Hasi R. Das

Evaluation of inhibitory activities of plant extracts on production of LPS-stimulated pro-inflammatory mediators in J774 murine macrophages

Whole plant methanolic extracts of 14 traditionally used medicinal herbs were evaluated for their anti-inflammatory activity. Extracts of Grindelia robusta, Salix nigra, Arnica montana, and Quassia amara showed up to 4.5-fold inhibition of nitric oxide (NO) production in the J774 murine macrophage cells challenged with LPS without cytotoxicity. These four selected extracts significantly reduced the protein levels of inducible NO synthase (iNOS) and the cyclooxygenase-2 (COX-2) as observed by Western blot analysis. Culture supernatants from cells treated with these extracts indicated 3–5-fold reduction of tumor necrosis factor-α (TNF-α). However, only G. robusta and Q. amara extracts significantly inhibited (by 50%) IL-1β and IL-12 secretions. Furthermore, all these plant extracts were shown to prevent the LPS-mediated nuclear translocation of nuclear factor-κB (NF-κB). All the above observations indicate the anti-inflammatory potential of these plant extracts.

Identification of Novel Autoantigen in the Synovial Fluid of Rheumatoid Arthritis Patients Using an Immunoproteomics Approach

Rheumatoid arthritis (RA) is a chronic, autoimmune and inflammatory joint disease with a poorly understood etiology. Despite widespread diagnostic use of anti-citrullinated protein antibodies and rheumatoid factor proteins there is a strong demand for novel serological biomarkers to improve the diagnosis this disease. The present study was aimed to identify novel autoantigens involved in rheumatoid arthritis (RA) pathogenesis through immune-proteomic strategy. Synovial fluid samples from clinically diagnosed RA patients were separated on two-dimensional gel electrophoresis (2-DE). Samples from patients with non-RA rheumatisms (osteoarthritis and trauma) were used as controls. Immunoreactive proteins were spotted by Western blotting followed by identification through Q-TOF mass spectrometer analysis. Forty Western blots were generated using plasma from ten individual RA patients and 33 reactive spots were identified, 20 from the high molecular weight (HMW) gel and 13 from the low molecular weight (LMW) gel. Among the 33 common immunogenic spots, 18 distinct autoantigens were identified, out of which 14 are novel proteins in this context. Expression analysis of five important proteins, vimentin, gelsolin, alpha 2 HS glycoprotein (AHSG), glial fibrillary acidic protein (GFAP), and α1B-glycoprotein (A1BG) by Western blot analysis using their specific antibodies revealed their higher expression in RA synovial fluid as compared to non-RA samples. Recombinantly expressed GFAP and A1BG protein were used to develop an in-house ELISA to quantify the amount of autoantibodies in the RA patients. RA patients revealed an increase in the expression of GFAP and A1BG in the plasma as compared to osteoarthritis patients. Therefore, GFAP and A1BG can be proposed as potential new autoantigens of diagnostic importance for RA subjects. Further characterization of these proteins in rheumatoid arthritis will be helpful in understanding the role of these proteins in the disease pathogenesis providing new diagnostic tool with better specificity and accurate detection of the disease.

Amelioration of collagen-induced arthritis by Salix nigra bark extract via suppression of pro-inflammatory cytokines and oxidative stress

Our study goals to investigate the anti-arthritic potential of Salix nigra bark methanol extract (SNME) against both inflammation and oxidative stress in the collagen-induced arthritis (CIA) rat model. Results showed that SNME exhibited maximum scavenging activity against superoxide, hypochlorous acid and hydrogen peroxide radicals along with the suppression of lipid peroxidation. Female wistar rats were immunized with porcine type II collagen and treated with SNME (100 mg/kg body weight) for 15 days starting on day 20. SNME significantly inhibited the paw swelling and arthritic score; exhibited maximum CIA inhibition of 93.7% by the end of the experimental period. Administration of SNME to arthritic rats significantly improved the histological findings in joints as evident by reduced infiltration of polymorphonuclear cells and smooth synovial lining. Roentgenograms of tibiotarsal joints of both SNME and indomethacin-treated rats showed protection against osteophyte formation, soft tissue swelling and bone resorption. Furthermore, levels of inflammatory mediators (nitric oxide, TNF-α, IL-1β, IL-6) measured in both plasma and joint exudates were significantly reduced by SNME treatment. Increased oxidative stress observed in the arthritic animals was also found to be significantly restored in SNME- treated rats. Taken together, our studies clearly indicate the potential of S. nigra as an anti-arthritic agent.

Association of mannose-binding lectin gene (MBL2) polymorphisms with rheumatoid arthritis in an Indian cohort of case-control samples

AbstractSingle nucleotide polymorphisms in the mannose-binding lectin (MBL2) gene, as well as the serum MBL2 level, have been associated with various autoimmune diseases. We investigated whether such polymorphisms and/or the serum MBL2 level were associated with rheumatoid arthritis (RA) in an Indian population. The frequency of the B variant (codon 54) of the MBL2 gene was quite frequent in the healthy Indian population and was significantly (P=6.35×10−6) lower in RA patients. We replicated this association (P=1.78×10−5) in an independent cohort of control individuals. Promoter polymorphism at −550 nt showed a significant overrepresentation (P=0.003) of the minor allele G in severe RA patients compared with the less severe group. Haplotype LYA frequency was significantly (P=0.03) high in the less severe group, while the frequency of the HYA haplotype was significantly (P=0.04) increased in the severe RA patients. No statistically significant difference in serum MBL2 was observed as a whole, but the individuals homozygous for the LYA haplotype had significantly lower (P=0.017) serum MBL2 levels compared with individuals homozygous for the HYA haplotype. Therefore, the B variant of the MBL2 gene may be associated with protection from RA in our study population, and the promoter polymorphism (−550 nt) seems to have some role in disease progression.

Isolation and characterization of a lectin from peanut roots

A glucose-specific lectin has been purified to apparent homogeneity from 7-day-old peanut (Arachis hypogaea) roots by affinity chromatography on a Sephadex G-50. The lectin has a 66 kDa native molecular mass and a 33 kDa subunit molecular mass as revealed by native and denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. The purified lectin, gives a single precipitin line with the antiserum produced against 7-day-old root extract and shows 5 bands in the pH range of 4.4-5.4 in the isoelectric focusing gel. The glucose-specific lectin activity in the peanut roots appears from the fourth day onwards. Lipopolysaccharides isolated from the host specific Rhizobium strain are a 68-fold more potent inhibitor of the lectin as compared to glucose.

Altered expression and glycosylation of plasma proteins in rheumatoid arthritis

Altered glycosylation of plasma proteins has been directly implicated in the pathogenesis of rheumatoid arthritis (RA). The present study investigated the changes in the Concanavalin-A (Con-A)-bound plasma proteins in the RA patients in comparison to that of the healthy controls. Two proteins (MW ∼32 kDa and ∼62 kDa) showed an alteration in expression while an altered monosaccharide profile (high mannose) was observed in the ∼62 kDa protein in the samples collected from RA patients. The 2-dimensional polyacrylamide gel electrophoresis analysis of the Con-A-bound plasma samples showed a large number of protein spots, a few of which were differentially expressed in the RA patients. Some unidentified proteins were detected in the RA patients which were absent in the control samples. The present study, therefore, enunciates the role of carbohydrates as well as that of the acute phase response in the disease pathogenesis.

