Characterization of autoantibodies against sulfatide from a V-gene phage-display library derived from patients with systemic lupus erythematosus

Sulfatide, ceramide galactosyl-3'-sulfate, is mainly present in nervous tissue, kidney, testis, red blood cells, platelets and granulocyte. Antibodies to sulfatide are present in many patients with demyelinating peripheral neuropathy, HIV infection and systemic lupus erythematosus and may account for some of the clinical manifestations. To evaluate the effect of such antibodies, we have constructed a phage-display antibody fragment library from the lymphocytes of patients with systemic lupus erythematosus. Sulfatide-reactive phage were selected by absorption and elution on sulfatide liposomes and soluble single chain variable fragment (ScFv) were isolated from individual colonies and tested in an ELISA assay for binding to bovine brain sulfatide. Five ScFv clones that bound sulfatide were isolated. Two of the clones, PH5 and PA38, bound sulfatide but not phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin or ceramide. These two clones also bound sulfatide from human red blood cells. The DNA encoding the fragments was sequenced, revealing predicted polypeptides of 19 kDa for PH5 containing only variable heavy (VH) sequences, and 31 kDa for PA38, with both VH and variable light (VL) sequences. Although they had similar antigen specificities, the VH domains of the two clones were derived from different heavy-chain families. The clustered mutational patterns in the complementarity-determining region (CDR) of the heavy chains in both clones suggest that the V-domains are the products of antigen-driven B cell clonal maturation leading to the development of sulfatide-binding specificity. These results show the presence of sulfatide-specific antibodies in lupus patients, and allow us to test the possibility that the interaction of the antibodies with sulfatide may contribute to some of the symptoms. In addition, the antibodies provide useful reagents to test the role of sulfatide in pathophysiological processes.     

Taking Up Subfascial Endoscopic Perforator Surgery for Patients of Lower Limb Varicose Veins with Below Knee Perforators,in A Government Medical College–A Review of Eleven Cases

Varicose veins are the most common manifestation of chronic venous insufficiency (CVI) worldwide. They interfere with the patients’ daily activities and hamper their professional endeavors, and therefore need to be addressed at the earliest with the best tools. Eleven patients of varicose veins of the lower limb with below knee perforators underwent subfascial endoscopic perforator surgery (SEPS) from October 2010 to December 2011 in our institute. The procedure was successfully completed in all 11 patients. The mean operating time was 78 min (60–96 min). The results showed a favorable outcome for the patients in terms of faster recovery and less morbidity.     

Prevalence of celiac disease in nutritional anemia at a tertiary care center

Background

While anemia occurs in 80 % to 90 % of patients with celiac disease (CD), it may be the sole manifestation of CD. The prevalence of CD in Indian patients with nutritional anemia is not known.

Patients and Methods

Adolescent and adult patients presenting with nutritional anemia were prospectively screened for CD using IgA anti-tissue transglutaminase antibody (anti-tTG Ab) followed, if positive, by upper gastrointestinal endoscopy and duodenal biopsy.

Results

Ninety-six patients [mean?±?SD age 32.1?±?13.1 years and median duration of anemia 11 months (range 1 to 144 months)] were screened. Of these patients, 80 had iron deficiency anemia, 11 had megaloblastic anemia, and 5 had dimorphic anemia. Seventy-three patients were on hematinics and 36.4 % had received blood transfusions. Nineteen had a history of chronic diarrhea and the mean?±?SD duration of diarrhea in them was 9.7?±?35.8 months. IgA anti-tTG Ab was positive in 13 patients, of whom 12 agreed to undergo duodenal biopsy. Ten patients had villous atrophy (Marsh grade 3a in three, 3b in one, and 3c in six) and two did not. Thus, 10 patients with nutritional anemia (iron deficiency 9, vitamin B₁₂ deficiency 1) were diagnosed to have CD. On multivariate logistic regression, age, duration of symptoms, and presence of diarrhea were found to be the predictors of CD. All the patients with CD were put on gluten-free diet and with iron and vitamin supplementations and showed a significant improvement in hemoglobin concentration.

