Search

PSMA, a transmembrane glycoprotein of approximately 100kD, is expressed in prostate epithelial cells (Horoszewicz et al., 1987; Israeli et al., 1993). In addition to being expressed in non-neoplastic prostate epithelium, PSMA is expressed by a very high proportion of prostate cancers. Expression is further increased in higher-grade cancers and metastatic disease, and in hormone-refractory prostate cancers (Wright et al., 1995). PSMA is a type II membrane protein with a short N-terminal cytoplasmic tail and a large C-terminal extracellular domain (Israeli et al., 1993). The extracellular domain of PSMA has several potential N-glycosylation sites and shows homology (54% at nucleic acid level) to the transferrin receptor. PSMA is homologous to glutamate carboxypeptidase II (85% at nucleic acid level) and has been suggested to have folate hydrolase activity and N-acetylated alpha-linked acidic dipeptidase (NAALADase) activity (Pinto et al. 1996; Luthi-Carter et al., 1998). Abundance of PSMA expression in prostate cancer cells suggests that PSMA expression might be associated with events involved in prostate cancer progression (Rajasekaran et al.,2005)., The original document contains color images.

A systematic comparison of the short RNA universe of two prostate cancer cell lines was performed by cloning and deep sequencing to identify microRNAs whose expression may correlate with androgen dependence/independence or microRNAs that are regulated by androgens. The results were confirmed by microarrays and by quantitative reverse transcription and polymerase chain reaction. We have identified five microRNAs that are repressed and three that are induced during progression of prostate cancer from androgen dependent, early stage to androgen independent advanced stage. We have also identified four microRNAs that are repressed by androgens in the androgen dependent cells. The "progression-repressed" microRNAs render the advanced cancer cells unable to grow in charcoal-stripped androgen depleted serum. We have also identified several non-micro-short RNAs that are expressed abundantly in prostate cancer cells and provide evidence that one of them, tRF-1001, is necessary for cell proliferation., The original document contains color images.

The goal of this grant was to gain insight into the molecular basis of prostate cancer. Preliminary evidence suggested that the taci gene is expressed in normal prostate tissues, but not in prostate tumor cells. We had proposed the APRIL provides a proliferative signal to normal prostate epithelial cells by means of an unknown receptor.

Regulation of the stability and activity of androgen receptor by Hsp90 provides a scientific basis for current chemotherapy of prostate cancer with Hsp90 inhibitors. However, how Hsp90 activity is regulated is still not fully understood. In this current research, we have investigated the role of HDAC6 in prostate cancer development and progression through regulation of Hsp90 activity. We have found that Hsp90-ATP association, a reliable indication of Hsp90 activity, is down-regulated in HDAC6-defient cells, and have provided evidence that loss of HDAC6 in cells also down-regulates protein levels of AR and other Hsp90 client proteins, probably through increased acetylation of Hsp90 and down-modulation of co-chaperone HOP. Requirement of HDAC6 for proper Hsp90 activity and AR activity is further substantiated by the fact that HDAC6-deficient LNCaP cells have slower growth and increased cell death (observation not yet documented)in medium with charcoal-stripped serum, which can not be reversed by androgen add-back. Another unique finding from this research is the identification of B-Raf, one of the key factors in Ras-Raf-MEK-ERKsignaling axis, as a potential Hsp90 client protein. Once this finding is verified, it will have wide implications in understanding tumorigenesis as well as in cancer therapy as a whole.

The present research has developed a non-viral gene targeting technology, whereby the effects of a neurotoxin on the brain can be reversed shortly after the intravenous injection of a therapeutic gene medicine without the use of viral vectors. The brain gene targeting technology developed in this work creates an artificial virus which is comprised of non-immunogenic lipids and proteins, wherein the therapeutic gene is packaged in the interior of the gene delivery vehicle, which is called a pegylated immunoliposome (PIL). The PIL carrying the gene is a 85 nm stealth nanocontainer, which is relatively invisible to the body s reticuloendothelial system, which normally removes nanocontainers from the blood. The surface of the nanocontainer is studded with a receptorspecific monoclonal antibody (MAb). This MAb acts as a moleculr Trojan horse, and triggers the transport of the stealth nanocontainer across the 2 biologicial membrane barriers that separate the blood from the interior of brain cells. These barriers are the brain microvascular endothelial wall, which forms the bloodbrain barrier in vivo, and the brain cell plasma membrane. Both barriers express the transferrin receptor, and the anti-receptor MAb enables the PIL to cross the membrane barriers via normal physiological transport processes usually used for endogeneous ligands such as transferrin. With this approach non-viral, non-invasive gene therapy of the brain is now possible., The original document contains color images.

The present research has developed a non-viral gene targeting technology, whereby the effects of a neurotoxin on the brain can be reversed shortly after the intravenous injection of a therapeutic gene medicine without the use of viral vectors. The brain gene targeting technology developed in this work creates an "artificial virus" which is comprised of non-immunogenic lipids and proteins, wherein the therapeutic gene is packaged in the interior of the gene delivery vehicle, which is called a pegylated immunoliposome (PIL). The PIL carrying the gene is a 85 nm "stealth" nanocontainer, which is relatively invisible to the body's reticuloendothelial system, which normally - removes nanocontainers from the blood. The surface of the nanocontainer is studded with a receptor-specific monoclonal antibody (MAb). This MAb acts as a molecule Trojan horse, and triggers the transport of the stealth nanocontainer across the 2 biological membrane barriers that separate the blood from the interior of brain cells. These barriers are the brain microvascular endothelial wall, which forms the blood-brain barrier in vivo, and the brain cell plasma membrane. Both barriers express the transferrin receptor, and the anti-receptor MAb enables the PIL to cross the membrane barriers via normal physiological transport processes usually used for endogeneous ligands such as transferrin. With this approach non-viral, non-invasive gene therapy of the brain is now possible.

