Detection of cytokines in nasal lavage samples of patients with cystic fibrosis: comparison of two different cytokine detection assays.

BACKGROUND: Cystic fibrosis (CF) is a genetic multisystem disorder. Inflammatory processes, which presumably begin early in infancy, play a crucial role in the progression of the disease. The detection of inflammatory biomarkers, especially in the airways, has therefore gained increasing attention. Due to improved treatment options, patients with CF produce less sputum. Nasal lavage samples therefore represent a promising alternative to induced sputum or bronchoalveolar lavage specimens. However, methodology of cytokine measurements is not standardised and comparisons of results are therefore often difficult. The aim of this study was to identify suitable detection methods of cytokines in nasal lavage samples by comparison of two different assays. METHODS: Nasal lavage samples were obtained from the same patient at the same time by trained respiratory physiotherapists using a disposable syringe and 10 ml of 0.9% sodium chloride per nostril during outpatient visits. The cytokines IL-17 A, IL-2, IL-6 and IL-10 were measured using two different assays (BD™ and Milliplex®), which have already been applied in sputum and nasal lavage samples, despite different lower detection limits. RESULTS: 22 participants were included in the study. In 95.5% of measurements, values were below the limit of detection with respect to the BD™ assay. Only IL-6 could be detected in approximately half of the patients. Individual cytokine levels were considerably higher when measured with Milliplex®, which is also reflected in a statistically significant manner (p = < 0.01). CONCLUSION: The right choice of analysis method is crucial for measuring inflammatory markers in nasal lavage samples. Compared to the literature, Milliplex® showed higher detection rates and similar concentrations to other studies. TRIAL REGISTRATION: Ethics approval was obtained from the ethics committee at Medical University of Innsbruck (EK Nr: 1055/2022).

Systemic and local responses of cytokines and tissue histology following intramammary lipopolysaccharide challenge in dairy cows.

During bovine mastitis, immune responses include the release of cytokines and the recruitment of leukocytes, resulting in profound structural and functional changes in the mammary gland. Our aims were to delineate systemic and local cytokine responses and to quantify histological changes in the mammary tissue of lactating cows after acute intramammary lipopolysaccharide (LPS) challenge. Ten multiparous dairy cows were paired to either treatment (TRT) or control (CON) groups. For TRT cows, one side of the udder was randomly assigned to receive treatment with LPS (50 µg in 10 mL of saline, TL) into both the front and rear quarters; the contralateral quarters received saline (10 mL). Udder-halves of CON cows were similarly assigned randomly to receive either saline (10 mL, CS) or no infusion (untreated). Temporal changes in the concentrations of 15 cytokines in the blood (0, 3, 6, 12, and 24 h relative to the LPS infusion) and in mammary tissue (0, 3, and 12 h) were determined, as were concomitant changes in mammary histology. The cytokines IL-6, IL-10, MCP-1, and MIP-1ß showed a systemic response as their concentrations were significantly different in the plasma of TRT cows as compared with CON cows after LPS challenge. The cytokines IL-1α, IL-1ß, IL-6, IL-8, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1ß, TNF-α, and VEGF-A showed a local response in TL glands, and 8 cytokines, IL-1ß, IL-6, IL-10, IL-17A, IL-36RA, IP-10, MIP-1ß, and VEGF-A showed systemic changes in the nonchallenged mammary glands adjacent to LPS-infused glands. Endotoxin challenge evoked changes in the histology of mammary tissue that included a 5.2- and 7.2-fold increases in the number of neutrophils in alveolar lumens at 3 h and 12 h, respectively. In summary, LPS challenge induced specific local and systemic responses in cytokine induction and elicited neutrophil infiltration in bovine mammary tissue.

Comparative Evaluation of Gingival Crevicular Fluid Interleukin-17, 18 and 21 in Different Stages of Periodontal Health and Disease.

