Ramazan Gunesacar

Analysis of CD28 and CTLA‐4 gene polymorphisms in Turkish patients with Behcet's disease

In this study we aimed to investigate IVS3 +17T/C single nucleotide polymorphism (SNP) of CD28 gene, +49A/G and −318C/T SNPs of CTLA‐4 gene in patients with Behçets disease (BD) and their potential association to the main clinical features of the disease. These polymorphisms were investigated in a Turkish population of 123 patients with BD and 179 healthy controls, by using PCR‐RFLP technique. HLA‐B*51 genotype was also studied in both groups by using PCR‐SSP. The frequency of IVS3 +17TC genotype of the CD28 gene was significantly increased in BD patients compared to controls (43.6% vs. 31.2%, OR = 1.663, 95% CI = 1.033–2.679, P = 0.039). CTLA‐4 +49GG genotype frequency was found to be significantly lower in patients with BD than those of healthy controls (4% vs. 10.6%, OR = 0.357, 95% CI = 0.130–0.983, P = 0.05). Genotype and allele frequencies of the CTLA‐4–318C/T polymorphism between the BD and healthy control groups were not significantly different (12.2% vs. 10.6%, OR = 1.170, 95% CI = 0.570–2.402, P = 0.713). There were no associations between the studied polymorphisms and the main clinical features of BD. The frequencies of HLA‐B*51 were 60.3% and 30.7% in BD and control groups, respectively (OR = 3.429, 95% CI = 2.115–5.559, P = 0.0001). Association between HLA‐B*51 and each studied polymorphism did not reach to significant levels (OR = 0.479, 95% CI = 0.228–1.004, P = 0.064 for CD28 IVS3 +17TT genotype; OR = 2.180, 95% CI = 1.025–4.639, P = 0.061 for TC genotype; OR = 1.570, 95% CI = 0.870–2.836, P = 0.146 for C allele). These results may suggest that CD28 IVS3 +17TC genotype may be a risk factor for the development of BD, on the contrary CTLA‐4 +49GG genotype may be protective in the studied Turkish population.

Interleukin-10 and -12 in human milk at 3 stages of lactation: a longitudinal study.

This study was undertaken to analyze postpartum changes in concentrations of interleukin (IL)-10 and IL-12 through the 3 stages of lactation. A total of 87 human milk samples were collected from 29 healthy mothers during the colostrum (0–3 days), early milk (14–17 days), and mature milk (44–47 days) phases. Enzyme-linked immunosorbent assay tests were performed on the milk samples. IL-10 was detected in 7 and IL-12 in 4 of the colostrum samples. In the transitional milk samples, IL-10 was present in 4 and IL-12 in 2; however, both of these cytokines became undetectable in mature milk samples. The decrease in concentrations of IL-10 and IL-12 was statistically significant during the postpartum period (P=.001 and P=.024, respectively). IL-10 levels in the colostrum samples were higher than in the transitional samples (P=.018, with use of the post hoc test). No statistically significant differences between IL-12 levels were noted in the colostrum samples and the transitional samples (P=.068, with use of the post hoc test). A negative correlation was observed between concentrations of IL-10 in colostrum and the total number of pregnancies (R=−.401;P=.031). The findings of the present study suggest that mean concentrations of IL-10 and IL-12 are decreased in human milk as lactation continues through its 3 phases.

Expression of soluble CD27 and interleukins-8 and -10 in B-cell chronic lymphocytic leukemia: Correlation with disease stage and prognosis

Investigators in this study explored levels of soluble CD27 (sCD27), interleukin (IL)-8, and IL-10 in B-cell chronic lymphocytic leukemia (B-CLL), and the correlation of these levels with disease stage and prognosis. Plasma IL-8, IL-10, and sCD27 levels were assessed with enzyme-linked immunosorbent assay tests in 22 healthy donors and 70 patients with B-CLL (49 men and 21 women). Mean patient age was 61.57 y (range, 44–75 y). Mean healthy donor age was 62.09 y (range, 40–72 y). In the study group, mean values were as follows: plasma IL-8, 284.758 pg/mL (0–1000 pg/mL); plasma IL-10, 26.152 pg/mL (0–100 pg/mL); sCD27, 731.357 U/mL (139.9–1000 U/mL); white blood cell count, 59.9 × 109/L (0.8–250.0 × 109/L); hemoglobin count, 11.2 g/dL (5.0–16.2 g/dL); platelet count, 162.5 × 109/L (29.8–317 × 109/L); B2 microglobulin (B2M) 3350.2 mg/L (274.7–7499.9 mg/L); CD38, 19.5%; and lactate dehydrogenase (count, 497.5 U/L (263.0–1507 U/L). Patients represented all Rai stages, with 22.9% at stage 0, 11.4% at stage I, 11.4% at stage II, 41.4% at stage III, and 12.9% at stage IV. Plasma levels of IL-8, IL-10, and sCD27 were correlated between study and control groups; significantly higher IL-8 (P=.001) and sCD27 (P=.000) levels were found, but the IL-10 level was not significant (P=.139). Plasma IL-10 (P=.01) and sCD27 (P=.008) were positively correlated with Rai stage, but IL-8 was not (P=.146). Levels of sCD27 were significantly correlated with values for B2M (P=.000), hemoglobin (P=.028), lactate dehydrogenase (P=.001), CD19 (P=.03), and IL-10 (P=.000). IL-8 was significantly correlated with white blood cell (P=.000) count, and CD38 (P=.001) and CD5 (P=.006) levels. IL-10 was significantly correlated with B2M (P=.017), CD19 (P=.000), platelet (P=.002), and CD27 (P=.000). In survival distributions for CD27, IL-8 and IL-10 were found to have more significant relationships for all parameters (P=.0000). In conclusion, the authors suggest that sCD27, IL-8, and IL-10 are more significant prognostic factors for B-CLL when compared with others, and these values should correlate with new prognostic factors (eg, zeta-associated protein-70, mutated/unmutated immunoglobulin variable heavy chain).

Evaluation of nitrite/nitrate levels in relation to oxidative stress parameters in liver cirrhosis

BACKGROUND AND OBJECTIVE Nitric oxide and reactive oxygen species have been implicated in several pathophysiological events leading to fibrosis and cirrhosis. The aim of the present study was to investigate the possible contribution of peroxynitrite (formed by the interaction of nitric oxide and superoxide anion) in the pathophysiology of cirrhosis. METHODS Twenty-six cirrhotic patients classified as Child-Pugh A, and seven as Child-Pugh B, were included in the study, and nine healthy volunteers served as controls. Levels of nitrite/nitrate (NOx), thiobarbituric acid-reactive substances (TBARS), nitrotyrosine (peroxynitrite marker), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were measured in blood samples. RESULTS NOx, TBARS, CAT, SOD and GSH levels were higher in cirrhosis patients than in the controls (NOx: 0.17 ± 0.02, 0.95 ± 0.12, 1.3 ± 0.1; TBARS: 2.0 ± 0.05, 4.6 ± 0.3, 5 ± 0.3; CAT: 1.8 ± 0.1, 4 ± 0.3, 4.5 ± 0.4; SOD: 1.8 ± 0.2, 4.8 ± 0.5, 7 ± 0.4; and GSH: 1.3 ± 0.05, 3.6 ± 0.3, 4.5 ± 0.6 in controls, and Child-Pugh A and B patients, respectively). However, there were no differences in nitrotyrosine levels across these groups (controls: 11.4 ± 0.4; Child-Pugh A: 11.1 ± 0.4; Child-Pugh B: 11.9 ± 1.6). NOx levels showed significant and strongly positive correlations with TBARS, SOD, CAT and GSH levels. In contrast, no correlations were found between either NOx or TBARS and nitrotyrosine levels. CONCLUSION Nitric oxide and reactive oxygen species, but not peroxynitrite, are overproduced in patients with cirrhosis in spite of evidence of an increase in antioxidant defenses. This suggests that therapeutic measures aimed at attenuating oxidative stress as well as increasing antioxidant defenses may well benefit patients with cirrhosis.

Correlation of serum adiponectin levels and hepatic steatosis in hepatitis c virus genotype 1 infection

Steatosis is an important cofactor in hepatitis C virus (HCV) because it is associated with fibrosis and reduces early and sustained virologic response. Recent studies suggest that HCV genotype 1 is not steatogenic if additional risk factors are not present. Because hypoadiponectinemia was found to be a feature of nonalcoholic steatohepatitis (NASH) independent of insulin resistance, its level in patients with hepatitis C genotype can reveal the optimal therapeutic strategy. This study was conducted to determine the role of the relationship between steatosis and serum adiponectin levels in the progression of liver damage in HCV genotype 1 without known risk factors for NASH. Patients (n=50) with biopsy-proven chronic hepatitis C (CHC), positive HCV RNA, and raised alanine aminotransferase were enrolled. They were carefully selected to rule out possible confounding factors for the presence of steatosis and additional systemic or liver disease. Associations between serum adiponectin levels and grade of steatosis, histologic activity index (HAI), fibrosis grade of liver biopsies, patient age, HCV viral load, and serum transaminase activities were studied. Also, adiponectin levels were compared with those of a control group of 30 healthy volunteers with normal ultrasound findings of the upper abdomen who had no known NASH risk factors. The investigators found that adiponectin levels in patients with CHC genotype 1 were similar to those in healthy subjects. No significant association was found between adiponectin levels and severity of steatosis, HCV RNA levels, HAI, transaminases, and fibrosis. Steatosis was present in 41 patients (82%) with CHC. Multivariate analysis of data on 50 patients revealed that severity of steatosis was independently related to age alone (P=.03). A correlation between HCV RNA load and HAI was observed (P=.02; r=0.712). HAI also was associated with stage of fibrosis (P=.00;r= 0.612). In cases of chronic HCV genotype 1 hepatitis, steatosis is a common histologic feature, although no risk factors are known. Results presented here cannot establish an association between adiponectin and severity of steatosis when risk factors for steatosis are unknown. Additional studies are needed to discover a metabolic treatment that would seek to improve the progression of hepatic steatosis in CHC infection.