Cell surface and other morphological changes accompanying growth inhibition in simian virus 40-transformed 3T3 mouse fibroblasts cells induced by glucocorticoids§

AbstractsA number of morphological changes accompany the G2 blockage caused by glucocorticosteroids in simian virus 40-transformed 3T3 mouse fibroblasts cells. Under phase contrast microscopy dexamethasone-treated cells have darkened and raised nuclear regions with ‘lines’ running over their cytoplasmic areas. They are more resistant to trypsinization and smaller in volume. Since inhibitors of DNA and protein synthesis prevent this ‘glucocorticoid morphology’, the ‘darkening’ may be due to the accumulation of macromolecules within G2-blocked cells and the induction of a protein(s) may be needed for the morphological changes. Colchicine and cytochalasin B do not bring about the glucocorticoid morphology, suggesting that it is not due to a general de-polymerization of microtubules or microfilaments. With scanning electron microscopy treated cells show a great reduction in the amount and a re-organization of microvilli and microplicae. Granules of lead precipitate at the periphery are also clearly evident in transmission electron micrographs. These observations reveal profound morphological alterations, including cell surface changes, induced by glucocorticosteroids.

Expression of TNF-α and Related Signaling Molecules in the Peripheral Blood Mononuclear Cells of Rheumatoid Arthritis Patients

We examined the role of tumor necrosis factor (TNF-α) and its related signaling intermediates leading to apoptosis/proliferation in the peripheral blood mononuclear cells (PBMCs) of RA patients. The constitutive expression of mRNA for TNF-α receptors (TNFR-I and TNFR-II) and the adapter molecules, such as the TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor interacting protein (RIP), and TNF receptor-associated factor 2 (TRAF-2) were analyzed by reverse transcriptase-PCR (RT-PCR) in PBMCs from control and RA cases. PBMCs of RA patients showed a significant increase in TNF-α and TNFR-I expression as compared with that from control subjects along with significantly increased constitutive expression of TRADD, RIP, and TRAF-2 mRNA. There was a decrease in expression of FADD in RA patients, but the difference was not significant as compared to controls. These data suggested enhanced signaling by the TNFR-I-TRADD-RIP-TRAF-2 pathway and suppressed signaling by the TNFR-I-TRADD-FADD pathway in PBMCs of RA patients. However, the regulatory mechanisms for TNF-α induced signaling may not be explained only by these pathways.

Jacalin Bound Plasma O-Glycoproteome and Reduced Sialylation of Alpha 2-HS Glycoprotein (A2HSG) in Rheumatoid Arthritis Patients

Glycosylation studies of plasma proteins can reveal information about the onset and progression of diseases, where in the glycan biosynthetic pathways are disturbed as in rheumatoid arthritis (RA). The present study was focused on analysis of O-linked glycoproteins of plasma in RA patients. Two dimensional gel electrophoresis of jacalin bound plasma of RA patients revealed a number of differentially expressed protein spots as compared to healthy controls. Eighteen protein spots were found to have statistically significant (p<0.05) difference in their expression level from four sets of gels and were identified by MALDI-TOF MS. Most of the identified proteins were predicted to be O-glycosylated proteins by Net–O-Gly 3.1 algorithm. Among these the alpha 2HS glycoprotein (A2HSG) was found to be down regulated whereas inter alpha trypsin inhibitor H4 (ITIH4) was up regulated and this was validated by Western blotting. The glycosylation studies showed the reduced N-linked sialylation of A2HSG in RA patients. Altered glycoprotein expression and functional as well as structural studies of glycans might help in the diagnosis of RA and understanding the disease pathogenesis.

Interaction of peanut root lectin (PRA II) with rhizobial lipopolysaccharides

Sugar specific binding of peanut root lectin (PRA II) to peanut specific bradyrhizobial lipopolysaccharides (LPS) was demonstrated by gel retardation assay and lectin based ELISA. Sephadex G-50 gel purified high molecular weight polysaccharides from NC 92 LPS bind PRA II most efficiently. Binding of NC 92 LPS only to PRA II and not to PNA, SBA and PSL by Western blot analysis suggests that this lectin-LPS interaction is tissue as well as species specific.