The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.
- MeSH
- celiakie krev MeSH
- dospělí MeSH
- imunologické faktory * krev imunologie farmakologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- pilotní projekty MeSH
- regulace genové exprese * účinky léků imunologie MeSH
- THP-1 buňky MeSH
- TNF-alfa * biosyntéza imunologie MeSH
- toll-like receptor 9 * biosyntéza imunologie MeSH
- volné cirkulující nukleové kyseliny * krev imunologie farmakologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Interleukin-23 (IL-23), a heterodimeric cytokine of covalently bound p19 and p40 proteins, has recently been closely associated with development of several chronic autoimmune diseases such as psoriasis, psoriatic arthritis or inflammatory bowel disease. Released by activated dendritic cells, IL-23 interacts with IL-23 receptor (IL-23R) on Th17 cells, thus promoting intracellular signaling, a pivotal step in Th17-driven pro-inflammatory axis. Here, we aimed to block the binding of IL-23 cytokine to its cell-surface receptor by novel inhibitory protein binders targeted to the p19 subunit of human IL-23. To this goal, we used a combinatorial library derived from a scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived p19-targeted variants, called ILP binders. From 214 clones analyzed by ELISA, Western blot and DNA sequencing, 53 provided 35 different sequence variants that were further characterized. Using in silico docking in combination with cell-surface competition binding assay, we identified a group of inhibitory candidates that substantially diminished binding of recombinant p19 to the IL-23R on human monocytic THP-1 cells. Of these best p19-blockers, ILP030, ILP317 and ILP323 inhibited IL-23-driven expansion of IL-17-producing primary human CD4+ T-cells. Thus, these novel binders represent unique IL-23-targeted probes useful for IL-23/IL-23R epitope mapping studies and could be used for designing novel p19/IL-23-targeted anti-inflammatory biologics.
- MeSH
- aktivace lymfocytů imunologie MeSH
- buněčné linie MeSH
- buňky Th17 imunologie metabolismus MeSH
- dendritické buňky imunologie metabolismus MeSH
- fagocyty imunologie metabolismus MeSH
- interleukin-23 - podjednotka p19 chemie metabolismus farmakologie MeSH
- interleukin-23 chemie metabolismus MeSH
- konformace proteinů MeSH
- lidé MeSH
- makrofágy imunologie metabolismus MeSH
- molekulární modely MeSH
- receptory interleukinů metabolismus MeSH
- rekombinantní proteiny MeSH
- signální transdukce MeSH
- T-lymfocyty - podskupiny imunologie metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Distinct cellular level of the Ca2+-binding chaperone calreticulin (CRT) is essential for correct embryonal cardiac development and postnatal function. However, CRT is also a potential autoantigen eliciting formation of antibodies (Ab), whose role is not yet clarified. Immunization with CRT leads to cardiac injury, while overexpression of CRT in cardiomyocytes induces dilated cardiomyopathy (DCM) in animals. Hence, we analysed levels of anti-CRT Ab and calreticulin in the sera of patients with idiopatic DCM and hypertrophic cardiomyopathy (HCM). ELISA and immunoblot using human recombinant CRT and Pepscan with synthetic, overlapping decapeptides of CRT were used to detect anti-CRT Ab. Serum CRT concentration was tested by ELISA. Significantly increased levels of anti-CRT Ab of isotypes IgA (p < 0.001) and IgG (p < 0.05) were found in patients with both DCM (12/34 seropositive for IgA, 7/34 for IgG) and HCM (13/38 seropositive for IgA, 11/38 for IgG) against healthy controls (2/79 for IgA, 1/79 for IgG). Titration analysis in seropositive DCM and HCM patients documented anti-CRT Ab detected at 1/1600 dilution for IgG and 1/800 for IgA (and IgA1) and at least at 1/200 dilution for IgA2, IgG1, IgG2 and IgG3. Pepscan identified immunogenic CRT epitopes recognized by IgA and IgG Ab of these patients. Significantly increased levels of CRT relative to healthy controls were found in sera of patients with HCM (p < 0.01, 5/19). These data extend the knowledge of seroprevalence of anti-CRT Ab and CRT, and suggest possible involvement of autoimmune mechanisms directed to CRT in some forms of cardiomyopathies, which are clinically heterogeneous.
