[Analysis of IFN-γR1 (CD119) and IL-12Rß1 (CD212) Deficiency by Flow Cytometry]. / IFN-γR1 (CD119) ve IL-12Rß1 (CD212) Eksikliginin Akan Hücre Ölçer ile Analizi.

Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare primary immune deficiency (PID). IL-12Rß1 deficiency is the most frequently observed of more than 16 genetic defects that have been identified for MSMD. Genetic and immunological tests are remarkable in the diagnosis of PID. In this study, it was aimed to determine the expression of IFN-γR1 and IL-12Rß1 in patients with MSMD, their relatives, and healthy individuals and to evaluate the importance of flow cytometry as a fast and reliable method in the diagnosis of MSMD. IFN-γR1 and IL-12Rß1 expression levels were analyzed in 32 volunteers including six patients, six relatives, and 20 healthy individuals. The normal range of IFN-γR1 and IL-12Rß1 levels among healthy individuals were determined. IL-12Rß1 expression level in lymphocytes was found to be low in one patient's relative, and less than 1% in three patients and in one patient's relative. It was observed that the IL-12Rß1 expression levels of the patient with STAT1 deficiency were increased compared to the healthy individuals. No difference was found in the expression levels of IFN-γR1 and IL-12Rß1 in one patient, but IFN-γR1 expression was decreased in one patient compared to healthy individuals. Our results show that the determination of IL-12Rß1 and IFN-γR1 deficiencies by flow cytometry can be used as a rapid and reliable method for the diagnosis of MSMD. The use of this method as a screening test will enable early diagnosis especially in patients whose genetic diagnosis has not been confirmed and clinically compatible with MSMD. In addition, it is thought that IL-12Rß1 and IFN-γR1 range data obtained from healthy individuals will be considered as a reference source in routine and research studies to be conducted with MSMD.

The role of cytokines and T-bet, GATA3, ROR-γt, and FOXP3 transcription factors of T cell subsets in the natural clinical progression of Type 1 Diabetes.

Th cells play an important role in pathogenesis of type 1 diabetes (T1D). Peripheral blood mononuclear cells were isolated from peripheral blood samples from newly diagnosed (ND), 1-year (1YD), and 5-year T1D (5YD) patients (n:8 of each group), 8 healthy controls (HC), and cultured for 24 h under unstimulated (US) and stimulated conditions. Cell ratios of Th1, Th2, Th17, Treg, and intracellular levels of IFN-γ, TNF-α, IL-10, TGF-ß, IL-5, IL-13, IL-17, and IL-21 cytokines were evaluated using the flow cytometry. mRNA expressions of transcription factors T-bet, GATA3, ROR-γt, and FOXP3 of these cells were determined by real-time PCR. Reduced CD4+CD25high cell ratios were detected in ND. CD4+CD25high cells were found to be reduced in ND and 1YD compared to HC under IL-2-stimulated conditions. Intracellular IFN-γ and TNF-α levels were low in all patients under US and IL-12-stimulated conditions. IL-17A and IL-21 were found to be high in patients with IL-6-stimulated conditions. Expressions of IL-10 and TGF-ß have been observed to be reduced in patients. Th1/Th2, Th17/Treg, and Th1/Treg ratios were higher in patient groups. FOXP3 and GATA3 mRNA expressions were found to be low in patients, while RORγt and T-bet mRNA levels were higher than HC. Th1, Th17, and Treg cells and their cytokines have been shown to be associated with type 1 diabetes.

Thrombotic risk assessment in antiphospholipid syndrome: do noncriteria antibodies contribute?