Background and Objectives: The elicitation of a host's immune−inflammatory responses to overcome oral bacterial biofilm challenges is mediated by numerous cytokines. We explored the role of three such cytokines, viz. interleukin (IL)-17, 18 and 21, by measuring their levels in the gingival crevicular fluid (GCF) of Indian individuals with healthy gingiva, chronic gingivitis, or chronic periodontitis. Materials and Method: Ninety systemically healthy individuals were enrolled in the study on the basis of predefined criteria and were categorized into three groups of 30 participants each. Groups A, B and C were composed of a control group with healthy gingiva, subjects with chronic gingivitis and subjects with chronic periodontitis, respectively. The periodontal disease status was assessed on the basis of a subject's gingival index, probing pocket depth, clinical attachment loss and radiographic evidence of bone loss. After the complete history-taking and identification of gingival sulcus/pocket depth areas for GCF collection, a sample was collected from each subject in all groups for an estimation of the cytokine levels using ELISA. Statistical analysis was performed using SPSS v 21.0. Intergroup comparisons were conducted using a post hoc Tukey's test. A value of p < 0.05 was considered to be statistically significant. Results: The mean IL-17, 18 and 21 concentrations in pg/mL was the greatest for Group C (99.67 ± 18.85, 144.61 ± 20.83 and 69.67 ± 12.46, respectively), followed by Group B (19.27 ± 2.78, 22.27 ± 2.43 and 22.74 ± 1.43, respectively) and finally by Group A (healthy control; 11.56 ± 0.99, 17.94 ± 1.24 and 12.83 ± 1.21 respectively). A statistically significant difference in the mean concentrations of two interleukins (IL-17 and IL-18) was observed between Groups A and C and also between Groups B and C. A statistically significant difference in the mean concentrations of IL-21 was observed between Groups B and C. Conclusions: Within the limitations of the present study, the findings revealed that the GCF levels of IL-17, IL-18 and IL-21 rose and correlated well with the severity of the disease. Thus, these cytokines present in GCF have the potential to be considered as biomarkers for periodontal tissue destruction. IL-21 in particular appears to be a promising biomarker for differentiating between gingivitis and periodontitis.

Salivary IL-4: A Bleeding Predictor on Probing in Descendants of Severe Periodontitis Patients.

OBJECTIVE: Periodontitis in younger patients can cause severe periodontal destruction, and cases are usually more numerous in members of the same family due to the sharing of susceptibility factors. Thus, the use of a familial study design could improve our understanding of initial alterations in periodontal tissue. This observational study aimed to evaluate the salivary inflammatory pattern in descendants of periodontitis patients and identify any correlation with the clinical periodontal condition. STUDY DESIGN: Fifteen children of Generalized Aggressive Periodontitis (GAgP) patients and 15 children with periodontally healthy parents were evaluated for their plaque index (PI), gingival index (GI), bleeding on probing (BoP), and probing depth (PD). The concentrations of interferon (IFN)-γ, interleukin (IL)-10, IL-17, IL-1ß, IL-4, and tumor necrosis factor (TNF)-α were measured in unstimulated saliva using the Luminex MAGPix platform. RESULTS: Children from the GAgP group presented higher probing depth (PD) and bleeding on probing (BoP) (p<0.05) and lower release of IL-4 in saliva (p<0.05) than the periodontally healthy group. The cytokines IL-10, IFN-γ, IL-17, and IL-4 were negatively correlated with the gingival index, while IL-4 was negatively correlated with BoP. A regression analysis revealed that salivary IL-4 and plaque were predictors of BoP. CONCLUSIONS: Children of GAgP parents presented lower salivary IL-4 and higher BoP and PD than children from periodontally healthy families. Additionally, salivary IL-4 was a predictor of bleeding on probing in the children, suggesting that the lower presence of this anti-inflammatory cytokine is related to higher clinical inflammation.

IL-17 is expressed on beta and alpha cells of donors with type 1 and type 2 diabetes.

PURPOSE: IL-17 is an important effector cytokine driving immune-mediated destruction in autoimmune diseases such as psoriasis. Blockade of the IL-17 pathway after the initiation of insulitis was effective in delaying or preventing the onset of type 1 diabetes (T1D) in rodent models. Expression of IL-17 transcripts in islets from a donor with recent-onset T1D has been reported, however, studies regarding IL-17 protein expression are lacking. We aimed to study whether IL-17 is being expressed in the islets of diabetic donors. METHODS: We stained human pancreatic tissues from non-diabetic (n = 5), auto-antibody positive (aab+) (n = 5), T1D (n = 6) and T2D (n = 5) donors for IL-17, Insulin, and Glucagon, and for CD45 in selected cases. High resolution images were acquired with Zeiss laser scanning confocal microscope LSM780 and analyzed with Zen blue 2.3 software. Cases stained for CD45 were also acquired with widefield slide scanner and analyzed with QuPath software. RESULTS: We observed a clear cytoplasmic staining for IL-17 in insulin-containing islets of donors with T1D and T2D, accounting for an average of 7.8 ± 8.4% and 14.9 ± 16.8% of total islet area, respectively. Both beta and alpha cells were sources of IL-17, but CD45+ cells were not a major source of IL-17 in those donors. Expression of IL-17 was reduced in islets of non-diabetic donors, aab+ donors and in insulin-deficient islets of donors with T1D. CONCLUSION: Our finding that IL-17 is expressed in islets of donors with T1D or T2D is quite intriguing and warrants further mechanistic studies in human islets to understand the role of IL-17 in the context of metabolic and immune stress in beta cells.

IL-11 Induces Encephalitogenic Th17 Cells in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis.

IL-11+CD4+ cells accumulate in the cerebrospinal fluid of patients with early relapsing-remitting multiple sclerosis (MS) and in active brain MS lesions. Mouse studies have confirmed a causal role of IL-11 in the exacerbation of relapsing-remitting experimental autoimmune encephalomyelitis (RREAE). Administration of IL-11 at the time of clinical onset of RREAE induced an acute exacerbation and increased clinical scores, which persisted during the entire course of the disease. IL-11 increased the numbers of spinal cord inflammatory foci, as well as the numbers of peripheral and CNS-infiltrating IL-17+CD4+ cells and IL-17A serum levels. Ag recall assays revealed that IL-11 induces IL-17A+, GM-CSF+, and IL-21+CD4+ myelin Ag-reactive cells. Passive transfer of these encephalitogenic CD4+ T cells induced severe RREAE with IL-17A+CCR6+ CD4+ and B cell accumulation within the CNS. Furthermore, passive transfer of nonmanipulated CNS-derived mononuclear cells from mice with RREAE after a single dose of IL-11 induced severe RREAE with increased accumulation of IL-17A+ and CCR6+ CD4+ cells within the CNS. These results suggest that IL-11 might serve as a biomarker of early autoimmune response and a selective therapeutic target for patients with early relapsing-remitting MS.

Immunological response and clinical profile in patients with recurrent dermatophytosis.

BACKGROUND: An alarming increase in the number of patients with chronic and recurrent dermatophytosis has invoked the need to study the immunological parameters of the host. OBJECTIVES: To evaluate delayed type of hypersensitivity (DTH) response and immediate hypersensitivity (IH) response by flow cytometry evaluation of immune cells from peripheral blood and intradermal trichophyton skin test in patients with recurrent dermatophytosis. METHODS: A hundred patients with recurrent dermatophytosis and 50 controls (healthy controls and acute dermatophytosis controls) were included. Relevant risk factors for recurrence were analysed, and serum IgE levels were estimated. Flow cytometry evaluation of immune cells in peripheral blood and intradermal trichophyton skin test was done. Dermatophyte pathogens were isolated, and antifungal susceptibility was performed. RESULTS: Trichophyton mentagrophytes complex (95.84%) and T. rubrum (4.16%) were isolated in culture. Serum IgE was elevated in 83.15% cases (p = .01). IFN-γ+ cells (p = .0501, p = .0001, p = .0014), Th1 cells (p = .1197, p = .0024, p = .0169), IL-17+ cells (p = .0127, p = .0006, p = .0007) and Th17 cells (p = .0634, p = .0001, p = .0054) were reduced, and IL-4+ cells (p = .0108, p = .0175, p = .0018) were increased in cases. Intradermal test demonstrated negative DTH response in all cases (p < .001, p < .001, p < .001), strongly positive IH response in 6%, and borderline positive IH response in 85% cases (p = .018, p < .001, p < .001). Topical corticosteroids application, undergarment types (tight fit), poor frequency of washing clothes, family history of tinea, sharing of towels were significant risk factors for recurrent dermatophytosis. CONCLUSIONS: Reduced IFN-γ+ , Th1, IL-17+ and Th17 cells population along with impaired DTH response by the intradermal test was observed in patients with recurrent dermatophytosis.

