Lactobacillus rhamnosus CNCM I-3690 and the commensal bacterium Faecalibacterium prausnitzii A2-165 exhibit similar protective effects to induced barrier hyper-permeability in mice

Impaired gut barrier function has been reported in a wide range of diseases and syndromes and in some functional gastrointestinal disorders. In addition, there is increasing evidence that suggests the gut microbiota tightly regulates gut barrier function and recent studies demonstrate that probiotic bacteria can enhance barrier integrity. Here, we aimed to investigate the effects of Lactobacillus rhamnosus CNCM I-3690 on intestinal barrier function. In vitro results using a Caco-2 monolayer cells stimulated with TNF-α confirmed the anti-inflammatory nature of the strain CNCM I-3690 and pointed out a putative role for the protection of the epithelial function. Next, we tested the protective effects of L. rhamnosus CNCM I-3690 in a mouse model of increased colonic permeability. Most importantly, we compared its performance to that of the well-known beneficial human commensal bacterium Faecalibacterium prauznitzii A2-165. Increased colonic permeability was normalized by both strains to a similar degree. Modulation of apical tight junction proteins expression was then analyzed to decipher the mechanism underlying this effect. We showed that CNCM I-3690 partially restored the function of the intestinal barrier and increased the levels of tight junction proteins Occludin and E-cadherin. The results indicate L. rhamnosus CNCM I-3690 is as effective as the commensal anti-inflammatory bacterium F. prausnitzii to treat functional barrier abnormalities.     

Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 30% to 40% in the presence of EDTA at 22 to 25 degrees C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP IIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to "strong" agonists exhibit an increased number of surface-oriented epitopes associated with GP IIb/IIIa. The GP IIb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.     

Association of tumour necrosis factor-alpha -308 G/A polymorphism with primary open-angle glaucoma

Background: Tumour necrosis factor‐alpha (TNF‐α) is an important proinflammatory cytokine driving axonal degeneration and retinal ganglion cell apoptosis in glaucoma. The aim of the study was to evaluate the association of TNF‐α ‐308 G/A and ‐238 G/A polymorphisms with primary open‐angle glaucoma (POAG). Design: A prospective, case–control study, university hospital setting. Participants: Eighty‐six POAG patients and 193 healthy unrelated controls. Methods: TNF‐α polymorphisms were screened by using direct gene sequencing. Main Outcome Measures: Frequency of TNF‐α ‐308 G/A and TNF‐α ‐238 G/A promoter polymorphisms in glaucoma and healthy subjects. Results: The frequencies of TNF‐α ‐308 GA genotype and ‘A’ allele were higher in patients with POAG (22.1% and 12.2%, respectively) in comparison with the control group (10.9% and 6%, respectively) (P = 0.046 and 0.02, respectively), with odds ratios of 2.45 (P = 0.01, 95% CI = 1.23–4.87) and 2.19 (P = 0.013, 95% CI = 1.18–4.08), respectively. Genotype distribution of the TNF‐α ‐238 variants did not yield a statistically significant difference between the two groups (P = 0.87). Conclusion: TNF‐α ‐308 G/A polymorphism seems to be associated with POAG in Turkish population. However, population‐based studies with large number of subjects and long‐term follow‐up are needed to verify the association of TNF‐α ‐308 G/A polymorphism with glaucoma susceptibility.     

Evaluation of serum interleukin-6 (IL-6), IL-13, and IL-17 levels and computed tomography finding in interstitial lung disease associated with connective tissue disease patients

Objective

Interstitial lung disease (ILD) is one of the most severe complications which is associated with connective tissue disease (CTD) and causes to morbidity and mortality. So, we aimed to determine serum levels of interleukin-6 (IL-6), IL-13, and IL-17, to investigate whether these cytokines are related to CTD-ILD, and to find their possible contribution to determining the prognosis of the disease.

Methods

A total of 150 participants, 80 patients diagnosed with CTD-ILD (mean age, 58.21?±?12.36) and 70 healthy controls (mean age, 57.07?±?9.60) were recruited from the rheumatology department between January 2016 and June 2019 in the study. High-resolution computed tomography (HRCT) findings were scored as similarly to previous studies. Serum IL-6, IL 13, and IL-17 levels were measured by ELISA test kits.

Results

The levels of IL-6, IL-13, and IL-17 in CTD patients were significantly higher than the healthy individuals (p?<?001), but the HRCT score’s relation were not determined. IL-6 was associated with disease duration and disease activity scores of DAS28, ESDAII, and dSSc. There was a significant relation between dSSc, HCRT fibrosis, and total score.CRP, hemoglobin, and platelets were associated with the HRCT inflammation pattern.

