tumour necrosis factor (TNF), IL-12p70 and IFN-γ in the supernatants. PBMCs from three untreated CAPS patients were cultured in the presence of the IL-1β blocker Anakinra. Fifty healthy individuals served as controls. CAPS patients had high spontaneous production of IL-1β, IL-6, TNF and IFN-γ by unstimulated cells. However, stimulation indexes (SIs, ratio of stimulated to unstimulated production) of these cytokines to PHA and LPS were low in NOMID patients compared to controls. Unstimulated IL-10 and IL-12p70 production was normal, but up-regulation after PHA and LPS was also low. LPS plus IFN-γ inadequately up-regulated the production of IL-1β, IL-6, TNF and IL-10 in CAPS patients. In-vitro but not in-vivo treatment with Anakinra improved SIs by lowering spontaneous cytokine production. However, in-vitro treatment did not improve the low stimulated cytokine levels. Activating mutations in NLRP3 in CAPS are correlated with poor SIs to PHA, LPS and IFN-γ. The impairment in stimulated cytokine responses in spite of IL-1β blocking therapy suggests a broader intrinsic defect in CAPS patients, which is not corrected by targeting IL-1β.

Exposure to dogs is associated with a decreased tumour necrosis factor‐α‐producing capacity in early life

It appears that contacts with furred animals early in life and already during gestation contribute to the immunological development in humans, but the mechanisms and relevant exposures are not clear. To investigate whether exposure to animals during pregnancy and the first year of life is associated with early immune development, determined as stimulated cytokine responses of children at birth and at age 1 year. Cord blood (n=228) and peripheral venous blood (n=200) samples 1 year after birth were collected and stimulated with Gram-positive superantigen Staphylococcal enterotoxin B, Gram-negative bacterial lipopolysaccharide (LPS) and the combination of mitogenic phorbol 12-myristate 13-acetate and calcium ionophore ionomycin (P/I) for 24 and 48 h. TNF-α, IFN-γ, IL-5, IL-8 and IL-10 responses were measured by ELISA. For each cytokine, the time-point with the highest response was chosen for further analyses. Animal contacts were surveyed by self-administered questionnaires. Dog ownership was associated with decreased TNF-α-producing capacity at birth (P/I: median 841 vs. 881 pg/10(6) WBC, P=0.05) and 1 year after birth (P/I: 1290 vs. 1530, P=0.01; LPS: 425 vs. 508, P=0.02). Associations remained significant after adjustment for potential confounders. Cat ownership was not associated with cytokine production. Having a dog in the household in infancy and already during pregnancy may be associated with reduced innate immune responses in early childhood. The observed attenuation of cytokine production may help in preventing exaggerated immune responses against harmless antigens later in life. Thus, intensive exposure to dogs in early life may be beneficial during normal immune maturation.

Maturation of cytokine‐producing capacity from birth to 1 yr of age

Little is known about the immunologic maturation in the early stages of life. The aim of this study was to investigate maturation of immune system from birth to 1 yr of age and to compare immune functions between mothers and their children. Also the effect of atopy to the immune responses of children was examined. Cord blood samples (n = 228) and peripheral blood samples of children (n = 200) and their mothers (n = 208) 1 yr after birth were collected. Whole blood samples were stimulated for 24 and 48 h with Staphylococcal enterotoxin B (SEB), lipopolysaccharide (LPS) and the combination of phorbol ester and ionomycin (P/I). Production of TNF-alpha, IFN-gamma, IL-5, IL-8 and IL-10 was determined using ELISA. Significant mother-to-child correlation was detected in cytokine-producing capacity at the age of 1 yr. TNF-alpha (P/I, SEB and LPS stimulation), IFN-gamma (P/I and SEB), IL-5 (P/I and SEB) and IL-10 (P/I, SEB and LPS) producing capacity increased from birth to 1 yr of age. In general, stimulated cytokine responses were higher in mothers' than in children's blood samples, except in the case of P/I and LPS-stimulated IL-8, which were highest at birth. Maternal inhalation atopy was associated with increased cord blood IL-5 (24 and 48 h) and IL-10 (48 h) production following P/I stimulation. Also children of food atopic mothers expressed elevated cord blood IL-10 (48 h, P/I) responses and decreased IFN-gamma/IL-5 ratio (24 h, P/I). In addition, the production of IFN-gamma (24 and 48 h, P/I) and the IFN-gamma/IL-5 ratio (24 h and 48 h, P/I) at the age of 1 yr was lower among children with food atopic mothers. In conclusion, our results suggest that both adaptive and innate immune responses increase from birth to 1 yr of age, but are still weak in comparison to adult responses. Cytokine responses of children begin to correlate with those of their mothers during the first year of life. Although only few associations were observed between atopy and cytokine-producing capacity, our results suggest that children of atopic mothers express T(h)2-polarized cytokine pattern.

