Culture Supernatants Of Mononuclear Cells Research Articles

TNF blockers alone and associated with Benznidazole impact in vitro cytokine dynamics in chronic Chagas disease.

Studies involving the immune response in Chagas disease suggest an imbalance in the immune response of symptomatic patients, with an inflammatory profile dominating in Chagas heart disease, mainly by tumour necrosis factor (TNF). TNF is considered a key cytokine in immunopathology in chronic carriers in several processes during the immune response. Our work aimed to evaluate regulatory (interleukin [IL]-4 and IL-10) and inflammatory (TNF, interferon-gamma [IFN-γ], IL-2 and IL-6) cytokines in peripheral blood mononuclear cells culture supernatants. of affected patients with undetermined clinical forms-IND (n = 13) mild heart form-CARD1 (n = 13) and severe cardiac form-CARD2 (n = 16), treated in vitro with two TNF blockers, Adalimumab (ADA) and Etanercept (ETA) alone or in association with Benznidazole (BZ). The results indicate that ADA was more competent in blocking TNF (compared to ETA) in all groups but with much lower levels in the CARD2 group. ETA statistically decreased TNF levels only in the CARD2 group. IFN-γ increased in the CARD2 group after treatment with ETA relative to ADA. IL-4 had its levels decreased when treated by both drugs. IL-2 was detected in cells from CARD2 carriers compared to the NEG group after treatment with both drugs. The association with BZ decreased levels of IL-2/TNF and increased IL-4. These data reinforce the participation of TNF in severe Chagas heart disease and bring perspectives on using these blockers in the immunological treatment of Chagas disease since the use of BZ is extremely limited in these patients.

Prune Consumption Attenuates Proinflammatory Cytokine Secretion and Alters Monocyte Activation in Postmenopausal Women: Secondary Outcome Analysis of a 12-Mo Randomized Controlled Trial: The Prune Study

BackgroundProinflammatory cytokines are implicated in the pathophysiology of postmenopausal bone loss. Clinical studies demonstrate that prunes prevent bone mineral density loss; however, the mechanism underlying this effect is unknown. ObjectiveWe investigated the effect of prune supplementation on immune, inflammatory, and oxidative stress markers. MethodsA secondary analysis was conducted in the Prune Study, a single-center, parallel-arm, 12-mo randomized controlled trial of postmenopausal women (55–75 y old; n = 235 recruited; n = 183 completed) who were assigned to 1 of 3 groups: “no-prune” control, 50 g prune/d and 100 g prune/d groups. At baseline and after 12 mo of intervention, blood samples were collected to measure serum high-sensitivity C-reactive protein (hs-CRP), serum total antioxidant capacity (TAC), plasma 8-isoprostane, proinflammatory cytokines [interleukin (IL)-1β, IL-6, IL-8, monocyte chemoattractant protein-1, and tumor necrosis factor (TNF)-α] concentrations in plasma and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) culture supernatants, and the percentage and activation of circulating monocytes, as secondary outcomes. ResultsPrune supplementation did not alter hs-CRP, TAC, 8-isoprostane, and plasma cytokine concentrations. However, percent change from baseline in circulating activated monocytes was lower in the 100 g prune/d group compared with the control group (mean ± SD, −1.8% ± 4.0% in 100 g prune/d compared with 0.1% ± 2.9% in control; P < 0.01). Furthermore, in LPS-stimulated PBMC supernatants, the percent change from baseline in TNF-α secretion was lower in the 50 g prune/d group compared with the control group (−4.4% ± 43.0% in 50 g prune/d compared with 24.3% ± 70.7% in control; P < 0.01), and the percent change from baseline in IL-1β, IL-6, and IL-8 secretion was lower in the 100 g prune/d group compared with the control group (−8.9% ± 61.6%, −4.3% ± 75.3%, −14.3% ± 60.8% in 100 g prune/d compared with 46.9% ± 107.4%, 16.9% ± 70.6%, 39.8% ± 90.8% in control for IL-1β, IL-6, and IL-8, respectively; all P < 0.05). ConclusionsDietary supplementation with 50–100 g prunes for 12 mo reduced proinflammatory cytokine secretion from PBMCs and suppressed the circulating levels of activated monocytes in postmenopausal women.This trial was registered at clinicaltrials.gov as NCT02822378.

Distinct CRTH2+CD161+ (peTh2) memory CD4+ T-cell cytokine profiles in food allergy and eosinophilic gastrointestinal disorders.

