Emel Eksioglu-Demiralp

L-Carnitine ameliorates methotrexate-induced oxidative organ injury and inhibits leukocyte death

Methotrexate (MTX), a folic acid antagonist widely used for the treatment of a variety of tumors and inflammatory diseases, affects normal tissues that have a high rate of proliferation, including the hematopoietic cells of the bone marrow and the gastrointestinal mucosal cells. To elucidate the role of free radicals and leukocytes in MTX-induced oxidative organ damage and the putative protective effect of L-carnitine (L-Car), Wistar albino rats were administered a single dose of MTX (20 mg/kg) followed by either saline or L-Car (500 mg/kg) for 5 days. After decapitation of the rats, trunk blood was obtained, and the ileum, liver, and kidney were removed for histological examination and for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity, and collagen content. Our results showed that MTX administration increased the MDA and MPO activities and collagen content and decreased GSH levels in all tissues, while these alterations were reversed in L-Car-treated group. The elevated serum TNF-α level observed following MTX treatment was depressed with L-Car. The oxidative burst of neutrophils stimulated by Annexin V was reduced in the saline-treated MTX group, while L-Car abolished this inhibition. Similarly, flow cytometric measurements revealed that leukocyte apoptosis was increased in MTX-treated animals, while L-Car reversed these effects. Severe degeneration of the intestinal mucosa, liver parenchyma, and glomerular and tubular epithelium observed in the saline-treated MTX group was improved by L-Car treatment. These results suggest that L-Car, possibly via its free radical scavenging and antioxidant properties, ameliorates MTX-induced oxidative organ injury and inhibits leukocyte apoptosis. Thus, supplementation with L-Carnitine as an adjuvant therapy may be promising in alleviating the systemic side-effects of chemotherapeutics.

Lithium-mediated downregulation of PKB/Akt and cyclin E with growth inhibition in hepatocellular carcinoma cells

We studied in vitro effects of glycogen synthase kinase 3β (GSK3β)‐inhibitor lithium on the growth of hepatocellular carcinoma (HCC) cells. Lithium induced strong growth inhibition (>70%) in 75% (n = 9 of 12) of cell lines, apparently independent from the status of major genes that are mutated in HCC including p53, p16INK4a, β‐catenin and Axin1. Comparative studies with a growth‐sensitive Huh7 and growth‐resistant Hep40 cell lines showed that lithium induces growth arrest in Huh7 cells but not in Hep40 cells. Lithium induced the accumulation of N‐terminally phosphorylated inactive form of GSK3β with concomitant increase in β‐catenin and β‐catenin/TCF transcriptional activity in both cell lines. This suggests that lithium‐mediated HCC growth inhibition is independent of its well‐known stimulatory effect on Wnt‐β‐catenin signaling. The main differences between Huh7 and Hep40 responses to lithium treatment were observed at the levels PKB/Akt and cyclin E proteins. Lithium induced depletion of both proteins in growth‐sensitive Huh7, but not in growth‐resistant Hep40 cells. PKB/Akt and Cyclin E are 2 major proteins that are known to be constitutively active in HCC. The targeting of both proteins with lithium may be the main reason why most HCC cells are responsive to lithium‐mediated growth inhibition, independent of their p53, retinoblastoma and Wnt‐β‐catenin pathways. The exploration of molecular mechanisms involved in lithium‐mediated growth inhibition in relation with PKB/Akt and cyclin E downregulation may provide new insights for therapy of liver tumors.

Leptin ameliorates burn-induced multiple organ damage and modulates postburn immune response in rats

The present study was designed to determine whether exogenous leptin reduces remote organ injury in the rats with thermal burn trauma. Leptin (10 microg/kg) or saline was administered intraperitoneally after burn injury, and the rats were decapitated at either 6 or 24 h. Plasma samples of 24-h burn group were assayed for the determination of monocyte and neutrophil apoptosis. Thermal injury increased tissue-associated myeloperoxidase (MPO) activity and microscopic damage scores in the lung, liver, stomach, colon and kidney of both 6- and 24-h burn groups. In the 6-h burn group, leptin reduced microscopic damage score in the liver and kidney only, while damage scores in the 24-h burn group were reduced in all the tissues except the lung. Also, in both burn groups, leptin reduced elevated MPO activity in all tissues except the lung. The percentage of mononuclear cells was significantly reduced at the 24 h of burn injury, while the granulocyte percentage was increased. Leptin treatment, however, had no significant effect on burn-induced reversal of white blood cell ratios. On the other hand, burn-induced increase in the death of mononuclear cells and granulocytes was significantly reduced in leptin-treated rats. The results of the present study suggest that leptin may provide a therapeutic benefit in diminishing burn-induced inflammation and associated multiple organ failure.

