Anticoagulant effect of Naja naja venom 5'nucleotidase: demonstration through the use of novel specific inhibitor, vanillic acid.

The snake venom proteins affect hemostasis by either advancing/delaying blood coagulation. Apart from proteases and phospholipase A(2)s (PLA(2)s), 5'nucleotidase is known to affect hemostasis by inhibiting platelet aggregation. In this study, the possible involvement of Naja naja venom 5'nucleotidase in mediating anticoagulant affect is evaluated. Vanillic acid selectively and specifically inhibited 5'nucleotidase activity among other enzymes present in N. naja venom. It is a competitive inhibitor as evident of inhibition relieving upon increased substrate concentration. Vanillic acid dose dependently inhibited the anticoagulant effect of N. naja venom up to 40%. This partial involvement of 5'nucleotidase in mediating anticoagulant effect is substantiated by concanavalin-A (Con-A) inhibition studies. Con-A, competitively inhibited in vitro protease and 5'nucleotidase activity up to 100%. However, it did not exhibit inhibitory activity on PLA(2). The complete inhibition of anticoagulant effect by Con-A upon recalcification time suggests the participation of both 5'nucleotidase and protease in mediating anticoagulant effect of N. naja venom. Vanillic acid and Con-A inhibition studies together suggest that probably 5'nucleotidase interacts with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Thus, this study for the first time demonstrates the involvement of 5'nucleotidase in mediating N. naja venom anticoagulant effect.     

Mizoribine--an inosine monophosphate dehydrogenase inhibitor--acts synergistically with cyclosporine A in prolonging survival of murine islet cell and heart transplants across major histocompatibility barrier

IntroductionMizoribine (MZR) is an inosine monophosphate dehydrogenase inhibitor. It has been widely used in Japan in the treatment of autoimmune diseases and is known to inhibit T and B cell proliferation. The aim of this study was to evaluate the efficacy of MZR as an immunosuppressive agent and determine its ability to synergize with a commonly used calcineurin inhibitor Cyclosporine A (CsA) in prolonging survival of murine islet cells and heart transplanted across major histocompatibility barrier.MethodsMurine allogeneic islet cell transplantation between Balb/c donor mice and C57BL/6 recipient mice and heterotopic heart transplantation was done between C3H/He donor mice and Balb/c recipient mice. Recipients were divided into groups based on immunosuppression: Group 1—No immunosuppression, Group 2—MZR alone (20 mg/kg/day), Group 3—CsA alone (20 mg/kg/day), Group 4—MZR + CsA (20 mg/kg/day). Donor specific IFN-γ, IL-10, IL-2, IL-4 secreting cells were enumerated by ELISpot. Serum cytokine and chemokine concentration was measured by Luminex.ResultsIslet cell allograft recipients treated with CsA and MZR had prolonged islet function compared to other groups [normoglycemia (blood glucose < 200 mg/dL) up to 32 ± 4 days, p < 0.05]. Similarly, heart allograft survival was significantly improved in mice treated with CsA and MZR compared to other groups (50% 30-day survival, p = 0.04). Donor specific IFN-γ, IL-4, IL-2 secreting cells were significantly decreased in recipients treated with CsA and MZR with marked increase in IL-10 secreting cells (p < 0.05). There was also an increase in serum IL-10 with decrease in IFN-γ, IL-4, IL-2, MCP-1, and IL-6 in mice treated with CsA and MZRConclusionMZR and CsA when used in combination are potent immunosuppressive agents in murine islet cell and heart transplantation models. These agents lead to a decrease in donor specific IFN-γ with increase in IL-10 secreting cells leading to improved allograft survival and function.     

Hepatoprotective action of Orthosiphon diffusus (Benth.) methanol active fraction through antioxidant mechanisms: An in vivo and in vitro evaluation

Ethnopharmacological relevance

Preparations of Orthosiphon diffusus (Benth.) have been used by folk medicinal practitioners in the Western Ghats of India for treating inflammation, hepatitis and jaundice for many years and their effectiveness is widely acclaimed among the tribal communities.

Aim of the study

To evaluate the mechanisms behind the antioxidant and hepatoprotective potential of Orthosiphon diffusus methanol active fraction (MAF) using in vivo (rat) and in vitro (cell culture) models.

