Intracellular Cytokine Detection Research Articles

Temperature impacts the bovine ex vivo immune response towards Mycoplasmopsis bovis

Although cattle are the mammalian species with most global biomass associated with a huge impact on our planet, their immune system remains poorly understood. Notably, the bovine immune system has peculiarities such as an overrepresentation of γδ T cells that requires particular attention, specifically in an infectious context. In line of 3R principles, we developed an ex vivo platform to dissect host–pathogen interactions. The experimental design was based on two independent complementary readouts: firstly, a novel 12–14 color multiparameter flow cytometry assay measuring maturation (modulation of cell surface marker expression) and activation (intracellular cytokine detection) of monocytes, conventional and plasmacytoid dendritic cells, natural killer cells, γδ T cells, B and T cells; secondly, a multiplex immunoassay monitoring bovine chemokine and cytokine secretion levels. The experiments were conducted on fresh primary bovine blood cells exposed to Mycoplasmopsis bovis (M. bovis), a major bovine respiratory pathogen. Besides reaffirming the tight cooperation of the different primary blood cells, we also identified novel key players such as strong IFN-γ secreting NK cells, whose role was so far largely overlooked. Additionally, we compared the host–pathogen interactions at different temperatures, including commonly used 37 °C, ruminant body temperature (38–38.5 °C) and fever (≥ 39.5 °C). Strikingly, working under ruminant physiological temperature influenced the capacity of most immune cell subsets to respond to M. bovis compared to 37 °C. Under fever-like temperature conditions the immune response was impaired compared to physiological temperature. Our experimental approach, phenotypically delineating the bovine immune system provided a thorough vision of the immune response towards M. bovis and the influence of temperature towards that immune response.

CYTOSPOT: Intracellular Staining and Detection of Cytokines by Flow Cytometer, an Alternative to LYMESPOT.

Intracellular cytokine staining is a versatile technique used to analyze cytokine production in individual cells by flow cytometry. This methodology has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation, and functional parameters pertaining to responding T cells. This methodology applied after short-term culture of cells, followed by fixation and permeabilization make this technique ideal for the assessment of T-cell immune responses induced by different challenges. Here we describe an intracellular staining method followed by flow cytometry after cell stimulation with immune-relevant antigens for Lyme disease.

Comparing Flow Cytometry and ELISpot for Detection of IL-10, IL-6, and TNF Alpha on Human PBMCs.

ELISpot and flow cytometry are two methods often utilized side-by-side for detecting secreted and intracellular cytokines, respectively. Each application has its own advantages and challenges. ELISpot is more sensitive compared to ELISA and appears to be more consistent in detecting IL-10 production than flow cytometry. ELISpot can be used for detecting the secretion of multiple cytokines but not from the same cells simultaneously, whereas flow cytometry allows for the concurrent detection of multiple intracellular cytokines by the same cells. Flow cytometry is a convenient technique allowing for the detection of many cytokines at the same time in a population of cells. The restimulation cocktails used for cytokine detection in flow cytometry are hard on cells and lead to decreased cell viability. Using a live dead dye allows for the exclusion of dead cells when analyzing data. We illustrated the differences between ELISpot and flow cytometry by stimulating cells with two toll-like receptor (TLR) agonists, LPS or Pam3CSK4. Both activators increase production of various cytokines, including IL-10, IL-6, and TNF-alpha. The TLR2 antagonist, MMG-11, was used to inhibit this increased cytokine production. We observed some inhibition of IL-6 and IL-10 from Pam3CSK4 stimulation in the presence of MMG-11 by flow cytometry. TNF-α remains largely unchanged as its basal expression is high, but there is some reduction in the presence of MMG-11 for both methods. However, IL-10 was difficult to detect by ELISpot given the low seeding density. Overall, both ELISpot and flow cytometry are good methods for detecting secreted and intracellular cytokines, respectively, and should be used as complimentary assays.

Oral commensal bacterial flora is responsible for peripheral differentiation of neutrophils in the oral mucosa in the steady state

ObjectivesCommensal bacteria in the host body play a fundamental role in the differentiation and maintenance of the immune system. Studies on intestinal immunity have revealed that, under steady-state conditions, microflora have an important role in the maintenance of health. However, the role of oral commensal bacteria on the oral immune system is still unclear. Here, we clarify the interactions between commensal bacteria and the oral mucosal immune system under steady-state conditions. MethodsWe used germ-free mice that had never been exposed to bacteria and conventional mice grown with normal bacterial flora. Oral cells were isolated from the oral mucosa, stained with specific antibodies, and analyzed by flow cytometry. For the detection of myeloperoxidase and intracellular cytokines, oral cells were stimulated with N-formyl-methionine-leucyl-phenylalanine and phorbol 12-myristate 13-acetate/ionomycin, respectively. ResultsWe found that the oral mucosa harbored more neutrophils in germ-free mice than in conventional mice. However, the majority of neutrophils in the germ-free oral mucosa exhibited an immature phenotype. Other immune cells, including macrophages, T cells, and B cells, in the oral mucosa of germ-free mice showed similar differentiation to those in conventional mice. These results indicate that in the steady-state oral mucosa, the normal commensal flora promote the peripheral differentiation of neutrophils. ConclusionsThe presence of commensal flora is critical for the development of adequate immune system in the oral mucosa.

Intracellular cytokine detection based on flow cytometry in hemocytes from Galleria mellonella larvae: A new protocol.

Invertebrates are becoming increasingly popular models for research on the immune system. The innate immunity possessed by insects shows both structural and functional similarity to the resistance displayed by mammals, and many processes occurring in insect hemocytes are similar to those that occur in mammals. However, the use of insects as research models requires the development of methods for working with hemocytes. The aim of this study was to develop a protocol for intracellular cytokine detection in Galleria mellonella larvae hemocytes based on flow cytometry. It describes the anticoagulant composition of the buffer, the optimal conditions for hemocyte permeabilization and fixation, as well as the conditions of cell centrifugation to prevent cell disintegration. A key element is the selection of staining conditions, especially the length of the incubation time with the primary antibody, which turned out to be much longer than recommended for mammalian cells. The development of these individual steps allowed for the creation of a reproducible protocol for cytokine detection using flow cytometry in wax moth hemocytes. This will certainly facilitate the development of further protocols allowing for wider use of insect cells in immunological research.

Immunogenicity and safety of the booster BNT162b2 vaccine in patients with axial spondyloarthritis treated with biological disease-modifying drugs.

BackgroundVaccination confers relatively short-term protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), indicating the need for booster doses. Immunocompromised individuals, including those with immune-mediated inflammatory diseases (IMIDs), may have pronounced immune response waning. Vaccine-boosted humoral and T-cell responses minimize poor coronavirus disease 19 (COVID-19) outcome without increasing adverse events (AE). There is limited evidence of third-dose vaccination in axial spondyloarthritis (AxSpA) patients. We investigated immune-response persistence after primary vaccination and immunogenicity and safety after the BNT162b2 booster vaccination.MethodsThis prospective observational study enrolled an AxSpA cohort treated with interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNFα) inhibitors. Serum SARS-CoV-2-specific and virus-neutralizing antibodies for humoral response and flow cytometric detection of intracellular cytokines following SARS-CoV-2-specific peptide-based stimulation for T-cell immune responses were assessed, and safety was evaluated via a clinical questionnaire.ResultsFifteen male AxSpA patients treated with TNFα (73·3%) or IL-17 (26·7%) inhibitors were enrolled and had humoral response persistence at 6 months: 905·6 ( ± 186·1 SD) and 409·1 ( ± 335·7) U/mL. Specific antibody concentrations further increased after booster vaccination to 989·7 ( ± 12·62) and 1000 U/mL and T-cell responders from 53·3% to 80%, with no differences between AxSpA (including “vaccination only” and “hybrid immunity” subgroups) and healthy control (HC) cohorts. No severe AE occurred; the AE spectrum was comparable to that of the general population.ConclusionImmune-response persistence after primary vaccination and immunogenicity after booster vaccination were unaffected by anti-IL17 or anti-TNFα therapy with similar AE as in the general population.