Conclusion

At the study, it has been observed that serum IL-13, IL-6 and IL-17 levels are increased in patients with CTD-ILD. Besides, IL-6 was associated with disease activity scores of DAS28, ESDAII, and dSSc. Also, HRCT fibrosis score is associated with dSSc. Further and comprehensive studies are needed to understand better the complex intersection of lung disease with systemic autoimmunity.

Molecular and cellular effects of antisickling concentrations of alkylureas

Alkylureas are capable of inhibiting sickling in vitro and the gelation of solutions of hemoglobin S at concentrations between 0.05 and 0.1 M with increasing effectiveness that is directly proportional to the length of the alkyl chain (butyl greater than propyl greater than ethyl greater than methyl). 6The inhibitory effect is independent of pH between 6.5 and 7.5 and is a process driven by entropy. The alkylureas at concentrations of 0.1 M have minimal effects on several erythrocyte functions. Oxygen equilibria, osmotic fragility, reduced glutathione content, and glutathione reductase activity are totally unaffected, while pyruvic kinase activity is decreased only by butylurea by about 20%, and glucose-6-phosphate dehydrogenase activity is decreased progressively to a maximum of 30% in direct proportion to the length of the alkyl chain. Alkylureas not only inhibit sickling but are also capable of desickling erythrocytes that have been maintained in the deoxygenated state. They have little effect on several erythrocyte functions at antisickling concentrations, but their toxicity must be evaluated before they can be examined as potential therapeutic agents for the treatment or prevention of acute episodes in sickle cell anemia.     

T-cell receptor gene rearrangement in T-cell large granular leukocyte leukemia: preferential V alpha but diverse J alpha usage in one of five patients

T-gamma lymphoproliferative disease (T-gamma LPD) is a chronic disorder of mature T cells that is associated with neutropenia and autoimmune phenomena. Although the progression of the lymphoproliferation is indolent, it is often associated with a monoclonal proliferation of T- cell-type large granular lymphocytes (LGL) that manifest multiple in vitro suppressor and cytotoxic activities. We considered the possibility that the granulocytopenia or anemia might represent an autoimmune disorder mediated by the monoclonal LGL via T-cell receptor (TCR) recognition of an antigen involved in hematopoiesis. Therefore, in an effort to characterize the usage of the TCR alpha- and beta-chain genes in patients with T-gamma LPD, we cloned and sequenced TCR alpha- and beta-chain mRNAs derived from the T-cell type LGL of five patients. The five patients studied did not use a common V alpha nor a common J alpha segment. However, an unusual finding was observed in one of the patients where the occurrence of a single variable-diversity-junctional (VDJ) rearrangement of the beta chain confirmed the monoclonal origin of the LGL proliferation. In accord with this evidence for monoclonality, many of the cells studied used a common V alpha (V alpha 19.1). In contrast to this common V alpha usage, there was a marked diversity of the J alpha segments and N-region addition that were associated with the V alpha 19.1 segment. This pattern of common V alpha usage associated with different N and J alpha segments suggests an immune-mediated selection process affecting the TCR alpha chain occurring after the transformation event that established the clone. We suggest that the T-cell-type LGL malignant clone might have developed autoreactivity conferred by the selected TCR alpha chain and that this autoreactivity might be implicated in this patient's anemia.     

Specificity of autoantibodies in autoimmune thrombocytopenia

In 42 patients with autoimmune thrombocytopenia (AITP) and a positive direct platelet suspension immunofluorescence test (PSIFT), the antigenic specificity of the autoantibodies was studied. Because the autoantibodies were often not detectable in the serum and additional HLA antibodies may disturb the reaction pattern with the platelet panel, we used eluates prepared from the patients' platelets for this study. Thirty-five patients had antibodies equally reactive with normal platelets, irrespective of their antigenic make-up, but not with the platelets from two Glanzmann's disease patients. Absorption and elution experiments in two patients showed that his was probably not due to the presence of a combination of anti-Zwa and anti-Zwb antibodies. Thus, the majority of autoantibodies against platelets seems to be directed against antigenic determinants not present on Glanzmann's disease platelets, but perhaps located on the platelet-membrane glycoproteins IIb and/or IIIa. In ten patients, antibodies of no, or still unknown, specificity were detected. Three of these had additional antibodies not reactive with the platelets of the two Glanzmann patients.     

Increased P Wave Dispersion and Maximum P Wave Duration after Hemodialysis

Background: Atrial fibrillation is a frequent arrhythmia in patients undergoing hemodialysis. The consequences of hemodialysis on P wave durations and P wave dispersion have not been fully understood. The objective of this study was to study the effect of dialysis on P wave maximum (P_max), P wave minimum (P_min), and P wave dispersion (P_d). Methods: We studied P_max, P_min, and P_d in 32 patients (17 men and 15 women, mean age 54 ± 18 years) with chronic renal failure undergoing hemodialysis. The difference between maximum and minimum P wave duration was calculated and defined as P wave dispersion (P_d= P_max? P_min). Results: There was a significant increase in P_max at the end of dialysis compared to the beginning (98 ± 13 ms vs. 125 ± 12 ms, P < 0.001). P_min did not show any significant change (71 ± 11 ms vs. 73 ± 10 ms, P = 0.42). P_d was significantly increased at the end of dialysis (27 ± 9 ms vs. 52 ± 11 ms, P < 0.001). There was a negative correlation between serum potassium, magnesium, phosphate, blood urea nitrogen, and creatinin at the end of dialysis and P_max and P_d, respectively (P < 0.05). A weak positive correlation was found between serum calcium, bicarbonate at the end of dialysis and P_max and P_d (P < 0.05). Conclusion: Hemodialysis ends with significant increase in P wave maximum duration and P wave dispersion, which might be responsible for the increased occurrence of atrial fibrillation in these groups of patients.     

Effect of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on hematopoietic regeneration as demonstrated in a nonhuman primate chemotherapy model

Using a recently developed hepsulfam-induced pancytopenia model in rhesus macaques, we have studied the effects of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on marrow regeneration. Control animals were given hepsulfam (1.5 g/m2 by a single 30-minute intravenous [i.v.] injection, n = 4), while study animals received hepsulfam followed by rhIL-6, rhIL-3, or a combination of rhIL-6 and rhIL-3 (n = 3 per study group). Each cytokine was administered by once- daily subcutaneous (SC) injection (15 micrograms/kg/d) for 3 weeks beginning the day after chemotherapy (days 2 through 22). Mean platelet counts in control animals were < 100,000/microL on days 15 through 24, with 50% of the counts < 50,000/microL and two of four animals requiring platelet transfusion. In the rhIL-6- and rhIL-6/rhIL-3- treated groups, the nadir mean platelet counts were 164,000 +/- 58,700/microL and 162,300 +/- 23,800/microL, respectively, and occurred on day 15. Platelet counts in the rhIL-3-treated group were similar to those in controls. Mean absolute neutrophil counts (ANCs) < 1,000/microL occurred on days 10 through 29 in control animals, days 8 through 15 in rhIL-6-treated animals, and days 6 through 8 and 13 in rhIL-6/rhIL-3-treated animals. The frequency of ANCs < 500/microL was significantly less in the rhIL-6- and rhIL-6/rhIL-3-treated groups versus control groups (2.7 +/- 0.6 and 2.0 +/- 1.0 vs 7.0 +/- 1.4 occurrences, respectively; P < .05). rhIL-3-treated animals had ANCs similar to those in controls; one animal died with septicemia on day 21. Monkeys receiving rhIL-6 were significantly more anemic during the cytokine administration period; however, the anemia resolved by day 24. Coadministration of rhIL-3 and rhIL-6 partially corrected the anemia. The data indicate that rhIL-6 prevents significant thrombocytopenia and shortens the neutropenic period in this chemotherapy model.     

Vimentin silencing effect on invasive and migration characteristics of doxorubicin resistant MCF-7 cells

Chemotherapy is one of the well-known treatments in cancer therapy. The effectiveness of chemotherapy is limited by several factors one of which is the emergence of multidrug resistance (MDR). One of the major mechanisms of MDR is the activity of several ATP binding cassette (ABC) transporters that pump drugs out of the cells. Doxorubicin intercalates and inhibits DNA replication; it is a powerful chemotherapeutic agent. However, it causes development of MDR in tumor cells. Vimentin is a type III intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and also poor prognosis of cancer. There are several studies that have shown the connection between expression level of vimentin and invasiveness of tumor cells. In this study, MCF-7 cell line which is a model for human mammary carcinoma, and a doxorubicin resistant subline (MCF-7/Dox) were used. The resistant subline was previously obtained by stepwise selection in our laboratory. In the resistant cells, high levels of vimentin expression were observed. The main purpose of this study was to investigate changes in invasive and migration characteristics of MCF-7/Dox cell line, after transient silencing of vimentin gene by specific siRNA.