Background/aim: In this cross-sectional study, it was aimed to test the predictive value of noncriteria antiphospholipid antibodies (aPL) in addition to the global antiphospholipid syndrome score (GAPSS) in predicting vascular thrombosis (VT) in a cohort of patients with APS and aPL (+) systemic lupus erythematosus (SLE). Material and methods: This study included 50 patients with primary APS, 68 with SLE/APS, and 52 with aPL (+) SLE who were classified according to VT as VT ± pregnancy morbidity (PM), PM only or aPL (+) SLE. Antiphospholipid serology consisting of lupus anticoagulant (LA), anticardiolipin (aCL) immunoglobulin G (IgG)/IgM/IgA, antibeta2 glycoprotein I (aß2GPI) IgG/IgM/IgA, antiphosphatidylserine/prothrombin (aPS/PT) IgG/IgM and antidomain-I (aDI) IgG was determined for each patient. The GAPSS and adjusted GAPSS (aGAPSS) were calculated for each patient, as previously defined. Logistic regression analysis was carried out with thrombosis as the dependent variable and high GAPSS, aCL IgA, aß2GPI IgA, and aDI IgG as independent variables. Results: The mean GAPSS and aGAPSS of the study population were 11.6 ± 4.4 and 9.6 ± 3.8. Both the VT ± PM APS (n = 105) and PM only APS (n = 13) groups had significantly higher GAPSS and aGAPSS values compared to the aPL (+) SLE (n = 52) group. The patients with recurrent thrombosis had higher aGAPSS but not GAPSS than those with a single thrombotic event. The computed area under the receiver operating characteristic curve demonstrated that a GAPSS ≥13 and aGAPSS ≥10 had the best predictive values for thrombosis. Logistic regression analysis including a GAPSS ≥13, aCL IgA, aß2GPI IgA, and aDI IgG showed that none of the factors other than a GAPSS ≥13 could predict thrombosis. Conclusion: Both the GAPSS and aGAPSS successfully predict the thrombotic risk in aPL (+) patients and aCL IgA, aß2GPI IgA, and aDI IgG do not contribute to high a GAPSS or aGAPSS.

The relationship between serum A proliferation-inducing ligand and B-cell activating factor levels with disease activity and organ involvement in systemic lupus erythematosus.

OBJECTIVES: We aim to investigate the association between serum B-cell activating factor (BAFF) and A proliferation-inducing ligand (APRIL) levels with disease activity and clinical findings in SLE patients. METHODS: Seventy-nine patients with SLE and 27 healthy controls were included into the study. Serum BAFF and APRIL levels were measured by using ELISA. In 19 patients with active disease at the time of the assessment, BAFF/APRIL levels were reassessed after 6 months of follow-up and disease activity was evaluated by using SLEDAI-2K. The relationship between renal histopathology index scores and lupus nephritis (LN) classes with serum BAFF/APRIL levels was examined in 16 patients who had recent renal involvement and underwent biopsy during the study. RESULTS: Although both BAFF/APRIL levels were higher in patients with SLE compared to the control group (p < 0.001), no correlation was found between BAFF/APRIL levels and SLEDAI scores. Serum BAFF levels were higher in patients with renal disease activity (p = 0.01), and there was a significant correlation between APRIL levels and proteinuria (r = 0.42, p = 0.02). A weak inverse correlation was observed between BAFF and C3 levels (r = 0.25, p = 0.02). No correlation was found between BAFF/APRIL levels and renal SLEDAI scores, renal histopathology, activity, and chronicity index scores. In the active disease group after treatment, there was no significant change in serum BAFF levels, but a significant increase in serum APRIL levels was observed. CONCLUSION: These results suggest that both cytokines are involved in the pathogenesis of SLE and that serum BAFF can be valuable as a biomarker in SLE especially in patients with renal activity.

Assessment of Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia: A Multicenter Study From Turkey.

