Platelet-derived endothelial cell growth factor was previously identified as the sole angiogenic activity present in platelets; it is now known to be thymidine phosphorylase (TP). Intense interest has recently centered on the angiogenic process, largely as a result of the realization that disregulated blood vessel growth plays a critical role in several disease states, including (inter alia) cancer (tumor angiogenesis), diabetic retinopathy, psoriasis, rheumatoid arthritis, and hyperproliferation of the vasa vasorum in atherosclerosis (1). Platelet-derived endothelial cell growth factor was described as a mitogenic and angiogenic factor present in platelets (2).Platelet-derived endothelial cell growth factor is now known to be thymidine phosphorylase (TP) (3) and the effects of TP on cellular uptake of [methyl-3H]thymidine when it is added exogenously to cells arise from its effect on the availability of thymidine in the extracellular culture medium (4-6). Several years prior to the isolation of the so-called plateletderived endothelial growth factor and demonstration of its angiogenic activity, TP had been identified as an enzyme whose level was significantly elevated in the plasma of cancer patients relative to that in healthy volunteers (7) and in the plasma of xenografted mice relative to that of controls (8) and expressed at a high level in tumors (9, 10). We have found (11) that expression of TP shows a strong correlation with ovarian malignancy and ovarian tumor blood flow.We report here that TP is angiogenic in the rat sponge model (12) and in a freeze-injured skin graft model (13). Expression of TP strongly correlated with malignancy in breast tumors, and while expression of TP had no effect on breast carcinoma cell growth in vitro, it greatly enhanced tumor growth in vivo. MATERIALS AND METHODSCell Isolation and Culture. Bovine aortic endothelial cells were isolated as described (14) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum. Low-passage MCF-7 cells (passage 53) were a gift from Marc Lippman (Georgetown University, Washington, DC).Antibodies to TP. Rabbit anti-TP antisera and mouse anti-TP monoclonal antibodies were raised against recombinant TP expressed in Escherichia coli (4).Site-Directed Mutagenesis and Protein Expression. Three site-directed mutants of TP were constructed by a two-step PCR (15). Recombinant proteins were expressed in E. coli using the expression vector pET-14b (ams Biotechnology, Witney, U.K.) and purified by metal chelate affinity chromatography. By gel filtration, recombinant TP was dimeric with a molecular mass of 100 kDa. Concentrations of pure TP were calculated by using a molecular mass of 100 kDa.Rat Sponge Angiogenesis Assay. Sterile circular polyether sponge discs with central cannula were implanted subcutaneously in male Wistar rats (180-200 g) after induction of neuroleptanalgesia by Hypnorm (12). Four sponges were used in each experimental group. Protein in 50 ,ul of PBS was injected daily into...
Tumor formation by V12 cells could provide a useful model for the assessment of anti-angiogenic drugs.
The outcome of human immunodeficiency virus type 1 (HIV-1) infection is related to the set-point plasma virus load (pVL) that emerges after primary HIV-1 infection (PHI). This set-point pVL generally remains stable but eventually increases with progression to disease. However, the events leading to loss of viremic control are poorly understood. Here, we describe an individual who presented with symptomatic PHI and subsequently progressed rapidly, after an initial period of 1 year during which viral replication was well controlled. Escalation of viral replication in this atypical case was preceded by the emergence of escape variants in many epitopes targeted by dominant CD8 + T cell responses and a marked decrease in HIV-1-specific CD4 + and CD8 + T cell frequencies. There were no changes in viral tropism, replication kinetics, or neutralizing antibody titers. These findings demonstrate the temporal relationship between viral escape from CD8 + T cell activity, decrease in HIV-1-specific T cell frequencies, and loss of control of viral replication.Substantial evidence indicates that the adaptive immune system plays a major role in containing viral replication during HIV-1 infection [1,2]. In particular, HIV-1-specific CD8 + T cell responses are thought to mediate the decrease in the initial viremia during primary HIV-1 infection (PHI) [3,4]. After PHI, a setpoint plasma virus load (pVL) emerges that, in general, remains relatively stable throughout the chronic asymptomatic phase of infection. The magnitude of this setpoint pVL, which is likely determined by early interactions between the virus and the immune system, is inversely related to the rate of progression to AIDS [5]. However, the factors that are responsible for the maintenance of the established set-point pVL are less well defined, and it is not clear which events eventually lead to loss of control of viremia. Identification of such events requires longitudinal and comprehensive analysis of adaptive immune responses and viral evolution in patients with untreated HIV-1 infection.Here, we studied the natural course of HIV-1 infection over a period of 2 years in an individual who presented with symptomatic PHI. Initially, viral replication
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