Gastric IL-1ß, IL-8, and IL-17A expression in Moroccan patients infected with Helicobacter pylori may be a predictive signature of severe pathological stages.

INTRODUCTION: Helicobacter pylori induces acute gastritis that can progress to serious diseases such as gastric cancer. H. pylori interacts with host cells within the gastric mucosa, resulting in activation of multiple innate immune signalling pathways, leading to pro-inflammatory cytokines production and immune cells recruitment. Various studies have shown that there are ethnic- and population-related differences in the expression of these cytokines. Although the H. pylori infection is a major public health problem in Morocco, to our knowledge, no study has been carried out in gastric cytokine expression from H. pylori-infected Moroccan patients. Thus we aimed to (i) determine the IL-1ß, IL-8 and IL-17A gene expression in gastric biopsies from Moroccan patients infected with H. pylori, and (ii) to determine the cytokine signature of each pathological stages associated with this infection. MATERIAL AND METHODS: 71 patients with epigastralgic pain were included in this study. The H. pylori detection on gastric biopsies was performed by histopathological and PCR analysis. The IL-1ß, IL-8 and IL-17A mRNA expression in the antrun and fundus biopsies was performed by RT-qPCR. RESULTS: The histopathological and PCR analyses revealed that 87.32% of the patients were infected with H. pylori. IL-1ß mRNA expression was significantly lower in the antral mucosa of H. pylori-infected patients (p = 0.0038) than in the uninfected while there was no significant difference in the expression of IL-8 and IL-17A mRNA. The expression of the three cytokines was higher in the fundic mucosa of H. pylori-infected patients than in the uninfected patients, but only IL-8 and IL-17A expression reached statistical significance (p = 0.042 and p = 0.0179 respectively). Furthermore, the multivariate predictive analysis highlighted a cytokine signature that may predict metaplasia during the infection progression that involves a specific down-regulation of IL17A and an up-regulation of IL1ß in antral and fundic metaplasia respectively.

Salivary Del-1, IL-17, and LFA-1 levels in periodontal health and disease.

OBJECTIVE AND BACKGROUND: Developmental endothelial locus-1 (Del-1), lymphocyte function-associated antigen-1 (LFA-1), and interleukin 17 (IL-17) play critical roles in transendothelial migration of neutrophils in periodontal diseases. The aim of this study was to evaluate salivary Del-1, IL-17, and LFA-1 protein levels in patients with gingivitis (G), chronic periodontitis (CP), and generalized aggressive periodontitis (GAP). METHODS: A total of 180 systemically healthy, non-smoking patients (45 periodontally healthy (H) and 45 G, 50 CP, and 40 GAP) individuals (between March 2014 and February 2016) were included in this study according to Armitage's (1999) classification. Clinical periodontal parameters, including clinical attachment level, probing depth, plaque index, and gingival index, were recorded. Del-1, IL-17, and LFA-1 protein expression levels were measured in unstimulated saliva samples collected from patients by using enzyme-linked immunosorbent assays. Kruskal-Wallis and Mann-Whitney U tests were used for multiple comparisons and post hoc statistical analyses, respectively. ROC curve analysis was used to evaluate the sensitivity and specificity of Del-1, IL-17, and LFA-1 in distinguishing periodontal disease from health and gingivitis. RESULTS: It was found a high level of IL-17 and a low level of Del-1 in the CP and GAP, as compared to the G and H groups (P < .001). Nevertheless, we found LFA-1 levels were higher in the GAP than in the CP or G groups (P = .00). Consistently, LFA-1 levels were lower in the H and G groups than in the CP and GAP groups (P = .00). The combination of three biomarkers was found as the best predictor yielded exhibited the highest AUC [0.893, 0.845-0.94 (%95 CI) P < .001] in discriminating periodontal disease from health and gingivitis. CONCLUSION: Salivary Del-1, LFA-1, and IL-17 levels might be useful markers for determining the clinical health and disease status of patients with periodontitis. However, further studies that evaluate the level of salivary Del-1, LFA-1, and IL-17 before and after periodontal therapy are required to understand the exact roles of these cytokines during the periodontal healing period.