Materials and methods

Neutralization of CCl₄-induced hepatotoxicity by MAF was evaluated in rats. Towards this, serum levels of hepatic injury markers (lactate dehydrogenase and alkaline phosphatase), antioxidant enzymes in the liver homogenates, and histological examination were performed. In in vitro studies, mechanisms of neutralization of H₂O₂-induced toxicity by MAF using MTT, Comet assay and up-regulation of antioxidant enzymes at genetic level (RT-PCR) was performed in HepG2 cells.

Results

Rats pre-treated with Orthosiphon diffusus MAF demonstrated significantly reduced levels of serum LDH (1.3-fold, p<0.05) and ALP (1.6-fold, p<0.05). Similarly, multiple dose MAF administration demonstrated significantly enhanced levels (p<0.05) of antioxidant enzymes in the liver homogenates. Histological analysis revealed complete neutralization of CCl₄-induced liver injury by the extract. The in vitro studies demonstrated that, pre-treatment of MAF effectively prevented H₂O₂-induced oxidative stress, genotoxicity and significantly enhanced (~6-fold, p<0.01) expression of genes for antioxidant enzymes.

Conclusions

Orthosiphon diffusus MAF demonstrated significant hepatoprotection against CCl₄-induced hepatotoxicity by antioxidant mechanisms comparable to silymarin. H₂O₂-induced oxidative stress was completely neutralized by MAF through enhanced expression of genes for antioxidant enzymes. Therefore, this study validates the use of Orthosiphon diffusus by folk medicinal practitioners in India. Further, MAF of Orthosiphon diffusus can serve as a strong candidate for the development of herbal hepatoprotective agents.     

Molecular profiling and bioactive potential of an endophytic fungus Aspergillus sulphureus isolated from Sida acuta: a medicinal plant

Context: Sida acuta Burm.f. (Malvaceae) extracts are reported to have applications against malaria, diuretic, antipyretic, nervous and urinary diseases. No fungal endophytes of S. acuta are reported.

Objective: Isolation, identification and evaluation of antibacterial, antioxidant, anticancer and haemolytic potential of fungal endophytes from the ethnomedcinal plant S. acuta.

Materials and methods: Sida acuta stem segments were placed on PDA medium to isolate endophytic fungi. The fungus was identified by genomic DNA analysis and phylogenetic tree was constructed using ITS sequences (GenBank) to confirm species. The antibacterial efficacy of Aspergillus sulphureus MME12 ethyl acetate extract was tested against Gram-positive and Gram-negative pathogenic bacteria. DPPH free radical scavenging activity, anticancer and DNA fragmentation against EAC cells, and direct haemolytic activity (100–500?μg/mL) using human erythrocytes were determined.

Results and discussion: The ethyl acetate extract of A. sulphureus (Fresen.) Wehmer (Trichocomaceae) demonstrated significant antibacterial potential against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella typhi compared to streptomycin. MIC against test pathogens was in the range of 15.6–62.5?μg/mL. The antioxidant results revealed significant RSA from 12.43% to 62.02% (IC₅₀?=?350.4?μg/mL, p?≤?0.05). MME12 offered considerable inhibition of EAC proliferation (23% to 84%, IC₅₀?=?216.7?μg/mL, p?≤?0.05) supported by DNA fragmentation studies. The extract also offered insignificant haemolysis (5.6%) compared to Triton X-100.

Conclusions: A single endophytic fungus, A. sulphureus MME12 was isolated and identified using molecular profiling. The above-mentioned findings support the pharmacological application of A. sulphureus MME12 extract and demand for purification of the active principle(s).     

PLA2 mediated arachidonate free radicals: PLA2 inhibition and neutralization of free radicals by anti-oxidants--a new role as anti-inflammatory molecule

