Protective effects of Acetobacter ghanensis against gliadin toxicity in intestinal epithelial cells with immunoregulatory and gluten-digestive properties.

PURPOSE: The aim of this study was to establish whether Acetobacter ghanensis, the probiotic characteristics of which were evaluated previously, attenuates gliadin-induced toxicity in intestinal epithelial cells with gluten-digestive and immunoregulatory properties. METHODS: A co-culture model of human intestinal epithelial cell (Caco-2) monolayers on top of peripheral blood mononuclear cells (PBMCs) obtained from patients with celiac disease (CD) was established. The gluten-digestive properties of A. ghanensis were determined by checking bacterial growth in a medium containing gluten as the main nitrogen source. The mRNA levels of genes encoding TJ-associated proteins were measured by quantitative real-time PCR (qRT-PCR). The concentrations of IL-6 and TNFα were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that PT-gliadin disrupted intestinal barrier integrity by modulating the expression of TJ-associated genes encoding zonulin (increased by ~ 60%), zonula occludens-1 (ZO-1) (decreased by ~ 22%), and occludin (decreased by ~ 28%) in Caco-2 cells. Furthermore, PT-gliadin treatment in Caco-2 cells was associated with increased concentrations of IL-6 (~ 1.6-fold) and TNFα (~ twofold) from PBMCs. These modulatory effects of PT-gliadin, however, were suppressed when Caco-2 cells were subjected to A. ghanensis in the presence of PT-gliadin. As a factor underlying these protective effects, we showed that A. ghanensis could digest gluten peptides. CONCLUSIONS: To our knowledge, the current study is the first to demonstrate that A. ghanensis improves intestinal barrier functions by attenuating the modulatory effects of PT-gliadin with immunoregulatory and gluten-digestive properties.

The effects of rivaroxaban, an oral anticoagulant, on human IVD primary cultures.

Introduction: The present study aimed to investigate the potential effects of rivaroxaban, an oral anticoagulant that inhibits the effects of factor Xa, on intact intervertebral disc tissue cells and the extracellular matrix (ECM). Material and methods: Rivaroxaban was applied to primary human cell cultures prepared from tissues of the intervertebral disc. Comparative molecular analyses were performed on non-drug-treated control group samples. Descriptive statistics were presented as the mean ± standard deviation. An analysis of variance test was performed to determine whether there were significant differences in the mean across the groups. When differences across groups were observed, Tukey's honestly significant difference post-hoc test was used for multiple pairwise comparisons. The significance of the obtained data was determined statistically. The α significance value was < 0.05. Results: The cells in the control group and in the rivaroxaban-treated group were viable, healthy, and proliferated (p < 0.05). However, the expression levels of the chondroadherin gene (CHAD), cartilage oligo matrix protein (COMP), matrix metalloproteinase (MMP)-13, and MMP-19 genes were changed (p < 0.05). Conclusions: Although rivaroxaban does not suppress cell proliferation due to morphological, biological, and biochemical changes in the intervertebral disc tissue, it may change the expression of genes that are related to ECM maintenance.

Alloreactive memory B cell detection by flow cytometric cross match using polyclonally activated memory B cell culture supernatants.

In addition to alloantibodies, alloreactive memory B cell (mBC) evaluation has a potential for immunological risk assessment during transplantation processes. For the alloreactive mBCs evaluation currently, direct Flow Cytometric (FC) analysis using the HLA tetramer staining is an option. Evaluation of alloantibodies produced by the polyclonally stimulated alloreactive mBCs in in vitro culture system seems to be another useful approach, but this needs further downstream applications. In this study, we investigated the usefulness of the Flow Cytometric Cross Match (FCXM-supernatant) in which in vitro polyclonally activated mBCs culture supernatants and potential donor's lymphocytes being used for the mBC detection. FCXM-supernatant assays were performed between culture supernatants of polyclonally activated mBCs obtained from 4 allosensitized multiparous women and 14 renal transplant patients, and their non-alloimmunized spouses' or donors' lymphocytes, and vice versa. HLA typing was performed by SSP method. Anti-HLA antibodies produced by in vitro activated alloreactive mBCs were also evaluated by the Luminex assays. The success of in vitro polyclonal activation of mBCs was evaluated by a total IgG ELISA test and antibody secreting cell analyses by FC. Donor specific alloreactive mBCs were detected by FCXM-supernatant in 45% of the 18 allosensitized cases. Detection rate was 85% (6 out of 7) in the strongly allosensitized cases. No alloreactive mBCs was detected in control cases without allosensitization. FCXM-supernatant negative results of the allosensitized cases were related to low level of allosensitization and insufficient polyclonal stimulation evaluated by total IgG antibody tests of the supernatants. We herein report a practical methodology for alloreactive mBC detection as a donor specific manner using the FCXM-supernatant assay so that this would easily be transformed into a routine test performed in tissue typing laboratories.