Assestment of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) is of utmost importance both for risk classification and tailoring of the therapy. The data of pediatric ALL patients that received treatment with Berlin-Frankfurt-Münster (BFM) protocols were retrospectively collected from 5 university hospitals in Turkey. Of the 1388 patients enrolled in the study 390 were treated according to MRD-based protocols. MRD assestment was with real time quantitative polymerase chain reaction (qPCR) in 283 patients and with multiparametric flow cytometry (MFC)-MRD in 107 patients. MRD monitoring had upstaged a total of 8 patients (2%) from intermediate risk group to high-risk group. Univariate analysis revealed age 10 years or above, prednisone poor response, PCR-MRD ≥10-3 on day 33 and on day 78 as poor prognostic factors affecting event-free survival (EFS). Detection of >10% blasts on day 15 with MFC (MFC-high-risk group) was not shown to affect EFS and/or overall survival (log-rank P=0.339). Multiple logistic regression analysis revealed PCR-MRD ≥10-3 on day 78 as the only poor prognostic factor affecting EFS (odds ratio: 8.03; 95% confidence interval: 2.5-25; P=0.000). It is very important to establish the infrastructure and ensure necessary standardization for both MRD methods for optimal management of children with ALL.

Effects of Tyrosine Kinase Inhibitor-masitinib Mesylate on Canine Mammary Tumour Cell Lines.

INTRODUCTION: Masitinib mesylate, a selective tyrosine kinase inhibitor of the c-KIT receptor, is used for the treatment of mast cell tumours in dogs. Masitinib has previously been investigated in various cancers; however, its potential anticancer effect in canine mammary tumours (CMTs) is unknown. In the present paper, we investigated the antiproliferative effect of masitinib in CMT cells and its possible mechanisms of action. MATERIAL AND METHODS: The effect of masitinib on the proliferation of CMT-U27 and CMT-U309 cells was assessed by MTT assay and DNA fragmentation. Flow cytometric analysis was used to measure the effect of masitinib on apoptosis and the cell cycle. Additionally, vascular endothelial growth factor levels (VEGF) were measured, and the proliferation marker Ki-67 was visualised in immunocytochemical stainings in CMT cells. RESULTS: Treatment with masitinib inhibited the proliferation of CMT cells in a concentration-dependent manner. Maximal apoptotic activity and DNA fragmentation were observed at approximately IC50 of masitinib in both cell lines. In addition, cell cycle distribution was altered and VEGF levels and Ki-67 proliferation indices were decreased in masitinib-treated cells in comparison with control cells. CONCLUSION: In this study, masitinib suppressed cell proliferation concomitantly via induction of apoptosis and cell cycle arrest by decreasing VEGF levels and the Ki-67 proliferation index in CMT-U27 and CMT-U309 cells in vitro, suggesting its potential as a therapeutic tool in the clinical setting of mammary cancer treatment in dogs.

Functions of NK and iNKT cells in pediatric and adult CVID, ataxia telangiectasia and agammaglobulinemia patients.

Primary immune deficiencies (PID) are known to be more than 400 genetic defects caused by the impairment in development and/or functions of the immune system. Common Variable Immunodeficiency (CVID), Ataxia Telangiectasia (AT) and Agammaglobulinemia (AG) are examples of the most common immunodeficiency syndrome. Natural killer (NK) cells are a component of innate immune system and play a major role in the host-rejection of both tumors and virally infected cells. iNKT cells have a role in autoimmune and infectious diseases and controlling of tumor rejection. In this study, NK and iNKT cells and their functions, and intracellular cytokine amount are aimed to determine in patients that suffer CVID, AT and AG. NKp30, NKp46, NKG2D, perforin and granzyme mRNA expression levels were analyzed using RT-PCR. Receptors, cytokine amount of NK cell subset and iNKT were analyzed by flow cytometry. Decreased CD3+ T and elevated NK cell subset in pediatric AT were found. Expression of NKp44 was decreased in adult AG, but not in pediatric patients. Low NKp44 expression in CD3-CD16+CD56dim NK cell subset was found in pediatric AT patients. High HLA-DR, perforin and granzyme expression were found in CD3-CD16+CD56dim NK cell subset of pediatric CVID and AT patients. Alteration of the number of NK subsets, NK receptor expression and cytokine production were observed in pediatric patients compared to healthy subjects.

Metformin reverses the effects of high glucose on human dermal fibroblasts of aged skin via downregulating RELA/p65 expression.

