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Introduction: The diagnosis of tuberculosis (TB) disease and TB infection (TBI) remains a challenge, and there is a need for non-invasive and blood-based methods to differentiate TB from conditions mimicking TB (CMTB), TBI, and healthy controls (HC). We aimed to determine whether combination of cytokines and established biomarkers could discriminate between 1) TB and CMTB 2) TB and TBI 3) TBI and HC. Methods: We used hemoglobin, total white blood cell count, neutrophils, monocytes, C-reactive protein, and ten Meso Scale Discovery analyzed cytokines (interleukin (IL)-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon (IFN)-ɣ, and tumor necrosis factor (TNF)-a) in TruCulture whole blood tubes stimulated by lipopolysaccharides (LPS), zymosan (ZYM), antiCD3/28 (CD3), and unstimulated (Null) to develop three index tests able to differentiate TB from CMTB and TBI, and TBI from HC. Results: In 52 persons with CMTB (n=9), TB (n=23), TBI (n=10), and HC (n=10), a combination of cytokines (LPS-IFN-ɣ, ZYM-IFN-ɣ, ZYM-TNF-a, ZYM-IL-1b, LPSIL-4, and ZYM-IL-6) and neutrophil count could differentiate TB from CMTB with a sensitivity of 52.2% (95% CI: 30.9%–73.4%) and a specificity of 100 % (66.4%- 100%). Null- IFN-ɣ, Null-IL-8, CD3-IL-6, CD3-IL-8, CD3-IL-13, and ZYM IL-1b discriminated TB from TBI with a sensitivity of 73.9% (56.5% - 91.3%) and a specificity of 100% (69.2-100). Cytokines and established biomarkers failed to differentiate TBI from HC with ≥ 98% specificity. Discussion: Selected cytokines may serve as blood-based add-on tests to detect TB in a low-endemic setting, although these results need to be validated. [ABSTRACT FROM AUTHOR]

IntroductionThe diagnosis of tuberculosis (TB) disease and TB infection (TBI) remains a challenge, and there is a need for non-invasive and blood-based methods to differentiate TB from conditions mimicking TB (CMTB), TBI, and healthy controls (HC). We aimed to determine whether combination of cytokines and established biomarkers could discriminate between 1) TB and CMTB 2) TB and TBI 3) TBI and HC. MethodsWe used hemoglobin, total white blood cell count, neutrophils, monocytes, C-reactive protein, and ten Meso Scale Discovery analyzed cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon (IFN)-ɣ, and tumor necrosis factor (TNF)-α) in TruCulture whole blood tubes stimulated by lipopolysaccharides (LPS), zymosan (ZYM), anti-CD3/28 (CD3), and unstimulated (Null) to develop three index tests able to differentiate TB from CMTB and TBI, and TBI from HC. ResultsIn 52 persons with CMTB (n=9), TB (n=23), TBI (n=10), and HC (n=10), a combination of cytokines (LPS-IFN-ɣ, ZYM-IFN-ɣ, ZYM-TNF-α, ZYM-IL-1β, LPS-IL-4, and ZYM-IL-6) and neutrophil count could differentiate TB from CMTB with a sensitivity of 52.2% (95% CI: 30.9%–73.4%) and a specificity of 100 % (66.4%-100%). Null- IFN-ɣ, Null-IL-8, CD3-IL-6, CD3-IL-8, CD3-IL-13, and ZYM IL-1b discriminated TB from TBI with a sensitivity of 73.9% (56.5% - 91.3%) and a specificity of 100% (69.2-100). Cytokines and established biomarkers failed to differentiate TBI from HC with ≥ 98% specificity.DiscussionSelected cytokines may serve as blood-based add-on tests to detect TB in a low-endemic setting, although these results need to be validated.

Recent developments in multiplex technologies enable the determination of a large nu\mber of soluble proteins such as cytokines in various biological samples. More than a one-by-one determination of the concentration of immune mediators, they permit the establishment of secretion profiles for a more accurate description of conditions related to infectious diseases or vaccination. Cytokine profiling has recently been made available for bovine species with the development of a Luminex® technology-based 15-plex assay. Independently from the manufacturer, we evaluated the bovine cytokine/chemokine multiplex assay for limits of detection, recovery rate, and reproducibility. Furthermore, we assessed cytokine secretion in blood samples from 107 cows upon stimulation with heat-killed bacteria and TLR2/4 ligands compared to a null condition. Secretion patterns were analyzed either using the absolute concentration of cytokines or using their relative concentration with respect to the overall secretion level induced by each stimulus. Using Partial Least Square-Discriminant Analysis, we show that the 15-cytokine profile is different under Escherichia coli , Staphylococcus aureus , and Streptococcus uberis conditions, and that IFN-γ, IL-1β, and TNF-α contribute the most to differentiate these conditions. LPS and E. coli induced largely overlapping biological responses, but S. aureus and S. uberis were associated with distinct cytokine profiles than their respective TLR ligands. Finally, results based on adjusted or absolute cytokine levels yielded similar discriminative power, but led to different stimuli-related signatures. [ABSTRACT FROM AUTHOR]

[Display omitted] • COVID-19 with and without non-invasive ventilation showed similar cytokine release. • Higher spontaneous release of IFNγ and IP-10 was detected in COVID-19 patients. • Lower production of MCP-1 was detected in COVID-19 patients after specific stimulus. • Mitogenic stimulus induced major differences between COVID-19 patients and controls. T cells are key players in the resolution of the infection by SARS-CoV-2. A delay in their activation can lead to severe COVID-19. The present work aimed to identify differences in cytokine release by T cells ex-vivo between COVID-19 patients in the acute phase, showing diverse disease severity. Concentrations of IFNγ, Granzyme B, IL-6, IL-10, IL-17A, IL-18, IP-10, MCP-1, and TNFα were evaluated after stimulation ex-vivo of whole blood samples with peptides from SARS-CoV-2 spike protein and a mitogen as well as without stimulation. Samples derived from hospitalized COVID-19 patients and SARS-CoV-2 vaccinated controls (CTR). Patients were classified on disease severity considering the necessity of non-invasive ventilation (NIV). Samples from patients requiring NIV revealed a similar release of cytokines compared with patients without NIV. COVID-19 patients showed higher spontaneous production of IFNγ and IP-10, lower production of MCP-1 after SARS-CoV-2 peptide stimulation and lower production of IFNγ, IL-10, IL-17A, Granzyme B, IP-10 after mitogenic stimulus compared with CTR. In conclusion, differences in T cell responses evaluated ex-vivo by a whole blood-based cytokine release assay do not appear to explain the need for non-invasive ventilation in COVID-19 patients. [ABSTRACT FROM AUTHOR]

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 (IFNγ) and tumour necrosis factor alpha (TNFα) were significantly activated by PMA/I stimulation and IL‐6, IL‐10 and TNFα by LPS stimulation, in the absence of immunosuppressive drugs. After ciclosporin treatment, IL‐10, IFNγ and TNFα production was significantly reduced after stimulation with PMA/I compared to pre‐treatment. After prednisone treatment, TNFα 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. [ABSTRACT FROM AUTHOR]

The immune system defends the body from infection and plays a vital role in a wide range of health conditions. Metabolism affects a series of physiological processes, including those linked to the function of human immune system. Cellular metabolism modulates immune cell activation and cytokine production. Understanding the relationship between metabolism and immune response has important implications for the development of immune-based therapeutics. However, the deployment of large-scale functional assays to investigate the metabolic regulation of immune response has been limited by the lack of standardized procedures. Here, we present a protocol for the analysis of immune response using standardized whole-blood stimulation with metabolism modulation. Diverse immune stimuli including pattern recognition receptor (PRR) ligands and microbial stimuli were incubated with fresh human whole blood. The metabolic inhibitors were used to modulate metabolic status in the immune cells. The variable immune responses after metabolic interventions were evaluated. We described in detail the main steps involved in the whole-blood stimulation and cytokines quantification, namely, collection and treatment of whole blood, preparation of samples and controls, cytokines detection, and stimulation with metabolic interventions. The metabolic inhibitors for anabolic pathways and catabolic pathways exert selective effects on the production of cytokines from immune cells. In addition to a robust and accurate assessment of immune response in cohort studies, the standardized whole-blood stimulation with metabolic regulation might provide new insights for modulating immunity., Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00114-0., Competing Interests: Conflict of interestYiran Zhao is the editorial operation team member of Phenomics, and she was not involved in reviewing this paper., (© International Human Phenome Institutes (Shanghai) 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Objectives: Sepsis is a complex clinical condition associated with high morbidity and mortality. A distinctive feature of sepsis is the reduced capacity of leukocytes to release proinflammatory cytokines in response to ex vivo stimulation. Cellular signaling events leading to immunosuppression in sepsis are not well defined. We investigated cell-specific signaling events underlying the immunosuppressed phenotype in sepsis.Design: Ex vivo study.Setting: ICU of an academic hospital.Patients: Nineteen patients with sepsis and 19 age-matched healthy controls.Interventions: None.Measurements and Main Results: The phosphorylation state of p38 mitogen activated protein kinase and nuclear factor kappa-light-chain-enhancer of activated B cells were determined in ex vivo stimulated CD4 T cells, CD8 T cells, B cells, monocytes, and neutrophils. Messenger RNA expression levels of p38 mitogen activated protein kinase and nuclear factor kappa-light-chain-enhancer of activated B cells and negative regulators tumor necrosis factor-α-induced protein 3 (A20) and mitogen activated protein kinase phosphatase-1 were determined in neutrophils and peripheral blood mononuclear cells. Upon ex vivo stimulation, monocytes of sepsis patients were less capable in phosphorylating nuclear factor kappa-light-chain-enhancer of activated B cells. Sepsis was also associated with reduced phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells in stimulated B cells, CD4 and CD8 T cells. Messenger RNA expression levels of nuclear factor kappa-light-chain-enhancer of activated B cells and A20 were diminished in peripheral blood mononuclear cells of sepsis patients, whereas p38 mitogen activated protein kinase messenger RNA was up-regulated. In neutrophils of sepsis patients, mitogen activated protein kinase phosphatase-1 messenger RNA levels were down-regulated.Conclusions: Sepsis-induced immunosuppression associates with a defect in the capacity to phosphorylate nuclear factor kappa-light-chain-enhancer of activated B cells in lymphoid cells and monocytes. [ABSTRACT FROM AUTHOR]

Control of human African trypanosomiasis (HAT) is highly dependent on the ability to detect and treat infected individuals. However, a number of individuals exposed to Trypanosoma brucei gambiense are able to control infection to undetectable levels in blood. They are long-term potential reservoirs and thus a threat for control strategies. Cytokine responses in whole blood stimulation assays were quantified in individuals with contrasting HAT status. Trypanosome-induced IFN-γ production was only observed in “trypanotolerant” subjects suspected of harboring latent infections. This result contributes new insights into the immune responses associated with infection control and opens novel diagnosis perspectives regarding HAT elimination. [ABSTRACT FROM AUTHOR]

Eicosanoids play an important role in homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca ionophores and Ca-ATPase inhibitors, as well as natural agonists such as formylmethionine-leucyl-phenylalanine (fMLP), can stimulate eicosanoid biosynthesis. The aims of this work were to develop a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was partially validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient ≥0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of ≤15 %, except for the lower limit of quantification, where these values were ≤20 %. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were tested. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli for the production or liberation of eicosanoids. We next compared the eicosanoid profiles of stimulated whole blood samples of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients and those of healthy subjects, mainly for 5-HETE, 12-HETE, LTB, LTE, TXB, and PGE. In conclusion, our analytical method can detect significant changes in eicosanoid profiles in stimulated whole blood, which will contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

β-Glucans are components of fungal cell walls and potent stimulants of innate immunity. The majority of research on biological activities of glucans has focused on β-(1→3)-glucans, which have been implicated in relation to fungal exposure-associated respiratory symptoms and as important stimulatory agents in anti-fungal immune responses. Fungi—and bacteria and plants—produce a wide variety of glucans with vast differences in the proportion and arrangement of their β-(1→3)-, -(1→4)- and -(1→6)-glycosidic linkages. Thus far, the pro-inflammatory potential of different β-glucans has not been studied within the same experimental model. Therefore, we compared the potency of 13 different glucan preparations to induce in vitro production of IL-1β, IL-6, IL-8 and TNF-α in human, whole blood cultures. The strongest inducers of all cytokines were pustulan [β-(1→6)-glucan], lichenan [β-(1→3)-(1→4)-glucan], xyloglucan [β-(1→4)-glucan] and pullulan [α-(1→4)-(1→6)-glucan]. Moderate-to-strong cytokine production was observed for curdlan [β-(1→3)-glucan], baker’s yeast glucan [β-(1→3)-(1→6)-glucan] and barley glucan [β-(1→3)-(1→4)-glucan], while all other glucan preparations induced very low, or no, detectable levels of cytokines. We therefore conclude that innate immunity reactions are not exclusively induced by β-(1→3)-glucans, but also by β-(1→6)- and β-(1→4)-structures. Thus, not only β-(1→3)-glucan, but also other β-glucans and particularly β-(1→6)-glucans should be considered in future research. [ABSTRACT FROM AUTHOR]

Within a neonatal vitamin A supplementation (VAS) trial, we investigated the effect of VAS on TNF-α, IL-10, IL-5 and IL-13 production after lipopolysaccharide, purified protein derivative (PPD) of Mycobacterium tuberculosis and phytohaemagglutinin stimulation using a whole blood culture protocol. We found that VAS recipients had lower unstimulated TNF-α concentrations than placebo recipients. In the present paper, we investigated whether the SNP TNF-α − 308, TNF-α − 238, IL-10 − 592, IL-10 − 1082 and toll-like receptor 4 (TLR4)+896 modified the effect of VAS on cytokine production. DNA and cytokine concentrations were available from 291 children. We found a significant interaction between TNF-α − 308 genotype and VAS for the unstimulated TNF-α production (Pinteraction = 0·04); among G homozygotes, TNF-α concentrations were significantly lower after VAS compared with placebo, whereas for A carriers, VAS did not appear to have any effect. For TNF-α − 238, there was a tendency towards an increase in PPD-stimulated TNF-α production after VAS for the G homozygotes, but the opposite tendency for A allele carriers (Pinteraction = 0·07). Stratification by sex revealed a significant VAS–genotype interaction for boys for TNF-α − 238. There was a borderline-significant three-way interaction (P = 0·05) between sex, VAS and TLR4+896 genotype. Although the present study had very limited representation of the genetic variation with potential for modification of the response to VAS, it adds to the efforts of untangling the diverse effects and impact of VAS. [ABSTRACT FROM PUBLISHER]

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: www.trialregister.nl/ NTR2152 [ABSTRACT FROM AUTHOR]

A widely applied method to study the activation of the innate immune system is in vitro stimulation of whole blood using lipopolysaccharide (LPS). However, it is unclear if in vitro cytokine production relates to in vivo cytokine levels elicited during experimental endotoxemia or sepsis. To determine the correlation between in vitro cytokine production and the in vivo inflammatory response, blood was obtained from 15 healthy volunteers for in vitro incubation with Escherichia coli LPS, immediately followed by experimental E. coli endotoxemia. Correlations of in vitro and peak in vivo cytokine concentrations were determined using Pearson correlation coefficient. In stimulated whole blood, tumor necrosis factor (TNF)-α, Interleukin (IL)-1b, IL-6, IL-10 and interferon (IFN)-γ were induced to 279±53, 392±64, 5312±624, 83±20 and 343±85 pg/ml, respectively, whereas in vivo cytokine induction led to cytokine levels of 603±123, 11±1, 4999±1228, 167±25 and 194±40 pg/ml, respectively. Correlation coefficients between the in vitro and in vivo cytokine concentrations were for TNF-α, IL-1β, IL-6, IL-10 and IFN-γ-0.10 (P=0.7), 0.09 (P=0.8), 0.36 (P=0.2), 0.19 (P=0.5) and 0.40 (P=0.1), respectively. Comparison between in vitro and in vivo stimulation with LPS shows no correlation between the amount of cytokines produced. In vitro cytokine production, therefore, does not predict the in vivo inflammatory response. [ABSTRACT FROM AUTHOR]