High affinity mouse-human chimeric Fab against hepatitis B surface antigen

Aim: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. Methods: We cloned the VH and VL genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and CL domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli. Results: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield. Conclusion: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a full-length chimeric antibody for therapeutic uses

CHEMICAL INVESTIGATION OF NEEM LEAF GLYCOPROTEIN USED AS IMMUNOPROPHYLACTIC AGENT FOR TUMOR GROWTH RESTRICTION

Objective: Unique immune modulatory function of an aqueous preparation of neem leaf (NLP) in relation to cancer has already been reported. The objective of this present study is to find out the active component present in NLP.Methods: NLP was exposed to a gradient of temperature, pH and enzymes to use for mice immunization before tumor inoculation. Glycoprotein extracted from NLP was purified and analyzed by using Folin's phenol reagent, polyacrylamide gel electrophoresis, scanning electron microscopy and amino acid analysis. Carbohydrate moiety of this protein was analysed by GLC-MS. Immunogenicity was checked by ELISA and immunoblotting.Results: Exposure of NLP to adverse temperature and pH causes significant reduction in tumor growth restricting function of NLP. Treatment of NLP with proteolytic enzymes results abolition of the tumor growth restriction in mice. Biochemical assays indicated the presence of a glycoprotein in NLP, designated as neem leaf glycoprotein (NLGP) which appeared in non-denatured PAGE as a single band, and as three bands in SDS-PAGE having molecular weights 48 Kda, 24 Kda and 15 Kda. NLGP constitutes the carbohydrate moiety of about 33% consisting of arabinose, galactose and glucose. This glycoprotein consisted of seventeen amino acids of which nine were essential. Immunogenecity of this protein was defined by strong reaction of the ant-NLGP sera with NLGP by ELISA and immunoblot.Conclusion: Overall results demonstrated the immense potential of newly identified NLGP, present in NLP as an immunoprophylactic agent for tumor growth restriction.Â

Executive Functions and Prefrontal Cortex: A Matter of Persistence?

Executive function is thought to originates from the dynamics of frontal cortical networks. We examined the dynamic properties of the blood oxygen level dependent time-series measured with functional MRI (fMRI) within the prefrontal cortex (PFC) to test the hypothesis that temporally persistent neural activity underlies performance in three tasks of executive function. A numerical estimate of signal persistence, the Hurst exponent, postulated to represent the coherent firing of cortical networks, was determined and correlated with task performance. Increasing persistence in the lateral PFC was shown to correlate with improved performance during an n-back task. Conversely, we observed a correlation between persistence and increasing commission error – indicating a failure to inhibit a prepotent response – during a Go/No-Go task. We propose that persistence within the PFC reflects dynamic network formation and these findings underline the importance of frequency analysis of fMRI time-series in the study of executive functions

Problems in using statistical analysis of replacement and silent mutations in antibody genes for determining antigen-driven affinity selection

The analysis of molecular signatures of antigen-driven affinity selection of B cells is of immense use in studies on normal and abnormal B cell development. Most of the published literature compares the expected and observed frequencies of replacement (R) and silent (S) mutations in the complementarity-determining regions (CDRs) and the framework regions (FRs) of antibody genes to identify the signature of antigenic selection. The basic assumption of this statistical method is that antigenic selection creates a bias for R mutations in the CDRs and for S mutations in the FRs. However, it has been argued that the differences in intrinsic mutability among different regions of an antibody gene can generate a statistically significant bias even in the absence of any antigenic selection. We have modified the existing statistical method to include the effects of intrinsic mutability of different regions of an antibody gene. We used this method to analyse sequences of several B cell-derived monoclonals against T-dependent antigens, T-independent antigens, clones derived from lymphoma and amyloidogenic clones. Our sequence analysis indicates that even after correcting for the intrinsic mutability of antibody genes, statistical parameters fail to reflect the role of antigen-driven affinity selection in maturation of many clones. We suggest that, contrary to the basic assumption of such statistical methods, selection can act both for and against R mutations in the CDR as well as in the FR regions. In addition we have identified different methodological difficulties in the current uses of such statistical analysis of antibody genes

A simple route to the syntheses of both enantiomers of trans-oak lactone and (+)-cis-oak lactone

Stereocontrolled syntheses of both enantiomers of trans-oak lactone and (+)-cis-oak lactone have been achieved from a common precursor

Domino ring-opening metathesis–ring-closing metathesis of bicyclo[2.2.2]octene derivatives: scope and limitations

Domino metathesis involving ring-opening metathesis–ring-closing metathesis (ROM–RCM) of a bicyclo[2.2.2]octene derivative having an appropriate alkene chain, expected to produce a 7/6 fused bicyclic system, provided a decalin system in contrast to ROM–RCM of the corresponding bicyclo[2.2.1]heptene analogues, which as expected produced the 7/5 fused bicycles. The expected 6/7 bicyclic system could, however, be made through RCM of the elusive ROM product prepared from the same bicyclo[2.2.2]octene analogue by a nonmetathetic route. A rationale to explain the difference in reactivity pattern between these two systems toward ROM–RCM has been forwarded

Microfilaria in pleural fluid cytology: A rare finding

Lymphatic filariasis is endemic in India and Southeast Asia. Detection of microfilaria is infrequently reported during cytological evaluation of various lesions or body cavity fluids. Presence of microfilaria in pleural fluid cytology is very rare finding even in endemic areas. Few cases of accidental finding of microfilaria have been reported in association with malignant pleural effusion. But pleural effusion of filarial origin is extremely rare manifestation. Here we report a classical case of microfilaria in pleural fluid cytology