Kix domain specific Immunoglobulin A can protect from adverse lung and cerebral pathology induced by Plasmodium berghei ANKA.

Plasmodium specific IgA has been detected in serum and breast milk among the endemic population but the role it can play in vivo is not clear. In this report, we demonstrate the utility of Malaria specific IgA, elicited by peptide sequences (referred as Mpep3 and Mpep4) of region VI of EBA-175 (PfrVI). Immunization of mice with KLH tagged or untagged peptides of Mpep3, Mpep4 or with PfrVI have resulted in specific IgA response that inhibits the in vitro invasion of Plasmodium falciparum merozoites. Mice having the IgA specific to Mpep4 have exhibited higher tolerance to Plasmodium berghei ANKA parasitemia, exhibited several fold lesser sequestration of infected RBC, lesser damage to microvasculature with no signs of perivascular haemorrhage and lesser lung inflammation in comparison to unimmunized mice. In addition, the immunized mice have B-cell population that secrete the IgA specific to PfrVI. These results suggest that the IgA specific to these malarial antigens can confer significant advantage to hosts and it may also reduce the severity of malaria infection.

Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment.

BACKGROUND: GAD65 (Glutamic acid decarboxylase 65 KDa isoform) is one of the most important auto-antigens involved in Type 1 diabetes induction. Although it serves as one of the first injury markers of ß-islets, the mechanisms governing GAD65 expression remain poorly understood. Since the regulation of GAD65 is crucial for the proper functioning of insulin secreting cells, we investigated the stress induced regulation of GAD65 transcription. RESULTS: The present study shows that SMAR1 regulates GAD65 expression at the transcription level. Using a novel protein-DNA pull-down assay, we show that SMAR1 binding is very specific to GAD65 promoter but not to the other isoform, GAD67. We show that Streptozotocin (STZ) mediated DNA damage leads to upregulation of SMAR1 and p53 expression, resulting in elevated levels of GAD65, in both cell lines as well as mouse ß-islets. SMAR1 and p53 act synergistically to up-regulate GAD65 expression upon STZ treatment. CONCLUSION: We propose a novel mechanism of GAD65 regulation by synergistic activities of SMAR1 and p53.

A monoclonal antibody against annexin A2 targets stem and progenitor cell fractions in tumors.

The involvement of cancer stem cells (CSCs) in driving tumor dormancy and drug resistance is well established. Most therapeutic regimens however are ineffective in targeting these regenerative populations. We report the development and evaluation of a monoclonal antibody, mAb150, which targets the metastasis associated antigen, Annexin A2 (AnxA2) through recognition of a N-terminal epitope. Treatment with mAb150 potentiated re-entry of CSCs into the cell cycle that perturbed tumor dormancy and facilitated targeting of CSCs as was validated by in vitro and in vivo assays. Epigenetic potentiation further improved mAb150 efficacy in achieving total tumor regression by targeting regenerative populations to achieve tumor regression, specifically in high-grade serous ovarian adenocarcinoma.

Correction to: Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment.

An amendment to this paper has been published and can be accessed via the original article.

Inactivation of polygalacturonase and pectate lyase produced by pH tolerant fungus Fusarium moniliforme NCIM 1276 in a liquid medium and in the host tissue.

Fusarium moniliforme NCIM 1276 produced pH dependent an extracellular polygalacturonase (PG) and pectate lyase (PL) at pH 5 and pH 8, respectively. In the extracellular medium about 20.3% PG and 54% of PL protein concentrations were present in the active state at pH 5 and pH 8, respectively, whereas in intracellularly, more than 86% of both protein contents remained in the active state at all pH tested. We found two possible reasons, end-product inhibition and effect of environmental pH on conformation of the proteins after their release into the medium. Additionally, in infected tomato and cauliflower plants, the fungus secreted similar proteins which were located near to the epidermal and vascular regions of the hypocotyls. In infected tissues, between 26.9% and to 41.5% of PG and only 0.84%-13.4% of PL protein concentrations were present in active state. Thus, the medium/cell sap pH and concentrations of substrate/end products seem to play an important role in fungal invasion during plant pathogenesis are discussed with current literature.