The use of crevicular fluid to assess markers of inflammation and angiogenesis, IL-17 and VEGF, in patients with solid tumors receiving zoledronic acid and/or bevacizumab.

PURPOSE: Crevicular fluid was used to assess interleukin-17 (IL-17) and vascular endothelial growth factor (VEGF) in cancer patients receiving zoledronic acid and/or bevacizumab. The markers were also assessed in the serum. METHODS: Twenty-five patients were included and comprised three groups: patients who received zoledronic acid (n = 9), patients who received bevacizumab (n = 9), and patients who received zoledronic acid combined with bevacizumab (n = 5). One patient received zoledronic acid and everolimus and another received zoledronic acid, bevacizumab, and temsirolimus. IL-17 and VEGF were measured by standard quantitative ELISA kits and assessed in two study points. RESULTS: Twenty-four patients maintained good periodontal health; one had asymptomatic osteonecrosis of the jaw. First assessment: 44 samples were collected; 21 from serum and 23 from crevicular fluid. Second assessment, 6 months later: 11 samples were collected; 6 from serum and 5 from crevicular fluid. IL-17 was detected in all samples, in serum and crevicular fluid, and remained unchanged at both time points. Serum VEGF in patients with bevacizumab alone or combined with zoledronic acid was significantly lower compared with that of patients who received zoledronic acid alone. VEGF was not detected in the crevicular fluid. CONCLUSIONS: Crevicular fluid might be an easy, non-invasive means to assess IL-17. The stable values of IL-17 in crevicular fluid and serum and the lack of VEGF in the crevicular fluid could be related to the good periodontal health of our patients. Further studies are needed to assess IL-17 and VEGF in the crevicular fluid in patients with and without periodontal disease.

Attenuation of Helper T Cell Capacity for TH1 and TH17 Differentiation in Children With Nontuberculous Mycobacterial Infection.

Nontuberculous mycobacteria (NTM) infect children with increasing frequency worldwide. Using blood and lymph node tissue from children with NTM lymphadenitis, and uninfected lymph node tissue from community controls, we evaluated helper T (TH) cells in functional assays of TH1/TH17 differentiation and measured the concentration of their associated cytokines at the site of infection. Circulating TH cells from infected children were attenuated in their TH1/TH17 differentiation capacity and expressed less interferon γ and interleukin 17 after polyclonal stimulation. Similar differences were observed at the site of infection, where most cytokine concentrations were unchanged relative to controls. Our data are consistent with a model wherein TH1/TH17 differentiation is attenuated in NTM-infected children.

IL-17A inhibits autophagic activity of HCC cells by inhibiting the degradation of Bcl2.

Hepatocellular carcinoma (HCC) is associated with poor prognosis due to many unknowns about its inflammatory microenvironment. As a pivotal proinflammatory cytokine, IL-17A exerts a protective effect on the survival and function of HCC cells. It is widely accepted that IL-17A plays an important role in regulating autophagy. Bcl2, a key molecule promoting the survival of HCC cells, also plays an indispensable role as an autophagy regulator. The aim of this study was to investigate the role of Bcl2 in IL-17A-regulated autophagy of HCC cells. The results showed that IL-17A not only inhibited autophagic activity, but also increased Bcl2 levels in HCC cells under starvation. Besides, IL-17A could prevent the dissociation of autophagy protein Beclin1 from Bcl2-Beclin1 complex upon starvation. Overexpression of Beclin1 rescued the autophagy deficiency of HCC cells in presence of IL-17A. Moreover, RNAi-induced Bcl2 silencing impaired the function of IL-17A in inhibiting the activation of autophagy, subsequently reducing the viability and migration of HCC cells, while the inhibition of Beclin1 by spautin-1 could reduce autophagic activity to a certain degree, thus restoring the viability and migration of HCC cells. In summary, it was suggested that the inhibition of Bcl2 degradation may be an important mechanism by which IL-17A inhibits autophagy response, subsequently maintaining the survival in HCC cells.

CCR6 defines a subset of activated memory T cells of Th17 potential in immune thrombocytopenia.

Current researches have determined the significance of C-C chemokine receptor (CCR)6 expression as either a marker of T helper cells (Th) or an effector and regulator of T cell function. However, the roles of CCR6 in the pathogenesis of immune thrombocytopenia (ITP) are unclear. In this study, we aimed to investigate the phenotype and functional characteristics of circulating CCR6+ T cells in blood from chronic ITP patients and healthy controls. We found that the frequency of CCR6+ CD4+ cells was higher in ITP patients than in healthy controls. Anti-CD3/anti-CD28 stimulation induced rapid expansion of CCR6+ CD4+ cells in ITP patients. CCR6+ CD4+ cells had a phenotype of activated cells and predominantly expressed CD45RO. Forkhead box protein P3 (FoxP3) and CD25-positive cells were exclusively detected within the CCR6+ CD4+ cells. In ITP patients, CCR6+ regulatory T cells (Treg ) were decreased and positively correlated with platelet counts and transforming growth factor (TGF)-ß plasma levels. In contrast to CCR6- counterparts, CCR6+ CD4+ cells produced higher levels of interleukin (IL)-17A. The frequency of CCR6+ Th17 was higher in ITP patients and positively correlated with IL-17A levels in supernatant. Most importantly, CCR6+ CD4+ cell subpopulations, but not CCR6- CD4+ , were closely correlated to treatment response of ITP patients. These findings suggest that circulating CCR6+ CD4+ cells in ITP patients have characteristics of activated memory Th17 phenotype and could be used to monitor disease activity and treatment response.

Noradrenaline through ß-adrenoceptor contributes to sexual dimorphism in primary CD4+ T-cell response in DA rat EAE model?

Males exhibit stronger sympathetic nervous system (SNS) activity, but weaker primary CD4+ T-cell (auto)immune responses. To test the role of catecholamines, major end-point SNS mediators, in this dimorphism, influence of propranolol (ß-adrenoceptor blocker) on mitogen/neuroantigen-stimulated CD4+ T cells from female and male EAE rat draining lymph node (dLN) cell cultures was examined. Male rat dLNs exhibited higher noradrenaline concentration and frequency of ß2-adrenoceptor-expressing CD4+ T lymphocytes and antigen presenting cells. Propranolol, irrespective of exogenous noradrenaline presence, more prominently augmented IL-2 production and proliferation of CD4+ lymphocytes in male than female rat dLN cell cultures. In neuroantigen-stimulated dLN cells of both sexes propranolol increased IL-1ß and IL-23/p19 expression and IL-17+ CD4+ cell frequency, but enhanced IL-17 production only in male rat CD4+ lymphocytes, thereby abrogating sexual dimorphism in IL-17 concentration observed in propranolol-free cultures. Thus, ß-adrenoceptor-mediated signalling may contribute to sex bias in rat IL-17-producing cell secretory capacity.

Natural killer cells protect white matter integrity in bipolar disorder.

BACKGROUND: Bipolar Disorder (BD) associates with disrupted white matter (WM) microstructure and functional connectivity, and with a perturbation of the immune system. Higher cytokines, and reduced T cells, correlated with WM disruption and fMRI responses. A core component of the innate immune system, natural killer (NK) cells were detected in brain parenchyma, but never studied in BD. METHODS: We studied Diffusion Tensor Imaging (DTI) measures of water diffusion, fMRI corticolimbic functional response and connectivity, and multi-parameter cytofluorometry analysis of NK (CD56+) subpopulations, in 30 inpatients with active Bipolar Disorder type I. NK cells were also obtained in 36 healthy controls. RESULTS: Patients had significantly higher circulating counts of CD56+GMCSF+, CD56+INFγ+, and CD56+IL17+. NK cell levels positively associated to fractional anisotropy (FA) measures. CD56+TNFα+, CD56+INFγ+, and CD56+GMCSF+ directly correlated with FA, and inversely with radial (RD) and mean (MD) diffusivity. Duration of lithium treatment associated with higher CD56+TNFα+, CD56+IL2+, and CD56+IL4+, and positively associated with FA in tracts were NKs had significant effects. A mediation model suggested a partial mediation of CD56+TNFα+ cells, higher in patients on lithium, on the effects of lithium on FA. Frequencies of the same cytokine-producing NK cells also influenced fMRI cortico-limbic functional connectivity during processing of both, emotional and non-emotional stimuli. DISCUSSION: Higher circulating cytokine-producing NK cells associated with lithium treatment, and with DTI measures of WM integrity, partially mediating the effect of lithium on WM. The same cells associated with fMRI responses and connectivity, thus suggesting an effect on structural and functional connectomics in BD.

Imbalanced spinal infiltration of Th17/Treg cells contributes to bone cancer pain via promoting microglial activation.

Increasing evidence suggests that T cells participate in the pathology of neuropathic pain, as well as the activation of microglia. However, whether T cells infiltrate into the spinal cord and contribute to the development of bone cancer pain (BCP) remains unknown. Here, we used a mouse model of BCP to show that numbers of T cells infiltrated into the spinal cord after sarcoma cell implantation with increased BCP, and most infiltrating T cells in the spinal cord were CD3+CD4+ T cells. Both Th17 and Treg subpopulations were analyzed by immunofluorescence. Treg cells in the spinal cord were transiently up-regulated, followed by an imbalance towards Th17 afterwards, and elevated IL-17/IL-17A levels were observed in both blood and spinal cord. Meanwhile, TGF-ß, IL-6, and IL-23, the factors which regulate Th17/Treg differentiation, increased their expressions during the development of BCP. Additionally, IL-17A receptor (IL-17AR) was found to be expressed on microglia, and the level of IL-17AR increased with activated microglia during BCP development. Furthermore, BCP was ameliorated when IL-17/IL-17A neutralizing antibodies were intrathecally injected, accompanied with inhibited Th17/Treg infiltration and suppressed microglial activation. In conclusion, T cells infiltrated into the spinal cord with the imbalance of Th17/Treg towards Th17 during the development of BCP, which could promote the microglial activation and further increased BCP, while neutralizing IL-17/IL-17A in the spinal cord could ameliorate BCP. Our results suggest that targeting the imbalanced Th17/Treg infiltration in the spinal cord could be a novel strategy for BCP therapy.

Recognition of Schistosoma mansoni egg-expressed ovalbumin by T cell receptor transgenic mice.

Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.

Dialysate cell-free mitochondrial DNA fragments as a marker of intraperitoneal inflammation and peritoneal solute transport rate in peritoneal dialysis.

BACKGROUND: Mitochondrial DNA (mtDNA) released into extracellular subsequent to cell injury and death can promote inflammation in patients and animal models. However, the effects of peritoneal dialysate cell-free mtDNA on intraperitoneal inflammation and peritoneal solute transport rate (PSTR) in peritoneal dialysis (PD) patients remain unclear. METHODS: We select the incident patients who began PD therapy between January 1, 2009, and December 30, 2010. Peritoneal dialysate was collected at the time of peritoneal equilibration test. The cell-free mtDNA, IL-6, IL-17A, TNF-α and IFN-γ were measured. All patients were followed till December 2017. The results were compared with PSTR and patient survival. RESULTS: One hundred and eighty-nine patients were included in the study. The average age was 47.1 ± 13.5 years, 55.6% of the patients were males. The average PSTR was 0.66 ± 0.12, the median dialysate mtDNA levels were 4325 copies/ul. The median concentrations of IL-6, IL-17A, TNF-α and IFN-γ were 25.9, 10.8, 25.8 and 17.9 pg/ml, respectively. We found that dialysate mtDNA was significantly correlated with PSTR (r = 0.461, P < 0.001), IL-6 (r = 0.568, P < 0.001), TNF-α (r = 0.454, P < 0.001) and IFN-γ (r = 0.203, P = 0.005). After adjustment for multiple covariates, dialysate mtDNA levels were independently correlated with IL-6 and PSTR. Dialysate mtDNA levels were not associated with patient survival. CONCLUSIONS: We found that dialysate mtDNA levels correlated with the degree of intraperitoneal inflammatory status in PD patients. Peritoneal effluent mtDNA was an independent determinant of PSTR but did not affect patient survival.

Celiac-Related Autoantibodies and IL-17A in Bulgarian Patients with Dermatitis Herpetiformis: A Cross-Sectional Study.

Background and objectives: Dermatitis herpetiformis (DH) is a blistering dermatosis, which shares common immunologic features with celiac disease (CD). The aim of the present study was to explore the performance of a panel of CD-related antibodies and IL-17A in Bulgarian patients with DH. Materials and Methods: Serum samples from 26 DH patients at mean age 53 ± 15 years and 20 healthy controls were assessed for anti-tissue transglutaminase (anti-tTG), anti-deamidated gliadin peptides (anti-DGP), anti-actin antibodies (AAA), and IL-17A by enzyme linked immuno-sorbent assay (ELISA), as well as anti-tTG, anti-gliadin (AGA), and anti-Saccharomyces cerevisiae antibodies (ASCA) using immunoblot. Results: The average serum levels of anti-tTG, anti-DGP, AGA, AAA, and the cytokine IL-17A were at significantly higher levels in patients with DH compared to the average levels in healthy persons which stayed below the cut-off value (p < 0.05). Anti-DGP and anti-tTG antibodies showed the highest diagnostic sensitivity and specificity, as well as acceptable positive and negative predictive value. None of the healthy individuals was found positive for the tested antibodies, as well as for ASCA within the DH group. All tests showed good to excellent correlations (r = 0.5 ÷ 0.9, p < 0.01). Conclusions: Although the diagnosis of DH relies on skin biopsy for histology and DIF, serologic testing of a panel of celiac-related antibodies could be employed with advantages in the diagnosing process of DH patients. Furthermore, DH patients who are positive for the investigated serologic parameters could have routine monitoring for gastrointestinal complications typical for the gluten-sensitive enteropathy.

Delayed Ischemic Preconditioning Attenuated Renal Ischemia-Reperfusion Injury by Inhibiting Dendritic Cell Maturation.

BACKGROUND/AIMS: Even though delayed ischemic preconditioning (DIPC) has been reported to produce renal protection, the underlying mechanism remains poorly understood. We reported that a 15-minute renal ischemic preconditioning (IPC) 4 days before subsequent ischemia-reperfusion attenuated renal injury Kidney dendritic cells (DCs) are abundant in the renal tubulointerstitium and, depending on their status, can induce immune activation or tolerance. The aim of the present study was to investigate the role of DCs in IPC of the kidney. METHODS: Mouse kidneys were challenged by transient brief episodes of sublethal ischemia followed by subsequent prolonged ischemia. DC abundance and maturation in the spleen and kidney were measured by flow cytometry and immunohistochemical staining. To confirm the function of mature DCs in the renoprotective effect of IPC on renal ischemia-reperfusion injury the A2 adenosine receptor (A2AR) antagonist SCH58261 was administered to stimulate DC maturation prior to assessment of renal functional and histological injury and the inflammatory reaction. RESULTS: Compared with sham-operated animals, preconditioned mice had a reduced injury with less CD11c+ cells, lower levels of the pro-inflammatory cytokine IL-17 and reduced expression of the mature DC marker CCR7. Preconditioned mice also produced more of the anti-inflammatory cytokine IL-10. Both renal cells and splenocytes from these mice had more DCs (CD45+/CD11c+/F4/80-), but fewer of these DCs were mature (CD45+/CD11c+/ F4/80-/MHC-II+/CD80+) compared with those from sham-treated animals, suggesting that the immunomodulatory effect of renal ischemic preconditioning is both local and systemic. Additionally, injection of the A2AR antagonist SCH58261 reversed IPC-induced inhibition of DC maturation and mitigated the protective effect of preconditioning, suggesting that DC maturation contributes to immune cell-mediated ischemic preconditioning. CONCLUSION: Our results show that DIPC of the kidney provides local and systemic immunosuppression by inhibiting DC maturation and hence mediates a renal protective effect.