Conclusions

CD screening should be included in the work up of otherwise unexplained nutritional anemia.     

Protection of arsenic-induced hepatic disorder by arjunolic acid

Arsenic is one of the ubiquitous environmental pollutants, which affects nearly all organ systems. The present study has been carried out to investigate the hepatoprotective role of arjunolic acid, a triterpenoid saponin, against arsenic-induced oxidative damages in murine livers. Administration of sodium arsenite at a dose of 10 mg/kg body weight for 2 days significantly reduced the activities of antioxidant enzymes, superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase and glutathione peroxidase as well as depleted the level of reduced glutathione and total thiols. In addition, sodium arsenite also increased the activities of serum marker enzymes, alanine transaminase and alkaline phosphatase, enhanced DNA fragmentation, protein carbonyl content, lipid peroxidation end-products and the level of oxidized glutathione. Studies with arjunolic acid show that in vitro it possesses free radical-scavenging and in vivo antioxidant activities. Treatment with arjunolic acid at a dose of 20 mg/kg body weight for 4 days prior to arsenic administration prevents the alterations of the activities of all antioxidant indices and levels of the other parameters studied. Histological studies revealed less centrilobular necrosis in the liver treated with arjunolic acid prior to arsenic intoxication compared to the liver treated with the toxin alone. Effects of a known antioxidant, vitamin C, have been included in the study as a positive control. In conclusion, the results suggest that arjunolic acid possesses the ability to attenuate arsenic-induced oxidative stress in murine liver probably via its antioxidant activity.     

Cytoprotective effect of arjunolic acid in response to sodium fluoride mediated oxidative stress and cell death via necrotic pathway

The present study was conducted to investigate the role of arjunolic acid (AA) against sodium fluoride (NaF)-induced cytotoxicity and necrotic cell death in murine hepatocytes. Dose-dependent studies suggest that incubation of hepatocytes with NaF (100mM) for 1h significantly decreased the cell viability as well as intracellular antioxidant power. Besides, NaF administration increased the activities of the membrane leakage enzymes and accumulation of intracellular reactive oxygen species; decreased the activities of the antioxidant enzymes, the glutathione (GSH) and total thiol contents; and elevated the level of oxidised glutathione (GSSG), lipid peroxidation end products as well as protein carbonyl content. In addition to the oxidative impairments, fluoride exposure caused hepatic cell death mainly via the necrotic pathway as supported by the flowcytometric and DNA fragmentation analyses. Incubation with AA (100 microg/ml) both prior to and in combination with NaF almost normalized the altered activities of antioxidant indexes. AA treatment enhanced the cellular antioxidant capability and protected hepatocytes against NaF-induced cytotoxicity and necrotic death. The cytoprotective activity of AA was found to be comparable to that of a known antioxidant, vitamin C. Combining, data suggest that AA plays a protective role against NaF-induced cellular damage and prevents hepatocytes from necrotic death.     

Streptozotocin induced activation of oxidative stress responsive splenic cell signaling pathways: Protective role of arjunolic acid

Present study investigates the beneficial role of arjunolic acid (AA) against the alteration in the cytokine levels and simultaneous activation of oxidative stress responsive signaling pathways in spleen under hyperglycemic condition. Diabetes was induced by injection of streptozotocin (STZ) (at a dose of 70 mg/kg body weight, injected in the tail vain). STZ administration elevated the levels of IL-2 as well as IFN-γ and attenuated the level of TNF-α in the sera of diabetic animals. In addition, hyperglycemia is also associated with the increased production of intracellular reactive intermediates resulting with the elevation in lipid peroxidation, protein carbonylation and reduction in intracellular antioxidant defense. Investigating the oxidative stress responsive cell signaling pathways, increased expressions (immunoreactive concentrations) of phosphorylated p65 as well as its inhibitor protein phospho IκBα and phosphorylated mitogen activated protein kinases (MAPKs) have been observed in diabetic spleen tissue. Studies on isolated splenocytes revealed that hyperglycemia caused disruption of mitochondrial membrane potential, elevation in the concentration of cytosolic cytochrome c as well as activation of caspase 3 leading to apoptotic cell death. Histological examination revealed that diabetic induction depleted the white pulp scoring which is in agreement with the reduced immunological response. Treatment with AA prevented the hyperglycemia and its associated pathogenesis in spleen tissue. Results suggest that AA might act as an anti-diabetic and immunomodulatory agent against hyperglycemia.     

