Development and comparative evaluation of a competitive ELISA with rose bengal test and a commercial indirect ELISA for serological diagnosis of brucellosis.

The development of a competitive ELISA for the detection of brucella-specific antibodies in bovines is described. Anti-brucella guinea pig serum was used as a source of competing antibodies. Lipo-polysaccharide purified from inactivated B. abortus S19 culture was used as antigen for the development of the assay. Sera from cattle were used in the competitive ELISA, rose bengal test and a commercial indirect ELISA. The following cattle sera were tested: (i) known positive sera (n = 80) (ii) known negative sera (n = 100) and (iii) field sera (n = 1184). Based on the receiver operating characteristics curve analysis and frequency distribution of the percentage of inhibition, 30% inhibition was considered the cut-off for positive and negative results. The sensitivity and specificity estimate on comparison with the commercial indirect ELISA was 94.87 and 92.12% respectively. The competitive ELISA described is a simple method for the routine screening of animal sera for detecting Brucella-specific antibodies.

Chimeric tymovirus-like particles displaying foot-and-mouth disease virus non-structural protein epitopes and its use for detection of FMDV-NSP antibodies.

Expression of Physalis mottle tymovirus (PhMV) coat protein (CP) in Escherichia coli (E. coli) was earlier shown to self-assemble into empty capsids that are nearly identical to the capsids formed in vivo. Aminoacid substitutions were made at the N-terminus of wild-type PhMV CP with single or tandem repeats of infection related B-cell epitopes of foot-and-mouth disease virus (FMDV) non-structural proteins (NSPs) 3B1, 3B2, 3AB, 3D and 3ABD of lengths 48, 66, 49, 51 and 55, respectively to produce chimeras pR-Ph-3B1, pR-Ph-3B2, pR-Ph- 3AB, pR-Ph-3D and pR-Ph-3ABD. Expression of these constructs in E. coli resulted in chimeric proteins which self-assembled into chimeric tymovirus-like particles (TVLPs), Ph-3B1, Ph-3B2, Ph-3AB, Ph-3D and Ph-3ABD as determined by ultracentrifugation and electron microscopy. Ph-3B1, Ph-3B2, Ph-3AB and Ph-3ABD reacted with polyclonal anti-3AB antibodies in ELISA and electroblot immunoassay, while wild-type PhMV TVLP and Ph-3D antigens did not react. An indirect ELISA (I-ELISA) was developed using Ph-3AB to detect FMDV-NSP antibodies in sera of animals that showed clinical signs of FMD. Field serum samples from cattle, buffalos, sheep, goats and pigs were examined by using these chimeric TVLPs for the differentiation of FMDV infected animals from vaccinated animals (DIVA). The assay was demonstrated to be highly specific (100%) and reproducible with sensitivity levels (94%) comparable to the Ceditest kit (P>0.05).

Anti-B7-1/B7-2 antibody elicits innate-effector responses in macrophages through NF-kappaB-dependent pathway.

Blocking T cell co-stimulatory signals by anti-B7-1/B7-2 mAb is an attractive approach to treat autoimmune diseases. However, anti-B7-1/B7-2 mAb treatment is known to exacerbate autoimmune diseases through mechanisms not fully understood. Tumor necrosis factor alpha (TNF-alpha) and reactive oxygen species (ROS) also play important roles in determining the clinical outcome of autoimmune diseases. In this study, we demonstrate that the anti-B7-1 and the anti-B7-2 mAbs activate macrophages for higher induction of TNF-alpha and other effector responses such as bacterial cytotoxicity and production of ROS. Nuclear factor-kappaB (NF-kappaB) was found to be increased with anti-B7-1/B7-2 mAb treatment. Inhibition of NF-kappaB activity by over-expression of phosphorylation-defective I-kappaB alpha in anti-B7-1/B7-2 mAb-treated macrophages decreased TNF-alpha production. These data indicate that anti-B7-1 and anti-B7-2 mAbs can trigger innate-effector responses in macrophages by activating NF-kappaB-signaling pathway. Our results suggest that the B7 molecules are not only essential for induction of adaptive immune responses but also play roles in activation of innate immune responses.

Clusters of PE and PPE genes of Mycobacterium tuberculosis are organized in operons: evidence that PE Rv2431c is co-transcribed with PPE Rv2430c and their gene products interact with each other.

About 10% of the coding capacity of the Mycobacterium tuberculosis (M. tb) genome is devoted to the PE/PPE family of genes scattered throughout the genome. We have identified 28 PE/PPE operons which are organized within the M. tb genome in such a way that most PE members are upstream to PPE members. One example of such a gene arrangement is the PPE gene Rv2430c, earlier shown by us to code for a highly antigenic protein eliciting strong B-cell responses in TB patients [Choudhary, R.K., Mukhopadhyay, S., Chakhaiyar, P., Sharma, N., Murthy, K.J.R., Katoch V.M. and Hasnain, S.E. (2003) PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B cell response. Infect. Immun. 71, 6338-6343], situated downstream to PE gene Rv2431c. Rv2431c and Rv2430c are transcribed as an operon. Expression of either rRv2431c or rRv2430c alone in E. coli limited their localization to the inclusion bodies. However, when they were co-expressed, both the proteins appeared in the soluble fraction. These two proteins interact with each other and form oligomers when alone, however, when present together they exist as heteromer.

Hydrogen peroxide inhibits IL-12 p40 induction in macrophages by inhibiting c-rel translocation to the nucleus through activation of calmodulin protein.

Although the antimicrobial activity of reactive oxygen species (ROSs) is well defined, the role of ROSs in regulating the immune response of the body is not well understood. We now provide evidence that hydrogen peroxide (H2O2), a major component of ROSs, inhibits interleukin-12 (IL-12) p40 and IL-12 p70 induction in murine macrophages and catalase pretreatment prevents H2O2-mediated down-regulation of IL-12. Endogenous accumulation of H2O2/ROSs in macrophages treated with alloxan resulted in IL-12 p40 inhibition. Although nuclear expression of both p50 and p65 NF-kappaB increased on H2O2 exposure, nuclear c-rel level was inhibited. Overexpression of c-rel restored IL-12 p40 on stimulation with lipopolysaccharide plus IFN-gamma during H2O2 treatment. H2O2 did not inhibit c-rel induction in cytosol; however, it prevented the transport of c-rel from cytosol to the nucleus. H2O2 activated calmodulin (CaM) protein in the cytosol, which subsequently sequestered c-rel in the cytosol preventing its transport to the nucleus. The CaM inhibitor trifIuoperazine increased both nuclear c-rel and IL-12 p40 levels in H2O2-treated macrophages, emphasizing a role of CaM in these processes. H2O2/ROSs thus down-regulate IL-12 induction in macrophages by a novel pathway inhibiting c-rel translocation to the nucleus through activation of CaM protein.

Molecular characterization of foot-and-mouth disease virus type C of Indian origin.

Comparison of nucleotide sequences of the partial 1D region of foot-and-mouth disease type C viruses of Indian origin with those of European, South American, and Southeast Asian viruses revealed that the Indian viruses form a distinct genotype. The vaccine strain C IND/51/79 belongs to this genotype and may be a prototype strain of this genotype.