Role of Adiantum philippense L. on Glucose Uptake in Isolated Pancreatic Cells and Inhibition of Adipocyte Differentiation in 3T3-L1 Cell Line.

BACKGROUND: Adiantum philippense (AP) is a pteridophyte that shows antihyperglycemic activity in vivo diabetic model, but the mechanism of action is unknown. OBJECTIVE: AP was found to play a pivotal role in minimizing the high blood glucose in alloxan-induced diabetic rats. Simultaneously, it was observed that it could maintain the normal lipid profile even in diabetic condition. To investigate its insulin-like activity along with its inhibitory role on adipocyte differentiation became the objective of our present study. MATERIALS AND METHODS: Glucose uptake potential of this fern was done in isolated pancreatic islets and inhibition of adipocyte differentiation was assessed in 3T3-L1 cell line. Before this, the cytotoxic concentration was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on L929 cell line. To determine its role in lipid metabolism, the oil droplets produced in adipocytes were stained with Oil 'O' red staining, and triglyceride levels of various drug treatments were measured spectrophotometrically. RESULTS: This fern extract was found to be actively utilizing glucose in the glucose uptake assay. Moreover, it was also involved in inhibiting differentiation of pro-adipocyte to adipocyte in the 3T3-L1 cell lines. The percentage inhibition as obtained from the absorbance showed that the ethanolic extract at the concentration of 200 µg/ml showed 32.48% inhibition. CONCLUSION: All the above-mentioned parameters when appraised indicated that this fern could be used as an alternative medicine in managing diabetes associated with obesity. SUMMARY: Adiantum phillippense (AP) is a pteridophyte that can work as antihyperglycemic agent by minimizing some adverse effects produced by diabetes. Diabetes produces oxidative stress, hampers normal glucose uptake in the pancreas, promotes adipocyte differentiation, and leads to obesity, and as a result, it generates catastrophic effect to the normal cells. The present study has shown that ethanolic extract of AP gives better protection rate against H2 O2-induced cytotoxicity, elicits insulinotropic activity in isolated mouse pancreatic glucose uptake assay. It also inhibits the preadipocytes to become mature adipocytes judged by morphology or lipid-specific Oil-Red-O staining of 3T3-L1 cell line. Abbreviations used: AP: Adiantum phillipense; MTT: (3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide); BSA: Bovine serum albumin; FCS: Fetal calf serum; DMEM: Dulbecco's minimum essential media; RPMI: Roswell park memorial institute medium; DTZ: Dithizone; TG: Triglyceride; PPARγ: Peroxisome proliferator-activated receptor gamma; IBMX: 3-isobutyl-1-methylxanthine; nm: Nanometer; GI: Growth Inhibition; ELISA: Enzyme linked immunosorbent assay.

Studies on wound healing potential of polyherbal formulation using in vitro and in vivo assays.

BACKGROUND: The use of herbal plant extracts in wound healing is known through decades, but it is necessary to provide scientific data through reverse pharmacology. OBJECTIVE: The aim of the present study is to find the mechanism behind the healing of wounds using in vitro and in vivo assays. MATERIAL AND METHODS: The study was designed to determine proliferation and mobilization of fibroblast and keratinocytes at the site of injury, angiogenesis at the site of healing and reduction in oxidative stress while healing. In our earlier studies it was observed that herbal extract of Vitex negundo L. (VN), Emblica officinalis Gaertn (EO), and Tridax procumbens L. (TP) showed rapid regeneration of skin, wound contraction and collagen synthesis at the site of injury in excision wound model. In the present study the cell mobilization was monitored in the scratch assay on L929 fibroblastic cell line and HaCaT keratinocytes cell line under the influence of aqueous plant extracts and its formulation. This formulation was also assessed for its angiogenic potential using CAM assay. Study was carried out to probe synergistic effect of polyherbal formulation using excision model in rat. RESULTS: The formulation was found to contain high amount of flavonoids, tannins and phenols which facilitate wound healing. At 20 µg/ml concentration of formulation, significant increase in tertiary and quaternary vessels were observed due to angiogenic potential of formulation. Formulation at the concentration of 3 µg/ml and 5 µg/ml showed significant mobilization of keratinocytes and fibroblasts respectively at the site of injury. Polyherbal formulation showed rapid regeneration of skin and wound contraction. Biochemical parameters like hydroxyproline, hexosamine and collagen turnover was increased in test drug treated animals as compared to untreated, whereas antioxidants such as catalase and GSH were increased significantly and decreased amount of tissue MDA was observed. CONCLUSION: Polyherbal formulation prepared from the plant extracts accelerates wound healing process by proliferation and mobilization of fibroblast and keratinocytes, and angiogenesis at the site of injury. It also shows fast contraction of wound with its beneficial improvement in tissue biochemical and antioxidant parameters.