The purpose of this project is to use phage antibody libraries to identify novel breast tumor antigens The antibodies could be used for breast cancer immunotherapy and the antigens could be used as cancer vaccines. In the first year, we used a model system to identify the factors allowing successful phage antibody library selection on tumor cell lines. Multivalent display of phage antibodies led to more efficient selection of cell binding antibodies, as did recovery of phage from within the cell after binding to an internalizing cell surface receptor. The methods were used to select a panel of phage antibodies which bound the breast tumor cell line SKBR3. Some of the antibodies bound ErbB2, some the transferrin receptor, and one a novel antigen overexpressed on breast tumor cells. All were efficiently endocytosed as native antibody fragments and. thus potentially useful for targeted cancer therapy. One of the ErbB2 antibodies is being commercialized for breast cancer therapy. To widen the utility of this approach, a large human phage antibody library was constructed in a true phage vector in which multiple copies of antibody fragment are displayed on each phage. We have validated the utility of this library and are continuing characterizing a large panel of breast tumor cell specific antibodies under other funding. We also developed a high throughput assay which allows rapid screening of unpurified antibody fragments for endocytosis into tumor cells.

This study describes a new fat-based vehicle (liposphere) that has been developed as a carrier for vaccines. Manufacture of lipospheres is accomplished by gently melting neutral fat in the presence of phospholipid and antigen, and then dispersing the melted mixture in an aqueous suspension by vigorous shaking. Upon cooling of this mixture a phospholipid-stabilized solid hydrophobic fat core containing antigen forms spontaneously. A typical composition of the lipid phase of the lipospheres consists of a neutral fat, such as tristearin or ethylstearate, and a phospholipid such as lecithin. A recombinant malaria antigen, R32NS 1, containing epitopes derived from the circumsporozoite protein of Plasmodium falciparum, was incorporated in lipospheres together with lipid A as an adjuvant. After intramuscular injection of the formulation in rabbits, high levels of specific IgG antibodies to the antigen were still present 12 weeks after primary immunization. Several chemical and physical parameters that might be expected to influence the liposphere vaccine formulation were investigated. The particle size distribution of lipospheres depended on the fat to phospholipid (F/PL) molar ratio, and the immune response to liposphere-encapsulated R32NSI was also dependent on the F/PL ratio. For F/PL ratios of < or = 0.75 most of the lipospheres had an average diameter of less than 10 micrometers, while at F/PL ratios of > or = 2.5 approximately 80 % of the particles had an average size of 73 micrometers. Among the ratios tested, a maximal level of IgG antibody production was obtained at a F/PL ratio of 0.75, while at larger ratios decreased antibody production was observed. The effects of antigen dose and phospholipid composition were also examined.

Swine have been used as a model for assessing the effects of exposure to T-2 toxin. Acute T-2 toxicosis is a cardiovascular shock syndrome characterized by reductions in cardiac output and blood pressure, and increased plasma concentrations of epinephrine, norepinephrine, thromboxane B2, 6-keto-PGF 1 alpha, and lactate. An initial leukocytosis is followed by a leukopenia. Serum bound calcium concentrations decrease while magnesium, phosphorus and potassium increase. Blood flow to the heart, brain, kidneys, spleen and stomach decreases, while blood flow to the adrenal glands, liver and intestines increases. Topical application of T-2 toxin in swine produces a severe necrotizing dermatitis with the healing process beginning on day 7. Morphologic changes in internal organs are minimal. Responses of purified peripheral blood lymphocytes to the T-cell mitogens, phytohemagglutinin and concanavalin A, are decreased between days 20 and 31 after dosing. Neutrophilia and fever are apparent during the first 2 weeks of exposure. Body weights of treated pigs decrease after the first week. There are significant reductions in serum albumin, alkaline phosphatase and glucose but increases in serum globulin in the first 2 weeks. Sublethal intravenous injection of T-2 toxin produce heart and pancreatic lesions in addition to the well-documented radiometric lesions. Numerous potential therapeutic agents have been tested for efficacy against T-2 toxicosis. Key words: Metabolism; Pathophysiology; Immunopathology; Hematology; Serum biochemistry; Fusarium.

Hepatitis A virus (HAV) infection is highly endemic in Israel and extensive use of immune serum globulin is used to prevent the disease in the military. With the recent advances in the development of the new HAV vaccines, it has become necessary to evaluate the possibility of a vaccine trial in the IDF. In order to identify candidate populations for the trial, two groups were evaluated in this study. The first study group comprised of a selected group of soldiers completing compulsory military service and believed to be at increased risk of contracting the disease. The soldiers were evaluated for HAV antibodies at the end of their three year compulsory service and an attempt was made to ascertain their antibody status at conscription, in order to estimate seroconversion rate. The prevalence of anti-HAV antibodies in this group at discharge was 45.3%. In addition 3.2% were hepatitis B surface antibodies. The second group comprised members of the permanent force in which the prevalence of anti-HAV antibodies was found to be 81.3%. These findings describe basic seroepidemiologic data on two populations in whom the new HAV vaccines can be tested., See also Part 1, ADA211765.