Although IgE-mediated food allergy (FA) and eosinophilic gastrointestinal disorders (EGID) are clinically distinct and treated differently, pathogenic effector Th2 (peTh2) cells are implicated in the pathogenesis of both FA and EGID. The aim of this study was to better characterize peTh2 cells in the context of FA and EGID and the overlap between these two conditions. Peripheral blood peTh2 cells (CD3+CD4+CD27-CD49d+CRTH2+CD161+) were profiled by intracellular cytokine flow cytometry in the following patient cohorts: patients with FA alone (n = 8), FA and food-triggered EGID (EGID+FA+FT, n = 7), food-triggered EGID alone (EGID+FT, n = 7), EGID without FA or specific food triggers (ONLY_EGID, n = 9), and healthy volunteers (HV, n = 7). Overnight peripheral blood mononuclear cell (PBMC) culture supernatants were assessed for cytokine production by multiplex analysis. CRTH2+CD161+ (peTh2) memory CD4+ T cells were significantly increased in both patients with FA and those with ALL_EGID (inclusive of EGID+FA+FT, EGID+FT and ONLY_EGID) when compared to HV. However, ALL_EGID patients, particularly those with EGID+FA+FT, had significantly elevated IL-5+IL-13+ peTh2 cells, whereas FA patients had significantly elevated IFN-γ or IL-17A-expressing peTh2 cells. This finding was supported by increased spontaneous IL-5 and IL-13 production in overnight cultures of PBMC from EGID+FA+FT patients compared to spontaneous IL-10 and IFN-γ production by PBMC from FA patients. FA patients had increased IL-9, IL-10, IL-17A, and IFN-γ production in overnight cultures of stimulated PBMC. EGID and IgE-mediated FA share a common cell subtype defined by specific surface markers and termed CRTH2+CD161+ (peTh2) memory CD4+ T cells. However, the cytokine profiles of these CRTH2+CD161+ (peTh2) memory CD4+ T cells are markedly different between the two disorders.

Mycobacterial Heat Shock Proteins in Sarcoidosis and Tuberculosis.

Pathological similarities between sarcoidosis (SA) and tuberculosis (TB) suggest the role of mycobacterial antigens in the etiopathogenesis of SA. The Dubaniewicz group revealed that not whole mycobacteria, but Mtb-HSP70, Mtb-HSP 65, and Mtb-HSP16 were detected in the lymph nodes, sera, and precipitated immune complexes in patients with SA and TB. In SA, the Mtb-HSP16 concentration was higher than that of Mtb-HSP70 and that of Mtb-HSP65, whereas in TB, the Mtb-HSP16 level was increased vs. Mtb-HSP70. A high Mtb-HSP16 level, induced by low dose-dependent nitrate/nitrite (NOx), may develop a mycobacterial or propionibacterial genetic dormancy program in SA. In contrast to TB, increased peroxynitrite concentration in supernatants of peripheral blood mononuclear cell cultures treated with Mtb-HSP may explain the low level of NOx detected in SA. In contrast to TB, monocytes in SA were resistant to Mtb-HSP-induced apoptosis, and CD4+T cell apoptosis was increased. Mtb-HSP-induced apoptosis of CD8+T cells was reduced in all tested groups. In Mtb-HSP-stimulated T cells, lower CD8+γδ+IL-4+T cell frequency with increased TNF-α,IL-6,IL-10 and decreased INF-γ,IL-2,IL-4 production were present in SA, as opposed to an increased presence of CD4+γδ+TCR cells with increased TNF-α,IL-6 levels in TB, vs. controls. Mtb-HSP modulating the level of co-stimulatory molecules, regulatory cells, apoptosis, clonal deletion, epitope spread, polyclonal activation and molecular mimicry between human and microbial HSPs may also participate in the induction of autoimmunity, considered in SA. In conclusion, in different genetically predisposed hosts, the same antigens, e.g., Mtb-HSP, may induce the development of TB or SA, including an autoimmune response in sarcoidosis.

In vitro detection of suspected drug in maculopapular drug reaction to antibiotics using secreted cytokines from drug-specific activated T cells.

Maculopapular drug eruption (MPE) in reaction to antibiotics is associated with enhanced expression of T-helper (Th)1 cytokines such as interferon gamma (IFN-gamma) or Th2 cytokines such as interleukin (IL)-5. Identifying the culprit drug usually involves rechallenge, which may not be forthcoming. Memory lymphocytes remain responsive to the culprit drug long after the reaction has resolved. On reactivation in vitro, there is increased proliferation and expression of certain markers, which provides us with an opportunity to predict the causal drug. The study aimed to assess drug-specific cytokine production (IL-5 and IFN-gamma) in peripheral blood mononuclear cell (PBMC) culture supernatants to predict the causal antibiotic in cases of MPE. PBMCs of 18 patients who developed MPE to 20 suspected antibiotics (2 patients had 2 suspected antibiotic allergies each), along with 11 drug-matched healthy controls, were incubated for 5 days with the respective drugs at 2 different concentrations. Secreted cytokines were measured in the supernatants, using enzyme-linked immunosorbent assay (ELISA) for IL-5 and IFN-gamma, at 6 h of incubation, then on day 2 and day 5. Drug-specific IL-5 and IFN-gamma production could be demonstrated in 65% and 74% of the cases, respectively. Maximal secretion of IL-5 and IFN-gamma was observed on day 5 and day 2 of incubation, respectively. The cut-off delta values, defined as the difference in cytokine concentration between drug-stimulated and unstimulated samples, were 4 pg mL-1 for IL-5 and 6 pg mL-1 for IFN-gamma. The measurement of drug-specific secretion of IL-5 and IFN-gamma using ELISA is a valuable method for detecting antibiotic-induced MPE.

TNF-induced metalloproteinase-9 production is associated with neurological manifestations in HTLV-1-infected individuals.

HTLV-1-infected individuals may develop a neurologic inflammatory condition known as HTLV-1-associated myelopathy (HAM/TSP), in which the high production of TNF is observed. These patients exhibit higher proviral loads, enhanced production of proinflammatory cytokines and lymphocyte proliferation in comparison to asymptomatic HTLV-1 carriers and those presenting overactive bladder (OAB-HTLV-infected). Metalloproteinases (MMPs) are known to degrade the components of the blood-brain barrier, favoring the migration of infected cells into the central nervous system. Moreover, the unbalanced production of MMPs and their inhibitors (TIMPs) has also been associated with tissue damage. The present work studied the production of MMP-9 and TIMPs in HTLV-1-infected individuals with and without neurological manifestations. HAM/TSP patients presented higher concentrations of MMP-9 in peripheral blood mononuclear cell (PBMC) culture supernatants, as well as a higher MMP-9/TIMP-3 ratio when compared to the other groups studied. MMP-9 levels positively correlated with proviral load and TNF in OAB-HTLV-infected individuals, and the in vitro neutralization of TNF significantly decreased MMP-9 levels in PBMC culture supernatants. Our findings indicate an association between MMP-9 production and the proinflammatory state associated with HTLV-1 infection, as well as HAM/TSP.

Toxoplasma gondii infection and peripheral-blood gene expression profiling of older people reveals dysregulation of cytokines and identifies hub genes as potential therapeutic targets

Infections of humans with the protozoan parasite Toxoplasma gondii (T. gondii) can lead to the disease's development, even in an asymptomatic status. However, the mechanisms that result in these clinical outcomes after infection are poorly understood. This study aimed to explore the molecular pathogenesis of toxoplasmosis-related inflammation through next-generation sequencing, to assess RNA expression profiles in peripheral blood from 5 female patients with chronic toxoplasmosis and 5 healthy female controls. All plasma samples were analyzed for anti-Toxoplasma IgG and IgM antibody titers by using electrochemiluminescence. Detection of acute and chronic toxoplasmosis was carried out using the ELISA IgG avidity. We evaluated the levels of INF-γ, IL-2, IL-12, TNF-α, IL-10, and IL-1β in culture supernatants of Peripheral Blood Mononuclear Cells infected with Toxoplasma lysate antigen (TLA) prepared with tachyzoites of strain T. gondii RH. Differential expression analysis was performed using DESeq2, pathway and enrichment analysis of DEGs was done on WEB-based Gene SeT AnaLysis Toolkit (WebGestalt) and Protein-protein interaction was carried out using NetworkAnalyst with STRING. In older people with chronic asymptomatic infection, a significant difference in the levels of inflammatory cytokines INF-γ and IL-2 was observed compared to seronegative individuals. Our results revealed differences in the regulation of critical biological processes involved in host responses to chronic T. gondii infection. Gene ontology analysis revealed several biologically relevant inflammatory and immune-related pathways.

Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant

The quantification of cytokines can improve our understanding of immune response and inflammation dynamics in dairy cows. Bead-based assays provide a sensitive, high-throughput platform, allowing for simultaneous quantification of multiple cytokines within a wide linear detection range. Our objective was to develop a multiplex bead-based assay using monoclonal antibodies for simultaneous quantification of bovine tumor necrosis factor (TNF)-α, IL-10, and IFN-γ in plasma and peripheral blood mononuclear cell (PBMC) culture supernatants. Recombinant cytokine standards produced in mammalian cells were used to determine the lower limit of detection and the linear detection range for each cytokine. The lower limit of detection was 110 pg/mL for IL-10, 95 pg/mL for TNF-α, and 20 pg/mL for IFN-γ. The linear quantification range was 110 to 241,000 pg/mL for IL-10, 95 to 620,000 pg/mL for TNF-α, and 20 to 130,000 pg/mL for IFN-γ. All 3 monoclonal capture and detection antibodies were specific for their respective cytokine analyte when using the recombinant IL-10, TNF-α, and IFN-γ standards. Intraassay and interassay coefficients of variation (CV) were <10% and <12%, respectively, for all analytes and samples matrices. Next, concentrations of native cytokines were determined in PBMC culture supernatants (n = 4) and in plasma from whole-blood samples (n = 6) with or without stimulation with Escherichia coli lipopolysaccharide or a mix of phorbol myristate acetate (PMA) and ionomycin. Peak concentrations of all 3 cytokines were secreted from PBMC after PMA/ionomycin stimulation (TNF-α, 8 h, range: 39,266–506,422 pg/mL; IL-10, 18 h, range: 15,770–63,415 pg/mL; IFN-γ 18 h, range: 189,977–492,659 pg/mL). In contrast, the highest concentrations in plasma from whole-blood stimulation were observed for IL-10 and TNF-α after LPS stimulation (TNF-α, 4 h, range: 1,764–13,460 pg/mL; IL-10, 24 h, range: 2,401–6,371 pg/mL), whereas PMA and ionomycin induced the highest secretion of IFN-γ (18 h, range: 53–20,215 pg/mL). In conclusion, the multiplex assay can quantify native IL-10, TNF-α, and IFN-γ across a broad concentration range in bovine plasma and cell culture supernatant, thereby providing a novel tool to evaluate inflammatory profiles in cattle and especially in dairy cows with inflammatory conditions. The existing multiplex assay can be expanded in the future by adding bead assays for additional bovine cytokines.

