Specific Immunoglobulin E and G to Common Food Antigens and Increased Serum Zonulin in IBS Patients: A Single-Center Bulgarian Study.

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder whose pathogenesis is considered multifactorial, including abnormal gut motility, visceral hyperreactivity, psychological factors, disturbances in the brain-gut axis, leaky gut, oxidative stress, etc. We aimed to investigate serum levels of specific immunoglobulin E and G to common food antigens and zonulin and to assess their use in clinical practice for patients with IBS. Material and methods. We included 23 participants, 15 with IBS (diagnosed according to the Rome IV criteria) and 8 healthy controls. We investigated serum levels of specific IgG antibodies to 24 food antigens, specific IgE antibodies to 20 food antigens, anti-celiac antibodies, fecal calprotectin and serum zonulin by ELISA. Results. Food-specific positive IgG antibodies were significantly higher in patients with IBS than in controls (p = 0.007). IgE-mediated allergic reactions were found in five patients with IBS; no one had anti-TG antibodies. One-third of IBS patients demonstrated a low degree of chronic inflammation (positive fecal calprotectin test > 50 ng/mL) without specific bacterial infection. Serum levels of zonulin in IBS patients were higher than in healthy controls (0.378 ± 0.13 vs. 0.250 ± 0.14 ng/mL, p = 0.0315). However, no correlations between clinical symptoms and zonulin levels were found. Conclusion. The mechanisms of IgG hypersensitivity and low degree inflammation in IBS and elevated zonulin may contribute to multifactor pathogenesis in IBS.

The Dynamic Changes in Soluble CD30 and Regulatory T Cells Before and After Solid Organ Transplantations: A Pilot Study.

Among multiple parameters, applied in the immunologic monitoring of transplantation, the levels of serum soluble CD30 (sCD30) and peripheral regulatory T cells (Tregs) are very promising. These are relatively new biomarkers, considered to reflect immune activation and tolerance in solid organ transplantation. Results are shown here from a preliminary study on the relevance of sCD30 and Tregs in the monitoring of the early post-transplantation period. Sixteen patients with chronic liver or kidney disease were examined. Nine of them were further selected for transplantation. Follow-up of sCD30 and Tregs was carried out during the first month after transplantation. Until day 30 (D30) after transplantation, a progressive decrease in sCD30 levels was observed in all patients. Conversely, the dynamic of Tregs was dependent on the transplanted organ: in liver recipients, an increase of Tregs was detected at day 7 (D7) followed by a gradual decrease until D30, whereas in kidney recipients, a sustained downward trend starting on D7 was observed. In liver recipients, the increase in Tregs preceded albumin normalization, whereas in kidney recipients, sCD30 was found to have predictive significance for the creatinine levels. Our results demonstrated that peripheral blood sCD30 and Tregs are valuable parameters in the immunologic monitoring of transplanted patients.

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.

Alterations in cytokine gene expression profile in colon mucosa of Inflammatory Bowel Disease patients on different therapeutic regimens.

Inflammatory bowel disease (IBD) is assumed to be caused by genetic and environmental factors that interact together in promoting intestinal immune dysregulation where cytokines have validated role. However, the underlying intimate mechanisms in the human IBD involving cytokines still needs to be supplemented especially in the clinical context. The aim of this study was to investigate the expression of some inflammatory and regulatory cytokines (IL-17A, IL-23, IL-6, TGFß1, and IL-10) as well as of the transcription factor FoxP3 in mucosal samples of IBD and non-IBD patients. We assessed the mRNA relative quantities (RQ) of the above-mentioned cytokines and the transcription factor FoxP3 in paired colonic samples (inflamed and adjacent normal mucosa) from 37 patients with IBD and in normal mucosal tissue in 12 persons without IBD by performing a qRT-PCR assay and tested the protein levels of target cytokines in serum samples. The patients were divided into three groups: without any therapy (n=10), on 5-ASA (n=11) and on immunosuppressants (Azathioprine±5-ASA/corticosteroids) (n=16) in order to compare the RQ values for each therapeutic group. All investigated genes were found upregulated in the inflamed mucosa of IBD patients in the following order: IL-6>FoxP3>TGFß1>IL-23>IL-17A>IL-10. We also observed that the gene expression of FoxP3 and IL-6 were substantially higher in the inflamed mucosal tissue of the IBD patients than the adjacent normal mucosa (p=0.035, p=0.03 respectively). Differences between higher mRNA expression of FoxP3 and IL-6 in inflamed tissue were considered significant in patients with ulcerative colitis (UC) (p=0.011, p=0.000 respectively) and with Crohn's disease (CD) (p=0.008, p=0.000 respectively) in comparison to the normal mucosa of non-IBD persons and we found increased TGFß1 in CD patients alone (p=0.041). Furthermore, IL-6 and TGFß1 were overexpressed (RQ>10) in non-inflamed mucosa from IBD patients compared to the normal mucosa from the controls. When we compared the gene expression for paired mucosa in the immunosuppressive treated group with the 5-ASA treated group we observed opposite changes in IL-6 and TGFß1 expression. Additionally, we found higher serum levels of IL-23 (p=0.008), TGFß1 and IL-6 in IBD patients compared to non-IBD patients. The obtained specific expression profile consisting of IL-6, TGFß1, IL-10 and FoxP3 may represent a transcriptional hallmark for IBD. Furthermore, we found that treatment with immunosuppressive therapy was more beneficial for driving cytokine expression to restore immune regulation in patients with IBD, unlike the 5-ASA therapy.

Nitroxidergic modulation of behavioural, cardiovascular and immune responses, and brain NADPH diaphorase activity upon morphine tolerance/dependence in rats.

Opioid and non-opioid effects of acute and chronic morphine administration on behaviour, cardiovascular responses, cell proliferation and apoptosis and nitric-oxide synthase (NOS) activity were studied in rats. A novel score-point scale was introduced to quantify the signs of opioid withdrawal syndrome. NOS inhibitor L-NAME (NG-nitro-L-arginine methyl ester) was applied to reveal the role of NOS/NO pathway in the modulation of morphine-induced in vivo and in vitro responses. The obtained data showed that chronic co-administration of L-NAME drastically attenuated naloxone-precipitated withdrawal syndrome and prevented the development of morphine tolerance to cardiovascular action of morphine. The apoptotic process was very much restricted by L-NAME supplementation of chronic morphine treatment, which resulted in few apoptotic cells, less low molecular weight genomic DNA and preservation of high molecular weight non-fragmented genomic DNA. The study provides new data for nitroxidergic modulation of opioid tolerance and dependence.

Conditioned medium from adipose tissue-derived mesenchymal stem cells induces CD4+FOXP3+ cells and increases IL-10 secretion.

Mesenchymal stem cells (MSCs) are a new and promising tool for therapy of autoimmune disorders. In recent years their possibility to take part in the modulation of the immune response is discussed. The exact mechanisms for immunoregulation realized by MSCs are not clear yet, but interactions with other immunoregulatory cells may be involved in this process. The investigation of the influence of MSCs on the expression of FoxP3 and cytokine secretion by T helper cells was the aim of this study. T helper cells were isolated from PBMCs by magnetic separation and MSCs were isolated from human adipose tissue, and CD4⁺ T cells were cultured with conditional medium of MSCs. The methods which were used include flow cytometry, ELISA, and Human Proteome profiler kits. The results demonstrated that secretory factors in MSCs conditional medium lead to increased expression of FoxP3 and increased secretion of IL-10 by T helpers. The obtained results give us opportunity to discuss the interaction between two kinds of immunoregulatory cells: MSCs and FoxP3⁺ T helpers. We suppose that this interaction leads to increased number of immunosuppressive helpers which secrete IL-10. MSCs provide some of their immunosuppressive functions acting on T regulatory cells, and we believe that IL-6 secreted by MSCs is involved in this process.

Mesenchymal stem cells from human bone marrow or adipose tissue differently modulate mitogen-stimulated B-cell immunoglobulin production in vitro.

Mesenchymal stem cells (MSC) have been characterized as multipotent cells which are able to differentiate into several mesodermal and nonmesodermal lineage cells and this feature along with their extensive growth and comprehensive immunomodulatory properties establish them as a promising tool for therapeutic applications, including cell-based tissue engineering and treatment of immune-mediated disorders. Although bone marrow (BM) is the most common MSC source, cells with similar characteristics have been shown to be present in several other adult tissues. Adipose tissue (AT), large quantities of which can be easily obtained, represents an attractive alternative to BM in isolating adipose tissue-derived MSC (AT-MSC). BM-MSCs and AT-MSCs share some immunomodulatory properties as they are both not inherently immunogenic and suppress the proliferation of alloantigen- or mitogen-stimulated T-cells. Our purpose was to comparatively examine under appropriate in vitro conditions, phenotypes, morphology and some functional properties of BM-MSCs and AT-MSCs, such as differentiation potential and especially the ability to suppress the immunoglobulin production by mitogen-stimulated B-cells. While the morphological, immunophenotypical, colony-forming and adipogenic characteristics of both types of cells were almost identical, AT-MSCs showed less potential for osteogenic differentiation than BM-MSCs. We found that AT-MSCs not only inhibited the Ig-production but also suppressed this B-cell function to a much greater extent compared to BM-MSC. This finding supports the potential role of AT-MSCs as an alternative to BM-MSCs for clinical purposes.