Impact of Platelets and Fresh Frozen Plasma in Contrast to Red Cell Concentrate on Unstimulated and Stimulated Cytokine Release in an In Vitro Model of Transfusion

Transfusion of blood may contribute to immunomodulation. Leuco-depleted standard blood products are supposed to result in less immunomodulation compared with whole blood. To determine the influence of leuco-depleted blood products on the cytokine response, red blood cell concentrates (RBC), fresh frozen plasma (FFP) and platelet concentrates (PC) were investigated in an in vitro model of blood transfusion. Leuco-depleted standard blood bank RBC, FFP and PC were mixed in vitro with AB0 compatible venous blood from healthy volunteers in ratios of 3:1, 1:1 and 1:3. Specimens were incubated in presence or absence of lipopolysaccharide, 1 mug/ml. After 24 h of incubation cytokine release of tumour necrosis factor (TNF)-alpha and interleukin-10 (IL-10) was measured in cell culture supernatants by means of enzyme-linked immunsorbent assay. Addition of RBC, FFP and PC to venous blood from healthy volunteers led to a significant and dose-dependent increase in spontaneous TNF-alpha and IL-10 release. After endotoxin stimulation, RBC, FFP and PC significantly suppressed the TNF-alpha response, while the stimulated release of IL-10 tended to increase, reaching significance only after high doses of FFP. Addition of leuco-depleted blood products changed the spontaneous and stimulated cytokine response in an in vitro model of transfusion. These data may suggest a possible contribution of transfused FFP and PC to immunomodulation after transfusion similar to RBC.

QS412. Effect of Tobacco Smoke on Arterial Cell Populations and Stimulated Cytokine Responses

QS412. Effect of Tobacco Smoke on Arterial Cell Populations and Stimulated Cytokine Responses

Maternal mood scores in mid-pregnancy are related to aspects of neonatal immune function

Background Although there are recognised associations between psychological and immune function, the effects of maternal depressive symptoms on fetal immune development have not been investigated. Methods This study examined the relationship between maternal depression scores as assessed by the Beck Depression Inventory (BDI) in the second trimester and measure of neonatal immune function measured in cord blood. This study was conducted in a cohort of women ( n = 83) who had received either fish oil containing 3.7 g/day n-3 polyunsaturated fatty acid ( n-3PUFA) or a placebo from 20 weeks gestation as part of a randomised controlled trial. Results At 20 weeks gestation, prior to the intervention, 22% of women in the study manifested mild to moderate depressive symptoms (BDI ⩾10). Neonates of these women had higher lymphoproliferative responses to a range of stimuli (including egg ovalbumin and cat allergen) compared with neonates of women with normal BDI scores (<10). These neonates also showed higher spontaneous cytokine production including (IL-6 and IL-10) and higher stimulated cytokine responses to both bacterial antigens and allergens. These patterns were evident after allowing for maternal age and education, parity, gestation, infant gender, delivery method and neonatal n-3/ n-6 PUFA status. Conclusion This exploratory study supports the notion that maternal mood in pregnancy may have the potential to influence fetal immune development. Further studies are needed to determine the significance of this.

Exposure to environmental bacteria may have differing effects on tumour necrosis factor‐α and interleukin‐6‐producing capacity in infancy

Our previous study showed an association between increased concentration of endotoxin in house dust and elevated IFN-gamma responses in neonates. The impact of other microbial agents on immune responses in infancy is poorly known. To examine whether stimulated cytokine responses of mothers and their children are associated with concentrations of other microbial markers in addition to endotoxin in house dust samples. Mitogen-stimulated production of IFN-gamma, IL-4, IL-6 and TNF-alpha was measured in cord blood and in peripheral blood of mothers (n=29) and their children (n=29) 3 months after birth. Gas chromatography mass spectrometric analysis was applied to measure the concentrations of ergosterol (marker of fungal biomass), muramic acid (indicating the presence of Gram-positive bacteria) and 3-hydroxy fatty acids (C(10:0)-C(14:0), indicating the presence of Gram-negative bacteria) in house dust. Endotoxin was determined with Limulus assay. Significant mother-to-child correlations were observed in stimulated production of TNF-alpha and IL-6 3 months after birth. 3-hydroxy fatty acid (C(10:0)-C(14:0)) levels in bed dust were inversely associated with the production of TNF-alpha and IL-6 in blood samples of mothers and their 3-month-old children. High concentrations of muramic acid in floor dust were related to increased production of TNF-alpha and IL-6 at the age of 3 months. In contrast to endotoxin, none of the other microbial markers were significantly associated with enhanced IFN-gamma-producing capacity from birth to 3 months. Exposure to Gram-negative bacteria and their components may be associated with down-regulated immune responses in early infancy, indicated as an impaired production of pro-inflammatory cytokines following mitogen stimulation. Gram-positive bacteria and their constituents seem to have opposite effects. Of the measured markers, exposure to bioactive endotoxin appears to have the strongest impact on T-helper type 1 responses.

Antibiotics modulate the stimulated cytokine response to endotoxin in a human ex vivo, in vitro model

Sepsis may lead to the suppression of stimulated cytokine release after Gram-negative stimuli, correlating with a fatal outcome. Treatment of sepsis includes adequate therapy with antibiotics. The aim of this study was to investigate the role of antibiotics in the modulation of the lipopolysaccharide (LPS)-stimulated cytokine response of human monocytes. In this ex vivo, in vitro study, whole blood samples were taken from 10 healthy volunteers, stimulated with LPS in the presence or absence of various antibiotics (penicillin, amoxicillin, cefuroxime, ceftazidime, cefotaxime, piperacillin/tazobactam, imipenem/cilastatin, gentamicin, netilmicin, ciprofloxacin, vancomycin) and cultured for 24 h. Thereafter, tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) were measured in the supernatants by enzyme-linked immunosorbent assay (ELISA). Furthermore, CD14 and HLA-DR expression on monocytes was assessed using flow cytometry. All cephalosporins decreased LPS-stimulated IL-10 release. Cefuroxime and cefotaxime also decreased the expression density of the LPS recognition molecule CD14 on monocytes. An increase in LPS-stimulated IL-10 release was observed with vancomycin. A suppression of LPS-stimulated TNF-alpha and IL-10 release was observed in the presence of ciprofloxacin. These results indicate a modulation of the expression density of CD14 on monocytes, together with a shift from a balanced to an inflammatory cytokine release pattern, by cefuroxime and cefotaxime. Vancomycin changes the response to an anti-inflammatory release pattern. After ciprofloxacin, a profound unresponsiveness of immune-competent cells to LPS stimulation is observed. Because of the critical role of a balanced innate immune response, these data may be of importance for the selection of antibiotics in septic patients.

Einfluss des Geschlechts auf die stimulierbare Zytokinantwort bei Patienten mit schwerer Sepsis

Studies suggest that female mice have lower mortality rates than males after sepsis or trauma-hemorrhage. This study investigated the impact of gender and disease severity on monocyte hyporesponsiveness in severe human sepsis. We prospectively investigated 49 (male n=28, female n=21) consecutive patients with severe sepsis. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) were assayed by ELISA in unstimulated whole blood cultures or after stimulation with lipopolysaccharide (LPS; E. coli 0111:B4) or Staph. aureus Cowan strain I (SAC-I) lysate at days 1, 2, 3, 4, and 8 after enrollment. Testosterone and estradiol levels were quantified by electrochemoluminescence immunoassays. Mortality was similar for males (35.7%) and females (42.9%). While disease severity was also comparable, septic patients showed a substantial suppression in stimulated TNF-alpha response compared to healthy controls who recovered within 8 days in surviving patients. Stimulated cytokine response recovered in female non-surviving patients, while it remained suppressed in non-surviving male patients and was significantly different compared to female non-surviving patients. Testosterone levels were substantially suppressed in male but not female septic patients compared to normal values but did not differ between surviving and non-surviving patients. Estradiol levels were elevated in female and male septic patients. Addition of different concentrations of testosterone and estradiol to whole blood obtained from younger (<35 years old) and older (>60 years old) male as well as from younger (proestrous premenopausal) and older (postmenopausal) female non-septic volunteers revealed no effect on LPS-stimulated TNF-alpha and IL-10 release. Severe sepsis leads to a substantial suppression of stimulated cytokine response. Prolonged suppression may serve as a marker of unfavourable outcome in male but not in female individuals suffering from severe sepsis. Furthermore, our data suggest that gender differences in cellular immunity described for young, sexually mature animals obviously persist in typical postmenopausal intensive care unit patients, although a direct interaction between testosterone or estradiol and LPS-stimulated cytokine response could not be demonstrated.

Modulation of endotoxin stimulated cytokine response by antibiotics in cultured human whole blood

Modulation of endotoxin stimulated cytokine response by antibiotics in cultured human whole blood

Impact of alloantigens and storage-associated factors on stimulated cytokine response in an in vitro model of blood transfusion.

Transfusion of blood may contribute to immunosuppression in major surgery. The authors assessed the impact of alloantigens and storage on function of peripheral blood mononuclear cells cultured in their physiologic environment. Blood units (whole blood, packed erythrocytes) were prepared with or without prestorage leukodepletion and stored for 24-26 days. Blood samples were coincubated with allogeneic fresh blood, autologous, or allogeneic stored blood. Endotoxin-stimulated release of tumor necrosis factor-alpha (TNF-alpha) and interleukin 10 (IL-10) was measured after 24 h of culture by enzyme-linked immunosorbent assay. Coincubation with equal amounts of allogeneic fresh blood showed almost no influence on TNF-alpha (-12%, not significant) and IL-10 (+11%, not significant) release. Stored allogeneic whole blood resulted in a significant TNF-alpha depression (-61%) and IL-10 induction (+221%). These effects were diminished but not prevented by prestorage leukodepletion (TNF-alpha -42%, IL-10 +110%) and required the presence of soluble factors (TNF-alpha suppression) and cellular components (IL-10 induction). TNF-alpha decrease and IL-10 increase were in the same order of magnitude (-40%, +134% with, -65%, +314% without leukodepletion) after coincubation with autologous blood. In contrast, allogeneic erythrocytes had only little effects (TNF-alpha -6%, IL-10 +36%) even at this high transfusion equivalent. These data suggest that banked whole blood has an immunosuppressive effect that is largely attributable to storage-dependent factors. These factors are partially removed by prestorage leukodepletion, while the contribution of alloantigens is of minor significance. Immunosuppressive effects are least apparent with leukodepleted erythrocytes, suggesting that the presence of plasma during storage is required for the immunosuppressive effect to develop.