Th17 and Th1 cells in systemic lupus erythematosus with focus on lupus nephritis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by T cells imbalance. Indeed, a correlation between levels of Th17 cells and disease activity has been reported. Our work aimed to study the functional association of subpopulations of Th cells and SLE with (lupus nephritis, LN) or without (lupus erythematosus, LE) renal involvement in Tunisian patients through the detection of intracellular cytokines and surface marker expression. The IL23R and RORC mRNA expression levels were evaluated. The level of Th17 and Th1 cells was higher in LE and LN patients compared to healthy controls (HC) (p = 0.007 and p = 0.018, respectively), while Th1/17 cells were increased only in LN patients compared to HC (p = 0.011). However, no significant difference was described in the mRNA expression levels of RORC and IL-23R between SLE and HC. Our findings suggest that the Th1/Th17 differentiation mechanisms are altered in SLE and that this imbalance should have an important influence on the development and severity of the disease.

Whole Blood Dendritic Cell Cytokine Production Assay.

This protocol outlines a method for the timely detection of intracellular cytokines produced by activated dendritic cells (DC) in human whole blood. The quantification of cytokines is used to measure DC immune responsiveness, providing information on the breadth, strength, and DC subtypes responding spontaneously and to specific stimulation with toll-like receptor (TLR) ligands or parasite-infected erythrocytes. DC subsets, plasmacytoid DC, CD1c+ DC, CD141+ DC, and CD16+ DC, are examined in their natural environment of plasma and blood cells (erythrocytes, neutrophils, platelets, and leukocytes) enabling disease, medication, nutritional, and hematological effects on DC function to be examined in vaccine studies, ageing, health, and disease.

Age-Associated Characteristics of CD4+ T-Cell Composition in Patients with Atherosclerosis

Background. We aimed to analyze the contents of the main CD4+ T-cell subsets in patients with atherosclerosis (AS) depending on age. Methods. Male patients with coronary and/or carotid AS, who are non-smokers, and who are receiving statins were divided into three age groups (I—<55 y.o. (n = 23), II—55–64 y.o. (n = 42), III—≥65 y.o. (n = 46)). Leukocyte phenotyping was performed by direct immunofluorescence and flow cytometry. For intracellular cytokine detection, blood mononuclear cells were pre-activated with phorbol 12-myristate 13-acetate and ionomycin in the presence of an intracellular vesicle transport blocker monensin. Results. The groups did not differ in traditional CVD risk factors and AS severity. The content of CD4+ T-cells was lower in group III and II than in group I. The content of CD4+CD25high Treg was lower in group III than in groups I and II. No differences in the quantities of the primed CD39+CD45RA− and CD278high Treg, CD4+INFγ+ Th1, CD4+IL17+ Th17, and CD4+IL17+INFγ+ Th1/17 were observed. There were negative correlations between the values of CD4+ T-cells, CD4+CD45RA+ T-cells, CD4+CD25high Treg, CD4+CD25highCD45RA+ Treg, and age. Conclusion. In patients with AS, the age-related depletion of naive CD4+ T-cells also extends to the regulatory compartment. This phenomenon should be considered when studying the impact of the immune cells on the progression of AS.

Exhausted PD-1hi T-Cells Are Enriched in Secondary Lymphoid Organs of CLL Patients