Medulloblastomas: a correlative study of MIB-1 proliferation index along with expression of c-Myc,ERBB2, and anti-apoptotic proteins along with histological typing and clinical outcome

Background  Medulloblastoma (MB) is the most common pediatric brain tumor. It is however rare in adults. The genetic and protein expression profile of medulloblastoma is complex, which is worthwhile in terms of prognostication and development or selection of targeted therapy. Aims and objectives  The aims and objectives to correlate the MIB-1 proliferation index and protein expression profiles of c-Myc, ERBB2, and anti-apoptotic proteins (Bcl2 and Bcl-xL) in tumor cells with histological subtypes and clinical outcome. Methods and material  In 50 cases, histopathological subtyping was done, and protein expression profiling by immunohistochemical technique was performed by stains for MIB-1, Bcl2, Bcl-xL, c-Myc, and ERBB2 in 30 cases. The findings were correlated with histological types and patient’s average follow-up data. Results  Histological subtypes were similar to that described in literatures. The average expression of Bcl2, Bcl-xL, MIB-1, c-Myc, and ERBB2 were as follows: 50.38%, 38.18%, 59.03%, 46.16%, and 59.62%, respectively. Bcl2 expression showed statistically significant correlation with progress-free survival (PFS) [p = 0.046], while ERBB2 and MIB-1 showed a trend of higher expression in progressive disease. The protein expression pattern did not correlate with histological subtypes. Conclusion  Though Bcl-2, ERBB2, and MIB-1 LI came out to be potential markers of aggressive behavior, c-Myc did not correlate with PFS in MB.     

Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage: Role of NF-κB, p38 and JNK MAPK pathway

Cardiac dysfunction is a major cause of morbidity and mortality worldwide due to its complex pathogenesis. However, little is known about the mechanism of arsenic-induced cardiac abnormalities and the use of antioxidants as the possible protective agents in this pathophysiology. Conditionally essential amino acid, taurine, accounts for 25% to 50% of the amino acid pool in myocardium and possesses antioxidant properties. The present study has, therefore, been carried out to investigate the underlying mechanism of the beneficial role of taurine in arsenic-induced cardiac oxidative damage and cell death. Arsenic reduced cardiomyocyte viability, increased reactive oxygen species (ROS) production and intracellular calcium overload, and induced apoptotic cell death by mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. These changes due to arsenic exposure were found to be associated with increased IKK and NF-κB (p65) phosphorylation. Pre-exposure of myocytes to an IKK inhibitor (PS-1145) prevented As-induced caspase-3 and PARP cleavage. Arsenic also markedly increased the activity of p38 and JNK MAPKs, but not ERK to that extent. Pre-treatment with SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated NF-κB and IKK phosphorylation indicating that p38 and JNK MAPKs are mainly involved in arsenic-induced NF-κB activation. Taurine treatment suppressed these apoptotic actions, suggesting that its protective role in arsenic-induced cardiomyocyte apoptosis is mediated by attenuation of p38 and JNK MAPK signaling pathways. Similarly, arsenic intoxication altered a number of biomarkers related to cardiac oxidative stress and other apoptotic indices in vivo and taurine supplementation could reduce it. Results suggest that taurine prevented arsenic-induced myocardial pathophysiology, attenuated NF-κB activation via IKK, p38 and JNK MAPK signaling pathways and could possibly provide a protection against As-induced cardiovascular burden.