Increased CD4+CD16+ and CD4+CD56+ T cell subsets in Behçet's disease

Abstract Behçets disease is a systemic vasculitis of unknown etiology. Various immune abnormalities have previously been shown in Behçets disease. We investigated T lymphocyte subsets associated with cytotoxic activity and natural killer (NK) cells by flow cytometry in 37 patients with Behçets disease, 38 healthy controls, and 17 diseased control patients. Compared to the healthy controls, CD4+CD16+ and CD4+CD56+ subsets were found to be higher in the Behçets disease group as well as in the disease control group (CD4+CD16+: BD=5 ± 3, DC=14 ± 14, HC= 3 ± 2, P=0.001; CD4+CD56+: BD=11 ± 5, DC= 18 ± 17, HC=8 ± 6, P=0.01). CD8+CD16+ and CD8+CD56+ T cell subsets were at normal levels in Behçets disease but found to be elevated in disease controls. Similarly, NK cells (CD16+CD56+) were high only in the disease control group. Significant increases in CD4+CD16+ and CD4+CD56+ cell subsets in Behçets patients and disease controls suggest that T cell activation patterns of these subsets in Behçets disease are similar to those in other inflammatory disorders.

Toll-like receptor expression in monocytes in patients with chronic kidney disease and haemodialysis: relation with inflammation

BACKGROUND Inflammation is one of the main contributors to atherosclerosis in haemodialysis (HD) patients. Activation of Toll-like receptors (TLRs) leads to inflammatory response. In this study, we aimed to evaluate the expression of TLRs on monocytes and relate their expression with inflammation in chronic kidney disease (CKD) and HD patients. METHODS Thirty-four age- and gender-matched controls and stage 3-4 CKD patients and thirty-two HD patients were included in each study group. The effect of HD on the expression of Toll-like receptor-2 (TLR-2) and Toll-like receptor-4 (TLR-4) on CD14( +) monocytes was determined at the beginning (baseline), during (120 min) and following (300 min and 24 h) HD and compared with control and stage 3-4 CKD groups. The HD procedure was performed by using low-flux polysulphone dialysers. In addition, serum IL-6 levels were evaluated in both groups at baseline and after a HD session. RESULTS The percentage of CD14( +) monocytes expressing TLR-2 were similar in all of the study groups, whereas the percentage of CD14( +) monocytes expressing TLR-4 were significantly lower in both stage 3-4 CKD and HD patients at baseline than in controls. The mean fluorescence intensities (MFI) of TLR-2 were significantly lower in controls than in stage 3-4 CKD and HD patients at baseline. The MFI of TLR-4 was similar in all of the groups. The percentage of CD14( +) monocytes expressing TLR-2 did not change during and after HD. The MFI of TLR-2 decreased at 120 min of HD compared with baseline (1837 ± 672 vs 1650 ± 578, P < 0.05), and recovered back to baseline values at 300 min and at 24 h post-HD. MFI of TLR-4 increased at 24 h compared with baseline (941 ± 294 vs 1087 ± 441, P < 0.05). Serum IL-6 levels correlated with MFI of TLR-2 and TLR-4 in stage 3-4 CKD patients and in HD patients at baseline and after HD in univariate analysis. Stepwise multiple regression analysis revealed that MFI of TLR-2 was an independent determinant of serum IL-6 concentrations in stage 3-4 CKD and in HD patients at baseline, at 300 min and at 24 h post-HD. Conclusions. Our study demonstrates that TLR-2 is associated with the inflammatory response of non-dialysed and dialysed CKD patients.

Betulinic Acid Protects against Ischemia/ Reperfusion-induced Renal Damage and Inhibits Leukocyte Apoptosis

The possible protective effect of betulinic acid on renal ischemia/reperfusion (I/R) injury was studied. Wistar Albino rats were unilaterally nephrectomized and subjected to 45 min of renal pedicle occlusion followed by 6 h of reperfusion. Betulinic acid (250 mg/kg, i.p.) or saline was administered at 30 min prior to ischemia and immediately before the reperfusion. Creatinine, blood urea nitrogen (BUN), lactate dehydrogenase (LDH) and TNF‐α as well as the oxidative burst of neutrophil and leukocyte apoptosis were assayed in blood samples. Malondialdehyde (MDA), glutathione (GSH) levels, Na+, K+‐ATPase and myeloperoxidase (MPO) activities were determined in kidney tissue which was also analysed microscopically. I/R caused significant increases in blood creatinine, BUN, LDH and TNF‐α. In the kidney samples of the I/R group, MDA levels and MPO activity were increased significantly, however, GSH levels and Na+, K+‐ATPase activity were decreased. Betulinic acid ameliorated the oxidative burst response to both formyl‐methionyl‐leucyl‐phenylalanine (fMLP) and phorbol 12‐myristate 13‐acetate (PMA) stimuli, normalized the apoptotic response and most of the biochemical indices as well as histopathological alterations induced by I/R. In conclusion, these data suggest that betulinic acid attenuates I/R‐induced oxidant responses, improved microscopic damage and renal function by regulating the apoptotic function of leukocytes and inhibiting neutrophil infiltration. Copyright

Resveratrol protects against irradiation-induced hepatic and ileal damage via its anti-oxidative activity