Metformin has been successfully used as an anti-aging agent but exact molecular mechanisms of metformin in anti-aging remain unknown. Hyperglycemia during skin aging not only causes oxidative damage to cellular macromolecules, like dermal collagen, but also modulates the activation of transcription factor nuclear factor kappa B (NF-kB). We aimed to investigate in vitro effects of high glucose (HG) and metformin treatment on proliferation and apoptosis of human primary dermal fibroblasts (HDFs), and the expression of COL1A1, COL3A1, and RELA/p65 genes. Effects of normal glucose (5.5 mM) and HG concentration (50 mM HG) on HDFs, with two doses of metformin (50 µM and 500 µM), were investigated by immunostaining. Apoptotic levels were analyzed by flow cytometry. Expression of COL1A1, COL3A1, and RELA/p65 genes was measured by quantitative real-time PCR. The proliferation of HDFs was decreased significantly (P < 0.01) and expression of COL1A1 was downregulated by HG without metformin, whereas proliferation was elevated and expression was upregulated with 500 µM metformin + HG compared to 5.5 mM glucose (P < 0.05). The expression of COL3A1 and RELA/p65 were upregulated (P < 0.01 for COL3A1), and percentage of late apoptotic cells increased significantly by HG without metformin (P < 0.001) while it decreased in two concentrations of metformin dramatically compared with 5.5 mM glucose (P < 0.01 for expressions and < 0.001 for apoptosis). Metformin not only significantly downregulated RELA/p65 expression, but also inhibited the apoptosis of HDFs from aged human skin at toxic glucose concentrations which could be inversely mediated via COL1A1 and COL3A1 expression.

Inflammatory status might direct ILC and NK cells to IL-17 expressing ILC3 and NK subsets in Behcet's disease.

Innate lymphoid cells (ILCs) are lymphoid cells that have important effector and regulatory functions in innate immunity and tissue remodeling. Uncontrolled activation and proliferation of ILCs can contribute to inflammatory autoimmune diseases. Behcet's disease (BD) is a complex systemic inflammatory disorder of unknown etiology. It has been shown that natural killer (NK) cells may play an immunoregulatory role in BD, however the role of ILCs is unknown. In this study, the levels and functions of ILCs and NK cell subsets in BD patients were investigated. Cell surface and cytotoxic granules (perforin and granzyme) expression of NK cells and ILCs were evaluated and labeled according to whole blood lysing protocol in peripheral blood samples obtained from the patients and healthy subjects. Cytokine levels of NK cells were investigated in stimulated peripheral blood mononuclear cells. All data were analyzed by flow cytometry. Total ILC and ILC3+ cells were increased in active BD patients compared to inactive BD patients and healthy subjects. There was no significant difference between the patients and healthy subjects regarding NK cell surface and intracellular molecule expression. Although, an increase in IFN-γ and IL-17, and a decrease in IL-4 levels were observed in CD56dim NK cell subset of BD patients. Recent studies showed increased neutrophilic infiltration and IL-17 secreting Th17 cells in BD patients. It is known that ILC3+cells are similar to Th17 subset regarding their cytokine profile and transcription factor expression patterns. Results of current study may suggest that inflammatory microenvironment in BD patients might direct ILC cells to differentiate into ILC3+ subset, and IL-17 released by NK cells might have a role in neutrophilic infiltration.

Immune alterations in subacute sclerosing panencephalitis reflect an incompetent response to eliminate the measles virus.