- MeSH
- autoantigeny krev imunologie MeSH
- autoimunita MeSH
- autoprotilátky krev imunologie MeSH
- biologické markery MeSH
- dilatační kardiomyopatie krev diagnóza imunologie MeSH
- dospělí MeSH
- ELISA MeSH
- hypertrofická kardiomyopatie krev diagnóza imunologie MeSH
- imunoglobulin A krev imunologie MeSH
- imunoglobulin G krev imunologie MeSH
- kalretikulin krev imunologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- senioři MeSH
- studie případů a kontrol MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
T regulatory cells (Tregs) are essential for maintaining tolerance and preventing autoimmune diseases, such as type 1 diabetes (T1D). In our study, we investigated CD25 + FoxP3 + Tregs and thymic FoxP3 + Helios + Tregs in large cohorts of children with T1D at onset and with long-term T1D, and further in their relatives and healthy controls. We observed significantly decreased numbers of CD25 + FoxP3 + Tregs, but not FoxP3 + Helios + Tregs, in long-term patients compared with the control group and T1D onset. Furthermore, long-term T1D patients exhibited highly significant decrease of CD25 expression on both CD25 + FoxP3 + Tregs and FoxP3 + Helios + Tregs, independently on age or the duration of diabetes. A similar reduction of CD25 expression was also found in T1D relatives, more significant in those with positive autoantibodies. Low CD25 expression was associated with impaired signal transducer and activator of transcription 5 (STAT5) phosphorylation after IL-2 exposure. Our results show that the frequency of Tregs is altered in a large cohort of long-term T1D patients, a profound decrease in CD25 expression and altered IL-2 signaling are typical features of Tregs populations in long-term diabetic patients and their relatives.
- MeSH
- biologické markery MeSH
- buněčná diferenciace MeSH
- diabetes mellitus 1. typu diagnóza etiologie metabolismus MeSH
- dítě MeSH
- forkhead transkripční faktory metabolismus MeSH
- fosforylace MeSH
- imunofenotypizace MeSH
- interleukin-2 metabolismus farmakologie MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- počet lymfocytů MeSH
- předškolní dítě MeSH
- receptor interleukinu-2 - alfa-podjednotka genetika metabolismus MeSH
- regulační T-lymfocyty cytologie imunologie metabolismus MeSH
- signální transdukce MeSH
- studie případů a kontrol MeSH
- thymocyty cytologie imunologie metabolismus MeSH
- transkripční faktor STAT5 MeSH
- věkové faktory MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Cachectic rheumatoid arthritis, the less frequent form of the disease, is associated with loss of fat mass and often more severe course of the disease. Its experimental model represents rat adjuvant arthritis (AA) characterized by edema, lack of appetite, sharp body weight and fat loss. As individual fat depots display functional differences, here we studied lipolytic activity and sensitivity to lipolytic stimuli of nodeless epididymal fat (eWAT) and perinodal mesenteric fat (mWAT) depots at the peak of AA. We also examined changes in catecholamine and cytokine levels involved in lipolysis in plasma and/or isolated adipocytes from both WATs to identify the contribution of local, adipocyte-based processes and/or systemic events to adiposity loss in cachectic rheumatoid arthritis. AA was induced to male Lewis rats by complete Freund's adjuvant. Groups of ad libitum-fed and pair-fed controls were used to distinguish the effects of food restriction from inflammation-induced cachexia. Adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and its phosphorylated form (pHSL) were analyzed by western blot. CRP and catecholamine levels in plasma or adipocyte lysates were determined using ELISA kits. Cytokine-induced neutrophil chemoattractant-1 (CINC-1/CXCL1), monocyte chemoattractant protein-1 (MCP-1/CCL2), IL-1β, IL-6, IL-10 and leptin in adipocyte lysate were analyzed by quantitative protein microarray. Plasma glycerol and FFA were measured spectrophotometrically. AA rats developed severe cachexia, with lower adiposity in mWAT compared to normal and pair-fed controls, whereas in eWAT the adiposity was similarly reduced in AA and pair-fed groups. ATGL levels in both WATs were not affected by AA or pair feeding. AA upregulated levels of HSL, pHSL and pHSL/HSL ratio in mWAT, whereas none of these parameters has changed in eWAT of AA rats or in either WATs of pair-fed rats. In AA rats plasma glycerol was elevated, whereas FFA concentration was reduced. Plasma norepinephrine and epinephrine were increased in AA compared with both groups of controls. In eWAT adipocytes, AA but not pair feeding, upregulated norepinephrine levels. In mWAT adipocytes, AA rats showed higher epinephrine levels than pair-fed controls. Leptin levels in both WATs were depleted in AA animals in accordance with body weight loss. None of the measured cytokines in eWAT and mWAT was enhanced. Our results demonstrate augmented lipolytic activity in mWAT and not eWAT during cachectic arthritis. The adipocyte-derived cytokines do not seem to contribute to activated lipolysis. We first demonstrated enhanced presence of norepinephrine in perinodal adipocytes that may contribute to the regulation of local lipolytic activity by auto/paracrine fashion and thus provide independent fuel supply to activated lymph nodes.
