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A pharmacodynamic assay has been previously developed to monitor ciclosporin treatment in dogs by assessing inhibition of cytokine transcription after whole blood stimulation with 12‐myristate 13‐1 acetate and ionomycin (PMA/I). In this study, whole blood stimulation with either PMA/I or lipopolysaccharide (LPS) was used to assess the effect of multiple drugs (azathioprine, ciclosporin, mycophenolate, leflunomide and prednisone) after a 7‐day treatment course on production of cytokines measured with a multiplex assay in healthy dogs (n = 4 for each treatment). Interleukin‐10 (IL‐10), interferon gamma (IFN γ) and tumour necrosis factor alpha (TNF α) were significantly activated by PMA/I stimulation and IL‐6, IL‐10 and TNF α by LPS stimulation, in the absence of immunosuppressive drugs. After ciclosporin treatment, IL‐10, IFN γ and TNF α production was significantly reduced after stimulation with PMA/I compared to pre‐treatment. After prednisone treatment, TNF α production was significantly reduced after stimulation with PMA/I or LPS compared to pre‐treatment. No significant change was observed after treatment with azathioprine, leflunomide or mycophenolate. This methodology may be useful to monitor dogs not only treated with ciclosporin, but also with prednisone or a combination of both. Further studies are needed to assess the use of this assay in a clinical setting.

SARS-CoV-2 specific T-cells responses are essential for virus clearance. We present a novel and simple whole-blood assay allowing the detection of interferon-gamma-producing antiviral T-cells following peptide stimulation. We show that unlike neutralizing antibodies, antiviral memory T-cells persist at least 6 months in convalescent Covid-19 individuals.

Critically ill patients show signs of immune suppression, which is considered to increase vulnerability to nosocomial infections. Whole-blood stimulation is frequently used to test the function of the innate immune system. We here assessed the association between whole-blood leukocyte responsiveness to lipopolysaccharide (LPS) and subsequent occurrence of nosocomial infections in critically ill patients admitted to the intensive care unit (ICU). All consecutive critically ill patients admitted to the ICU between April 2012 and June 2013 with two or more systemic inflammatory response syndrome criteria and an expected length of ICU stay of more than 24 h were enrolled. Age- and sex-matched healthy individuals were included as controls. Blood was drawn the first morning after ICU admission and stimulated ex vivo with 100 ng/mL ultrapure LPS for 3 h. Tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 were measured in supernatants. Seventy-three critically ill patients were included, of whom 10 developed an ICU-acquired infection. Compared with healthy subjects, whole-blood leukocytes of patients were less responsive to ex vivo stimulation with LPS, as reflected by strongly reduced tumor necrosis factor-α, IL-1β, and IL-6 levels in culture supernatants. Results were not different between patients who did and those who did not develop an ICU-acquired infection. The extent of reduced LPS responsiveness of blood leukocytes in critically ill patients on the first day after ICU admission does not relate to the subsequent development of ICU-acquired infections. These results argue against the use of whole-blood stimulation as a functional test applied early after ICU admission to predict nosocomial infection.

Lauran Vogelaar, 1,* Colin de Haar,2,* Bas RJ Aerts,1 Maikel P Peppelenbosch,1 Reinier Timman,3 Bettina E Hanssen,1 C Janneke van der Woude1 1Department of Gastroenterology and Hepatology, Erasmus MC, Rotterdam, 2Applied Tumor Immunology, Laboratory of Translational Immunology, UMC Utrecht, Utrecht, 3Department of Psychiatry, Section of Medical Psychology and Psychotherapy, Erasmus MC, Rotterdam, the Netherlands *These authors contributed equally to this work Background: Although it is well recognized that fatigue is an important problem in many of the quiescent inflammatory bowel disease (IBD) patients, it is unknown whether the immune status is different in fatigued versus non-fatigued patients. In this study, we contrasted various characteristics of the immune system in fatigued against non-fatigued patients with IBD in clinical remission.Patients and methods: Patients with IBD in clinical remission were phenotyped according to the Montreal classification, and the checklist individual strength-fatigue (CIS-fatigue) was used to assess fatigue (CIS-fatigue ≥ 35). Flow cytometry on peripheral blood samples was used to investigate differences in leukocyte subsets. The expression of various cytokines was determined in stimulated whole blood and serum samples using enzyme-linked immunosorbent assay. Differences between fatigued and non-fatigued patients with IBD were assessed.Results: In total, 55 patients were included in the fatigue group (FG) and 29 patients in the non-fatigue group (NFG). No differences in demographic and clinical characteristics were observed between the groups. Flow cytometry data showed a significantly lower percentage of monocytes (p = 0.011) and a higher percentage of memory T-cells (p = 0.005) and neutrophils (p = 0.033) in the FG compared with the NFG. Whole blood stimulation showed increased TNF-α (p = 0.022) and IFN-γ (p = 0.047) in the FG. The median serum level was significantly higher for IL-12 (p&nbsp

Functional immune responses are increasingly important for clinical studies, providing in depth biomarker information to assess immunotherapy or vaccination. Incorporating functional immune assays into routine clinical practice has remained limited due to challenges in standardizing sample preparation. We recently described the use of a whole blood syringe-based system, TruCulture (R), which permits point-of-care standardized immune stimulation. Here, we report on a multi-center clinical study in seven FOCIS Centers of Excellence to directly compare TruCulture to conventional PBMC methods. Whole blood and PBMC5 from healthy donors were exposed to LPS, anti-CD3 anti-CD28 antibodies, or media alone. 55 protein analytes were analyzed centrally by Luminex multi-analyte profiling in a CLIA-certified laboratory. TruCulture responses showed greater reproducibility and improved the statistical power for monitoring differential immune response activation. The use of TruCulture addresses a major unmet need through a robust and flexible method for immunomonitoring that can be reproducibly applied in multi-center clinical studies. One sentence summary: A multi-center study revealed greater reproducibility from whole blood stimulation systems as compared to PBMC stimulation for studying induced immune responses. (C) 2017 Published by Elsevier Inc.

Estradiol, progesterone, prolactin and cortisol concentrations are substantially increased during pregnancy. Also, cortisol and prolactin levels are elevated during stress. In the present study, we exposed peripheral blood to estradiol, progesterone, prolactin and cortisol alone or in combination for 24 h before stimulation with T-dependent (phytohemagglutinin, PHA) and independent activators (lipopolysaccharide, LPS) to study their immunomodulatory role in interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and IL-10 production in a whole blood model. This should be similar to in vivo exposure conditions such as long-term stress, preovulatory or pregnancy periods. The present study showed that the stress-induced and preovulatory levels of prolactin and estradiol, respectively, increased the production of IFN-gamma and IL-12 levels (and IL-10 in the case of estradiol) in PHA + LPS-stimulated whole blood, and inhibited a hydrocortisone (100 nmol/l) suppressive effect on IFN-gamma, IL-12 and IL-10 productions. In LPS-stimulated whole blood, however, prolactin enhanced only IL-10 production levels in a non-concentration-dependent manner. Higher prolactin levels as in pregnancy did not modulate any of the cytokines, but pregnancy estradiol concentrations only induced higher IL-10 levels in PHA + LPS-stimulated whole blood. All progesterone levels tested revealed no effect on any of the cytokines following whole blood stimulation. Our results indicate that (1) a long exposure time of prolactin and estradiol to whole blood modulates the production of cytokines in a concentration- and stimulus-dependent manner; (2) stress-induced levels of prolactin and preovulatory estradiol concentrations can regulate cortisol-induced cytokine suppression, and (3) even though the cytokine pattern is different, pregnancy estradiol and cortisol levels decreased the IFN-gamma/IL-10 ratio, thereby keeping the anti-inflammatory IL-10 levels favored during pregnancy, which could be useful in regulating inflammatory-mediated autoimmune diseases.

Background Helium inhalation protects myocardium, brain and endothelium against ischemia/reperfusion injury in animals and humans, when applied according to specific “conditioning” protocols. Before widespread use of this “conditioning” agent in clinical practice, negative side effects have to be ruled out. We investigated the effect of prolonged helium inhalation on the responsiveness of the human immune response in whole blood ex vivo. Methods Male healthy volunteers inhaled 30 minutes heliox (79%He/21%O2) or air in a cross over design, with two weeks between measurements. Blood was withdrawn at T0 (baseline), T1 (25 min inhalation) and T2-T5 (1, 2, 6, 24 h after inhalation) and incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), T-cell stimuli anti-CD3/ anti-CD28 (TCS) or RPMI (as control) for 2, 4 and 24 hours or not incubated (0 h). An additional group of six volunteers inhaled 60 minutes of heliox or air, followed by blood incubation with LPS and RPMI. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-γ (IFN-γ) and interleukin-2 (IL-2) was analyzed by cytometric bead array. Statistical analysis was performed by the Wilcoxon test for matched samples. Results Incubation with LPS, LTA or TCS significantly increased TNF-α, IL-1β, IL-6, IL-8, IFN-γ and IL-2 in comparison to incubation with RPMI alone. Thirty min of helium inhalation did not influence the amounts of TNF-α, IL-1β, IL-6, IL-8, IFN-γ and IL-2 in comparison to air. Sixty min of helium inhalation did not affect cytokine production after LPS stimulation. Conclusions We conclude that 79% helium inhalation does not affect the responsiveness of the human immune system in healthy volunteers. Trial registration Dutch Trial Register:http://www.trialregister.nl/ NTR2152

Ex vivo whole blood stimulation with endotoxin has proved to be a useful method for quantitative evaluations of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) response ability in humans. In the present study, a dose- and time-response study was carried out in order to develop an ex vivo whole blood stimulation assay for the quantification of TNF-alpha production in pigs. The TNF-alpha response was enhanced with increasing endotoxin stimulation dose. The maximum TNF-alpha response occurred after 2-8 h of stimulation. Subsequently, the assay was used to evaluate the TNF-alpha response in pigs (n=32) in relation to weaning. The TNF-alpha response was 332 pg ml(-1) (+/-59 pg) plasma 2 days before weaning and 127 pg ml(-1) (+/-23 pg) plasma 2 days after weaning, which was a significant reduction (p0.001). Total and differential counts of leucocytes were the same before and after weaning. Thus the lower TNF-alpha response may be due to reduced monocyte responsiveness to the endotoxin, rather than caused by a reduction in monocyte numbers. The reduced TNF-alpha response in piglets after weaning may be a factor of importance to the increased disease susceptibility seen in piglets in this period.

Accumulating data suggest a protective effect of obesity in the case of severe infections. Higher baseline levels of the proinflammatory cytokine TNFα as well as more pronounced TNFα release following whole blood stimulation with endotoxin are reported in patients with a higher body mass index (BMI). This more pronounced proinflammatory response in obese patients may enable a rapid and more effective clearance of microbial pathogens. The effect of the body mass index on the innate immune response in vivo has not been assessed.

1912 Vol 349 • June 28, 1997 production with low interleukin-10 production. The figure shows that in healthy volunteers the maximum production of interleukin-10 is independent of the production of TNFwhen determined in a whole blood stimulation assay in vitro. TNFis a double-edged sword, it is a necessary mediator in the host response to infection but high circulating concentrations can be harmful. We propose a complete switch in the understanding of proinflammatory and anti-inflammatory cytokines that contribute to sepsis syndrome. Our data suggest that patients with low production of TNFand high production of interleukin10 early in fulminant meningococcal disease have an insufficient host response to infection and end up with a large bacterial burden, high circulating concentrations of cytokines, and a poor outcome.

The purpose of our study was to determine the acute inflammatory response of critically ill (ICU) and haemodialysis (HD) patients and to compare the cytokine profiles induced by different classes of pathogens in these two patient populations vulnerable to infections.