Serum RANTES, MIP-1α, and MCP-1 levels in Behçet’s disease

Behçet’s disease (BD) is a multisystemic vasculitis of unknown etiology characterized by oral and genital ulceration and eye, skin, joint, gastrointestinal, and neurological manifestations. Besides elevated levels of tumor necrosis factor alpha (TNF-a) in sera of patients with BD [1], increased TNF-a and interleukin (IL)-1 production has been shown in lipopolysaccharide-stimulated peripheral blood monocytes [2]. Additionally, TNF-a and IL-1b mRNA have been shown to be increased in attack-free periods in patients with familial Mediterranean fever (FMF) [3]. It is generally accepted that certain chemokines such as regulated-on-expression, normal-T-cell-expressedand-secreted (RANTES), macrophage inflammatory protein (MIP)-1, and monocyte chemotactic protein (MCP)-1 are stimulated in response to more proximal mediators such as TNF-a and IL-1 [4]. Elevated plasma and whole blood MCP-1 levels have been reported in patients with BD, regardless of activity [5]. To gain information about the role of chemokines in the inflammation of patients with BD and their relation to disease activity, serum levels of the CC chemokines RANTES, MIP-1a, and MCP-1 were measured. Patients with familial Mediterranean fever (FMF) and healthy study participants were taken as controls. Serum samples from 21 patients with BD (mean age 38.95±1.6 years, ten females and 11 males, 11 active and ten inactive) meeting the criteria of the International Study Group for BD were taken. Except for two patients from the active and inactive disease groups, all BD patients were on colchicine (0.5–1.5 mg daily). Those having new onset or exacerbation of at least one of the clinical manifestations including genital ulcer, uveitis, arthritis, erythema nodosum, thrombophlebitis, and central nervous system or gastrointestinal disease were considered as active. Patients using corticosteroids or immunosuppressives were excluded. Twenty-seven patients with FMF meeting Tel Hashomer criteria for the disease (mean age 34.3± 10.2 years, 14 females and 13 males, 13 during attack and 14 in attack-free periods) and 27 healthy participants (mean age 37±6.6 years, 13 females and 14 males) were taken as the control groups. The study was conducted at the rheumatology outpatient clinic of Cukurova University Hospital in Adana, Turkey. Written informed consent was taken from all patients, and the study was approved by the institutional review board. It was performed in compliance with the Helsinki Declaration. RANTES, MIP-1a, and MCP-1 levels were measured from the serum samples by enzyme-linked immunosorbent assay (ELISA) at one occasion by the same person. Commercial human ELISA kits (Endogen, USA) were used for each chemokine measurement. Assay ranges were 51.2–2000 pg/ml, 0–1000 pg/ml, and 51–2000 pg/ ml for RANTES, MIP-1a, and MCP-1, respectively. Four exon-10 mutations of the MEFV gene (M694V, M680I, V726A, and M694I) were sought by amplification refractory mutation [6]. Kruskal-Wallis analysis of variance (ANOVA) was used for comparison among three groups, and the Mann-Whitney-U test was used for comparing two groups. Correlation analyses were done by nonparametric Spearman’s correlation test. Mean serum MIP-1a levels were found to differ among the groups (P=0.012, ANOVA). The level in BD (mean±SEM 126.80±36.10 pg/ml) was significantly higher (P=0.005) than in the controls (80.93±43.38 pg/ ml) but not significantly different from that of the FMF patients (78.94±29.24 pg/ml). Mean MIP-1a levels of H. T. E. Ozer (&) Æ E. Erken Æ R. Gunesacar Rheumatology-Immunology Division, Cukurova University Faculty of Medicine, Adana, 01330 Turkey E-mail: teozer@cu.edu.tr Tel.: +90-533-7178442 Fax: +90-322-3386721

Clinical significance of hepatocyte growth factor, platelet-derived growth factor-AB, and transforming growth factor-α in bone marrow and peripheral blood of patients with multiple myeloma

Angiogenesis is a process that plays an important role in the growth and progression of cancer; growing evidence suggests that neovascularization is important in hematologic malignancies. Increased angiogenic potential has been identified in multiple myeloma (MM). In this study, investigators simultaneously measured the levels of hepatocyte growth factor (HGF), platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-alpha (TGF-/ga) through enzyme-linked immunosorbent assay in the bone marrow (BM) and peripheral blood (PB) of 30 patients with MM and 10 healthy controls. Differences in HGF values in BM sera were significant (P=.001) between patients and controls. In detailed analyses of HGF, PDGF-AB, and TGF-α, according to disease stage, a significant correlation was found between disease stage and BM HGF (P=.047), BM TGF-α (P=.021), and PB PDGF-AB (P=.006), respectively. When correlations between all other parameters were analyzed, significance was noted between PB TGF-α and lactate dehydrogenase (P=.02), PB TGF-α and PB HGF (P=.002), BM TGF-α and CD38 (P=.046), BM TGF-α and BM HGF (P=.000), BM TGF-α and BM PDGF-AB (P=.048), BM HGF and PB HGF (P=.044), and BM PDGF-AB and PB PDGF-AB (P=.000). BM HGF levels had a significant effect on overall survival, with disease severity assessed in terms of disease stage (P=.0018, log-rank test). These data show that in patients with MM, high levels of BM HGF, BM TGF-α, and PB PDGF-AB were associated with advanced disease stage; in addition, HGF played a significant role in disease processing and was related to disease severity. These findings have also led to the concept of a symbiotic relationship between the growth of myeloma cells and HGF, TGF-α, and PDGF-AB in BM.

Analysis of vascular endothelial growth factor gene 936 C/T polymorphism in patients with familial Mediterranean fever.

Vascular endothelial growth factor (VEGF) is a cytokine that promotes endothelial cell proliferation, leucocyte chemotaxis and expression of adhesion molecules and is a major mediator of vascular permeability. It has been demonstrated that VEGF directly activates neutrophils and it could promote acute recruitment of leucocytes. It is known that neutrophils are the major cell population involved in acute inflammation in familial Mediterranean fever (FMF) and the role of VEGF in these cells may be crucial. The aim of this study was to investigate whether the 936 C/T functional polymorphism of the VEGF gene is associated with susceptibility to FMF and its relationship with the main clinical features of the disease. Polymerase chain reaction–restriction fragment length polymorphism technique was used to determine 936 C/T polymorphism within the VEGF gene in 75 patients with FMF and 122 non‐related healthy controls. Genotype and allele frequencies of the VEGF 936 C/T polymorphism between patients with FMF and healthy control groups were not significantly different (OR = 0.74, 95% CI = 0.40–1.37, P = 0.335 for CT genotype; OR = 1.11, 95% CI = 0.67–1.83, P = 0.700, for T allele). Although VEGF 936 TT genotype was found to be more frequent in patients with FMF than in healthy controls (6.7% vs. 1.6%, respectively), the difference was not significant (OR = 4.28, 95% CI = 0.81–22.67, P = 0.108). No associations were found between the studied polymorphism and either the clinical features such as arthritis, abdominal pain, pleuritis, myalgia, arthralgia and erysipelas‐like erythema of the disease or the four common studied exon 10 mutations (M694V, M680I, V726A, M694I) of the Mediterranean fever gene. Present results suggest that VEGF gene 936 C/T polymorphism does not seem to be associated with susceptibility to FMF and its clinical manifestations.

Levels of serum and cerebrospinal fluid soluble CD27 in the diagnosis of leptomeningeal involvement of hematolymphoid malignancies.

Reportedly, soluble CD27 (sCD27) is a sensitive and specific marker for leptomeningeal involvement (LI) of CD27-expressing lymphoproliferations, such as B-cell non-Hodgkin’s lymphoma and chronic B-lymphocytic leukemia. On morphologic analysis of cerebrospinal fluid (CSF), one third of patients suspected of LI have false negatives, so a diagnostic marker for LI in B-cell non-Hodgkin’s lymphoma or B-lymphocytic leukemia would be extremely valuable. sCD27 was detected in the serum and CSF samples from 35 selected patients in whom 18 cases of acute lymphoblastic leukemia (ALL) (3 with LI), 7 of non-Hodgkin’s lymphoma, and 5 of acute myelogenous leukemia (3 with LI) were submitted for (immuno)morphologic detection of malignant cells and intrathecal therapy, along with samples from 5 control patients (2 submitted for epidural hemorrhage, 3 for lumbar disc protrusion). Concentrations of CSF-sCD27 were determined by enzyme-linked immunosorbent assay (PeliKine Compact Human Soluble CD27 ELISA Kit, Cat. No. M1960; Research Diagnostics Inc., Concord, Mass). The cutoff value was 350 U/mL. Serum and CSF-sCD27 concentrations above the cutoff value were not detected. Although it is unlikely that LI would be present in patients with chronic lymphoproliferation who have normal sCD27 concentrations in CSF samples, the determination of CSF-sCD27 is not sufficiently specific to allow it to serve as a reliable tumor marker.