In subacute sclerosing panencephalitis (SSPE) the persistence of measles virus (MeV) may be related to the altered immune response. In this study, cytokine responses of lymphocytes and monocytes were evaluated in SSPE compared to controls with non-inflammatory (NICON) and inflammatory (ICON) diseases. Patients with SSPE (n = 120), 78 patients with ICON and 63 patients with NICON were included in this study. Phenotypes of peripheral blood mononuclear cells (PBMC) have been analyzed by flow cytometry. CD3 and CD28, and S. aureus Cowan strain I (SAC) stimulated and unstimulated cells were cultured and IL-2, IL-10, IFN-γ, IL-12p40, IL-12p70 and IL-23 were detected in supernatants by ELISA. MeV peptides were used for MeV-specific stimulation and IFN-γ secretion of PBMC was measured by ELISPOT. Spontaneous and stimulated secretions of IL-10 were lower in SSPE compared to both control groups. T cell stimulation induced lower IFN-γ production than ICON group, but higher IL-2 than NICON group in SSPE. Stimulated PBMC produced lower IL-12p70 in SSPE and had decreased CD46 on the cell surface, suggesting the interaction with the virus. IFN-γ responses against MeV peptides were not prominent and similar to NICON patients. The immune response did not reveal an inflammatory activity to eliminate the virus in SSPE patients. Even IL-10 production was diminished implicating that the response is self-limited in controlling the disease.

Expression of Akt1 and p-Akt1 in peripheral T cell subsets of multiple sclerosis patients.

Multiple sclerosis is an autoimmune disorder induced by the infiltration of autoreactive immune cells into the central nervous system. Akt/PKB signaling pathway is crucially involved in T cell development and survival. We aimed to determine whether Akt1 expression levels of regulatory T (Treg) cells are altered in MS and are associated with disease activity. Relapsing-remitting multiple sclerosis (RR-MS, n = 17) patients and healthy individuals (n = 20) were enrolled. Peripheral blood mononuclear cells were isolated and anti-CD3, -CD4, -CD8, -CD25, -CD127 monoclonal antibodies were used to identify the T cell subsets. After stimulation with phorbol myristate acetate/ionomycin, the Akt1 and phosphorylated-Akt1 (p-Akt1) levels of T cell subsets were detected with intracellular staining using flow cytometry. Total Akt1 and p-Akt1 expression levels were found to be suppressed in CD4+ T cell and Treg populations of RR-MS patients. Progression indices were positively correlated with Akt1 expression levels of Tregs indicating that the Akt pathway might partake in the progression of multiple sclerosis. Flow cytometry may effectively be used for the evaluation of the Akt pathway activity. Our findings suggest that the magnitude of suppression of the Akt pathway might serve as a biomarker for the prognosis of multiple sclerosis.

Impact of fingolimod on CD4+ T cell subset and cytokine profile of relapsing remitting multiple sclerosis patients.

Fingolimod inhibits the egress of lymphocytes from lymphatic tissues and also directly affects their functions by modulation of the sphingosine-1-phosphate receptor 1 (S1P1). Our aim was to evaluate the impact of fingolimod on diverse CD4+ T cell subsets, and cytokines. Sixty-six relapsing remitting multiple sclerosis (RRMS) patients were treated with oral fingolimod (0.5 mg) for 6 months, and blood samples were collected at baseline, 3 months, and 6 months. Serum levels of seven cytokines and five chemokines were measured by multiplex immunoassay, and frequencies of peripheral blood mononuclear cell subsets were assessed by flow cytometry, and compared with those of 60 healthy controls. CCL2 (p = 0.039), and CCL5 (p = 0.001) levels were significantly higher in fingolimod-treated patients than healthy controls, whereas end-of-study serum levels of IL-6, IL-8, IL-17A, IL-22, IL-23, TNF-α, CXCL10, and CXCL13 were comparable to the baseline levels. Six months of fingolimod treatment reduced CD3+ T cell (mean ±â€¯standard deviation, 72.9% ±â€¯5.5 vs. 60.1% ±â€¯11.1, p < 0.001), CD4+ T cell (62.2% ±â€¯8.5 vs. 24.6% ±â€¯12.9, p < 0.001), CD4+CD25hi regulatory T cell (Treg) (3.4% ±â€¯1.3 vs. 2.0% ±â€¯1.4, p < 0.01), and CD19+ B cell (13.2% ±â€¯5.8 vs. 5.3% ±â€¯2.7, p < 0.001) frequencies, while CD8+ T cells (31.8% ±â€¯7.8 vs. 57.8% ±â€¯13.2, p < 0.001) were increased, and NK and NKT cells remained unchanged. The proportions of intracytoplasmic IL-4, IL-10, IFN-γ, and TNF-α-producing T cells were increased, whereas IL-17-producing cells remained relatively constant as measured by flow cytometry. Fingolimod appears to primarily diminish lymphocyte subsets involved in antigen presentation (CD19+ B and CD4+ T cells) rather than immune cells (CD8+ T, NK, and NKT cells) in charge of host defense against pathogens. In contrast, a relative increase is observed in pro- and anti-inflammatory cytokine-producing T helper subsets (IFN-γ, TNF-α, IL-4, and IL-10-producing CD4+ T cells), suggesting that effector T cells are suppressed to a lesser degree by S1P1 modulation.