- MeSH
- adrenalin biosyntéza MeSH
- artritida experimentální imunologie metabolismus MeSH
- biologické markery MeSH
- C-reaktivní protein MeSH
- epididymis metabolismus MeSH
- humorální imunita MeSH
- krysa rodu rattus MeSH
- lipolýza MeSH
- mezenterium metabolismus MeSH
- modely nemocí na zvířatech MeSH
- sterolesterasa metabolismus MeSH
- tukové buňky metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Apoptosis of tissues of fetal origin is thought to be one of the main sources of cell-free fetal DNA (cffDNA) in maternal circulation, impaired apoptosis is also involved in the mechanisms contributing to recurrent spontaneous miscarriages (RSM) associated with antiphospholipid syndrome (APS). The APS increases the risk for preeclampsia nine times. In preeclampsia, the elevated levels of cffDNA were described by different authors. To our knowledge, cffDNA in pregnant patients with APS was never studied. In our pilot study, we focused on the levels of cffDNA in four pregnant patients with treated primary APS and compared them with values obtained in twenty-one healthy subjects of comparable gestation age (the third trimester of pregnancy). We supposed that the increase of cffDNA concentration in our treated patients would signalize the elevated apoptosis of fetal tissues as in other pathological changes of placentation. The aim of our pilot study was to determine cffDNA concentrations in patients with treated APS and to compare them with values detected in healthy pregnant women of comparable gestation age in order to discover potential non-physiological elevations in patients. The elevated values of cffDNA were not observed in our patients (p value = 0.4363, Mann-Whitney test). All patients delivered healthy children. The measurement of concentrations of cffDNA seems to be a promising tool for monitoring of therapy effectiveness in pregnant women with APS but evaluation of randomized controlled trials would be necessary to determine the specificity and the sensitivity of this test.
- MeSH
- antifosfolipidové protilátky krev imunologie MeSH
- antifosfolipidový syndrom krev diagnóza genetika MeSH
- DNA krev MeSH
- dospělí MeSH
- komplikace těhotenství krev diagnóza genetika MeSH
- lidé MeSH
- pilotní projekty MeSH
- plod imunologie metabolismus MeSH
- těhotenství MeSH
- třetí trimestr těhotenství krev imunologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Heat shock proteins (Hsps) have been repeatedly implicated to participate in the pathogenesis of rheumatoid arthritis (RA). METHODS: Herein, Hsp70 cell surface and mRNA expression were studied in human fibroblast-like synovial cells, dermal fibroblasts and peripheral blood leukocytes derived from 24 RA patients, who underwent synovectomy by using flow-cytometric analysis and real-time quantitative reverse-transcriptase polymerase chain reaction. For comparison, peripheral blood leukocytes of 17 healthy controls were tested. RESULTS: Significantly higher Hsp70 membrane positivity was found on fibroblast-like synovial cells in RA patients (average 18.3%, median 16.5%) than on autologous and healthy control peripheral blood lymphocytes (RA patients: average 4.7%, median 2.9%, p = 0.002; healthy controls: average 6.0%, median 4.5%, p = 0.002) and/or autologous dermal fibroblasts (average 5.1%, median 4.3%, p < 0.001). Strong Hsp70 cell surface expression was also found on peripheral blood monocytes of RA patients (average 53.0%, median 58.1%) and healthy controls (average 49.4%, median 47.5%, p = 0.52). Peripheral blood granulocytes of healthy controls (average 41.8%, median 41.4%) showed significantly increased Hsp70 expression comparing with RA patients (average 10.7%, median 6.4%, p = 0.005). Significantly higher Hsp70 gene expression was observed in synovial cells of RA patients (average 2.04, median 1.7) when compared with autologous peripheral blood leukocytes (average 0.75, median 0.68; p < 0.001). However, the difference in Hsp70 gene expression between RA-derived synovial cells and healthy control peripheral blood leukocytes (average 1.69, median 1.64) was not observed (p = 0.83). We also found significantly lower relative gene expression in peripheral blood leukocytes of RA patients in comparison with healthy controls (p < 0.001). Interestingly, we found that Hsp70 gene expression in RA non-affected skin dermis gained from the operation wound was 3.7-fold higher in average (average 7.6, median 8.3) when compared to autologous RA-affected synovial tissue (p < 0.001); 10.1-fold higher in average when compared to autologous peripheral blood leukocytes (p < 0.001) and 4.5-fold higher in average comparing to control peripheral blood leukocytes (p < 0.001). CONCLUSION: Hsp70 gene expression in RA-affected synovial tissue is followed by Hsp70 cell surface expression on fibroblast-like synovial cells growing from RA synovial tissue. Hsp70 may be translocated to the cell surface from the cytosol and/or Hsp70 released from inflamed synovial tissue may be captured onto the membrane of synovial cells from the extracellular space via Hsp receptors. As a physiological response to potentially harmful enviromental stress factors, skin dermis produces higher levels of Hsp70 comparing to the cells of internal organs and tissues.
- MeSH
- buněčná membrána metabolismus MeSH
- dospělí MeSH
- fibroblasty metabolismus MeSH
- financování organizované MeSH
- kůže cytologie metabolismus MeSH
- leukocyty mononukleární metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteiny tepelného šoku HSP70 metabolismus MeSH
- průtoková cytometrie MeSH
- revmatoidní artritida imunologie metabolismus patofyziologie MeSH
- senioři MeSH
- škára cytologie metabolismus MeSH
- synoviální membrána cytologie metabolismus MeSH
- synoviální tekutina cytologie metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
OBJECTIVE: A role of autoreactive T cells for type 1 diabetes pathogenesis is considered crucial. In our pilot study we addressed if autoreactive mononuclear cells are present also in peripheral blood of patients with other specific forms of diabetes as cystic fibrosis related diabetes (CFRD). METHODS: Cellular immune responses to a known beta-cell autoantigen (GAD65 and GAD65 derived peptides) were analysed by ELISPOT (IFN-gamma) and by protein microarray analysis in four patients suffering from CFRD, in four cystic fibrosis (CF) patients without diabetes, in eight type 1 diabetes patients (without CF) and in four healthy controls. RESULTS: Response to the autoantigen GAD65 (protein and peptides) was observed in 7/8 patients suffering from CF and in all type 1 diabetes patients. Post-stimulation production of Th1 cytokines (IFN-gamma, TNF-beta) was observed in 2/4 CFRD, 1/4 CF patients and in 7/8 type 1 diabetes patients. All these patients carry prodiabetogenic HLA-DQ genotype. Th2- and Th3 type of cytokine pattern was observed in 2/4 CF patients. Production of IL-8 was observed in the third CFRD as well as in the third CF patient and in 1/8 type 1 diabetes patient and borderline production of this chemokine was also observed in 2/4 healthy controls. No reaction was observed in the other 2/4 healthy controls and in the fourth CFRD patient who carried a strongly protective genotype and did not produce autoantibodies. The most potent peptide of GAD65 was amino acids 509-528. CONCLUSIONS: We consider our observations as a sign of a reaction directed against the self-antigen GAD65 that are closely connected to type 1 diabetes. In CF patients who do not develop diabetes autoreactive mechanisms are very probably efficiently suppressed by immune self-tolerance mechanisms. CFRD patients are a heterogenic group. To disclose those who may display features of autoimmune diabetes could have an impact for their therapy and prognosis.
- MeSH
- autoprotilátky imunologie krev MeSH
- čipová analýza proteinů MeSH
- cystická fibróza imunologie komplikace MeSH
- diabetes mellitus etiologie imunologie MeSH
- dítě MeSH
- dospělí MeSH
- financování organizované MeSH
- glutamát dekarboxyláza imunologie MeSH
- HLA-DQ antigeny imunologie MeSH
- interferon gama krev MeSH
- interleukin-8 krev MeSH
- izoenzymy MeSH
- leukocyty mononukleární imunologie MeSH
- lidé MeSH
- lymfotoxin-alfa krev MeSH
- mladiství MeSH
- pilotní projekty MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
