Analysis of peripheral lymphocyte subsets and T-cell exhaustion in SARS-CoV-2 Infection.

Introduction: : Immune responses against Coronavirus (SARS-CoV-2) may be highly complex. It has been suggested that T-cell fatigue develops due to continuous stimulation of T-cells by SARS-CoV-2 in Coronavirus disease-2019 (COVID-19). It was aimed to assess peripheral lymphocyte subsets and T-cell exhaustion in various clinical courses of the disease in patients diagnosed with COVID-19. Materials and Methods: This study included 150 patients who were assigned into the "mild-to-moderate disease" group, or "severe disease" group based on their clinical and laboratory characteristics. Peripheral lymphocyte subsets and T-cell exhaustion markers [programmed cell death protein 1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (Tim-3)] were determined in the peripheral blood using flow cytometry. Result: Mean (±SD) age was 53.3 ± 14.5 years, and female to male ratio was 55/95. In the mild-to-moderate disease (MMD) group, 55 patients had pneumonia and 20 patients had COVID-19 without pneumonia. In the severe disease (SD) group, 43 patients had severe pneumoniae and 32 patients were in critical condition. Lymphocyte counts were less than 1.0 x 109/L in 69.3% of the patients in the SD group, and the difference between the MMD group and SD group was statistically significant (p= 0.001). Total T cells, CD4+ and CD8+ T-cell counts were significantly lower in the SD group vs. MMD group (p< 0.001, p< 0.001, p< 0.001, respectively). PD-1 expression by CD8+ and CD4 T+ cells was higher (p= 0.042, p= 0.029, respectively) and Tim-3 expression from CD4 T+ cells was lower (p= 0.000) in the SD group vs. MMD group. Serum IFN-γ levels were not statistically different in the MMD and SD groups (p= 0.2). Conclusions: T-cell counts may be significantly reduced along with an increased expression of the T-cell exhaustion marker PD-1 in severe COVID-19, but Tim-3 expression was not increased in our study patients.

Evaluating the effect of donor anxiety levels and lifestyle characteristics on the activation of platelet concentrates.

BACKGROUND: Smoking, alcohol use, performing regular physical exercise, dietary habits, and anxiety level may cause platelet activation. We aimed to evaluate the anxiety levels, smoking status, alcohol intake, and sportive habits of donors, and determine their impact on the quality of apheresis-platelets. METHODS: State and Transient Anxiety Inventory (STAI) was used to determine the level of donors' anxiety. STAI has two subscales: S-anxiety scale (STAI-I) and T-anxiety scale (STAI-II), each comprising 20 questions rated on a 4-point Likert scale. Data on smoking, alcohol consumption, and performing regular physical exercise were obtained from a questionnaire filled out before donation. Flow cytometric analysis was used to quantify activated platelets. RESULTS: The STAI-I level of 86 participants was normal, while that of 12 was higher. No significant difference was found in the active platelet absolute count [1.8×1011 (2.7) and 1.4×1011 (1.3), respectively; P=0.665] between donors with normal STAI-I levels and those with higher STAI-I levels. Of 98 donors, 42 had normal STAI-II levels, while 56 had higher STAI-II levels. No significant difference was found in the active platelet absolute count [2.3×1011 (3.1) and 1.5×1011 (2.3), respectively; P=0.224] between donors with normal STAI-II levels and those with higher STAI-II levels. Platelet counts of individuals who perform regular physical exercise were significantly higher than those of individuals who did not perform regular physical exercise (6.3±1.4×1011 vs. 5.5±1.4×1011). CONCLUSION: The quality of apheresis platelets is not affected by anxiety levels and lifestyle characteristics of blood donors. There is no need to organize apheresis blood donor pool considering with these subjects.

Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity.

OBJECTIVE: In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs), platelet degranulation, and release of platelet-derived growth factors (PDGFs). We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation. MATERIALS AND METHODS: Apheresis platelet concentrates (APCs) from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL) were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing. RESULTS: The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/µL vs. 319.9±80.5/µL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively), but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<.001) than those of fresh APCs. The mean endogenous thrombin potential (ETP) of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001). Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014). CONCLUSION: Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused by the freezing process and the scarcity of evidence for their in vivo superiority, frozen platelets should be considered for use in austere environments, reserving fresh platelets for prophylactic use in blood banks.

