Sera and biopsies of cervical lesions from 55 women with benign or malignant disease were analyzed for evidence of infection with herpes simplex virus type 2 (HSV-2) or human papillomavirus (HPV). In addition, information regarding known risk factors for cervical cancer was obtained by interview. The sera were tested for HSV-2 antibodies and the biopsies were tested for HPV or HSV DNA sequences by Southern blot hybridization. HSV-2 sequences were detected in 2/13 (15%) invasive neoplasms and in 1/12 (7%) benign lesions. Under non-stringent conditions of hybridization, reactions with HPV DNA were detected in biopsies of 2/17 (12%) inflammatory lesions, 6/12 (50%) intraepithelial neoplasms and 13/20 (65%) invasive neoplasms. All but one of the positive biopsies of invasive cancer, but only 4/11 biopsies of non-invasive lesions, contained HPV-16 DNA as determined by stringent hybridization conditions. Women with cervical cancer possessed the risk factors associated with the disease. Cigarette smoking and the presence of HPV-16 DNA were the most prominent risk factors. No evidence of an interaction between HSV-2 and HPV-16 was found among the cases of invasive cervical cancer.

BackgroundProduction of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope.ResultsAn antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner.ConclusionsThis strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C.

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic β-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic β-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand.