This initiative is fostering study of factors responsible for angiogenesis (abnormal blood vessel growth) in Kaposi's sarcoma in AIDS patient. These studies should provide knowledge that will have broad application for understanding the aberrations of vascular cell proliferation in a variety of diseases in which capillary cell dysfunction plays a key role. (Diabetic retinopathy and neo-vascularization in atherosclerotic plaques are two examples.)
R01 HL48491 - John Thompson - University of Pennsylvania, Philadelphia, PA
R01 HL48496 - Steven Miles - University of CA, Los Angeles AIDS Center, CA
R01 HL48493 - Marlys Witte - University of Arizona, Tucson, AZ
R01 HL48499 - .Parkash Gill - University of Southern California, Los Angeles, CA
R01 HL48495 - Marvin Karasek - Stanford University, Palo Alto, CA
R01 HL48547 - Susan Mallery - Ohio State University, Colombia, OH
R01 HL48503 - Stephen St. Jeor - University of Nevada, Reno, NV
R01 HL48497 - Mary Moyer - University of Texas, San Antonio, TX
Grantees have concentrated on work designed to identify KS progenitor cells and to elucidate the molecular and cellular characteristics of KS cells. One grantee has shown that vascular smooth muscle cells infected with HIV or transfected with the tat-gene exhibit changes in morphology reminiscent of KS cells. Another grantee has identified Oncostatin M as a major mitogen for AIDS-KS cells. The elucidation of its action through the IL-6 receptor pathway suggests new avenues of therapeutic approach. These include the modulation of these receptors by retinoic acid derivatives, modulation of the post-receptor pathway by geldanamycin and other protein kinase inhibitors, or the inhibition of the pathway with the soluble IL-6 gp130 receptor subunits of OncoM alpha chain receptor subunits. The results of these studies have directly led to the development of clinical trials of R024-7429 (tat inhibitor), IL-4 (inhibits IL-6 production), pentoxifylline (inhibits TNF secretion), all-trans retinoic acid plus alpha interferon and the pre-clinical development of soluble OncoM receptors, soluble IL-6 receptors as well as the identification of a series of novel retinoic acid derivatives with anti-KS activity. These studies have led to a fundamental shift in therapy for patients with KS.
Heparin-binding growth factor, an in vivo initiator of angiogenesis, has been found in the nuclei of vascular cells in KS and could be used as an indicator of early KS lesions.
Evidence has accumulated to confirm the hypothesis that human cytomegalovirus is an important cofactor in KS. The mechanism appears to be through induction of cytokines required for development of KS lesions. Mycoplasma has also been identified as a cofactor for KS.
Many autoimmune-like serological abnormalities have been described in HIV-1-seropositive individuals, e.g. hypergammaglobulinemia and circulating immune complexes. In addition, researchers have described autoimmune processes such as: immune thrombocytopenic purpura; rheumatologic disorders such as: Reiter's syndrome, Sjogren's-like syndrome, and Lupus-like syndromes; bulbous disease and a variety of antibodies against non-T lymphocytes. These conditions are frequently most prominent in patients with AIDS and low CD4 counts. Longitudinal serologic studies have suggested that autoimmunity is already present in subgroups of patients and may be exacerbated by concurrent HIV-1 core proteins p17 and p24 and by one or more uncharacterized myocyte antigens.
Investigators have identified a number of targets for treatment of Kaposi's sarcoma (KS) based on observations of factors that promote or retard growth of KS cells. These include: glucocorticoid hormones stimulate KS cell proliferation and the glucocorticoid receptor is upregulated in KS; chorionic gonadotropin kills KS cells; Il-1 receptor antagonist inhibits KS spindle cell growth but has no effect on normal endothelial cells; a chimeric fusion toxin DAB398-IL-6 is toxic to cells expressing IL-6 receptor by reason of its ability to suppress protein synthesis in AIDS-KS spindle cells. One grantee has succeeded in competitive renewal of her application to study oral KS. The grant will fund attempts to devise site-specific delivery systems of chemotherapeutic agents and angiostatic drugs (doxorubicin, bleomycin, vincristine and TNP-470). After testing in rabbits phase I clinical trials in patients will begin. If the site-specific delivery system is proved to be efficacious then it will be possible to treat other forms of AIDS-KS such as those occurring in internal organs such as the lung.
This was a two-year extension of a contract program to develop and evaluate new methods for the direct detection of HIV in cadaveric organ and tissue donors. The assays being developed must provide results within 2-3 hours since transplantation, particularly of organs, is generally done as early as possible after harvesting. Also, it is important that these assays can be readily conducted in clinically oriented laboratories in order to be well received by the transplantation community. The need for assays that can rapidly and simply detect HIV in organ/tissue donors has been underscored by the many reports of antibody-negative donors per ELISA who turn out to be HIV-infected and transmit the virus to recipients. Such donors are in the so-called "seronegative window of infectivity," a period of time between infection and the first appearance of detectable antibodies which may vary from a few weeks to a few months.
NO1 HB67021 - Bernard Poiesz - State University of New York, Stony Brook, NY
NO1 HB67022 - Robert Singer - University of Massachusetts, Boston, MA
Dr. Bernard Poiesz focussed on PCR-based assays that can detect DNA and/or RNA. Efforts have centered on sample selection and preparation, primer and detector optimization, and amplification and detection formats. The contractor's non-enzymatic PCR detection is based on the recognition of HIV gag gene using the primers SK38/SK39. After 35 cycles of amplification taking approximately 1 hour, this system, coupled with liquid hybridization (about 4 hours), can detect 1 copy of HIV gag DNA or 10 copies of genomic RNA by Poisson distribution. In another approach, the contractor has developed an EIA based on 96-well non-isotopic detection assay that has the sensitivity of a standard liquid hybridization assay. Also, Dr. Poiesz is evaluating a rapid pouch-based assay system. This system involves introducing DNA or lysed material into a one-way valve in a thin "pouch" approximately the size of a credit card.
The sample is inserted into a programmable thermocycler to both amplify and subsequently detect the targeted sequences. All of the components of the assay are contained in small bubbles within the pouch and specimens are sequentially moved from one bubble to the next at each step of the process. Ultimately, the amplified DNA is captured by bound detectors and an enzyme-based color dye reaction occurs. The entire process takes less than an hour. Dr. Robert Singer utilized an in situ hybridization procedure that can be analyzed in a secondary procedure by an imaging microscope. The instrument is called the Discovery System, which is a software-controlled digital image processor and image database electronically linked, by computer, to a microscope stage control, two light sources, filter blocks and three high-resolution video cameras. The data can be displayed in three modes: 1) list, 2) histogram and 3) scatterblot. This automated approach provides considerable convenience to the user. Most importantly, it quantitates not only the number or percentage of HIV-positive cells but it also measures the amount of signal per cell, thus permitting the calibration of the number of HIV copies per call. The in situ hybridization performed on frozen sections from clinical specimens can be done in less than 3 hours from the time the sections are received in the laboratory.
In order to increase the speed and sensitivity of the in situ hybridization procedure, the contractor combined the hybridization and detection steps by the use of chemically synthesized oligonucleotides directly labeled non-isotopically by linking an enzyme to the 5' end of the oligonucleotide probe. Dr. Singer has constructed 40 conjugated probes covering approximately 1,200 nucleotides of the HIV target. The enzyme most commonly linked to these probes is alkaline phosphatase, which develops a permanent red color when the appropriate substrate is present, and this is easily detectable by the Discovery System. The assay system developed by Dr. Singer is very promising and, once quantitative parameters are set on the discovery, sample analysis can be automated.
Although these assays are promising, their licensed use still requires considerable work.