Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry.

T-20, a synthetic peptide corresponding to a region of the transmembrane subunit of the HIV-1 envelope protein, blocks cell fusion and viral entry at concentrations of less than 2 ng/ml in vitro. We administered intravenous T-20 (monotherapy) for 14 days to sixteen HIV-infected adults in four dose groups (3, 10, 30 and 100 mg twice daily). There were significant, dose-related declines in plasma HIV RNA in all subjects who received higher dose levels. All four subjects receiving 100 mg twice daily had a decline in plasma HIV RNA to less than 500 copies/ml, by bDNA assay. A sensitive RT-PCR assay (detection threshold 40 copies/ml) demonstrated that, although undetectable levels were not achieved in the 14-day dosing period, there was a 1.96 log10 median decline in plasma HIV RNA in these subjects. This study provides proof-of-concept that viral entry can be successfully blocked in vivo. Short-term administration of T-20 seems safe and provides potent inhibition of HIV replication comparable to anti-retroviral regimens approved at present.

Plasma assay of gelatinase B: tissue inhibitor of metalloproteinase complexes in cancer.

BACKGROUND: Matrix metalloproteinases (MMPs), especially gelatinase A and gelatinase B (GLB), are believed to be important components of the metastatic process. Tissue Inhibitors of Metalloproteinases (TIMPs) form complexes with MMPs and inhibit cancer dissemination. After local secretion, MMPs and their complexes with TIMPs leach into the blood stream where their concentration can be measured, thereby serving as surrogate markers of disease. Elevated plasma gelatinase B levels have been detected in gastrointestinal cancer and breast cancer. The goal of this study was to determine whether plasma GLB:TIMP complexes also are increased in cancer and whether these tests have potential use as prognostic tumor markers. METHODS: An enzyme-linked immunosorbent assay (ELISA) was developed to measure the plasma concentration of GLB:TIMP complexes in patients with cancer. Correlation between ELISA results and clinical outcome was sought. RESULTS: Plasma GLB:TIMP complexes were significantly increased in patients with gastrointestinal cancer and gynecologic cancer, but not in patients with breast cancer. When results from plasma GLB:TIMP complexes and plasma GLB assays were combined (GLB/complexes), abnormal levels of one or both assays were found in 36% and 65% of patients with gastrointestinal and gynecologic cancer, respectively. In Stage IV gastrointestinal cancer, patient survival was shorter (P < 0.001) in the group with increased plasma GLB/complexes than for those with normal plasma levels (4 months vs. 20 months, respectively). CONCLUSIONS: The assay of plasma gelatinase B and GLB:TIMP complexes may be clinically useful in predicting survival in subsets of patients with cancer. The possibility of using these assays in early stage cancer to predict metastasis should be studied.

Comparison of techniques for measurement of gelatinases/type IV collagenases: enzyme-linked immunoassays versus substrate degradation assays.

Radiolabeled substrate degradation assays and gelatin zymography are routinely employed to assay 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) in biological fluids. Enzyme-linked immunosorbent assays (ELISA) have recently been developed for the quantitation of these matrix metalloproteinases (MMP). In this study, we have compared ELISA to standard substrate degradation assays for measurement of MMP-2 and MMP-9 in human plasma and tumor-conditioned media. Gelatin Sepharose chromatography and gel filtration chromatography were employed as partial purification procedures for MMP-2 and MMP-9. The ELISA data for MMP-2 and MMP-9 are linear on a log:log regression curve over a wide range of MMP concentrations and are specific for the designated gelatinase, with no overlap detected with related metalloproteinases. The minimum detectable concentrations of MMP-2 and MMP-9 were approximately 0.5 ng/ml and 0.2 ng/ml, respectively, in the ELISA as compared to 4 ng/ml and 3 ng/ml, respectively, in gelatin zymography. The [3H]gelatin degradation assay required a combination of > 50 ng/ml of MMP-2 and MMP-9 for detection. Although gelatin zymography was less sensitive than ELISA (primarily due to the smaller sample volume employed) and was more difficult to quantitate, this procedure offers the important advantage of being able to distinguish between latent and activated gelatinases.

Purification and characterization of a novel cytotoxic protein from transformed fibroblasts.

The mechanism by which cancer cells overwhelm normal parenchymal cells during cancer invasion remains obscure. In this article, we describe the purification of a potent cytotoxic protein from plasma membranes of ras oncogene transformed fibroblasts. Tumor cytotoxic protein was purified from detergent extracted tumor membranes by anion exchange and gel filtration chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the hemolytic fractions contained a single protein with an apparent molecular weight of 62,000. A higher concentration of tumor cytotoxic protein was required to lyse fibroblasts as compared to RBC. Based on plasma membrane localization, immunological identity, and biological characteristics, tumor cytotoxic protein is a novel cytolysin which is capable of killing normal cells during cancer invasion.

Human pleural effusions are rich in matrix metalloproteinases.

We identified and characterized type IV collagenase and gelatinase activity in pleural fluid from 32 patients. The capacity to substantially degrade type IV collagen was demonstrated in every pleural sample. Comparable results were also noted for the degradation of a radiolabeled gelatin substrate. Gelatin gel zymography of the pleural fluids revealed two prominent zones of lysis at 66 kDa and 92 kDa. These were identified by specific polyclonal antibodies as human matrix metalloproteinases MMP-2 and MMP-9. The concentration of MMP-2 in pleural fluid, as measured by enzyme-linked immunoassay, averaged 1,622 ng/ml whereas those of MMP-9 were 210 ng/ml. Substrate degradation activity was compared in both serum and pleural fluid from three patients and found to be similar. In serum this enzymatic activity was primarily due to MMP-9 whereas in pleural fluid, the predominant gelatinase was MMP-2. This was confirmed by immunoassay that showed that MMP-2 levels were two to five times higher in pleural fluid than in serum. We conclude that substantial amounts of MMP-2 and, to a lesser degree, MMP-9 are present in pleural effusions. The bioactivity and the immunoactivity of these enzymes did not help to distinguish among pleural fluids characterized as transudates, nonmalignant exudates, or malignant exudates. The differences in the distribution of these enzymes in pleural fluid and blood suggest that their presence is not due simply to the ultrafiltration of plasma, but rather to synthesis by the resident cells at the pleural surfaces.

Detergent extraction and characterization of tumor hemolytic factor from plasma membranes of oncogene transformed fibroblasts.

Cancer cells have the capacity to lyse erythrocytes by a cell-contact-requiring phenomenon. Subcellular fractionation procedures have revealed that the hemolytic principle resides in the cancer cell plasma membrane. In this study we report the detergent extraction of a potent hemolytic factor from the plasma membranes of ras-oncogene-transformed fibroblasts. Ammonium-sulfate partitioning (60-100%) of detergent-extracted proteins was used to enrich hemolytic activity. Tumor membrane Hemolytic Factor (mTHF) was inactivated by treatment with papain, suggesting that it is a protein. mTHF was inhibited by serum, but was unaffected by extremes of temperature and pH, also by metal chelation with EDTA. Surface radio-iodination of tumor cells and isolation of cell organelles was used to characterize the outer plasma membrane localization of mTHF. mTHF retained hemolytic activity when reconstituted into stable phospholipid vesicles. Pre-incubation of mTHF with red cell ghosts led to an abrogation of hemolytic activity. mTHF-induced hemolysis consists of a 2-stage phenomenon: an early binding step, followed by hemolysis after 4 hr.