Is Favipiravir a Potential Therapeutic Agent in the Treatment of Intervertebral Disc Degeneration by Suppressing Autophagy and Apoptosis?

AIM: To evaluate the effects of favipiravir (FVP) on cell viability and cytotoxicity in human degenerated primary intervertebral disc (IVD) tissue cell cultures. Furthermore, the protein expressions of hypoxia-inducible factor 1 alpha (HIF-1α), nuclear factor-kappa-b (NF-kB), and interleukin-1 beta (IL-1ß) were also examined. MATERIAL AND METHODS: Untreated cell cultures served as the control group, named group 1. Cell cultures treated with FVP served as the study group, named group 2. Pharmacomolecular analyses were performed in all groups at 0, 24, 48, and 72 hours (h). Obtained data were evaluated statistically. RESULTS: Cell proliferation was suppressed in the FVP-treated samples compared to the control group samples at 24 and 72 h, and this was statistically significant (p < 0.05). Decreased or increased protein expression levels of HIF-1α, NF-κB, and IL-1ß in FVPtreated samples may be an indication of suppression in anabolic events as well as proliferation in IVD cultures. FVP administration showed that AF/NP cells in a culture medium may induce a strong inflammatory response to FVP. This strong inflammatory response is likely to cause slowed proliferation. It may also be a trigger for many catabolic events. NF-κB expression increased within the first 24 h and then decreased rapidly. Based on the data obtained, it may be suggested that the rapidly increasing NF-kB may have stimulated the expression of many antiproliferative genes. CONCLUSION: The suppression of IL-1ß and NF-kB protein expressions in IVD cells treated with FVP is important in the treatment of IVD degeneration (IDD). If the protein expression of HIF-1α could be increased along with the suppression of IL-1ß and NF-kB, FVP would perhaps be a promising pharmacological agent in the treatment of IDD.

A Study of the Effects of Metformin, a Biguanide Derivative, on Annulus Fibrosus and Nucleus Pulposus Cells.

AIM: To investigate the effects of metformin, a drug used widely for the treatment of type 2 diabetes mellitus, on human primary cell cultures prepared from uninjured segment of disc material intervertebral disk tissues. MATERIAL AND METHODS: Primary cell cultures were prepared using the tissues of six patients (three males and three females) who had undergone lumbar microdiscectomy and sequestrectomy. Untreated samples served as the control group, and metformintreated samples served as the experimental group. All the samples were evaluated using an inverted light microscope, acridine orange/propidium iodide staining (AO/PI), and a fluorescence microscope. The cytostatic and cytotoxic effects of metformin, which was administered to the samples using a commercial MTT assay kit, were also evaluated. The data obtained were statistically assessed, and the alpha significance value was accepted as less than 0.05. In addition, for the groups’ changes in the expressions of chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1β (IL-1β) matrix metalloproteinase 7 (MMP-7), and matrix metalloproteinase 19 (MMP-19), genes related to the extracellular matrix synthesis and degradation were determined using gene-specific TaqMan Gene Expression Assays. RESULTS: The administration of the drug adversely affected nucleus pulposus (NP)/annulus fibrosus (AF) cells and extracellular matrixâ€"like structures. This was statistically significant (p < 0.05). CONCLUSION: Clinicians should not disregard the adverse effects of metformin, which is used widely in clinical practice, on the components of intervertebral disk tissues.