Whole Blood Stimulation Assays Research Articles

Immune response profiling of patients with spondyloarthritis reveals signalling networks mediating TNF-blocker function in vivo

ObjectivesAntitumour necrosis factor (TNF) therapy has revolutionised treatment of several chronic inflammatory diseases, including spondyloarthritis (SpA). However, TNF inhibitors (TNFi) are not effective in all patients and the biological basis...

In vitro responses of multiple cytokines to purified protein derivative in healthy and naturally Mycobacterium tuberculosis-infected rhesus monkeys (Macaca mulatta).

As the widely used biomarker of whole-blood stimulation assays for tuberculosis diagnosis, the release of IFN-γ might be affected by multiple factors, such as immunosuppression and some infectious agents. Here, we evaluated additional cytokines as diagnostic biomarkers. Forty-three cytokines were measured by Luminex xMAP technologies in 30 healthy and 10 naturally Mycobacterium tuberculosis (MTB)-infected rhesus monkeys pre- and post-stimulation by purified protein derivative (PPD). After stimulation, production of 23 and 38 cytokines was markedly increased in healthy and MTB-infected macaques, respectively. A comparison of the stimulation index (SI) between MTB infections and healthy macaques showed that the SIs of 32 cytokines in MTB-infected macaques were significantly higher than those in healthy macaques. Pooling the results, eight cytokines were suggested as ideal biomarkers for a whole-blood stimulation assay for MTB diagnosis. PPD could induce multiple cytokine responses in either healthy or MTB-infected monkeys, and eight cytokines had reliable predictive capacity as diagnostic biomarkers of MTB infection.

A preliminary study assessing cytokine production following ex vivo stimulation of whole blood with diet in dogs with chronic enteropathy

BackgroundEx vivo whole blood stimulation assays (WBSA) have been used to characterize the cytokine response to diet in cats. The present study aimed to use this assay to determine the cytokine response to diets being fed at the time of diagnosis to dogs with chronic enteropathy (CE) and to compare this to a control group of dogs presented for non-gastrointestinal (GI) causes.ResultsDogs with chronic GI signs and dogs presented for non-GI causes were prospectively recruited. For each case, residual blood following diagnostic sampling was placed into heparin. WBSAs were performed using crude extracts of the diet currently being fed and provided by the owner. Supernatants were collected and analyzed for tumor necrosis factor (TNF)-alpha, interleukin (IL)-10 and IL-4 using an enzyme-linked immunosorbent assay. The case group consisted of 22 dogs with CE diagnosed on histopathology of GI biopsy and 9 with suspected CE. The non-GI group consisted of 18 dogs. Of the diets being fed at or prior to diagnosis, hydrolyzed protein diets elicited significantly lower IL-10 and TNF-alpha concentrations compared to commercial intact protein diets in dogs with confirmed or suspected CE (P-value 0.004 and < 0.001, respectively). Six out of 15 dogs with detectable IL-4 concentrations in the confirmed CE group had IL-4 to IL-10 ratios that exceeded the 95% confidence interval (CI) of the mean of the non-GI group (non-GI: 95% CI of IL-4:IL-10 = 0.64–2.71; confirmed CE: IL-4:IL-10 in 6 dogs = mean 22.40, range 2.77–89.11).ConclusionsHydrolyzed protein diets elicited a significantly reduced cytokine response when incubated with patient whole blood ex vivo compared to commercial intact protein diets in dogs with CE. The IL-4 to IL-10 ratio as a marker of dietary responsiveness warrants further investigation, together with assessment of the cytokine response to diet at the intestinal mucosal surface.

Mycobacteria-Specific Mono- and Polyfunctional CD4+ T Cell Profiles in Children With Latent and Active Tuberculosis: A Prospective Proof-of-Concept Study.