Novel Th17 Lymphocyte Populations, Th17.1 and PD1+Th17, are Increased in Takayasu Arteritis, and Both Th17 and Th17.1 Sub-Populations Associate with Active Disease.

PurposeWe evaluated T helper lymphocyte profile, including novel Th17 subsets Th17.1 (secrete IFN-γ, associate with corticosteroid resistance) and PD1+Th17 (secrete TGF-β1, implicated in fibrosis), and related cytokines in peripheral blood of Takayasu arteritis (TAK).Materials and MethodsWe evaluated circulating Th1, Th2, Th17, Th17.1, PD1+CD4+ T lymphocytes, PD1+Th17, and Treg lymphocytes, inflammatory (IFN-γ, IL-4, IL-6, IL-17A, IL-23, IL-1β, TNF-α) and regulatory (IL-10, TGF-β1) cytokines in peripheral blood of TAK (n = 57; median age 35 (interquartile range 26–45) years; 40 females) in a cross-sectional design. We studied inflammatory and regulatory cytokines in culture supernatant of peripheral blood mononuclear cells (PBMCs) from TAK following stimulation with anti-CD3/anti-CD28 and their modulation by tacrolimus (immunosuppressive) with/without tadalafil (anti-fibrotic). Furthermore, we followed up immunosuppressive-naïve active TAK (n = 16) and compared T helper lymphocyte populations and cytokines before and after immunosuppressive therapy. Healthy controls (HC, n = 21) and sarcoidosis (disease control, n = 11) were compared against TAK.ResultsTAK had higher Th17, Th17.1 and PD1+Th17 lymphocytes than HC (p < 0.001), and higher PD1+CD4+ T lymphocytes than sarcoidosis (p < 0.001). Th17 lymphocytes associated with active TAK after multivariable-adjusted logistic regression (p = 0.008). TAK had greater cytokine secretion from PBMCs (IFN-γ, IL-17A, IL-10 versus HC; IL-6, TNF-α, IL-1β versus HC or sarcoidosis) (p < 0.05). In-vitro, PBMCs from TAK showed reduced secretion of all inflammatory cytokines with tacrolimus, with synergistic reduction in IL-17A, IL-6, IL-1β and IL-10 following addition of tadalafil to tacrolimus. Serial follow-up of immunosuppressive-naïve TAK (n = 16) showed reduction in serum IL-6 and TGF-β1 (p < 0.05) and IL-6 in culture supernatant (p < 0.05) following immunosuppressive therapy.ConclusionNovel Th17 sub-populations (Th17.1 and PD1+Th17) are elevated in TAK. Th17 lymphocytes associate with active TAK. In-vitro experiments on cultured PBMCs suggest promise for further evaluation of a combination of immunosuppressive tacrolimus with anti-fibrotic tadalafil (or other anti-fibrotic therapies) in clinical trials of TAK.

Ex vivo study of IL-6 expression and function in immune cell subsets from human periapical lesions.

Even though IL-6 is a key inflammatory cytokine in periapical lesions (PLs), its function in stable periapical disease is unknown. Therefore, the aim of this study was to investigate the following: first, the ex vivo production of IL-6 by clinically different PLs; next, subsets of immune cells expressing IL-6 in PLs according to their inflammatory status and finally, modulatory effect of IL-6 on T-cell cytokine production in cell cultures. Inflammatory cells were isolated from a total of 95 human PLs. Detection of IL-6+ cells within the myeloid and lymphoid populations was performed by multicolour flow cytometry. ELISA and FlowCytomix Microbeads Assay were used to measure cytokine levels in culture supernatants. To study the role of IL-6 in PLs, mononuclear cells (MNC) from symptomatic (Sy) or asymptomatic (Asy) PLs were treated with IL-6 or Tocilizumab, an IL-6R blocking antibody. The differences between groups were tested by unpaired t-test, Mann-Whitney or Friedman tests. The levels of IL-6 in PL MNC culture supernatants were significantly higher compared with total PL cells and PL granulocytes (p<.001). MNC from Sy PLs produced significantly higher levels of IL-6 than MNC from Asy PLs (p<.001). Flow cytometry analysis showed that NKT cells, CD8+ T cells and M2 macrophages (MØ), were dominant IL-6+ cells, in contrast to CD4+ T cells. However, CD8+ and CD4+ T cells contributed the most to IL-6 production. IL-6hi producing MNC cultures had higher levels of Th1 (IFN-γ), Th17 (IL-17A), Tfh (IL-21) and RANKL, whereas Th2 (IL-4) and Tregs cytokines (IL-10, TGF-β) were lower compared with IL-6low -producing cultures. Exogenous IL-6 stimulated 17A, IL-21 and RANKL, independently of PL activation status, but decreased IFN-γ, IL-4 and IL-33 levels in IL-6hi -producing cultures. Tocilizumab increased IL-10 and TGF-β in IL-6low -producing cultures. All differences were p<.05. Most immune cells from Sy PLs expressed higher levels of IL-6 compared with Asy PLs, which correlated with IL-6 production in culture. Analysis of cytokines suggested a dominant pro-inflammatory T-cell response and osteodestructive function of IL-6 in PLs judging by Th17/Tfh cell activation, Tregs inhibition and increased RANKL/OPG ratio. Excessive IL-6 production decreased Th1/Th2 responses.

РОЛЬ ИММУНОЛОГИЧЕСКИХ ФАКТОРОВ В НЕБЛАГОПРИЯТНОМ ИСХОДЕ ХРОНИЧЕСКОЙ ОБСТРУКТИВНОЙ БОЛЕЗНИ ЛЕГКИХ

Aim. The aim of the research was to study the role of immunological mechanisms in the unfavorable outcome of chronic obstructive pulmonary disease (COPD). Material and methods. The research included 116 patients hospitalized in 2005-2006 in the pulmonology department of patients with the exacerbation of mild and moderate COPD. Research protocol: general clinical and special (immunological) methods of examination were performed in patients with COPD. Instrumental research methods included: spirography, ECG, EchoCG, fibrobronchoscopy, chest X-ray. After diagnostic bronchoscopy, bronchoalveolar fluid (BAF) was taken for cytological and immunological examination. On the first and second days of inpatient treatment, patients underwent immunological studies: immunophenotyping of mononuclear cells (MNC), assessment of phagocytic activity of leukocytes (latex test, nitroblue tetrazolium (NBT) test) in blood and BAF, determination of the content of immunoglobulins in blood serum and BAF, study of cytokine levels in blood serum, BAF and MNC culture supernatant under conditions of their spontaneous production and activation in the mitogen-stimulated (phytohemagglutinin "Difco" 5 mg/ml) proliferation. Statistical data processing was carried out using the Statistica 10.0 program. In 2020, the long-term survival of COPD patients was assessed. The cohort of patients was divided into two groups. Survivors were included in the first group (n=44), and patients who died by 2020 were included in the other group (n=72). The retrospective comparison of the studied indicators was determined at the time of COPD exacerbation in 2005- 2006 in these patient groups. Results and discussion. In the surviving patients, the BAF cytogram was distinguished by the higher macrophage content and the increased number of NBT-positive cells in this population with the reduced neutrophil content in both blood and BAF. The deceased had elevated levels of IgM and IgA in the blood serum and BAF. The results of the study of cytokine production in MNC cultures in vitro indicate that the ratio of IFN-γ/IL-4 in the conditions of spontaneous cytokine production was higher in the deceased compared with the indicators of the surviving patients. This fact indicates the polarization of the immune response of the deceased in the direction of the cell type mediated by Th1 cells, which is confirmed by the peculiarities of the BAF cytokine profile in the deceased – the significant increase in the value of IFN-γ/IL-4 relative to the indicators in the survivors. Conclusion. The unfavorable outcome of COPD is associated with the increase in the number of neutrophil cells in BAF, in the blood, with the predominance of Th1-type activation of adaptive immunity at the local level and activation of both cellular and humoral mechanisms of adaptive immunity at the systemic level. The increased activity of innate immunity in COPD exacerbation, manifested by the increase in the number and metabolic activity of macrophages, is associated with the long-term survival of patients.

Algorithm Design for a Cytokine Release Assay of Antigen-Specific In Vitro Stimuli of Circulating Leukocytes to Classify Leprosy Patients and Household Contacts.

BackgroundImmunological biomarkers have often been used as a complementary approach to support clinical diagnosis in several infectious diseases. The lack of commercially available laboratory tests for conclusive early diagnosis of leprosy has motivated the search for novel methods for accurate diagnosis. In the present study, we describe an integrated analysis of a cytokine release assay using a machine learning approach to create a decision tree algorithm. This algorithm was used to classify leprosy clinical forms and monitor household contacts.MethodsA model of Mycobacterium leprae antigen-specific in vitro assay with subsequent cytokine measurements by enzyme-linked immunosorbent assay was employed to measure the levels of tumor necrosis factor (TNF), interferon-γ, interleukin 4, and interleukin 10 (IL-10) in culture supernatants of peripheral blood mononuclear cells from patients with leprosy, healthy controls, and household contacts. Receiver operating characteristic curve analysis was carried out to define each cytokine’s global accuracy and performance indices to identify clinical subgroups.ResultsData demonstrated that TNF (control culture [CC]: AUC = 0.72; antigen-stimulated culture [Ml]: AUC = 0.80) and IL-10 (CC: AUC = 0.77; Ml: AUC = 0.71) were the most accurate biomarkers to classify subgroups of household contacts and patients with leprosy, respectively. Decision tree classifier algorithms for TNF analysis categorized subgroups of household contacts according to the operational classification with moderate accuracy (CC: 79% [48/61]; Ml: 84% [51/61]). Additionally, IL-10 analysis categorized leprosy patients’ subgroups with moderate accuracy (CC: 73% [22/30] and Ml: 70% [21/30]).ConclusionsTogether, our findings demonstrated that a cytokine release assay is a promising method to complement clinical diagnosis, ultimately contributing to effective control of the disease.

Four weeks of spice consumption lowers plasma proinflammatory cytokines and alters the function of monocytes in adults at risk of cardiometabolic disease: secondary outcome analysis in a 3-period, randomized, crossover, controlled feeding trial

Numerous studies demonstrate acute anti-inflammatory properties of individual spices, but none have examined the effect of longer-term consumption of a spice blend incorporated in a meal. We investigated the effect of longer-term spice consumption on inflammatory cytokines and monocyte subsets [classical (CM), intermediate (IM), nonclassical (NCM)] in adults at risk of cardiometabolic disease. A 3-period, randomized, crossover, controlled feeding trial was conducted. Participants (n = 71 recruited; n = 63 completed) randomly consumed diets differing in terms of the quantity of spices: 0.547 g (low-dose spice diet; LSD), 3.285 g (medium-dose spice diet; MSD), or 6.571 g (high-dose spice diet; HSD) · d-1 · 2100 kcal-1, for 4 wk with a ≥2-wk washout between diets. At baseline and after each diet period, proinflammatory cytokines (IL-1β, IL-6, IL-8, monocyte chemoattractant protein-1, and TNF-α) in plasma and LPS-stimulated peripheral blood mononuclear cell culture supernatants, and the phenotype and function of monocyte subsets, were measured in fasted participants. Postprandial proinflammatory cytokines also were quantified at baseline by consumption of a low-spice-dose test meal, and after each diet period by consumption of a test meal containing a spice dose corresponding to daily spice consumption during the preceding 4-wk diet period. Fasting plasma IL-6 was reduced (mean ± SEM: -118.26 ± 50.63 fg/mL; P < 0.05) after MSD compared with baseline. Postprandial plasma IL-1β, IL-8, and TNF-α were lower (mean ± SEM : -9.47 ± 2.70 fg/mL, -0.20 ± 0.05 pg/mL, and -33.28 ± 12.35 fg/mL, respectively) after MSD compared with LSD (main diet effect; P < 0.05). CM adherence was reduced (mean ± SEM: -0.86 ± 0.34; P = 0.034) after HSD compared with LSD. IM migration was reduced after MSD and HSD compared with LSD (mean ± SEM: -0.39 ± 0.09 and -0.56 ± 0.14, respectively; P < 0.05). Four weeks of MSD consumption reduced fasting plasma IL-6 and postprandial plasma IL-1β, IL-8, and TNF-α as well as altering monocyte function.This trial was registered at clinicaltrials.gov as NCT03064932.

Development, Characterization, and Immunomodulatory Evaluation of Carvacrol-loaded Nanoemulsion

Carvacrol (CV) is an essential oil with numerous therapeutic properties, including immunomodulatory activity. However, this effect has not been studied in nanoemulsion systems. The objective of this study was to develop an innovative carvacrol-loaded nanoemulsion (CVNE) for immunomodulatory action. The developed CVNE comprised of 5% w/w oily phase (medium chain triglycerides + CV), 2% w/w surfactants (Tween 80®/Span 80®), and 93% w/w water, and was produced by ultrasonication. Dynamic light scattering over 90 days was used to characterize CVNE. Cytotoxic activity and quantification of cytokines were evaluated in peripheral blood mononuclear cell (PBMC) culture supernatants. CVNE achieved a drug loading of 4.29 mg/mL, droplet size of 165.70 ± 0.46 nm, polydispersity index of 0.14 ± 0.03, zeta potential of −10.25 ± 0.52 mV, and good stability for 90 days. CVNE showed no cytotoxicity at concentrations up to 200 µM in PBMCs. CV diminished the production of IL-2 in the PBMC supernatant. However, CVNE reduced the levels of the pro-inflammatory cytokines IL-2, IL-17, and IFN-γ at 50 µM. In conclusion, a stable CVNE was produced, which improved the CV immunomodulatory activity in PBMCs.

Diet-induced obesity aggravates NK cell-mediated contact hypersensitivity reaction in Rag1-/- mice.