Is one-size-fits-all strategy adequate for platelet storage?

BACKGROUND: Platelet suspensions (PSs) are stored at room temperature. However, recent reports show that PSs stored at 4 °C possess superior hemostatic properties. We compared the viabilities and thrombin generation capacities of PSs stored either at 4 °C or 22 °C hours. MATERIALS AND METHODS: Twenty units of apheresis derived platelets (ADPs) from 20 male donors and 20 units of random platelet suspensions (RPSs) from another 20 male donors were obtained. Half of the ADPs and half of the RPSs (10 units/per group) were stored at 4 °C, the other halves of ADPs and RPSs (10 units/per group) were stored in agitators at 22 °C for 48 hours. The flow cytometric viability tests and thrombin generation tests of the PSs were assessed. RESULTS: The viabilities of both ADPs and RPSs group platelets, stored either at 4 °C or 22 °C for 48 hours, were not statistically significantly different. The ADPs and RPSs stored at 4 °C generated significantly higher peak thrombin levels than the platelets stored at 22 °C. Moreover, the ADPs group stored at 4 °C showed significantly shorter time to thrombin generation and reach peak levels. CONCLUSION: The PSs stored at 4 °C showed higher and faster thrombin generation capacities than the room temperature PSs. Given the superior hemostatic properties of refrigerated platelets, creating different storage temperature capabilities for specific transfusion purposes may be a prudent approach, especially for improving the outcome of bleeding trauma casualties.

Killer-cell immunoglobulin-like receptor and human leukocyte antigen-C genes in common variable immunodeficiency.

BACKGROUND: We aimed herein to investigate the killer-cell immunoglobulin-like receptor (KIR) genes and human leukocyte antigen (HLA)-C alleles in patients with common variable immunodeficiency (CVID), and to reveal their differences from those in healthy population. METHODS: In all, 18 patients who have been diagnosed with CVID and 15 living donors of kidney transplant recipients were enrolled in the study. Polymerase chain reaction-sequence-specific primer (PCR-SSP) typing method was used in molecular genetic analysis. The frequencies of the genes in the study groups were statistically compared with each other using chi-square or Fisher exact tests, whichever were appropriate. RESULTS: Although there was no significant difference between both study groups with respect to distribution of KIR and HLA-C2 group genes, HLA-Cw7 allele frequency in patients with CVID was significantly lower than that in healthy population (P = 0.008). CONCLUSION: This present study results support that HLA-Cw7 allele, an inhibitor of KIR ligand, may play a role in the pathogenesis of CVID.

Freezing of Apheresis Platelet Concentrates in 6% Dimethyl Sulfoxide: The First Preliminary Study in Turkey.

OBJECTIVE: Transfusion of platelet suspensions is an essential part of patient care for certain clinical indications. In this pioneering study in Turkey, we aimed to assess the in vitro hemostatic functions of platelets after cryopreservation. MATERIALS AND METHODS: Seven units of platelet concentrates were obtained by apheresis. Each apheresis platelet concentrate (APC) was divided into 2 equal volumes and frozen with 6% dimethyl sulfoxide (DMSO). The 14 frozen units of APCs were kept at -80 °C for 1 day. APCs were thawed at 37 °C and diluted either with autologous plasma or 0.9% NaCl. The volume and residual numbers of leukocytes and platelets were tested in both before-freezing and post-thawing periods. Aggregation and thrombin generation tests were used to analyze the in vitro hemostatic functions of platelets. Flow-cytometric analysis was used to assess the presence of frozen treated platelets and their viability. RESULTS: The residual number of leukocytes in both dilution groups was <1x106. The mean platelet recovery rate in the plasma-diluted group (88.1±9.5%) was higher than that in the 0.9% NaCl-diluted group (63±10%). These results were compatible with the European Directorate for the Quality of Medicines quality criteria. Expectedly, there was no aggregation response to platelet aggregation test. The mean thrombin generation potential of post-thaw APCs was higher in the plasma-diluted group (2411 nmol/L per minute) when compared to both the 0.9% NaCl-diluted group (1913 nmol/L per minute) and the before-freezing period (1681 nmol/L per minute). The flow-cytometric analysis results for the viability of APCs after cryopreservation were 94.9% and 96.6% in the plasma and 0.9% NaCl groups, respectively. CONCLUSION: Cryopreservation of platelets with 6% DMSO and storage at -80 °C increases their shelf life from 7 days to 2 years. Besides the increase in hemostatic functions of platelets, the cryopreservation process also does not affect their viability rates.