Chronic lymphocytic leukemia (CLL) is associated with increased numbers of T-cells that exhibit substantial alteration in composition and function. But their role in CLL pathogenesis remains largely elusive. On the one side, T-cells provide proliferation and pro-survival signals to CLL cells, but on the other side, they show features of activation and exhaustion, including overexpression of inhibitory molecules, like PD-1, suggesting their involvement in anti-tumoral activity. Since CLL T-cells retain the ability to produce cytokines and cytotoxic molecules, they were termed pseudo-exhausted. Results of previous studies were mostly based on blood samples of CLL patients, allowing only limited conclusions as interactions of immune cells and CLL cells mainly occur in lymphoid organs.In the present study, we aimed at deciphering differences in phenotype, differentiation and functional status of T-cells in paired peripheral blood (PB) and lymph node (LN) samples of CLL patients, particularly focusing on T-cell exhaustion. We applied 12-color flow cytometry and ex vivo stimulation of primary CLL T-cells followed by intracellular cytokine and cytotoxic molecule detection. In addition, we quantified transcription factors that are known to regulate T-cell differentiation by intracellular flow cytometry.In line with published data, we detected an increased proportion of CD45RO- CCR7- CD8+ effector T-cells as well as increased numbers of PD-1+ CD8+ T-cells in PB of CLL patients (n=22), compared to age-matched healthy individuals (n=19), and a positive correlation of these numbers with disease load. Despite of their enhanced expression of PD-1 and other inhibitory receptors, PB CD8+ T-cells from CLL patients exhibited increased production of granzyme B (GzmB), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in comparison to cells from age-matched healthy donors, indicating disease-associated activation of PB CD8+ T-cells with intact functional capacity.Subsequently, we compared CD8+ T-cells from paired LN and PB samples of CLL patients (n=7). This analysis revealed that LNs were enriched in CD45RO+ CCR7- effector memory T-cells (TEM cells) and contained significantly smaller numbers of terminally differentiated effector memory cells re-expressing CD45RA (TEMRA cells). Moreover, LN CD8+ T-cells strongly expressed the activation marker CD69, yet presented with higher levels of PD-1 compared to PB CD8+ T-cells. In line with high PD-1 expression, LN CD8+ T-cells exhibited a significantly lower production of IFNγ and TNFα, and a substantially reduced cytotoxic capability as indicated by lower GzmB production compared to PB T-cells. TEM cells in CLL patients' LNs exhibited different levels of PD-1 expression, and we observed a significantly higher percentage of cells with high PD-1 expression (PD-1hi) in LNs compared to PB, which was mainly composed of cells with intermediate PD-1 expression (PD-1int), (Figure 1). Comparison of PD-1hi and PD-1int subsets in LNs showed that PD-1hi cells expressed higher levels of CD69 compared to PD-1int cells, but were substantially inferior in GzmB production. Furthermore, PD-1hi cells expressed significantly higher levels of EOMES, a transcription factor that is known to drive terminal differentiation and exhaustion of T-cells in chronic infections. In contrast, PD-1int cells showed higher expression of the transcription factors T-BET and TCF7, which mediate long-term endurance of T-cells in chronic situations.In summary, this study shows that exhausted T-cells are enriched in LNs rather than PB of CLL patients. Our data suggests that the increased leukemia-associated activation of CD8+ T-cells in patients' LNs drives the accumulation of PD-1hi exhausted T-cells. A better understanding of these processes will be essential for the development of novel immunotherapeutic approaches for CLL.Figure 1:(A) CD8+ T-cells from paired peripheral blood (PB) and lymph node (LN) samples of CLL patients (n=7) were analyzed by flow cytometry for the expression of PD-1 (representative of seven samples). (B) The relative abundance of PD-1low, PD-1int and PD-1hi subsets within the CD45RO+ CCR7- CD8+ effector memory (EM) population in PB and LN was quantified. [Display omitted] DisclosuresStilgenbauer:AbbVie: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding.