PLA2 enzyme catalyses the hydrolysis of cellular phospholipids at the sn-2 position to liberate arachidonic acid and lysophospholipid to generate a family of pro-inflammatory eicosanoids and platelet activating factor. The generation of pro-inflammatory eicosanoids involves a series of free radical intermediates with simultaneous release of reactive oxygen species (superoxide and hydroxyl radicals). Reactive oxygen species formed during arachidonic acid metabolism generates lipid peroxides and the cytotoxic products such as 4-hydroxy nonenal and acrolein, which induces cellular damage. Thus PLA2 catalyzes the rate-limiting step in the production of pro-inflammatory eicosanoids and free radicals. These peroxides and reactive oxygen species in turn activates PLA2 enzyme and further attenuates the inflammatory process. Therefore scavenging these free radicals and inhibition of PLA2 enzyme simultaneously by a single molecule such as antioxidants is of great therapeutic relevance for the development of anti-inflammatory molecules. PLA2 enzymes have been classified into calcium dependent cPLA2 and sPLA2 and calcium independent iPLA2 forms. In several inflammatory diseases sPLA2 group IIA is the most abundant isoform identified. This isoform is therefore targeted for the development of anti-inflammatory molecules. Many secondary metabolites from plants and marine sponges exhibit both anti-inflammatory and antioxidant properties. Some of them include flavonoids, terpenes and alkaloids. But in terms of PLA2 inhibition and antioxidant activity, the structural aspects of flavonoids are well studied rather than terpenes and alkaloids. In this line, molecules having both anti-oxidant and PLA2 inhibitions are reviewed. A single molecule with dual activities may prove to be a powerful anti-inflammatory drug.     

Procoagulant activity of Calotropis gigantea latex associated with fibrin(ogen)olytic activity.

The latex of Calotropis gigantea is a rich source of useful components that has medicinal properties and one of the main applications is in controlling bleeding. The crude latex extract contained many proteins, which are highly basic in nature and exhibited strong proteolytic activity. The crude extract hydrolyses casein, human fibrinogen and crude fibrin clot in a dose-dependent manner. The hydrolyzing activity was completely inhibited by IAA indicating they belong to the super family, cysteine proteases. Crude extract hydrolyses Aalpha, Bbeta and gamma subunits of fibrinogen. Among all the subunits the preferential subunit to get hydrolyzed was Aalpha followed by Bbeta and gamma subunit is highly resistant and hydrolyzed at higher protein concentration or over a prolonged incubation time. The crude extract hydrolysis crude fibrin clot strongly compared to trypsin and papain. Pharmacologically the crude extract is hemorrhagic and induces skin hemorrhage at >75 microg and reduces the coagulation time of citrated plasma from 150 to 47 s and promotes blood coagulation. Procoagulation and blood clot hydrolysis are important in wound healing process. This is due to unique cysteine proteases of plant latex and is responsible for the pharmacological actions observed in folk medicine.     

Synthesis and evaluation of trimethoxyphenyl isoxazolidines as inhibitors of secretory phospholipase A2 with anti-inflammatory activity

A series of trimethoxyphenyl isoxazolidine derivatives, 5a(i-v) and 5b(i-v), bearing different constituents at the 5th position of the isoxazolidine ring were synthesized and evaluated in vitro and in vivo for their inhibitory activity against purified group I and II phospholipase A2 (PLA2) enzymes from snake venom and human inflammatory synovial fluid. Irrespective of modification to the pharmacophore (isoxazolidine ring), they exhibited greater specificity for group II PLA2. The length of alkyl or aryl group at the 5th position, which alters the hydrophobic and aromatic property, was responsible for enhancing the inhibition towards PLA2 enzymes. All of the compounds quench the fluorescent property of the purified PLA2 enzyme, and quenching increases with the increase in length of alkyl or aryl group. The inhibitory effect of compounds appeared to be due to the direct interaction of compounds with the enzyme. Inhibition is substrate-dependent, and the inhibitor likely competes with the substrate for the same binding site of the enzyme. The IC50 value for the most potent interacting inhibitor 5b(v) was 54.8 microM. The most active interacting compounds 5a(v) and 5b(v) from in vitro inhibition of PLA2 activity showed similar potency in in vivo neutralization of PLA2-induced mouse paw edema and hemolytic activity.     

Interplay between immune responses to HLA and non-HLA self-antigens in allograft rejection

Recent studies strongly suggest an increasing role for immune responses against self-antigens (Ags) which are not encoded by the major histocompatibility complex in the immunopathogenesis of allograft rejection. Although, improved surgical techniques coupled with improved methods to detect and avoid sensitization against donor human leukocyte antigen (HLA) have improved the immediate and short term function of transplanted organs. However, acute and chronic rejection still remains a vexing problem for the long term function of the transplanted organ. Immediately following organ transplantation, several factors both immune and non immune mechanisms lead to the development of local inflammatory milieu which sets the stage for allograft rejection. Traditionally, development of antibodies (Abs) against mismatched donor HLA have been implicated in the development of Ab mediated rejection. However, recent studies from our laboratory and others have demonstrated that development of humoral and cellular immune responses against non-HLA self-Ags may contribute in the pathogenesis of allograft rejection. There are reports demonstrating that immune responses to self-Ags especially Abs to the self-Ags as well as cellular immune responses especially through IL17 has significant pro-fibrotic properties leading to chronic allograft failure. This review summarizes recent studies demonstrating the role for immune responses to self-Ags in allograft immunity leading to rejection as well as present recent evidence suggesting there is interplay between allo- and autoimmunity leading to allograft dysfunction.     