Anticardiolipin Antibodies in Children with Helicobacter pylori Infection.

BACKGROUND: Anticardiolipin (aCL) antibodies are associated with thrombosis and have an important role in the etiology of diseases such as stroke and myocardial infarction whose etiologies were based on thrombosis. H. pylori has been proposed to be responsible for the pathophysiology of some diseases including stroke, myocardial infarction, thrombosis, and autoimmune diseases. From this point of view, we hypothesized a possible relationship between H. pylori infection and aCL antibodies and initially aimed to determine the prevalence of aCL antibody positivity in children with H. pylori infection. MATERIALS AND METHODS: Anticardiolipin antibodies were studied in 84 patients before and after eradication therapy and in a control group including 40 children. RESULTS: The pretreatment aCL IgA (median 12.78 APL/mL), aCL IgM (median 21.60 MPL/mL), and aCL IgG antibody levels (median 14.22 GPL/mL) were significantly higher than those of post-treatment results (median 5.38 APL/mL, 7.02 MPL/mL, and 6.64 GPL/mL, respectively) and controls (median 5.90 APL/mL, 4.80 MPL/mL, and 4.81 GPL/mL, respectively). Anticardiolipin antibodies revealed no significant differences between the study group after therapy and the control group. CONCLUSIONS: In our particular experience, H. pylori can cause aCL antibody positivity in children and eradication of H. pylori provides the disappearance of these antibodies.

Elevated Urinary T Helper 1 Chemokine Levels in Newly Diagnosed Hypertensive Obese Children.

OBJECTIVE: Increasing evidence suggests that T helper (Th) cells play a significant role in the pathogenesis of hypertension. The aim of this study was to evaluate the effect of obesity and anti-hypertensive treatment on urinary Th1 chemokines. METHODS: The study groups consisted of three types of patients: hypertensive obese, healthy, and non-hypertensive obese. Pre-treatment and post-treatment samples of the hypertensive obese group and one sample from the other two groups were evaluated for urinary chemokine: regulated on activation, normal T cell expressed and secreted (RANTES), interferon-gamma-inducible protein 10 (IP10), and monokine induced by interferon-gamma (MIG). In the hypertensive obese group, urine microalbumin: creatinine ratio was examined before and after treatment. We recommended lifestyle changes to all patients. Captopril was started in those who could not be controlled with lifestyle changes and those who had stage 2 hypertension. RESULTS: Twenty-four hypertensive obese (mean age 13.1), 27 healthy (mean age 11.2) and 22 non-hypertensive obese (mean age 11.5) children were investigated. The pre-treatment urine albumin: creatinine ratio was positively correlated with pre-treatment MIG levels (r=0.41, p<0.05). RANTES was significantly higher in the pre-treatment hypertensive and non-hypertensive obese group than in the controls. The urinary IP10 and MIG levels were higher in the pre-treatment hypertensive obese group than in the non-hypertensive obese. Comparison of the pre- and post-treatment values indicated significant decreases in RANTES, IP10, and MIG levels in the hypertensive obese group (p<0.05). CONCLUSION: Th1 cells could be activated in obese hypertensive children before the onset of clinical indicators of target organ damage. Urinary RANTES seemed to be affected by both hypertension and obesity, and urinary IP10 and MIG seemed to be affected predominantly by hypertension.

