Publications

Hepatocyte growth factor (HGF) is a plasminogen-like protein with an alpha chain linked to a trypsin-like beta chain without peptidase activity. The interaction of HGF with c-met, a receptor tyrosine kinase expressed by many cells, is important in cell growth, migration, and formation of endothelial and epithelial tubes. Stimulation of c-met requires two-chain, disulfide-linked HGF. Portions of an alpha chain containing an N-terminal segment and four kringle domains (NK4) antagonize HGF activity. Until now, no physiological pathway for generating NK4 was known. Here we show that chymases, which are chymotryptic peptidases secreted by mast cells, hydrolyze HGF, thereby abolishing scatter factor activity while generating an NK4-like antagonist of HGF scatter factor activity. Thus, chymase interferes with HGF directly by destroying active protein and indirectly by generating an antagonist. The site of hydrolysis, Leu480, lies in the alpha chain on the N-terminal side of the cysteine linking the alpha and beta chains. This site appears to be specific for HGF because chymase does not hydrolyze other plasminogen-like proteins, such as macrophage-stimulating protein and plasminogen itself. Mast cell/neutrophil cathepsin G and neutrophil elastase generate similar fragments of HGF by cleaving near the chymase site. Mast cell and neutrophil peptidases are secreted during tissue injury, infection, ischemia, and allergic inflammation, where they may oppose HGF effects on epithelial repair. Thus, HGF possesses an "inactivation segment" that serves as an Achilles' heel attacked by inflammatory proteases. This work reveals a potential physiological pathway for inactivation of HGF and generation of NK4-like antagonists.

BACKGROUND

Human immunodeficiency virus (HIV) infection may increase the burden of malaria by increasing susceptibility to infection or by decreasing the response to antimalarial treatment. We investigated the seroprevalence rate of HIV-1 infection and its effect on antimalarial treatment outcomes in adults and children with uncomplicated falciparum malaria in Uganda.

METHODS

This retrospective study included 1965 patients > or =18 months old who were randomized to receive 1 of 3 antimalarial regimens at 7 sites in Uganda. HIV-1 testing was performed using 2 enzyme-linked immunosorbent assays and Western blot analysis of stored blood spots. The primary study outcome was clinical treatment failure at 28 days after antimalarial treatment. Molecular genotyping was used to distinguish clinical treatment failures due to new infections from those due to recrudescences.

RESULTS

The HIV-1 seroprevalence rate was 2.5% in 1802 patients <18 years old and 31% in 163 patients > or =18 years old presenting with malaria. HIV-1 infection was associated with a >3-fold (hazard ratio [HR], 3.28 [95% confidence interval [CI], 1.25-8.59]) increased risk of clinical treatment failure for adults, but there was no increased risk for HIV-1-infected children. Molecular genotyping revealed that clinical treatment failures were due to new infections (HR, 6.35 [95% CI, 1.64-24.5]) rather than to recrudescences (HR, 1.51 [95% CI, 0.27-8.58]).

CONCLUSIONS

The HIV-1 seroprevalence rate was surprisingly high in adults presenting with malaria. This finding supports the implementation of routine HIV counseling and testing for adults with uncomplicated falciparum malaria. HIV-1 infection increased the susceptibility to new malarial infections but did not increase the risk of recrudescences in adults.

The human organic anion transporter, OAT3 (SLC22A8), plays a critical role in renal drug elimination, by mediating the entry of a wide variety of organic anions, including a number of commonly used pharmaceuticals, into the renal proximal tubular cells. To understand the nature and extent of genetic variation in OAT3, and to determine whether such variation affects its function, we identified OAT3 variants in a large, ethnically diverse sample population and studied their transport activities in cellular assays. We identified a total of 10 distinct coding-region variants, which altered the encoded amino acid sequence, in DNA samples from 270 individuals (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). The overall prevalence of these OAT3 variants was relatively low among the screened population, with only three variants having allele frequencies of >1% in a particular ethnic group. Clones of each variant were created by site-directed mutagenesis, expressed in HEK-293 cells, and tested for function using the model substrates, estrone sulfate (ES) and cimetidine (CIM). The results revealed a high degree of functional heterogeneity among OAT3 variants, with three variants (p. Arg149Ser, p. Gln239Stop, and p. Ile260Arg) that resulted in complete loss of function, and several others with significantly reduced function. One of the more common variants (p. Ile305Phe), found in 3.5% of Asian-Americans, appeared to have altered substrate specificity. This variant exhibited a reduced ability to transport ES, but a preserved ability to transport CIM. These data suggest that genetic variation in OAT3 may contribute to variation in the disposition of drugs.

Hematopoietic stem cells can be accurately identified by the side population (SP) phenotype. It has been previously shown that hematopoietic stem cells are cell cycle arrested, but the mechanisms involved are currently poorly understood. In the present study, results from quantitative real-time RT-PCR show that while SP cells have increased expression of various cyclins and cyclin-dependent kinases, the increased expression of cyclin-dependent kinase inhibitors, in particular p57(Kip2), is responsible for the observed cell cycle arrest. In addition, gene expression analysis of c-kit(+/)/Sca-1(+)/Lineage- SP (KSL-SP) cells demonstrates that only p57(Kip2) shows both higher expression compared to both SP and non-SP cells. Furthermore, immunostaining also demonstrates significantly higher protein expression in KSL-SP cells. These results demonstrate that the maintenance of bone marrow SP cells in G0/G1 may be carefully controlled by p57(Kip2).