Gary Kent Dr Mcmaster

TGF-β: upregulator of HIV replication in macrophages

Abstract TGF-β at physiological concentrations, when added to monocyte-derived macrophages following HIV1 infection, has an enhancing effect upon the rate of virus production. This effect is observed with the monocytotropic isolate ADA, as well as with HIV1 IIIB, which poorly replicates in macrophages.

Localization of transforming growth factor-β1, -β2 and -β3 gene expression in bovine mammary gland

Abstract We have studied the expression of transforming growth factor (TGF)-β1, -β2, and -β3 in the non-lactating and lactating bovine mammary gland by in situ hybridization. All three isoforms were expressed in the lobuloalveolar framework of the non-lactating and lactating gland although marked differences were apparent in their spatial distribution. TGF-β1 was expressed predominantly by the epithelial cells of the lobules although expression was also observed in the intralobular stroma cells lining the epithelium. In contrast, TGF-β2 expression was only observed in the epithelial cells. TGF-β3 transcripts were expressed at the highest levels and were observed in almost all cells of the lobule. No TGF-β signals were found in the interlobular regions of the mammary gland. The possible regulatory functions of these molecules in development of the mammary gland and on differentiation processes in the neonate are discussed.

In situ hybridization analysis of cytokine, proto-oncogene and tumour suppressor gene expression in psoriasis.

The purpose of this study was to investigate and to compare, by in situ hybridization, gene expression of IL-1Β, IL-8, TGF-Β1, TGF-Β2, TGF-Β3, TGF-α, p53 and c-myc in lesions and in non-involved skin of patients with psoriasis. All lesional skin biopsies showed overexpression of IL-1Β, IL-8 and TGF-α mRNAs. IL-1Β hybridization signals were strong in a small number of cells localized predominantly in the dermal papillae and in the suprapapillary epidermis. Overexpression of TGF-α was observed in all suprabasal keratinocytes, whereas strongly elevated IL-8 mRNA expression was found to be restricted to clusters of suprabasal keratinocytes. TGF-Β3, p53 and c-myc transcripts were clearly detected in the epidermis of all biopsies, although expression levels were comparable in lesional and non-lesional skin.

New reporter cell lines to study macrophage-tropic HIV envelope protein-mediated cell-cell fusion.

The infection of human cells by HIV-1 virus can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 (huCD4) and appropriate human chemokine receptors. In this study, a macrophage-tropic (M-tropic) HIV cell-cell fusion assay was established that utilized huCD4, human CCR5 (huCCR5), and HIV ADAgpl60 as fusion components and a Gal4/VP16-activated luciferase as a reporter system. By combining CHO cells expressing huCD4 and huCCR5 with CHO cells expressing HIV ADAgpl60, a 300-fold increase in luciferase activity could be elicited relative to control. No luciferase activity was detected when HXB2gpl60 (T-tropic) was used instead of ADAgpl60 (M-tropic) as the fusion partner in the assay. Addition of anti-huCD4 (RPA-T4) or anti-huCCR5 (2D7) monoclonal antibodies in the assay significantly inhibited the fusion event; in contrast, an anti-CXCR4 (12G5) monoclonal antibody had little effect, indicating that the fusion assay was huCD4 and huCCR5 dependent. The cell-cell fusion occurred in a time-dependent manner; the maximum luciferase activity was detected about 8 hr after mixing the cells. The fusion events could also be monitored by another reporter system in which Gal4/VP16 activated green fluorescent protein (GFP) was used as the reporter instead of luciferase. In combination with fluorescence microscopy, the GFP reporter system allowed visualization of the fusion events in real time. Compared with previously described HIV fusion models, this system has several advantages, including simplicity, sensitivity, and the ability to allow continuous monitoring of the HIV cell-cell fusion event. Finally, this cell-cell fusion system is easily adapted to study other HIV fusion events.

Wound healing in aged animals — Effects of locally applied Transforming Growth Factor beta 2 in different model systems

Local application of a growth factor which could stimulate cell turnover, extracellular matrix synthesis and blood vessel formation in the skin should improve and accelerate wound healing processes which are often impaired in old age. We demonstrate the effects of TGF-beta 2 in promoting wound repair in old animals where normal healing responses are shown to be naturally slower. The potential use of TGF-beta s for the treatment of wound injuries, including chronic non-healing ulcers in the elderly, is discussed.

Crystallization and preliminary X-ray analysis of recombinant human transforming growth factor β2

Recombinant human transforming growth factor β2 (TGF‐β2) was cloned and expressed in E. coli. The protein was isolated from inclusion bodies, renatured and purified to a single component as judged by reversed‐phase HPLC. The recombinant TGF‐β2 was shown to have a biological activity equal to that of native TGF‐β2 in a fibroblast migration assay. Pure, active recombinant TGF‐β2 has been crystallized from polyethylene glycol 400. The trigonal crystals of spacegroup P3121 or P3221 have unit cell dimensions of a=b=60.6 Å, c=75.2 Å and diffract beyond 2.0 Å.