The expression of transmembrane and soluble CXCL16 and the relation with interferon-alpha secretion in patients with Behçet's disease.

OBJECTIVES: CXCL16 is a member of CXC chemokine, which is synthesised in plasmacytoid dendritic cell as a transmembrane molecule. Transmembrane CXCL16 on plasmacytoid dendritic cell plays a role in binding, uptaking and accumulation of CpG D ODN in early endosomes rather then lysosomal vesicles, thereby causing a high level of interferon-alpha secretion. Previously, we disclosed pronounced interferon-alpha production from these cells in patients with Behçet's disease. The aim of this study was to investigate the relation between the secretion of IFN-α and the expression of CXCL16 on surface of plasmacytoid dendritic cell from patients with Behçet's disease, and compare it with patients with ankylosing spondylitis and healthy controls. METHODS: The study population consisted of 73 cases (35 with Behçet's disease, 19 with ankylosing spondylitis and 19 controls). We investigated the expression of CXCL16 on surface of plasmacytoid dendritic cells by flow cytometry, and the serum levels of IFN-α and CXCL16 with ELISA. RESULTS: Serum levels of IFN-α in patients with Behçet's disease were significantly higher than the controls (p=0.009), and than patients with ankylosing spondylitis, but not statistically significant (p=0.124). Serum levels of CXCL16 in patients with Behçet's disease and patients with ankylosing spondylitis were significantly higher than controls (p=0.009, p=0.003, respectively). We found no difference in the percentage and MFI of plasmacytoid dendritic cells and CD123+CXCL16+ cells determined by flow cytometry among the study and control groups. In patients with Behçet's disease, a positive correlation was found between the percentage of plasmacytoid dendritic cells and CD123+CXCL16+ cells (p<0.001). Furthermore, there was also a positive correlation between the percentage of plasmacytoid dendritic cells and serum levels of CXCL16 in patients with ankylosing spondylitis (p=0.001). In addition, there was a positive correlation between the percentage of CD123+CXCL16+ cells and serum levels of IFN-α in Behçet's disease group (p=0.034). We could not find any significant difference in other comparisons. CONCLUSIONS: We suggested that the expression of transmembrane CXCL16 on surface of plasmacytoid dendritic cell might contribute to high serum IFN-α levels seen in patients with BD.

A polymorphism in ERAP1 is associated with susceptibility to ankylosing spondylitis in a Turkish population.

We assessed the role played by the ERAP1 gene in Turkish patients with ankylosing spondylitis (AS) in terms of disease susceptibility, clinical manifestations, and disease severity. We included 150 consecutive AS patients who met the modified New York classification criteria and 150 healthy controls. We documented the presence of 10 ERAP1 single-nucleotide polymorphisms (SNPs) and HLA-B27 in these patients. ERAP1 SNPs were genotyped using competitive allele-specific polymerase chain reaction. Differences between genotype and allele frequencies were compared using the Pearson's Chi-square test. The associations between ERAP1 SNPs, on the one hand, and with disease severity and clinical findings, on the other, were determined. One SNP, rs26653, was significantly associated with AS susceptibility (OR 1.609, 95% CI 1.163-2.226; p = 0.004). The population-attributable risk of possession of the rs26653 SNP allele was 23.4%. No relationship was noted between HLA-B27 positivity and the distribution of rs26653 genotype frequency. No associations were seen between disease severity measures and clinical manifestations of AS. In summary, an ERAP1 polymorphism was associated with AS in a Turkish population. The contributions of HLA-B27 and the rs26653 SNP to AS pathogenesis appear to be independent.

Immunosuppressive effects of multipotent mesenchymal stromal cells on graft-versus-host disease in rats following allogeneic bone marrow transplantation.

OBJECTIVE: Graft-versus-host disease (GVHD) is a major obstacle to successful allogeneic bone marrow transplantation (allo-BMT). While multipotent mesenchymal stromal cells (MSCs) demonstrate alloresponse in vitro and in vivo, they also have clinical applications toward prevention or treatment of GVHD. The aim of this study was to investigate the ability of MSCs to prevent or treat GVHD in a rat BMT model. MATERIALS AND METHODS: The GVHD model was established by transplantation of Sprague Dawley rats' bone marrow and spleen cells into lethally irradiated (950 cGy) SDxWistar rat recipients. A total of 49 rats were randomly assigned to 4 study and 3 control groups administered different GVHD prophylactic regimens including MSCs. After transplantation, clinical GVHD scores and survival status were monitored. RESULTS: All irradiated and untreated control mice with GVHD died. MSCs inhibited lethal GVHD as efficiently as the standard GVHD prophylactic regimen. The gross and histopathological findings of GVHD and the ratio of CD4/CD8 expression decreased. The subgroup given MSCs displayed higher in vivo proportions of CD25+ T cells and plasma interleukin-2 levels as compared to conventional GVHD treatment after allo-BMT. CONCLUSION: Our results suggest that clinical use of MSCs in both prophylaxis against and treatment of established GVHD is effective. This study supports the use of MSCs in the prophylaxis and treatment of GVHD after allo-BMT; however, large scale studies are needed. CONFLICT OF INTEREST: None declared.

Proteasome inhibitor bortezomib increases radiation sensitivity in androgen independent human prostate cancer cells.

OBJECTIVES: To investigate the effects of a strong proteasome inhibitor, bortezomib alone or in combination with radiotherapy on androgen-independent DU145 human prostate cancer cells. Proteasomes play important roles in cell cycle, proliferation, apoptosis, angiogenesis, and cellular resistance to chemotherapy and radiotherapy. METHODS: Increasing concentrations of bortezomib alone or in combination with radiation were applied to DU145 cells and IC(50) values that inhibited cell growth by 50% were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium-bromide assay. Apoptosis was determined using annexin V staining by flow cytometry. mRNA levels of proapoptotic caspase-3 and antiapoptotic Bcl-2 genes were examined by reverse transcriptase polymerase chain reaction. RESULTS: The IC(50) value of bortezomib was found to be 28 microm although 400- and 800-cGy radiation decreased the cell proliferation by 14% and 28%, respectively. In 400- and 800-cGy radiation applied DU145 cells, IC(50) value of bortezomib decreased to 23- and 12 microm, respectively. Exposure to 5 microm bortezomib for 48 hours caused apoptosis in 35% of the population whereas 800-cGy radiation resulted apoptosis in 14% of cells. However, 42% of DU145 cells that were exposed to 800 cGy and 5 microm bortezomib underwent apoptosis. Reverse transcriptase polymerase chain reaction results showed a significant decrease in mRNA levels of antiapoptotic Bcl-2 gene and an increase in proapoptotic caspase-3 gene expression in the combination group compared to control group. CONCLUSIONS: Bortezomib increases radiation sensitivity in androgen-independent human DU145 prostate cancer cells through inhibition of Bcl-2 and induction of caspase-3 genes.

Platelet-derived microparticle levels in women with recurrent spontaneous abortion.

OBJECTIVE: To investigate the significance of platelet-derived microparticles (PMPs) in women with recurrent spontaneous abortion. METHODS: We measured platelet P-selectin (CD62P) as a platelet activation marker and CD42b(+) microparticles as PMPs by flow cytometry in whole blood of 20 women with recurrent spontaneous abortion and 20 age-matched healthy controls with no previous history of spontaneous abortion. RESULTS: PMP levels in women with recurrent spontaneous abortion were higher than in women in the control group (4.79+/-1.18% vs 3.06+/-0.92%; P<0.000). CD62P levels were not significantly higher in the study group compared with the control group (13.78+/-8.62% vs 10.78+/-7.35%; P>0.05). CONCLUSION: Our findings suggest that PMPs may have a role in the pathogenesis of recurrent spontaneous abortion.

Diagnostic utility of anti-cyclic citrullinated peptide and anti-modified citrullinated vimentin antibodies in rheumatoid arthritis.

Several autoantibodies found in RA are directed to epitopes in citrullinated proteins. One of them is anti modified citrullinated vimentin (Anti-MCV). We tested the value a newly developed ELISA for the detection of antibodies against a genetically modified citrullinated vimentin (anti-MCV) in comparison with an anti-CCP based ELISA system for the diagnosis of RA. Thirty-five patients with RA (mean age; 42.6 +/- 10.87 years, mean disease duration; 9.37 +/- 3.98 years) were enrolled in this study. Twenty -five ankylosing spondylitis (mean age; 35.88 +/- 6.64 years, mean disease duration; 10.25 +/- 4.61 years), and 19 healthy subjects (mean age; 40.26 +/- 5.11 years) served as controls. Anti-CCP antibodies and Anti-MCV antibodies were measured using ELISA. In all RA patients, mean anti- CCP level was 69.07 +/- 90.43 U/ml and anti-MCV level was 665.77 +/- 1040.19 U/ml. In patients with AS, the mean anti-CCP level was 10.7 +/- 5.22 U/ml and anti-MCV level was 40.54 +/- 20.15 U/ml. In healthy controls, the mean anti-CCP level was 11.11 +/- 7.65 U/ml, anti-MCV level was 23.12 +/- 12.04 U/ml. In patients with active RA, the mean serum anti-CCP level was 100.54 +/- 98.07 U/ml and anti-MCV level was 998.74 +/- 1154.93 U/ml. In patients with inactive RA, the mean serum anti-CCP level was 8.77 +/- 1.55 U/ml and anti-MCV level was 27.59 +/- 23.10 U/ml. According to these results; In patients with RA, the mean serum anti-MCV and anti-CCP levels were significantly high compared to patients with AS and healthy controls (p=0.002, p=0.001, p=0.002, p=0.001 respectively). The mean serum anti-MCV and anti- CCP levels were significantly higher in active patients with RA than in inactive patients with RA patients (p=0.001 and p=0.001 respectively). In inactive patients with RA, the mean serum anti-MCV and anti-CCP levels were similar in patients with AS and patients (p=0.484, p=0.308, p=0.09 and p=0.222 respectively). The mean serum anti-MCV levels were correlated with DAS 28 (r=0.531, p=0.001), VAS score (r=0.332, p=0.01), ESR (r=0.458, p=0.001), serum CRP levels (r=0.568, p=0.01), serum RF levels (r=0.529, p=0.001), swollen joints number (r=0.525, p=0.001) and tender joints number (r=0.638, p=0.001). As a result; measurement of serum anti-MCV levels is useful for diagnosis of RA and combined use of anti-MCV and RF may be more useful prognostic factor than either method alone, RF and anti-CCP.

Optimization of transfection of green fluorescent protein in pursuing mesenchymal stem cells in vivo.

OBJECTIVE: Green Fluorescent Protein (GFP) has been used as a marker of gene expression and a single cell marker in living organisms in cell biology studies. The important areas that GFP is used are expression levels of different genes in different organisms by inserting GFP in these genes and as a marker in living cells. In this study, we tried to optimize transfection of mesenchymal stem cells, (MSCs) used for regeneration of damaged tissues in animals, by GFP containing plasmid vector by which MSCs can be followed in vivo. METHODS: To this aim, phM-GFP plasmid vector carrying GFP gene and effectene transfection reagent were used. RESULTS: The data revealed that twice transfection of MSCs resulted in higher expression of GFP for longer times as compared to once transfected MSCs. On the other hand, leaving the chemical transfection agents in the medium induced apoptosis after a while. CONCLUSION: As a conclusion we suggest the transfection of MSCs twice with 48 hours interval and removal of transfection agents after 8 hours which removed toxic and apoptotic effects of the chemicals.

Cytotoxic effects of four different endodontic materials in human periodontal ligament fibroblasts.