Nicotine reduces effectiveness of doxorubicin chemotherapy and promotes CD44+CD24- cancer stem cells in MCF-7 cell populations.

Breast cancer is the most common type of cancer in females and the second most common cause of cancer mortality after lung cancer. Cancer stem cells represent a novel approach to target cancer and reduce cancer recurrence and metastasis. Many patients with breast cancer continue to smoke after receiving their diagnosis. Nicotine is a key factor in tobacco addiction and also changes some cellular functions, such as activation of mitogenic pathways, angiogenesis and cell proliferation. In the present study, the impact of nicotine was assessed in a population of MCF-7 human breast cancer cells. Cluster of differentiation (CD)44+CD24- cancer stem cell population of MCF-7 cells were evaluated using flow cytometry and scanning electron microscopy. Chemoresistance effects of nicotine were demonstrated in these cells. These findings demonstrated harmful effects of nicotine following metastasis of cancer, owing to the chemoresistance produced through uninterrupted smoking, which may impact the effectiveness of treatment.

The protective effects of metformin in an in vitro model of aging 3T3 fibroblast under the high glucose conditions.

Metformin is the most widely used anti-diabetic drug in the world. It reduces advanced glycation end product (AGEs)-induced ROS generation in high glucose condition. Protein glycation contributes to skin aging as it deteriorates the existing collagen by crosslinking. The progressive increase of AGE during aging not only causes oxidative damage to cellular macromolecules but also modulates the activation of transcription factors nuclear factor kappa-B(NF-kB). However, it is still unclear whether metformin can change collagen production and NF-kB activity induced by high glucose conditions in 3T3 fibroblast. The effects of metformin on proliferation, apoptosis, and collagen levels and NF-kB activity of in vitro cell aging model of 3T3 fibroblast cells in high glucose conditions. At first, we investigated the effects of 50 mM high glucose concentration, with or without metformin, on 3T3 fibroblast proliferation, by BrdU immunostaining for cell proliferation. Apoptotic levels were analyzed by flow cytometric assay. NF-kB(p65) activity was measured by transcription factor assay kit and collagen I and III levels by Collagen Estimation Assay through ELISA. We observed that metformin exposure leads to decreased apoptosis levels and increased proliferation of 3T3 fibroblast in high glucose media. We also determined that metformin exposure leads to increased production of collagen I-III and decreased activation of NF-kB(p65) activity. The data are consistent with the observation that metformin has a protective effect in this in vitro model of aging 3T3 fibroblasts under high glucose conditions inducing cell proliferation, collagen I and III production, protection from apoptosis, and reducing NF-kB(p65) activity.

The immune system as a chronotoxicity target of the anticancer mTOR inhibitor everolimus.