Female sex steroid hormones modify some regulatory properties of monocyte-derived dendritic cells.

PROBLEM: Monocyte-derived dendritic cells (mDCs) are present in human decidua during the first month of pregnancy where they experience the effect of the increased concentration of progesterone (Pg) and estradiol (Estr). The aim of our study was to assess the effect of these reproductive hormones on the immunomodulatory role of mDCs. METHODS OF STUDY: The mDCs cultured in vitro in the presence of sex steroid hormones and characterized for their apoptosis and secretion of cytokines by flow cytometry, enzyme linked immunosorbent assay and proteome profiler kit. RESULTS: Female sex steroid hormones enhance the life cycle of mDCs, thus increasing the maturation and apoptosis, they also increase the production of interleukin (IL)-10 and IL-27 but only Pg increases the production of IL-13 and down regulates the secretion of IL-23. CONCLUSION: The Pg and Estr participate in the regulation of the immune response during pregnancy by influencing apoptosis and the cytokine profile of the mDCs.

Progesterone-modulated phosphatidylserine externalization in apoptosis and activation of Jurkat cells.

PROBLEM: During pregnancy the elevated levels of progesterone (Pg) have immunomodulating effects. It is important to follow-up Pg effects on basic biological processes at cell level as apoptosis and activation which was the aim of this study. METHODS OF STUDY: Jurkat cells cultured in the presence or absence of Pg were used as a model system. Apoptosis was induced by H(2)O(2) and activation by phorbol myriastate acetate. The induced changes in the phosphatidylserine (PS) externalization and cell surface CD69 expression were followed by fluorescence-activated cell sorter and immunofluorescence. RESULTS: After the induction of apoptosis PS externalizes in 52.3% of Jurkat cells. Cells cultured with Pg show tendency to a decrease of PS positive cells (42%). The opposite effect is observed in activated cells--PS externalization increase from 33.8% of control cells to 40.1% of Pg-treated cells. CONCLUSIONS: These findings would suggest that by increasing activation and decreasing apoptosis Pg could regulate local immune system during pregnancy.

Changes in cytokine profile during local IL-2 therapy in cancer patients.

BACKGROUND: Exogenous interleukin 2 (IL-2) can influence the complex cytokine network in vivo. This study investigated the cytokine profile of patients with different malignancies before and after local IL-2 administration. PATIENTS AND METHODS: The human TH1 / TH2 cytometric bead array (CBA) kit was used to investigate IL-2, IFNgamma, TNFalpha, IL-4, IL-5 and IL-10 in a control group and in 13 patients. RESULTS: The baseline serum IL-4 levels in patients were lower than in healthy controls, while the baseline ascitic IL-10 levels in patients was higher than in serum. The IL-2 applications induced a strong serum increase in IL-2 and IL-5 and even more in ascites, while IL-10 increased weakly and mainly locally. One month after the start of therapy, the serum IFNgamma had increased in patients, reaching the level of the control group. CONCLUSION: After local injection, IL-2 probably leaks into the blood circulation. The higher increases of IL-2, IL-5 and IL-10 in ascites compared to the serum suggests that the injected cytokines and their effects are mainly local. The minor increase of the immunosuppressive IL-10 could explain the therapeutic difference between local and systemic IL-2 therapy since IL-10 levels markedly increase after systemic IL-2 therapy. IL-5 was always increased after IL-2 therapy and, consequently, may be a downstream mediator of antitumour responses.

CD83 monocyte-derived dendritic cells are present in human decidua and progesterone induces their differentiation in vitro.

PROBLEM: Dendritic cells (DCs) play an important role in antigen presentation and immunoregulation. Modifications of the immune response during pregnancy require the participation of DC. The aim of this study was to follow-up the changes of DCs in human decidua and their correlations to progesterone (Pg) concentrations. METHODS OF STUDY: Blood leukocytes were isolated from human decidua and analyzed by flow cytometry for the expression of HLA-DR and CD83 markers. Blood monocytes were cultured to differentiate and mature to DCs in the presence of Pg, estradiol or testosterone and analyzed by flow cytometry. RESULTS: The percentage of mature DCs is increased in human decidua during the 1st trimester. When Pg is added to DCs cultured in vitro the percentage of cells expressing both markers increased in specific and dose-dependent manner. CONCLUSIONS: Pg increases the numbers of monocytic DCs locally and it may be speculated that these cells help the Th1/Th2 switching in pregnancy.

Humoral immune response in prostate cancer patients after immunization with gene-based vaccines that encode for a protein that is proteasomally degraded.