Role of defensins in the pathogenesis of chronic lung allograft rejection

Chronic rejection predominantly manifested as bronchiolitis obliterans syndrome (BOS), still remains a major problem affecting long-term outcomes in human lung transplantation (LTx). Donor specific antibodies (DSA) and infiltration of neutrophils in the graft have been associated with the development of BOS. This study determines the role of defensins, produced by neutrophils, and its interaction with α-1-antitrypsin (AAT) towards induction of airway inflammation and fibrosis which are characteristic hallmarks of BOS. Bronchoalveolar lavage (BAL) and serum from LTx recipients, BOS+ (n = 28), BOS− (n = 26) and normal healthy controls (n = 24) were analyzed. Our results show that BOS+ LTx recipients had higher α-defensins (HNP1–3) and β-defensin2 HBD2 concentration in BAL and serum compared to BOS-DSA-recipients and normal controls (p = 0.03). BOS+ patients had significantly lower serum AAT along with higher circulating concentration of HNP–AAT complexes in BAL (p = 0.05). Stimulation of primary small airway epithelial cells (SAECs) with HNPs induced expression of HBD2, adhesion molecules (ICAM and VCAM), cytokines (IL-6, IL-1β, IL-13, IL-8 and MCP-1) and growth-factor (VEGF and EGF). In contrast, anti-inflammatory cytokine, IL-10 expression decreased 2-fold (p = 0.002). HNPs mediated SAEC activation was completely abrogated by AAT. In conclusion, our results demonstrates that neutrophil secretory product, α-defensins, stimulate β-defensin production by SAECs causing upregulation of pro-inflammatory and pro-fibrotic signaling molecules. Hence, chronic stimulation of airway epithelial cells by defensins can lead to inflammation and fibrosis the central events in the development of BOS following LTx.     

Emerging trends in the epidemiology of human astrovirus infection among infants,children and adults hospitalized with acute watery diarrhea in Kolkata,India

Human astroviruses (HAstVs) have now emerged as another common cause of non-bacterial acute gastroenteritis (AGE) in humans worldwide. This study investigated the epidemiology and genetic diversity of human astrovirus strains circulating among infants, younger children (up to 6 years), older children and adolescents (>6–17 years) and adults (18 years and above) hospitalized for diarrhea and their role in AGE in Kolkata, India. A total of 2535 fecal samples were screened for the presence of known enteric viral, bacterial and parasitic etiologies by conventional microbiological assays and molecular methods. The overall incidences of sole or mixed infection of HAstV with known enteric viral, bacterial and parasitic pathogens were detected in 60 cases (2.4%) among all age groups. The clinical symptoms of astrovirus-associated acute watery diarrhea cases were recorded for all sole and mixed infection cases. A high number of sole (n = 13/60 [21.7%]) and mixed infection cases (n = 22/60 [36.7%]) were observed in adults (18 years old or more). Considering all age groups, 18 sole infection cases (n = 18/60 [30%]) and 42 mixed infection cases (n = 42/60 [70%]) with Rotavirus (n = 11/25 [44%]), Vibrio cholerae O1 (n = 6/24 [25%]) Cryptosporidium spp and Giardia lamblia (n = 5/13 [38.4%]) were observed. Further, eleven HAstV samples from infants and children (up to 6 years), children and adolescents (>6–17 years) and adults (18 years and above) were analyzed for their sequences of overlap region between ORF1b (RdRp) and ORF2 (capsid). Among these, ten strains were found to have close genetic relatedness to the Japanese strain HAstV_G1 [AB009985]. Additionally, the IDH2211 Kolkata strain showed a close genetic match with the Thai HAstV_G3 strain [EU363889]. Our study reports show that HAstVs as the sole agent and as mixed infection with other known enteric viral, bacterial, parasitic pathogens are also responsible for AGE among infants, children, adolescents and adults in Kolkata, India.