Background: Current immune-based TB tests, including the tuberculin skin test (TST) and interferon-gamma release assays (IGRA), have significant limitations, including the inability to distinguish between latent TB infection (LTBI) and active TB. Few biomarkers with the potential to discriminate between these two infection states have been identified.Objective: To determine whether functional profiling of mycobacteria-specific T cells can distinguish between TB-infected and -uninfected children, and simultaneously discriminate between LTBI and active TB.Methods: One hundred and forty-nine children with suspected active TB or risk factors for LTBI were recruited at the Royal Children's Hospital Melbourne. Whole-blood stimulation assays, using ESAT-6, CFP-10, PPD, and heat-killed M. tuberculosis as stimulants, were done, followed by intracellular cytokine staining and flow cytometric analysis.Results: Eighty-two participants in the well-defined diagnostic categories ‘uninfected individuals’ (asymptomatic, TST 0 mm / IGRA-; n = 61), LTBI (asymptomatic, TST ≥10 mm / IGRA+, normal chest radiograph; n = 15), or active TB [microbiologically-confirmed (n = 3) or fulfilling stringent criteria (n = 3)] were included in the final analysis. The proportions of mycobacteria-specific single-positive TNF-α+ and double-positive IFN-γ+/TNF-α+ CD4+ T cells were significantly higher in participants with active TB than in those with LTBI and uninfected individuals. Additionally, the frequency of IL-17-expressing CD4+ T cells, predominately with single-positive IL-17+ and double-positive IL-2+/IL-17+ phenotypes, was higher in participants with active TB than in the other two groups.Conclusions: The frequencies and functional profiles of mycobacteria-specific CD4+ T cells differ significantly both between TB-infected and TB-uninfected children, and between LTBI and active TB. Although confirmation in further studies will be required, these findings indicate that functional profiling of mycobacteria-specific CD4+ T cells could potentially be exploited for novel immune-based TB assays that enable the distinction between infection states based on a blood sample alone.

Genetic Variation in Pattern Recognition Receptors and Adaptor Proteins Associated With Development of Chronic Q Fever.

Q fever is an infection caused by Coxiella burnetii. Persistent infection (chronic Q fever) develops in 1%-5% of patients. We hypothesize that inefficient recognition of C. burnetii and/or activation of host-defense in individuals carrying genetic variants in pattern recognition receptors or adaptors would result in an increased likelihood to develop chronic Q fever. Twenty-four single-nucleotide polymorphisms in genes encoding Toll-like receptors, nucleotide-binding oligomerization domain-like receptor-2, αvβ3 integrin, CR3, and adaptors myeloid differentiation primary response protein 88 (MyD88), and Toll interleukin 1 receptor domain-containing adaptor protein (TIRAP) were genotyped in 139 patients with chronic Q fever and in 220 controls with cardiovascular risk-factors and previous exposure to C. burnetii. Associations between these single-nucleotide polymorphisms and chronic Q fever were assessed by means of univariate logistic regression models. Cytokine production in whole-blood stimulation assays was correlated with relevant genotypes. Polymorphisms in TLR1 (R80T), NOD2 (1007fsX1), and MYD88 (-938C>A) were associated with chronic Q fever. No association was observed for polymorphisms in TLR2, TLR4, TLR6, TLR8, ITGAV, ITGB3, ITGAM, and TIRAP. No correction for multiple testing was performed because only genes with a known role in initial recognition of C. burnetii were included. In the whole-blood assays, individuals carrying the TLR1 80R-allele showed increased interleukin 10 production with C. burnetii exposure. Polymorphisms in TLR1 (R80T), NOD2 (L1007fsX1), and MYD88 (-938C>A) are associated with predisposition to development of chronic Q fever. For TLR1, increased interleukin 10 responses to C. burnetii in individuals carrying the risk allele may contribute to the increased risk of chronic Q fever.

Evaluation of Gamma Interferon Immune Response Elicited by the Newly Constructed PstS-1(285-374):CFP10 Fusion Protein To Detect Mycobacterium tuberculosis Infection

The PstS1 antigen is highly immunogenic, principally when combined with CFP10 during both latent and active TB infection. In the present study, a selected pstS1 gene fragment was cloned, fused with CFP10, and expressed in Escherichia coli. The product [PstS-1(285-374):CFP10] was compared to the recombinant fused RD1 (region of deletion 1) protein (ESAT-6:CFP10) in detecting Mycobacterium tuberculosis infection in 108 recent contacts of pulmonary tuberculosis (TB) cases, considering a positive tuberculin skin test (TST) to be the baseline. The release of gamma interferon (IFN-γ) in 22-h whole-blood and 5-day lymphocyte stimulation assays primed with each antigen was determined. All contacts were clinically followed for up to 1 year, and 87% of the tuberculin skin test-positive (TST(positive)) patients accepted preventative treatment. Concerning the IFN-γ response to PstS-1(285-374):CFP10 in the 22-h and 5-day assays, a slight increase in contact-TST(positive) detection was observed (23/54 and 26/54) compared to the level seen with the RD1 protein (18/54 and 24/54) whereas in the TST(negative) group, similarly lower numbers (≤5/48) of responders were achieved for both antigens, except for RD1 in the 5-day assay (8/48). By combining the IFN-γ responders to both antigens in the 5-day assays, slightly higher increases in positivity were found in the TST(positive) (32/54) and TST(negative) (10/48) groups. Two of 12 untreated TST(positive) contacts progressed to active TB and were concordantly positive in all assays, except for one contact who lacked positivity in the RD1 5-day assay. We demonstrated for the first time that PstS-1(285-374):CFP10 slightly increased contact positivity and detection of active disease progression, suggesting its potential application as a TB infection marker.

C-Reactive Protein Elicits White Blood Cell Activation in Humans

Objective Consistent epidemiologic evidence suggests that acute infections increase the risk for acute cardiovascular events. We tested in humans whether activation of peripheral leukocytes in reaction to the administration of recombinant human C-reactive protein (rhCRP) may provide a mechanism for infectious diseases to promote atherosclerotic disease. Methods and Results By using quantitative real-time polymerase chain reaction analysis, whole-blood expression profiles were analyzed for 95 inflammatory markers before and after infusion of 1.25 mg/kg rhCRP in 5 male volunteers. Relevant transcript levels were measured at baseline and 4 and 8 hours after rhCRP-infusion. CRP caused significant up-regulation of matrix metalloproteinase (MMP)-9, monocyte chemoattractant protein (MCP)-1, plasminogen activator urokinase, macrophage inflammatory protein 1α, and nuclear factor of kappa B inhibitor mRNAs in peripheral leukocytes. mRNA up-regulation of MMP-9 and MCP-1 was 17- and 11-fold, respectively. The corresponding increase in plasma protein levels of MMP-9 (78 ± 32 ng/mL to 109 ± 41 ng/mL; P = .014) and MCP-1 (312 ± 92 pg/mL to 2590 ± 898 pg/mL; P = .007) closely mirrored mRNA findings. Also, in whole-blood culture stimulation assays, CRP induced proinflammatory changes. Notably, heat inactivation abolished the capacity of CRP to evoke these proinflammatory changes, excluding a role for contaminants within the purified CRP preparation. Conclusion CRP elicits activation of peripheral leukocytes with ensuing secretion of plaque-destabilizing mediators. These findings are consistent with the hypothesis that infectious diseases trigger manifestations of atherosclerosis, in which CRP elevation might contribute to the onset of cardiovascular events.

Performance of the whole-blood stimulation assay for assessing innate immune activation under field conditions

Innate propensity of immune activation is reflected in production of pro- and anti-inflammatory cytokines upon stimulation of Toll-like receptors (TLR) in whole-blood stimulation assays. The validity of the whole-blood stimulation assay under field conditions has not been evaluated extensively. Here, we have determined correlation of individually repeated whole-blood stimulation assays in a field-study in Ghana and compared it with that of two Dutch populations performed under optimal conditions. We also examined cytokine production to various TLR-agonists in order to create an assay that would mimic general innate immune responses. Under field conditions repeated assessments of lipopolysaccharide-induced Tumor Necrosis Factor-α (TNFα) production were poorly correlated ( r = 0.15, p = 0.087). Correlation was relatively high for production of Interleukin-10 (IL10) ( r = 0.48, p < 0.001) and comparable to that observed in the Dutch population under optimal conditions. Combined stimulation with lipopolysaccharide and zymosan resulted in cytokine production profiles that were similar to that attained after stimulation with a mixed culture of bacteria. Here, we conclude that variation of a whole-blood assay performed in field setting is large in general but that production of IL10 seems to better reflect an innate pro- or anti-inflammatory tendency whereas production of TNFα may predominantly reflect recent immunological challenges. Furthermore, simultaneous stimulation of several Toll-like receptors may mimic general innate immune activation.

The Measurement of Cytokine Production Capacity during Dialysis - a New Dynamic Method for the Evaluation of Biocompatibility?

The activation of monocytes and other immunocompetent cells during hemodialysis can be attributed to their contact with immunogenic structures such as membranes, blood lines, and endotoxins. The simple measurement of cytokines in blood cannot completely describe the whole dimension of this event. Stimulation of monocytes and other immunocompetent cells in whole blood with lipopolysaccharides (LPS) for IL-6 and phytohemagglutinine (PHA) for TNF at the start and end of dialysis may make it possible to better analyze cellular response during dialysis. Ten healthy volunteers and 10 patients suffering from chronic renal failure were tested with the commercial whole-blood stimulation assays "Dynamix"-IL-6-DIA and -TNF-alpha-DIA (Biosource Diagnostics, Ratingen, Germany). Then 24 patients undergoing hemodialysis with hemophane (n=12) and polyamide (n=12) membranes were examined before and after dialysis treatment. The unpaired Wilcoxon t- test was used for statistical analysis. Healthy volunteers and patients with chronic renal failure showed no statistical differences in concentrations of TNF-alpha and IL-6 before or after whole blood stimulation (WBS). In comparison to patients with chronic renal failure, pre-WBS concentrations of both cytokines (p<0.034) were increased in patients of each membrane group before dialysis. After whole blood stimulation, no differences were observed. At the end of dialysis treatment, the pre- and post-WBS IL-6 values were both significantly higher in the hemophane group (p=0.049 and p=0.0038, respectively) TNF-alpha concentrations were unchanged. No significant differences in the polyamide group were found between the start and end of treatment for either cytokine. A comparison of these membrane groups showed that only the pre-WBS IL-6 concentration in the hemophane group was elevated (p=0.022) after dialysis. In conclusion, the presence of uremia alone could not influence the cytokine production and release capacity. In our patients, dialysis elevated pre-WBS concentrations of TNF-alpha and IL-6, and increased IL-6 release from immunocompetent cells after whole blood stimulation in the hemophane group. The use of polyamide membranes decreased the action of monocytes and other immunocompetent cells, but could not completely prevent this phenomenon. The whole blood stimulation assays for measurement of TNF-alpha and IL-6 may represent a new, dynamic method for evaluating biocompatibility.

Presence of high levels of leukocyte-associated interleukin-8 upon cell activation and in patients with sepsis syndrome.

In inflammatory and infectious diseases, the presence of circulating cytokines in plasma strongly suggests, following their exacerbated production, that saturation of specific binding sites has occurred or that an equilibrium between receptor-bound and free cytokines has been reached. In this report, we demonstrate that in addition to circulating interleukin-8 (IL-8), high levels of cell-associated IL-8 were detected in blood samples from patients with sepsis syndrome. The following analysis will reveal that in addition to erythrocytes, which have been dubbed a "sink" for IL-8, peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) contributed to the detection of cell-associated IL-8. On a per cell basis, 2,000 to 7,000 times the amount of IL-8 was found associated with PMN than with erythrocytes. In addition, circulating cells may well be the source of the leukocyte-associated form of IL-8. Similarly, in vitro experiments, such as whole-blood stimulation assays or the addition of exogenous IL-8 in blood samples, demonstrated that a large proportion of the IL-8 was associated with leukocytes. This suggests that the trapping of free cytokines onto the cell surface and the internalization of the IL-8 bound to its receptor, occurring both in vitro and in vivo, allows the detection of this cell-associated form. This analysis of cell-associated cytokines was extended to IL-1ra, another component of the inflammatory response, which, in contrast to IL-8, has been demonstrated to exist as an intracellular form. Indeed, cell-associated IL-1ra was also detected in septic patients. The measurement of cell-associated proinflammatory and anti-inflammatory cytokines in patients is clearly a more reliable reflection of their production than is the simple measurement in plasma and may provide useful indication to further understand the inflammatory process.