A novel CD28 mRNA variant and simultaneous presence of various CD28 mRNA isoforms in human T lymphocytes.

The primary transcript of the human CD28 gene in T lymphocytes, encoding for a costimulatory molecule, is known to undergo alternative splicing, and different small sets of variant isoforms have been reported. This report presents the novel simultaneous presence of eight different mRNA isoforms, all observed together in normal human T cells; this is an interesting finding in the study of CD28 mRNA structural variants. A similar pattern was found in a total of four individuals. In addition, we also report the occurence and sequence of a new CD28 mRNA isoform, one of the above eight, which is a novel variant generated by the use of a new combination of splice donor and acceptor sites.

Dense cataract and microphthalmia--new spontaneous mutation in BALB/c mice.

We describe a new spontaneous mutation in BALB/c mice that causes abnormal phenotype, such as congenital cataract and microphthalmia. This abnormality was found to be inheritable because offspring with the same abnormality were produced by backcrossing the abnormal male to its normal female parent. Results of various crosses made to determine the mode of inheritance indicated that this abnormality is attributable to mutation of an autosomal recessive gene. Slit lamp examination of the mutant eyes revealed total lenticular opacity, disturbed typical iris pattern, and abnormal pupillary muscle development. Histologic changes in mutant eyes between gestation day 13 and postnatal day 1 indicated various eye and lens abnormalities, including microphthalmia; underdeveloped iris, optic stalk, cornea, and retina; degenerated lens fibers with lost fibrillar structure; and vacuoles of various sizes at the posterior border of the lens. Mild opacity of the lens was found to progress with age and became denser, resembling mature cataract, and occupying the lens completely at the age of six to eight weeks. We, therefore, temporarily designated this abnormality as dense cataract and microphthalmia, with the gene symbol dcm.

Disabling complement regulatory activities of vaccinia virus complement control protein reduces vaccinia virus pathogenicity.

Poxviruses encode a repertoire of immunomodulatory proteins to thwart the host immune system. One among this array is a homolog of the host complement regulatory proteins that is conserved in various poxviruses including vaccinia (VACV) and variola. The vaccinia virus complement control protein (VCP), which inhibits complement by decaying the classical pathway C3-convertase (decay-accelerating activity), and by supporting inactivation of C3b and C4b by serine protease factor I (cofactor activity), was shown to play a role in viral pathogenesis. However, the role its individual complement regulatory activities impart in pathogenesis, have not yet been elucidated. Here, we have generated monoclonal antibodies (mAbs) that block the VCP functions and utilized them to evaluate the relative contribution of complement regulatory activities of VCP in viral pathogenesis by employing a rabbit intradermal model for VACV infection. Targeting VCP by mAbs that inhibited the decay-accelerating activity as well as cofactor activity of VCP or primarily the cofactor activity of VCP, by injecting them at the site of infection, significantly reduced VACV lesion size. This reduction however was not pronounced when VCP was targeted by a mAb that inhibited only the decay-accelerating activity. Further, the reduction in lesion size by mAbs was reversed when host complement was depleted by injecting cobra venom factor. Thus, our results suggest that targeting VCP by antibodies reduces VACV pathogenicity and that principally the cofactor activity of VCP appears to contribute to the virulence.

Dense cataract and microphthalmia (dcm) in BALB/c mice is caused by mutations in the GJA8 locus.