Prostate-specific membrane antigen (PSMA), whose expression is upregulated in poorly differentiated, metastatic, and hormone refractory prostate cancer, could be targeted by gene-based vaccines. The aim of this study was to characterize the humoral immune response against PSMA in prostate carcinoma patients who have been vaccinated against PSMA with gene-based vaccines. Sera from prostate cancer patients who had been immunized repeatedly with plasmid DNA and a recombinant adenoviral vector, both carrying an expression cassette for human PSMA, and sera from healthy donors were tested for anti-PSMA antibodies by Western blot analysis and immunofluorescence. PSMA-producing LNCaP cells, recombinant PSMA protein, and a specific antibody against PSMA were used as positive controls. Specific anti-PSMA antibodies were detected by both Western blot and immunofluorescence in the sera of patients who had been vaccinated against PSMA with plasmid and recombinant adenoviral vectors. The specificity of the anti-PSMA antibodies was confirmed by preincubation and blocking experiments. Positive reactions were detected in 86% of the vaccinated prostate cancer patients. Anti-PSMA antibodies were not detected either in the patients' sera prior to vaccination or in the sera from healthy men and women. These data demonstrate that PSMA, a specific marker for prostate cancer, is a target for humoral immune response induced by gene-based PSMA vaccination. Detection of anti-PSMA antibodies by immunoblot analysis and by indirect immunofluorescence could be used to monitor the vaccination effect.

Depletion of CD25+ cells from human T-cell enriched fraction eliminates immunodominance during priming with dendritic cells genetically modified to express a secreted protein.

The ability of dendritic cells (DCs), genetically modified with one of two types of plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses, is compared. The first type, also called "secreted" vaccine (sVac), encodes for the full length of the human prostate-specific antigen (PSA) with a signal peptide sequence so that the expressed product is glycosylated and directed to the secretory pathway. The second type, truncated vaccines (tVacs), encodes for either hPSA or human prostate acidic phosphatase (hPAP), both of which lack signal peptide sequences and are retained in the cytosol and degraded by the proteasomes following expression. Monocyte-derived dendritic cells are transiently transfected with either sVac or one of two tVacs. The DCs are then used to activate CD25+-depleted or nondepleted autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs, peptide-pulsed DCs or monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tVacDCs and sVacDCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted towards one of the three antigen-derived epitopes when priming and boosting is performed with sVacDCs. In contrast, tVac-transfected DCs prime T cells towards all antigen-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance. While CD25+ cell depletion prior to priming with sVacDCs alleviates immunodominance, cotransfection of dendritic cells with GITR-L does so in some but not all cases.

Calcium-dependent changes in potassium currents in guinea-pig coronary artery smooth muscle cells after acute cobalt loading in vivo.

The aim of the present study was to determine whether cobalt poisoning induces haem oxidase isoenzyme-1 (HO-1) in coronary artery smooth muscle, or accounts for any changes in coronary smooth muscle cell (SMCs) membrane ionic currents that could result from this type of heavy metal poisoning. In SMCs isolated from cobalt-treated guinea-pig coronaries, K+ channel currents (IK) were much smaller than those in cells isolated from non-treated animals. Haemin (HO substrate) increased IK concentration dependently. This effect was mimicked by 1% CO and was abolished by pretreatment of cells with a competitive HO inhibitor, by inhibitors of guanylyl cyclase, protein kinase G or phospholipase C, as well as by blocking inositol trisphosphate-dependent Ca release, or sarcoplasmic reticulum Ca-ATPase, or by bathing cells in Ca-free external solution. Expression of the Na/Ca exchanger-1 (NCX-1) protein was reduced substantially in SMCs from coronary arteries of cobalt-treated animals. No expression of HO-1 was detected. It is concluded that acute cobalt poisoning in vivo depresses Ca-sensitive K currents via CO-dependent modulation of intracellular calcium availability, most probably by suppressing the expression of NCX-1 protein.

Human dendritic cells genetically engineered to express cytosolically retained fragment of prostate-specific membrane antigen prime cytotoxic T-cell responses to multiple epitopes.

The ability of two plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses is demonstrated. The first vaccine (truncated; tPSMA) encodes for only the extracellular domain of prostate-specific membrane antigen (PSMA). The product, expressed following transfection with this vector, is retained in the cytosol and degraded by the proteasomes. For the "secreted" (sPMSA) vaccine, a signal peptide sequence is added to the expression cassette and the expressed protein is glycosylated and directed to the secretory pathway. Monocyte-derived dendritic cells (DCs) are transiently transfected with either sPSMA or tPSMA plasmids. The DCs are then used to activate autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs or with peptide-pulsed monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tPSMA DCs and sPSMA DCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted toward one of the four PSMA-derived epitopes when priming and boosting is performed with sPSMA. In contrast, tPSMA-transfected DCs prime T cells toward several PSMA-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance.