Detection of hypoxic cells with the 2-nitroimidazole, EF5, correlates with early redox changes in rat brain after perinatal hypoxia-ischemia.

The hypoxia-dependent activation of nitroheterocyclic drugs by cellular nitroreductases leads to the formation of intracellular adducts between the drugs and cellular macromolecules. Because this covalent binding is maximal in the absence of oxygen, detection of bound adducts provides an assay for estimating the degree of cellular hypoxia in vivo. Using a pentafluorintated derivative of etanidazole called EF5, we studied the distribution of EF5 adducts in seven-day-old rats subjected to different treatments which decrease the level of oxygen in the brain.

Quantification of tumour vasculature and hypoxia by immunohistochemical staining and HbO2 saturation measurements.

Despite the possibility that tumour hypoxia may limit radiotherapeutic response, the underlying mechanisms remain poorly understood. A new methodology has been developed in which information from several sophisticated techniques is combined and analysed at a microregional level. First, tumour oxygen availability is spatially defined by measuring intravascular blood oxygen saturations (HbO2) cryospectrophotometrically in frozen tumour blocks.

Permanent anatomic closure of the ductus arteriosus in newborn baboons: the roles of postnatal constriction, hypoxia, and gestation.

Permanent closure of the ductus arteriosus requires loss of cells from the muscle media and development of neointimal mounds, composed in part of proliferating endothelial cells. We hypothesized that postnatal ductus constriction produces hypoxia of the inner vessel wall; we also hypothesized that hypoxia might lead to cell death and the production of vascular endothelial cell growth factor (VEGF), a hypoxia-inducible growth factor that stimulates endothelial proliferation. We mapped the distribution of hypoxia in newborn baboons and correlated it with the appearance of cell death (TUNEL technique), VEGF expression, and endothelial proliferation (proliferating cell nuclear antigen expression).

Regulation of human prostate-specific antigen gene expression in transgenic mice: evidence for an enhancer between the PSA and human glandular kallikrein-1 genes.

The human prostate-specific antigen (PSA) and glandular kallikrein-1 (hGK-1, also known as hK2) genes are tandemly located on chromosome 19, separated by a 12-kb intergenic region. The coordinate regulation of these two genes suggests the presence of common regulatory elements responsible for tissue specificity and/or levels of expression within this region. To identify such regulatory elements, we generated two sets of transgenic mice, which had incorporated either the PSA gene alone or together with the intergenic region.

A transgenic strategy for analyzing the regulatory regions of the human prostate-specific antigen gene: potential applications for the treatment of prostate cancer (Review).

Prostate-specific antigen (PSA) has been used clinically as a marker for the diagnosis and staging of prostate cancer due to its specific expression in prostate epithelial cells. In addition to its medical importance, its complex hormonal and tissue-specific regulation makes it an attractive model to study gene regulation. Two approaches have been applied to the identification of regulatory regions which confer this specific expression pattern.

Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube.

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (beta-D-glucosyl)3 Yariv phenyl-glycoside [(beta-D-Glc)3] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth.

IL-3 enhances both presentation of exogenous particulate antigen in association with class I major histocompatibility antigen and generation of primary tumor-specific cytolytic T lymphocytes.

Recent studies have reported that APC can present particulate exogenous Ag in the context of class I MHC to CD8+ CTL, and our laboratory demonstrated that IL-3 could enhance CTL generation to exogenous Ag. In this paper, we wished to determine whether presentation of particulate Ag could be enhanced by IL-3. A T cell hybridoma, B3Z86/90.

IL-3-mediated enhancement of particulate antigen presentation by macrophages.

Mice were exposed to interleukin- (IL-) 3 in vivo by injection of tumor cells transfected with the IL-3 gene. At 10 days post tumor injection, bone marrow cells were recovered, pulsed with particulate antigen in the form of ovalbumin (Ova)-coated magnetic beads, and tested for their ability to present antigen via class I to an Ova/class I-restricted T cell hybridoma. Cells from IL-3-stimulated mice exhibited a marked increase in antigen presentation compared with cells from mice injected with control non-cytokine-secreting tumor cells.

Tumor immunotherapy: cytokines and antigen presentation.

Increasing the ability of tumor-reactive T cells to mediate tumor regression in vivo has been a major goal of tumor immunologists. Progress toward this goal has been aided by the identification of tumor-associated antigens on both experimental mouse tumors and human tumors. However, the self-like nature and low immunogenicity of these antigens has made it clear that other measures to enhance the effectiveness of the T cells reactive to these antigens are essential if immunotherapy is to be clinically effective.

Interleukin 2 expression by tumor cells alters both the immune response and the tumor microenvironment.

Microenvironmental conditions within solid tumors can have marked effects on the growth of the tumors and their response to therapies. The disorganized growth of tumors and their attendant vascular systems tends to result in areas of the tumors that are deficient in oxygen (hypoxic). Cells within these hypoxic areas are more resistant to conventional therapies such as radiation and chemotherapy.

Expression of B7-1 by Pam 212 squamous cell carcinoma enhances tumor cell interactions with dendritic epidermal cells but does not affect in vivo tumor growth.

Direct antigen presentation of tumor-associated antigens by tumor cells to T lymphocytes may induce clonal anergy as a mechanism of escape from immune surveillance. B7-1 is a costimulatory molecule for the activation of both CD4+ and CD8+ T lymphocytes that prevents the induction of clonal anergy. Thus, the transfer of B7-1 genes into tumor cells can induce protective immunity and lead to tumor rejection of some tumors in model systems of in vivo tumor growth; however, there is no information on whether stable expression of B7-1 can affect the in vivo growth of squamous cell carcinoma, a common skin cancer.

Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53.

Significant numbers of myocytes die by apoptosis during myocardial infarction. The molecular mechanism of this process, however, remains largely unexplored. To facilitate a molecular genetic analysis, we have developed a model of ischemia-induced cardiac myocyte apoptosis in the mouse.

Interlaboratory variation in oxygen tension measurement by Eppendorf "Histograph" and comparison with hypoxic marker.

Background And Objectives: The median of pO2 values in tumor measured by Eppendorf "Histograph" with a needle-type electrode has been used as a prognostic indicator in cancer patients. However, it is not established that a pretreatment measured pO2 value can be used as a universal predictor of local control probability, because the variation in pO2 values, especially in hypoxic tissue, among institutes may not allow comparison of measured "absolute pO2 values." The purpose of this study was to examine the variation in oxygen tension measurement by Eppendorf "Histograph" among six laboratories using a single batch of mice and tumors and the same detailed protocol.

Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy.

Human prostate-specific antigen (PSA) has been widely used as a serum marker for cancer of the prostate. The cell type-specific expression of PSA also makes it a potential tumor antigen for prostate cancer immunotherapy. Study of the immunological aspects of PSA within either normal or malignant prostate tissue has been hampered by the lack of a mouse model, because no PSA counterpart has been identified in mice.

Chondrocytes in the endochondral growth cartilage are not hypoxic.

From an anatomic viewpoint, the blood supply to chondrocytes in the growth cartilage is limited. As a result, it has been suggested that these cells are hypoxic and that this condition regulates chondrocyte maturation and cartilage mineralization. We examined the state of chondrocyte oxygenation in the chick growth plate using a hypoxia-sensing drug, EF5.

Direct relationship between radiobiological hypoxia in tumors and monoclonal antibody detection of EF5 cellular adducts.

While the potential importance of hypoxia in limiting the sensitivity of tumor cells to ionizing radiation has long been appreciated, methods for accurately quantifying the number of radiation-resistant hypoxic cells within tumors have been lacking. We have used the pentafluorinated derivative [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acet amide] of etanidazole (EF5), which binds selectively to hypoxic cells. The adducts formed between EF5 and cellular proteins in the hypoxic cells were detected using the specific monoclonal antibody (MAb), ELK3-51 conjugated to the flurochrome Cy3, and the number of hypoxic cells was quantified by flow cytometry.

Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes.

Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells.

Immunocytochemical labelling of aerobic and hypoxic mammalian cells using a platinated derivative of EF5.

The monoclonal antibody ELK3-51 was previously developed to detect adducts of the 2-nitroimidazole EF5. Direct immunofluorescence was used to detect adducts of EF5 or of a platinated derivative cis-[PtCl2(NH3)EF5] in SCCVII cells treated under aerobic or hypoxic conditions. Fluorescence measurements of these cells using both image and flow cytometric methods were compared, giving similar profiles.

Characterization and linkage mapping of ten sheep microsatellite markers derived from a sheep x hamster cell hybrid.

A cosmid library has been constructed from a sheep x hamster cell hybrid containing sheep chromosome t1, rob (6;24). Clones containing sheep DNA were identified by hybridizing to a total sheep genomic DNA probe. Small fragments (< 500 bp) containing (AC)n microsatellites were subcloned and sequenced.

The linkage map of sheep Chromosome 6 compared with orthologous regions in other species.

The genetic linkage map of sheep Chromosome (Chr) 6 has been extended to include 35 loci with the addition of 11 RFLP and 12 microsatellite loci. The sex-averaged linkage map now spans 154 cM from phosphodiesterase cyclic GMP beta polypeptide (PDE6B) to OarCP125, an anonymous sheep microsatellite. The male and female map lengths, at 180 cM and 132 cM respectively, did not differ significantly.