A spontaneous mutation in BALB/c mice that causes congenital dense cataract and microphthalmia (dcm) was reported previously. This abnormality was found to be inheritable and the mode of inheritance indicated that this phenotype is due to mutation of an autosomal recessive gene. We performed genetic screen to identify the underlying mutations through linkage analysis with the dcm progenies of F(1) intercross. We identified the region of mutation on chromosome 3 and further mapping and sequence analysis identified the mutation in the GJA8 gene that encodes for connexin 50. The mutation represents a single nucleotide change at position 64 (G to C) that results in a change in the amino acid glycine to arginine at position 22 (G22R) and is identical to the mutation previously characterized as lop10. However, the phenotype of these mice differ from that of lop10 mice and since it is one of the very few genetic models with recessive pattern of inheritance, we propose that dcm mice can serve as a useful model for studying the dynamics and interaction of the gap junction formation in mouse eye development.

Pancreatic islets are very poor in rectifying oxidative DNA damage.

OBJECTIVE: Free radicals that escape scavenging by antioxidant defense damage lipids, proteins, and DNA. Damage to DNA can be repaired. Therefore, both cells' antioxidant defense and their ability to repair oxidatively damaged DNA decide its fate to survive oxidative stress. Pancreatic islets cells with poor antioxidant defense were checked for their ability to remove oxidative damage form DNA. METHODS: For ex vivo DNA repair, assay-cultured pancreatic islets and liver slices were treated with 1 and 10 mM H2O2, respectively, for 30 minutes. After incubation for different time intervals, 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA of these cells was estimated using monoclonal antibody raised against 8-OHdG by competitive enzyme-linked immunosorbent assay. For in vitro DNA repair assay, oxidatively damaged pBR322 was incubated with nuclear extracts of islet and liver cells, and 8-OHdG retained in the plasmid was quantitated. RESULTS: Oxidative damage induced by H2O2 was removed quickly and efficiently from DNA by liver cells compared with islet cells. The repair of oxidatively damaged plasmid DNA in vitro was also performed more efficiently (P < 0.05) by nuclear extracts from liver cells compared with islet cell. CONCLUSIONS: We clearly demonstrate that in addition to their low antioxidant defense, islets are very poor in rectifying the oxidative DNA damage.

Kinetic analysis of the interactions between vaccinia virus complement control protein and human complement proteins C3b and C4b.

The vaccinia virus complement control protein (VCP) is an immune evasion protein of vaccinia virus. Previously, VCP has been shown to bind and support inactivation of host complement proteins C3b and C4b and to protect the vaccinia virions from antibody-dependent complement-enhanced neutralization. However, the molecular mechanisms involved in the interaction of VCP with its target proteins C3b and C4b have not yet been elucidated. We have utilized surface plasmon resonance technology to study the interaction of VCP with C3b and C4b. We measured the kinetics of binding of the viral protein to its target proteins and compared it with human complement regulators factor H and sCR1, assessed the influence of immobilization of ligand on the binding kinetics, examined the effect of ionic contacts on these interactions, and sublocalized the binding site on C3b and C4b. Our results indicate that (i) the orientation of the ligand is important for accurate determination of the binding constants, as well as the mechanism of binding; (ii) in contrast to factor H and sCR1, the binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions as predicted earlier; (iii) VCP has a 4.6-fold higher affinity for C4b than that for C3b, which is also reflected in its factor I cofactor activity; (iv) ionic interactions are important for VCP-C3b and VCP-C4b complex formation; (v) VCP does not bind simultaneously to C3b and C4b; and (vi) the binding site of VCP on C3b and C4b is located in the C3dg and C4c regions, respectively.

The Th1-specific costimulatory molecule, m150, is a posttranslational isoform of lysosome-associated membrane protein-1.

In an earlier report, we had shown a 150-kDa protein termed as M150, isolated from the surface of activated macrophages, to possess costimulatory activity for CD4(+) T cells. Significantly, this protein was found to specifically elicit Th1 responses. In this study, we characterize M150, which belongs to a unique subset of the lysosome-associated membrane protein-1 glycoprotein. Interestingly, the costimulatory activity of M150 depends on its posttranslational modification, which has a distinct glycosylation pattern restricted to macrophages. Furthermore, it has been demonstrated that in addition to stimulating Th1-specific responses, M150 is also capable of driving differentiation of naive CD4(+) T cells into the Th1 subset. This altered posttranslational modification of housekeeping protein appears to represent a novel pathway by which APCs can additionally regulate T cell responses.