Aging is accompanied by a decrease in the number of naive Tregs

Abstract Funding Acknowledgements Type of funding sources: None. Background. Age-related changes in the immune system are an important factor contributing to the maintenance of chronic inflammatory status. There are undoubted data on the decrease of the number of T-lymphocytes with age caused by thymus involution, but there are currently no unambiguous data on changes of minor T-cell subpopulations, in particular, regulatory T-cells (Treg). The aim of this study was to analyze the content of effector and regulatory CD4+ T cell subsets in patients with coronary and/or carotid atherosclerosis depending on age. Methods. 111 patients (men, median age 63 (55;69)) with coronary and/or carotid atherosclerosis, without smoking anamnesis, were enrolled. Mononuclear leuocytes were isolated from blood by gradient centrifugation, and CD4 + CD25high and CD4 + Foxp3+ Treg, CD4 + IL17+ T-helpers (Th) 17 and CD4 + INFγ Th1 were evaluated by direct immunofluorescence and flow cytometry. For intracellular cytokine detection cells were pre-activated in vitro in the presence of PMA/ionomycin/brefeldin A. In 74 patients cells were additionally stained with CD39, CD278, CD45RA Mabs to reveal naïve and primed T-cells. Results. According to age the patients formed three groups: I – <55 y.o. (n = 23), II – 55-64 y.o. (n = 42), III - ≥65 y.o. (n = 46). All patients were taking statins at baseline. The groups were comparable in traditional risk factors of CVD (BMI, arterial hypertension, diabetes mellitus, previous myocardial infarction anamnesis). The absolute content of CD4+ T cells was lower in group III (646.3 (516.0;806.4)) compared to groups I (903.0 (585.6;1113.8), p = 0.03) and II (745.4 (502.2;924.0), p = 0.06). The absolute content of CD4 + CD25high Treg was lower in group III (24.2 (18.4;35.2)) compared to groups I (35.0 (28.7;54.4), p = 0.01) and II (31.0 (21.1;43.6), p = 0.03). There were no differences in Th1, Th17, CD39 + CD45RA- and CD278+ Treg content between groups. A negative correlation was found between age and the content of CD4+ T cells (r= -0.28), CD4 + CD25high Treg (r= -0.27), p < 0.05. A negative correlation was found between age and CD4 + CD25highCD45RA+ Treg (r= -0.24) and CD4 + CD45RA+ T cells (r= -0.36), CD4 + CD45RA+/CD4 + CD45RA- T-cells ratio (r= -0.24), p < 0.05. Conclusion. Here we demonstrate an age-dependent decrease of total CD4+ T cell population and Treg subset in patients with atherosclerosis. The changes observed were primary due to the deficiency of CD45RA+ naïve T cells. The effector cell Th1 and Th17 quantities were at the same levels. Future research will show whether the identified immunological patterns can contribute to the progression of atherosclerosis and other chronic inflammatory diseases.

Attenuation of GARP expression on regulatory T cells by protein transport inhibitors

An integrated understanding of the functional capacities of cells in the context of their physical parameters and molecular markers is increasingly demanded in immunologic studies. Regulatory T cells (Tregs) are a subpopulation of T cells involved in immune response modulation and mediating tolerance to self-antigen with their absence leading to a loss of tolerance. Glycoprotein repetitions A predominant (GARP) is a key marker for activated Tregs, but its detection may also be useful in determining the functional capacities of the cell. This study aims to deduce the optimal stimulation period and the impact of protein transport inhibitors (PTIs), commonly used in the detection of intracellular cytokines, on GARP detection. Through flow cytometric analysis we analyzed different cell culture conditions for optimal GARP expression on activated Tregs. Healthy donor PBMCs were stimulated with either Staphylococcal Enterotoxin B (SEB) or PMA/Ionomycin (PMA/Iono), in the presence and absence of PTIs monensin and/or brefeldin A (BFA) and GARP expression was assessed on CD4+ CD25+ FOXP3+ Tregs. The optimal stimulation period for the detection of GARP was highest at 24-h. Furthermore, we determined that GARP expression on Tregs is significantly reduced when cells are treated with the PTIs monensin and/or BFA following PMA/Iono stimulation. This effect was not seen following SEB stimulation. Therefore, due to the effects of PTIs, alternative methods should be considered when performing simultaneous analysis for cytokine expression and GARP expression on Tregs.

Unique expansion of IL-21+ Tfh and Tph cells under control of ICOS identifies Sjögren’s syndrome with ectopic germinal centres and MALT lymphoma

ObjectivesTo explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren’s syndrome (SS)...

Uncovering Distinct Primary Vaccination-Dependent Profiles in Human Bordetella Pertussis Specific CD4+ T-Cell Responses Using a Novel Whole Blood Assay.

To advance research and development of improved pertussis vaccines, new immunoassays are needed to qualify the outcome of Bordetella pertussis (Bp) specific CD4+ T-cell differentiation. Here, we applied a recently developed whole blood assay to evaluate Bp specific CD4+ T-cell responses. The assay is based on intracellular cytokine detection after overnight in vitro Bp antigen stimulation of diluted whole blood. We show for the first time that CD4+ T-cell memory of Th1, Th2, and Th17 lineages can be identified simultaneously in whole blood. Participants ranging from 7 to 70 years of age with different priming backgrounds of whole-cell pertussis (wP) and acellular pertussis (aP) vaccination were analyzed around an acellular booster vaccination. The assay allowed detection of low frequent antigen-specific CD4+ T-cells and revealed significantly elevated numbers of activated and cytokine-producing CD4+ T-cells, with a significant tendency to segregate recall responses based on primary vaccination background. A stronger Th2 response hallmarked an aP primed cohort compared to a wP primed cohort. In conclusion, analysis of Bp specific CD4+ T-cell responses in whole blood showed separation based on vaccination background and provides a promising tool to assess the quantity and quality of CD4+ T-cell responses induced by vaccine candidates.

Toll-like receptor 7-adapter complex modulates interferon-α production in HIV-stimulated plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (PDCs) and their production of interferon-alpha (IFN-α) are believed to play an important role in human immunodeficiency virus, type I (HIV-1) pathogenesis. PDCs produce IFN-α and other proinflammatory cytokines through stimulation of Toll-like receptor 7 (TLR7) and TLR9 present in endosomal compartments. TLR7 recognizes single-stranded viral RNA, while TLR9 recognizes unmethylated DNA. In this study, we examined the mechanisms that may underlie variations in IFN-α production in response to HIV, and the impact of these variations on HIV pathogenesis. In four distinct cohorts, we examined PDC production of IFN-α upon stimulation with inactivated HIV-1 particles and unmethylated DNA. The signaling cascade of TLR7 bifurcates at the myeloid differentiation protein 88 (MyD88) adaptor protein to induce expression of either IFN-α or TNF-α. To determine whether variations in IFN-α production are modulated at the level of the receptor complex or downstream of it, we correlated production of IFN-α and TNF-α following stimulation of TLR7 or TLR9 receptors. Flow cytometry detection of intracellular cytokines showed strong, direct correlations between IFN-α and TNF-α expression in all four cohorts, suggesting that variations in IFN-α production are not due to variations downstream of the receptor complex. We then investigated the events upstream of TLR binding by using lipid-like vesicles to deliver TLR ligands directly to the TLR receptors, bypassing the need for CD4 binding and endocytosis. Similar tight correlations were found in IFN-α and TNF-α production in response to the TLR ligands. Taken together, these results strongly suggest that differences in IFN-α production depend on the regulatory processes at the level of the TLR7 receptor complex. Additionally, we found no association between IFN-α production before HIV infection and disease progression.

Optimized Protocol for the Detection of Multifunctional Epitope-Specific CD4+ T Cells Combining MHC-II Tetramer and Intracellular Cytokine Staining Technologies.

Analysis of multifunctional CD4+ T cells is fundamental for characterizing the immune responses to vaccination or infection. Major histocompatibility complex (MHC)/peptide tetramers represent a powerful technology for the detection of antigen-specific T cells by specific binding to their T-cell receptor, and their combination with functional assays is fundamental for characterizing the antigen-specific immune response. Here we optimized a protocol for the detection of multiple intracellular cytokines within epitope-specific CD4+ T cells identified by the MHC class II tetramer technology. The optimal procedure for assessing the functional activity of tetramer-binding CD4+ T cells was based on the simultaneous intracellular staining with both MHC tetramers and cytokine-specific antibodies upon in vitro restimulation of cells with the vaccine antigen. The protocol was selected among procedures that differently combine the steps of cellular restimulation and tetramer staining with intracellular cytokine labeling. This method can be applied to better understand the complex functional profile of CD4+ T-cell responses upon vaccination or infection.

Circulating versus lipopolysaccharide-induced inflammatory markers as correlates of subthreshold depressive symptoms in older adults

Objectives: Circulating cytokines have been associated with depression, but their detection has limitations, which may be overcome by direct detection of intracellular cytokines (ICCs) after lipopolysaccharide (LPS) stimulation in vitro. This study compared circulating versus LPS-induced inflammatory markers as correlates of subthreshold depressive symptoms.Methods: Secondary data analysis of a cross-sectional insomnia study in healthy community-dwelling older adults was conducted. In 117 participants (≥55 years), plasma tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP) and in vitro LPS-induced monocyte production of IL-6 and TNF-α were assayed. Depressive symptoms were assessed using the clinician-rated Inventory of Depressive Symptomatology (IDS-C). Multivariate linear regression was conducted to test the associations between inflammatory markers and subthreshold depressive symptoms in the entire sample as well as in subgroups stratified into higher and lower inflammation levels.Results: LPS-induced TNF-α (adjusted β = 0.28, p = .04), IL-6 (0.29, p = .03) and TNF-α + IL-6 (0.43, p = .001) significantly positively correlated with subthreshold depressive symptoms only in higher inflammation subgroups. No circulating biomarkers positively correlated in any subgroups. In the entire sample, no biomarkers were significantly associated with subthreshold depressive symptoms.Conclusions: LPS-induced cytokines may be more sensitive correlates of subthreshold depressive symptoms than circulating cytokines, particularly in older adults with higher systemic inflammation.Clinical Trials Registry: ClinicalTrials.gov NCT00280020.

Prophylactic and therapeutic inhibition of allergic airway inflammation by probiotic Escherichia coli O83

Prophylactic and therapeutic inhibition of allergic airway inflammation by probiotic Escherichia coli O83

Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites.

Interleukin-6 (IL-6) expression and secretion, induced by inflammatory processes, stimulate the acute phase response cascade. The overexpression of IL-6 contributes to a variety of inflammatory diseases, e.g. rheumatoid arthritis, Castleman's disease, multiple myeloma, and prostate cancer. Screening for high amounts of IL-6 in the patients' blood serum can be crucial for an adequate treatment. In this study, five novel murine monoclonal antibodies (mAbs) reactive to human IL-6 were generated. The mAbs were characterized for potential diagnostic purposes and recombinant antibodies were derived thereof. Initial epitope mapping using a combination of blocking experiments and Hyper-IL-6, a fusion protein consisting of IL-6 and the soluble IL-6 receptor revealed distinct but overlapping binding sites. At least one of the mAbs was found to interact with the region of IL-6/ IL-R complex formation. Three mAbs were applied successfully in intracellular staining by flow cytometry, whereas one of the mAbs showed comparable binding as a reference reagent. Furthermore, the mAbs were tested for applications in various immunological assays such as ELISA, Western blot and surface plasmon resonance spectroscopy (SPR), using IL-6 from commercial sources as well as in-house produced protein (IL-6_IME). The limit of detection was determined by sandwich ELISA (0.5 ng/mL, SD ±0.005). Our results also demonstrated that the recombinant IL-6 produced was functional and correctly folded. These findings support the use of the generated mAb clones as promising candidates for application in various immunological assays for diagnostic and scientific purposes.

Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T Cells.

In a recent unit in this series, protocols for the isolation, cryopreservation, thawing, and immunophenotyping of HPBMC isolated from peripheral whole blood using cell surface marker (CSM) staining and multi-color flow cytometry analysis were presented. The current procedure describes the detection and quantification of CSM and intracellular markers (ICM), including transcription factors and cytokines, following activation and differentiation of CD4+ T-cells using multi-color flow cytometry. Results indicated that repeatable and robust detection of ICM could be obtained in surface marker-defined T cells that identify functional subsets of cells. There were no observed differences between fresh and cryopreserved HPBMC in eight phenotypes analyzed (T-CD3, Th-CD4, Tmem-CD45RO, activated T-CD3/CD25, Treg- Foxp3/CD25, Th1-IFNγ, Th2- IL-4, Th17-IL-17A). There was an observed difference in activated T- CD3/CD69 in the short term (30-90 days) cryopreserved samples as compared to the freshly isolated samples, which may have resulted from the variance in controls or small sample size. © 2017 by John Wiley & Sons, Inc.