No abstract

This study reports on the direct effect of the envelope glycoprotein (gp120) of the human immunodeficiency virus type 1 (HIV-1) on human monocyte function. Addition of preparations of purified gpl20 from the HIV-1 to human monocytes resulted in the production ofinterleukin 1 (IL-1) and arachidonic acid metabolites from the cyclooxygenase and lipoxygenase pathways. Quantification of prostaglandin E2 (PGE2) and IL-1 revealed an increase in both mediators with 50 ng of gpl20 per ml and an increase of 12-and 30-to 40-fold with 200-400 ng of gp120 per ml, respectively. Unlike native gpl20, the recombinant nonglycosylated gp120 fragments PB1-RF and PB1-IIIB, as well as one of the core structural proteins of HIV-1, p24, did not increase arachidonic acid metabolism or IL-1 activity. Cytofluorometric analysis revealed that gpl20 blocked the binding of OKT4A to the CD4 on monocytes, whereas OKT4 binding was unaffected. Involvement of the CD4 in signal transduction was further demonstrated by the ability of OKT4 and OKT4A monoclonal antibodies to increase monocyte PGE2, IL-1 activity, and nanogram amounts of IL-113.

Poloxamers are triblock copolymers with a center block of hydrophobic polypropylene oxide (PPO) flanked by two hydrophilic polyethyleneoxide (PEO) blocks. Among this family of copolymers, poloxamer 407 is a non-ionic surfactant with reversible gelation properties above a particular polymer concentration and a particular temperature. Easy preparation of poloxamer 407 based sterile injectable formulations have made this copolymer a good candidate for drug delivery, specifically when controlled release of the drug is required. Previously, the applications of compendial poloxamer 407 preparations were demonstrated; however, low viscosity, poor elasticity, and sol-to-gel transition temperature (Tsol-gel) over a wide temperature range were observed. A purification process was introduced to eliminate impurities and low molecular weight copolymer molecules from the compendial poloxamer 407 resulting in higher viscosity values with Tsol-gel in a narrow temperature range. Here, poloxamer 407 was purified based on the proposed process and the rheological and analytical evaluation of the purified poloxamer 407 was conducted and compared to unpurified, compendial poloxamer 407. Then, the impact of poloxamer 407 concentration on gel formation was evaluated. For drug delivery applications, the effect of relevant buffer salts and the effect of addition of ethanol to the poloxamer 407 solutions were rheologically evaluated.

In addition to inhibiting the proteolytic activity of the matrix metalloproteinases, tissue inhibitors of metalloproteinases (TIMPs) promote the growth of cells in the absence of other exogenous growth factors. TIMP-2 stimulates the proliferation of fibrosarcoma (HT-1080) cells and normal dermal fibroblasts (Hs68) in a dose-dependent manner. This response is evident as early as 2 h and persists up to 48 h after treatment with recombinant TIMP-2 (rTIMP-2). The specificity of this response is demonstrated by the ability of affinity-purified polyclonal anti-TIMP-2 antibodies to ablate TIMP-2 mitogenesis and by the lack of response to TIMP-1. This response is also blocked by the presence of an adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (SQ22536). Although SQ22536 did not affect untreated fibroblasts or fibrosarcoma cells, this inhibitor completely abrogates the proliferative response induced by rTIMP-2. Treatment of these cells with rTIMP-2 also stimulates the production of cAMP in a time-dependent manner that differs for the two cell lines. Moreover, treatment of purified cell membranes with rTIMP-2 suppresses cholera toxin-mediated ADP-ribosylation of the GTP-binding protein, Gs alpha subunit. These results indicate that the alpha beta gamma heterotrimer is dissociated by treatment with rTIMP-2, which may facilitate the Gs alpha-mediated activation of adenylate cyclase and subsequent production of cAMP. Since cAMP binds to the regulatory subunit of cAMP-dependent protein kinase and activates kinase activity, we evaluated how treatment with rTIMP-2 affected both these parameters. We demonstrate in this report that the cAMP produced in response to treatment with rTIMP-2 binds to the type I regulatory subunit of cAMP-dependent protein kinase and stimulates kinase activity. These results are the first demonstration that TIMP-2 directly activates adenylate cyclase to produce cAMP, which increases cAMP-dependent protein kinase activity, resulting in stimulation of fibroblast mitogenesis.