The purpose of this study was to compare the cytotoxicity, induced apoptosis and/or necrosis, and apoptotic mechanisms in human periodontal ligament (PDL) fibroblasts treated with four different endodontic materials: White ProRoot mineral trioxide aggregate (MTA) (MTA/Dentsply; Tulsa Dental, Memphis, TN), Diaket (ESPE, Seefeld, Germany), Endion (VOCO, Cuxhaven, Germany), and CYMED 8410 (NANO, Kaohsiung, Taiwan). The effects of these four materials on the viability of PDL fibroblasts were determined by MTT (3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-SH-tetrazolium bromide) assay. Apoptotic pathways were evaluated via several mechanisms. Exposure to MTA for 24, 48, and 72 hours resulted in no significant differences in MTT reduction and viable cell number compared with controls. However, treatment of PDL fibroblasts with Diaket, Endion, and CYMED 8410 for 24, 48, and 72 hours resulted in cytotoxicity with MTT and a reduction of viable cell number with trypan blue dye exclusion test compared with controls (from p < 0.05 to p < 0.001). Annexin V-FITC/PI staining showed that Diaket, Endion, and CYMED 8410 induced higher percentages of apoptosis and/or necrosis than in controls (45.6%, 25.5%, and 6.3%, respectively). Results of cell-cycle analyses were concordant with annexin V-FITC/PI staining findings. These results suggest that MTA is a very biocompatible filling material. However, Diaket, Endion, and CYMED 8410 are toxic to PDL fibroblasts in vitro. The main form of cell death induced by these filling materials was determined to be apoptosis and/or necrosis.

Synovial angiostatic non-ELR CXC chemokines in inflammatory arthritides: does CXCL4 designate chronicity of synovitis?

In our previous studies, we found higher synovial fluid (SF) levels of angiogenic ELR(+) CXC chemokines such as CXCL1, CXCL5, CXCL6 and CXCL8, which play an important role in neutrophil migration and angiogenesis, and more abundant synovial CXCR2 chemokine receptor expression in patients with rheumatoid arthritis (RA) than those with Behçet's disease (BD), familial Mediterranean fever and osteoarthritis (OA). As a continuation of our previous studies, we investigated synovial levels of angiostatic non-ELR CXC chemokines (CXCL4, CXCL9 and CXCL10) in patients with RA, BD, spondyloarthritis (SpA), and OA. Seventy (17 RA, 15 BD, 19 SpA, and 19 OA) patients were enrolled in the study. The levels of CXCL4, CXCL9, and CXCL10 were measured by ELISA. The SF levels of CXCL4 in patients with RA were higher than those of the patients with BD, SpA, and OA (P = 0.007, P = 0.022, and P = 0.017, respectively). No difference was found with respect to CXCL4 levels among the BD, SpA, and OA patients. The synovial CXCL9 levels of patients with RA and SpA were found to be higher than those of the patients with OA (P = 0.002 and P = 0.005, respectively), while no statistically significant difference was detected among the other groups. With regard to SF CXCL10 levels, patients with RA had higher levels as compared to patients with OA (P = 0.002), but no significant difference was found among the other groups. CXCL9 correlated with CXCL4 and CXCL10 (P < 0.05 for both) in patients with RA. No correlation was found in other parameters. The angiostatic non-ELR CXC chemokines were expressed in synovial inflammation. We proposed that angiostatic non-ELR CXC chemokines may increase to balance angiogenic ELR (+) CXC chemokines in which increased levels were shown in patients with inflammatory arthritides and CXCL4 may contribute to designate the chronicity of synovitis in patients with RA. In addition, as CXCL-9 and CXCL-10 play crucial role in inflammation characterized by Th1 polarization, we suggested that they may contribute to the commencement and the perpetuation of synovitis seen in these groups of arthritides.

Serum proinflammatory cytokines directing T helper 1 polarization in patients with familial Mediterranean fever.

Th1 type polarization has been implicated in the pathogenesis of familial Mediterranean fever (FMF). Interleukin-12 (IL-12) and IL-10 are proinflammatory cytokines, which play crucial role in Th1 and Th2 type immune response, respectively. IL-18 has a dual effect on T cell response: it was recognized as an IFN-gamma-inducing factor in T cells; acting in synergy with IL-12, leading to the development of Th1 type immune responses. But, in the absence of IL-12, IL-18 can promote the production of Th2 cytokines and take part in allergic inflammation. The aim of this study is to measure serum levels of IL-10, IL-12, and IL-18 in patients with FMF, and to investigate the relationship of their expressions with FMF attacks. Serum IL-10, IL-12, and IL-18 levels from patients with FMF were investigated. Thirty-one FMF patients with attack-free, 24 FMF patients with attack and 20 healthy controls were enrolled in the study. The levels of IL-10, IL-12p70 and IL-18 were measured by ELISA. Serum IL-10 levels were not different in FMF patients with attack and attack-free, and healthy controls. Serum IL-12 levels in FMF patients both with attack and attack-free were significantly higher than healthy controls (P = 0.002 and P = 0.047, respectively). There were no differences between FMF patients with attack and attack-free with regard to serum IL-12 levels. Serum IL-18 levels in FMF patients with attack and attack-free were significantly higher than healthy controls (P < 0.001 for both groups). With respect to serum IL-18 levels, no difference was found between FMF patients with attack and attack-free. Our results suggest that IL-12 and IL-18 contribute to the establishment of Th1 polarization seen in FMF and play a part in its pathogenesis. Detection of increased levels of IL-12 and IL-18 in patients with inactive disease implies that they seem to assist Th1 activation and subclinical inflammation persisting during the attack-free period of the disease.

Expression of CXCR-1 and CXCR-2 chemokine receptors on synovial neutrophils in inflammatory arthritides: does persistent or increasing expression of CXCR-2 contribute to the chronic inflammation or erosive changes?

OBJECTIVE: To analyze the CXCR-1 and CXCR-2 chemokine receptor expression on peripheral blood neutrophils (PBN) and synovial fluid neutrophils (SFN) of patients with rheumatoid arthritis (RA) and Behçet's disease (BD) (characterized by erosive and non-erosive arthritis, respectively), and to compare them with those of patients with osteoarthritis (OA). METHODS: We used flow cytometry to investigate the expression of CXCR-1 and CXCR-2 chemokine receptors on PBN and SFN of fifty-five (22 RA, 22 BD and 11 OA) age and sex-matched patients. RESULTS: In respect to chemokine receptor expression on neutrophils isolated from patients with RA, mean fluorescein intensity (MFI) of CXCR-1 chemokine receptors on PBN from active and inactive RA patients, and SFN from patients with RA were 151 (90-395), 129 (81-539) and 136 (64-220), respectively, and there were not statistically significant difference each other. But MFI of CXCR-2 chemokine receptors on SFN of patients with RA was 18 (10-32), and significantly higher than PBN of active and inactive RA patients (MFI: 10 (6-15) and 12 (7-16), P=0.002 and 0.037, respectively). In respect to chemokine receptor expression on neutrophils isolated from patients with BD, MFI of CXCR-1 chemokine receptors on PBN of active BD patients was 245 (97-844), and higher than PBN of active RA patients and SFN of BD patients (MFI: 151 (90-395) and 134 (61-231), P=0.047 and 0.017, respectively). MFI of CXCR-2 chemokine receptors on PBN of active and inactive BD patients, and SFN of patients BD were 10 (6-14), 10 (2-16), and 12 (8-24), respectively, there were not statistically significant difference each other. MFI of CXCR-1 chemokine receptors on SFN from patients with RA, BD, and OA were 136 (64-220), 134 (61-231), and 114 (60-180), respectively, and there was no difference between the study groups. MFI of CXCR-2 chemokine receptors on SFN of patients with RA was 18 (10-32), and higher than patients with BD and OA (MFI: 12 (8-24) and 11 (9-18), P=0.037 and 0.005, respectively), though there was no difference between last two groups. CONCLUSION: Our study points that CXCR-1 and CXCR-2 chemokine receptors of SFN may have diverse functions in the course of inflammatory arthritides. These results indicate that CXCR-2 chemokine receptor might play more critical role in long lasting accumulation of neutrophils within the synovial fluid of patients with RA.