The circadian timing system controls many biological functions in mammals including xenobiotic metabolism, detoxification, cell proliferation, apoptosis and immune functions. Everolimus is a mammalian target of rapamycin inhibitor, whose immunosuppressant properties are both desired in transplant patients and unwanted in cancer patients, where it is indicated for its antiproliferative efficacy. Here we sought whether everolimus circadian timing would predictably modify its immunosuppressive effects so as to optimize this drug through timing. C57BL/6J mice were synchronized with light-dark 12h:12h, with L onset at Zeitgeber Time (ZT) 0. Everolimus was administered orally to male (5 mg/kg/day) and female mice (15 mg/kg/day) at ZT1, during early rest span or at ZT13, during early activity span for 4 weeks. Body weight loss, as well as hematological, immunological and biochemical toxicities, were determined. Spleen and thymus were examined histologically. Everolimus toxicity was less severe following dosing at ZT13, as compared to ZT1, as shown with least body weight inhibition in both genders; least reductions in thymus weight both in males (p < 0.01) and females (p < 0.001), least reduction in female spleen weight (p < 0.05), and less severe thymic medullar atrophy both in males (p < 0.001) and females (p < 0.001). The mean circulating counts in total leukocytes, total lymphocytes, T-helper and B lymphocytes displayed minor and non-significant changes following dosing at ZT13, while they were decreased by 56.9% (p < 0.01), 45.5% (p < 0.01), 43.1% (p < 0.05) and 48.7% (p < 0.01) after everolimus at ZT1, respectively, in only male mice. Chronotherapy of everolimus is an effective way to increase the general tolerability and decrease toxicity on the immune system.

Response of cultured normal canine mammary epithelial cells to deracoxib-doxorubicin combination.

Currently, there is a growing interest in combining anticancer drugs with the aim to improve outcome in patients suffering from tumours and reduce the long-term toxicity associated with the current standard of treatment. In this study, we evaluated the possible role of deracoxib against the toxicity of doxorubicin on normal canine mammary epithelial cells. The effect of deracoxib and doxorubicin combination on cell viability was determined by MTT assay. Apoptosis was characterised by flow cytometry. Cell nitrite concentrations were measured with the Griess reaction. Deracoxib (50 and 100 µM) treatment decreased the cytotoxic action of doxorubicin at 0.9 µM in the cells, from 33.63% to 13.4% and 25.82%, respectively. Our results also showed that the reverse effect of deracoxib on doxorubicin-induced cytotoxic activity in the cells was associated with a marked (3.04- to 3.57-fold) decrease in apoptosis. In additional studies identifying the mechanism of the observed effect, deracoxib exhibited an activity to prevent doxorubicin-mediated overproduction of nitric oxide in the cells. Our in vitro study results indicate that deracoxib (50 and 100 µM) can be beneficial in protecting normal cells from the toxic effect of doxorubicin in conjunction with apoptosis by the modulation of nitric oxide production.

Which is better Stainless Steel or Titanium alloy?

AIM: To investigate immunologic reactions after implantation of stainless steel (SS) alloy and titanium (Ti) alloy in a rat model. Macrophage and cytokine responses have been reported after the in vivo and in vitro application of different biomaterials. MATERIAL AND METHODS: Wistar albino rats after an exploration of the thoracolumbar paravertebral muscle tissue of the subjects, group I underwent a sham surgery, and groups II and III were implanted Ti alloy and SS alloy rods respectively. The CD4, CD8, CD25 (IL-2R) (lymphocyte and CD4 gate), CD4+CD8+ and CD4+CD25+Foxp3+ (Tregs), IL-4, IL-10, IL-6, IL-17A, TGF-ß, TNF-α in the blood were analyzed. RESULTS: CD4, CD25 (IL-2R), CD4+CD8+ and Tregs levels were lower in the Group III compared to the sham and Group IIs. IL-6, IL-17A, TGF-ß and TNF-α levels in the G III showed a significant increase on all days in comparison with the sham and Group II. IL-4 and IL-10 levels, were lower in the Group III than those in the Group II; and a significant decrease was observed in the IL-10 level. While there was a reduction in IL-6 and IL-17A levels in the Group II as opposed to the sham group. CONCLUSION: As opposed to SS alloy, Ti alloy suppresses the development of inflammations by inhibiting proinflammatory response; strengthens the humoral immune system by intensifying the antibody-dependent immune response; triggers the development of immune tolerance by regulating the immune response; and activates the mechanism that prevents immune response-related damage from occurring.

CD133+ cells are associated with ADIPOCYTOKINES and endothelial dysfunction in hemodialysis patients.

BACKGROUND: Hemodialysis (HD) patients have increased risk of cardiovascular disease (CVD). Impaired stem cell health and adipocytokine metabolism may play important roles in the complex pathophysiological mechanisms of CVD in this patient population. We aimed to investigate the relationships between CD133+ cell counts, adipocytokines and parameters of endothelial dysfunction and atherosclerosis in HD patients. METHODS: In 58 chronic HD patients (male/female:28/30, mean age:58 ± 14 years), serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), leptin, adiponectin and resistin were measured by ELISA. Left ventricular mass index (LVMI), carotid intima-media thickness (CIMT), flow-mediated dilatation (FMD) of the brachial artery were measured. CD133+ cells were counted by flow cytometry (BD FACSCalibur-BD Bioscience,CA). RESULTS: CD133+ cell counts were inversely associated with FMD (r = -0.39, p = 0.007) and positively correlated with serum resistin (r = 0.45, p < 0.001) and serum TNF-α (r = 0.31, p = 0.02). Serum leptin levels were higher in high CD133 group compared to low CD133 group [32.37(12.74-72.29) vs 15.50(5.38-37.12)ng/mL, p = 0.03]. Serum leptin levels were correlated with TNF-α(r = 0.35, p = 0.009). Serum adiponectin levels were negatively correlated with serum leptin (r = -0.28, p = 0.03). Serum resistin levels were associated with TNF-α (r = 0.54, p < 0.001) and leptin (r = 0.29, p = 0.03). Serum IL-6 levels were significantly associated with LVMI (r = 0.31, p = 0.03). Serum IL-6 levels were significantly higher in patients with carotid plaque compared to patients without plaque [12.75(9.91-28.68) vs 8.27(5.97-14.04) pg/mL, p = 0.02]. In multiple linear regression analysis to determine the factors predicting LogFMD; dialysis vintage, LVMI and LogCD133+ cell counts were included as independent variables(R = 0.57, adjusted R-square = 0.27, p = 0.001). CD133+ cell count and LVMI were found to significantly predict FMD (p = 0.03 and p = 0.04 respectively). CONCLUSION: CD133+ cells were associated with inflammation and endothelial dysfunction in HD patients. Serum leptin, resistin and TNF-α levels were positively related to CD133+ cell count. Impaired regulation of undifferentiated stem cells and adipocytokines might contribute to endothelial dysfunction in HD patients.

A novel pathogenic frameshift variant of CD3E gene in two T-B+ NK+ SCID patients from Turkey.

Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiency, which is characterized by the dysfunction and/or absence of T lymphocytes. Early diagnosis of SCID is crucial for overall survival, and if it remains untreated, SCID is often fatal. Next-generation sequencing (NGS) has become a rapid, high-throughput technology, and has already been proven to be beneficial in medical diagnostics. In this study, a targeted NGS panel was developed to identify the genetic variations of SCID by using SmartChip-TE technology, and a novel pathogenic frameshift variant was found in the CD3E gene. Sanger sequencing has confirmed the segregation of the variant among patients. We found a novel deletion in the CD3E gene (NM000733.3:p.L58Hfs*9) in two T-B+ NK+ patients. The variant was not found in the databases of dbSNP, ExAC, and 1000G. One sibling in family I was homozygous and the rest of the family members were heterozygous for this variant. T cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) analyses were performed for T and B cell maturation. TRECs were not detected in both patients and the KREC copy numbers were similar to the other family members. In addition, heterozygous family members showed decreased TREC levels when compared with the wild-type sibling, indicating that carrying this variant in one allele does not cause immunodeficiency, but does effect T cell proliferation. Here, we report a novel pathogenic frameshift variant in CD3E gene by using targeted NGS panel.