The present study was undertaken to determine whether resveratrol (RVT) could ameliorate ionizing radiation-induced oxidative injury. After a 10-days pre-treatment with RVT (10 mg/kg/day p.o.), rats were exposed to whole-body IR (800 cGy) and the RVT treatment was continued for 10 more days after the irradiation. Irradiation caused a significant decrease in glutathione level, while malondialdehyde levels, myeloperoxidase activity and collagen content were increased in the liver and ileum tissues. Similarly, plasma lactate dehydrogenase and pro-inflammatory cytokine levels, 8-hydroxy-2′-deoxyguanosine and leukocyte apoptosis were elevated, while antioxidant-capacity was reduced in the irradiated rats as compared with the control group. Furthermore, Na+, K+-ATPase activity was inhibited and DNA fragmentation was increased in the ileal tissues. Resveratrol treatment reversed all these biochemical indices, as well as histopathological alterations induced by irradiation. In conclusion, supplementing cancer patients with adjuvant therapy of resveratrol may have some benefit for a more successful radiotherapy.

Salivary levels of antimicrobial peptides Hnp 1-3, Ll-37 and S100 in Behcet's disease

BACKGROUND Oral ulcer is the cardinal clinical sign and increased neutrophilic activity is a part of the pathogenesis in Behcets disease (BD). Saliva, as a part of the innate immune response, contains antimicrobial peptides (AMPs) that are derived from both oral epithelial cells and neutrophils. The aim of this study was to investigate the associations between salivary levels of AMPs HNP 1-3, LL-37 and S100 and disease course in patients with Behcets disease (BD). METHODS Fifty-three patients with BD and 44 healthy controls (HC) were included in the study. Disease severity score reflecting organ involvement was calculated. Salivary HNP 1-3, LL-37 and S100 levels were measured in unstimulated saliva samples by ELISA. RESULTS Salivary HNP 1-3 and S100 levels in BD patients (2715.2 ± 1333.4 μg/ml and 430.6 ± 203.9 ng/ml) were significantly higher compared to HC (1780.6 ± 933.2 μg/ml and 365.3 ± 84.7 ng/ml) (p = 0.000 and p = 0.004, respectively). Although LL-37 levels were also higher in BD than HC (190.9 ± 189.1 vs 143.1 ± 128.9 ng/ml), no significant difference was observed (p = 0.53). Salivary HNP 1-3 and LL-37 levels were associated with the severity of BD (mild disease: 1975.1 ± 1174.2 μg/ml and 115.9 ± 109.4 ng/ml vs severe disease: 2955.7 ± 1305.6 μg/ml and 215.3 ± 203.8 ng/ml, p=0.020 and p=0.031, respectively). Salivary LL-37 levels also correlated with the number of monthly oral ulcers (r = 0.5 p = 0.000). CONCLUSION An increase in salivary HNP 1-3 and S100 levels might be associated with enhanced local and systemic innate responses in BD.

Activation of the JAK/STAT pathway in Behcet’s Disease

Th1/Th17-type T-cell responses are upregulated in Behcet’s disease (BD). However, signaling pathways associated with this aberrant immune response are not clarified. Whole-genome microarray profiling was performed with human U133 (Plus 2.0) chips using messenger RNA of isolated CD14+ monocytes and CD4+ T cells from peripheral blood mononucleated cell (PBMC) in patients with BD (n=9) and healthy controls (HCs) (n=9). Flow cytometric analysis of unstimulated (US) and stimulated (phytohaemagglutinin) signal transducer and activator of transcription (STAT3) and pSTAT3 expressions of PBMCs were also analyzed (BD and HC, both n=26). Janus family of kinase (JAK1) was observed to be upregulated in both CD14+ monocytes (1.95-fold) and CD4+ T lymphocytes (1.40-fold) of BD patients. Using canonical pathway enrichment analysis, JAK/STAT signaling was identified as activated in both CD14+ monocytes (P=9.55E−03) and in CD4+ lymphocytes (P=8.13E−04) in BD. Interferon signaling was also prominent among upregulated genes in CD14+ monocytes (P=5.62E−05). Glucocorticoid receptor signaling and interleukin (IL-6) signaling were among the most enriched pathways in differentially expressed genes in CD14+ monocytes (P=2.45E−09 and 1.00E−06, respectively). Basal US total STAT3 expression was significantly higher in BD (1.2 vs 3.45, P<0.05). The JAK1/STAT3 signaling pathway is activated in BD, possibly through the activation of Th1/Th17-type cytokines such as IL-2, interferon (IFN-γ), IL-6, IL-17 and IL-23.

Phagocytosis and oxidative burst by neutrophils in patients with recurrent furunculosis

Neutrophil phagocytosis of fluorescently labelled Staphylococcus aureus and oxidative burst by the neutrophils were assessed by flow cytometry in 22 patients with recurrent furunculosis and in 17 controls. Phagocytosis and oxidative burst were not found to be significantly different between the patients and controls. Low serum iron concentrations were demonstrated in six patients (27%). In these patients with hypoferraemia, oxidative burst was significantly lower than in the patients without hypoferraemia and in the controls. These data suggest that hypoferraemia may be an important predisposing factor in a subgroup of patients with recurrent furunculosis in impairing oxidative killing by neutrophils.