Previous studies showed that natural killer (NK) cells mediate contact hypersensitivity (CHS) reaction. Many reports are showing that obesity promotes several inflammatory diseases. It was shown that diet-induced obesity (DIO) aggravates classical T cell-mediated CHS in mice. To determine whether the high-fat diet (HFD)-induced obesity modulates antigen-specific NK cell-mediated response. We evaluated the effect of DIO on NK cell-mediated CHS reaction using a model of dinitrofluorobenzene (DNFB)-induced CHS in Rag1-/- mice. Rag1-/- mice fed HFD for 8 but not for 4 weeks developed aggravated CHS reaction determined by ear swelling measurement when compared to animals kept on normal diet (ND) prior to DNFB sensitization. The obese Rag1-/- mice presented the adipose tissue inflammation. Furthermore, in vitro analysis showed that feeding with HFD significantly increases interferon γ (IFN-γ) and interleukin (IL)-12p70 and decreases adiponectin concentration in liver mononuclear cell (LMNC) culture supernatants. The flow cytometry analysis of LMNC revealed that HFD treatment prior to DNFB sensitization increases the percentage of NK1.1+ IFN-γ+ cell population and affects the development and maturation of NK1.1+ cells. In summary, current results suggest that the DIO significantly modulates the local and systemic inflammatory response, contributing to exacerbation of the CHS response mediated by liver NK cells.

Cytokines status in multiple sclerosis patients with comorbid recurrent depressive disorder

IntroductionThe presence of some common immunological pathogenesis mechanisms in multiple sclerosis and depression suggests the possibility of comorbid depressive disorder formation in multiple sclerosis patients, which significantly worsens their quality of life and patient’s compliance. In this regard, the depressive pathology diagnosis in people suffering from multiple sclerosis acquires important scientific and practical value.ObjectivesThe aim of the study was the cytokine status peculiarities identification in multiple sclerosis patients with comorbid recurrent depressive disorder (F33).MethodsThe cytokines content in patient’s blood mononuclear cells culture supernatants was carried out by ELISA. The recurrent depressive disorder diagnosis was established based on ICD-10 criteria. The depressive disorders symptoms severity was determined according to the M. Hamilton and A.T. Beck depression scales, as well as during the clinical interviewResultsA higher production of IL-6 was noted in multiple sclerosis patients with mild recurrent depressive disorder (F33.00), in contrast to patients without the affective symptoms. The IL-1β, TNF-α, IL-6 contents in the blood mononuclear cells culture supernatants of patients with severe recurrent depressive disorder (F33.2) exceeded the corresponding parameters of patients with mild depressive symptoms. A direct correlation between the depression severity and IL-1β, TNF-α, IL -6 spontaneous production by blood mononuclear cells of patients with multiple sclerosis was found.ConclusionsThe severity of recurrent depressive disorder correlates with a change in the parameters of the cytokine status: severe depressive symptoms are accompanied by a change in the functional activity of immune cells and an increase in the production of cytokines synthesized by type I T-helpers.DisclosureNo significant relationships.

Effects of Cytokines IFN-γ and TGF-β on the Functional Activity of Blood Mononuclear Cells against Giardia lamblia.

Background:This study aimed to analyze cultures of mononuclear (MN) cells with Giardia lamblia to determine the levels of the cytokines IFN-γ and TGF-β and the functional activity of MN cells after incubation with cytokines.Methods:This study was conducted in 2018 in Barra do Garças, Mato Grosso State, Brazil. Blood samples were collected from 60 healthy volunteer donors to obtain leukocytes. The levels of IFN-γ and TGF-β were quantified in trophozoite cell culture supernatants. Superoxide release, phagocytosis, microbicidal activity, apoptosis and intracellular calcium release were analyzed.Results:The cytokines evaluated were detected in the culture supernatant of MN cells and G. lamblia. Regardless of the type of cytokine, MN cells increased superoxide release in the presence of G. lamblia. Phagocytosis, microbicidal activity and apoptosis were higher when MN phagocytes were treated with cytokines. The highest microbicidal activity and apoptosis rates were observed in MN cells cultured with TGF-β. IFN-γ increased the release of intracellular calcium by MN phagocytes.Conclusion:Cytokines play a beneficial role in the host by activating MN cells against G. lamblia. In addition, phagocytosis causes G. lamblia death and that the modulation of the functional activity of blood MN phagocytes by cytokines is an alternative mechanism for eliminating G. lamblia.

Fatty-Acid-Binding Protein 4 as a Novel Contributor to Mononuclear Cell Activation and Endothelial Cell Dysfunction in Atherosclerosis

Background—Elevated circulating fatty-acid-binding protein 4 (FABP4) levels may be linked with cardiovascular events. This study aimed to investigate the mechanistic role of FABP4 in atherosclerosis. Methods—We recruited 22 patients with angiographically proven coronary artery disease (CAD) and 40 control subjects. Mononuclear cells (MNCs) and human coronary endothelial cells (HCAECs) were used for in vitro study. Results—Patients with CAD were predominantly male with an enhanced prevalence of hypertension, diabetes, and smoking history. FABP4 concentrations were up-regulated in culture supernatants of MNCs from CAD patients, which were positively correlated with the patients’ age, waist–hip ratio, body mass index, serum creatinine, type 2 diabetes, and the presence of hypertension. The adhesiveness of HCAECs to monocytic cells can be activated by FABP4, which was reversed by an FABP4 antibody. FABP4 blockade attenuated the oxidized low-density lipoprotein (oxLDL)-induced expression of ICAM-1, VCAM-1, and P-selectin. FABP4 impaired the tube formation and migration via the ERK/JNK/STAT-1 signaling pathway. FABP4 suppressed phosphorylation of eNOS and expression of SDF-1 protein, both of which can be reversed by treatment with VEGF. Blockade of FABP4 also improved the oxLDL-impaired cell function. Conclusion—We discovered a novel pathogenic role of FABP4 in MNC activation and endothelial dysfunction in atherosclerosis. FABP4 may be a therapeutic target for modulating atherosclerosis.

Functional expression cloning of molecules inducing CD34 expression in bone marrow-derived stromal myofibroblasts

We have previously shown a fraction of stromal fibroblasts/myofibroblasts (Fibs) from leukemic bone marrow cells expresses leukemia-specific transcripts along with hematopoietic and Fib-related markers. Normal bone marrow-derived Fibs (nFibs) do not express CD34 or CD45; however, nFibs may express hematopoietic markers with some specific stimulations. CD34 expression was detected in nFib cultures following the addition of a culture supernatant of blood mononuclear cells stimulated with phytohemagglutinin (PHA)-P. To identify the molecules responsible for inducing CD34 expression in nFibs, cDNA clones were isolated using functional expression cloning with a library constructed from PHA-P-stimulated human blood mononuclear cells. Positive clones inducing CD34 transcription in nFibs were selected. We confirmed that an isolated positive cDNA clone encoded human interleukin (IL)-1 beta (β). CD34 expression was observed in the nFib cultures with recombinant human (rh) IL-1β protein. And CD34 transcription was suppressed when a rhIL-1β neutralizing antibody was added to the IL-1β-stimulated nFib cultures. nFibs expressed gp130 and IL-6 receptors, and CD45 expression was detected in nFibs cultured with rhIL-1β and rhIL-6. Chronic myelogenous leukemia (CML) cells reportedly respond well to IL-1β. When CML-derived Fibs were cultured with rhIL-1β and rhIL-6, CD45-positive cells increased in number. Cell fate may be influenced by an external specific stimulation without gene introduction.

Immunoregulatory effects of flavonoid-containing medicinal herbs in human peripheral blood mononuclear cells

Plant-derived medicinal products provide a prominent advantage due to their low toxicity to humans and combined effects of biologically active substances, mainly presented by polysaccharides, flavonoids and terpenoids. One of the mechanisms undedrlying effects from medicinal plants on the immunoregulationrelated events is mediated via controlled production of certain cytokines. Here we examined immunoregulatory activity of water extracts derived from medicinal plant raw materials (LRS) containing polyphenolic compounds – flavonoids (rutin, quercetin, called P-vitamins). The aim of the study was to assess profile and level of cytokines secreted by human peripheral blood mononuclear cedlls exposured to flavonoid-containing LRS water extracts. LRS (1:10) water extracts of the following species were used: black currant leaves (Ribes nigrum L.), field horsetail grass (Equisetum arvense L.), common yarrow grass (Achillea millefolium L.), licorice roots (Glycyrrhiza uralensis Fisch.), sand immortelle flowers (Helichrysum arenarium (L.) Moench), wild strawberry leaves (Fragaria vesca L.), fruit common bird cherry (Padus avium Mill.), tansy flowers (Tanacetum vulgare L.) and oat grass (Avena sativa L.) (all purchased at the pharmacy). Production of pro – (TNFα, IL-8, IL-1β) and anti-inflammatory (IL-10) cytokines was measured by using ELISA kits (“Cytokine”, Russia) in mononuclear cell culture supernatant treated with / without LRS (experiment and control group, respectively). Amount of flavonoids contained in flowers and leaves was quantified aftedr complexation reaction with aluminum chloride on UV-3600 spectrophotometer (Shimadzu, Japan). It was found that LRS water extracts predominantly inhibited production both of pro- (TNFα, IL-8, IL-1β) and anti-inflammatory (IL-10) cytokines so that magnitude of such suppressive effect ranged from 51.5±3.4 to 99.5±4.1% compared to untreated control samples (p 0.05). Total flavonoid level in the LRS samples diirectly correlated with intensity of related immunoregulatory activity on cytokine secretion particularly TNFα (r = 0.65), IL-8 (r = 0.4), IL-1β (r = 0.48) and IL-10 (r = 0.68). The data of our study allow to conclude that extracts from the examined medicinal plant raw materials can be considered as promising components while developing new drugs with exhibiting immunoregulatory and antiflogogenic effects.