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Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 1993-1994

Summary Of Research Grants

Development of Differentiated Sertoli Cell Lines for Testing Male Reproductive Toxicity
Kim Boekelheide, MD, PhD
Brown University, Providence, Rhode Island
In this second year grant, Boekelheide and colleagues continue their development of a Sertoli cell line for in vitro evaluation of testicular toxicants. The rat-derived cell lines will be screened by morphology, cytochemistry, immunocytochemistry and by secretion of the Sertoli cell protein, transferrin. Clones which express the most differentiated phenotype will be evaluated for various media supplements and culture substrates. The cell lines will then be evaluated previously validated parameters of Sertoli cell toxicity.
An In Vitro Model for Neurotoxicity
Joseph Bressler, PhD
Kennedy Krieger Research Institute, Baltimore, Maryland
Bressler and colleagues are developing two model systems for the blood brain barrier (BBB) in order to devise an in vitro system for identifying toxins capable of passing through the BBB. In the cell culture model, the investigators will induce astrocytes to produce protein kinase C and other factors required by brain capillary endothelial cells to express a tight barrier (electrical resistance of 500 ohms/cm2). In the second system, they will attempt to identify the mechanisms responsible for brain microvessel uptake of zinc and mercury, two metals which can cross the BBB. The microvessel preparations are isolated directly from brain and express many properties found in vivo.
Development of Differentiated Sertoli Cell Lines for Testing Male Reproductive Toxicity
Nancy L.R. Bucher, MD
Boston University School of Medicine, Boston, Massachusetts
Bucher and colleagues will investigate the suitability of highly differentiated rat hepatocytes, freshly isolated and cultured suspended spheroidal aggregates, for toxicity testing. The researchers suggest that this system may prove a better model for hepatocytes in vivo than adherent culture systems due to the spheroid's 3-D structure, more normal cell-cell and cell-matrix interactions and avoidance of extraneous matrix components.
A microgravity device developed by NASA, the JSC Bioreactor, will be used to culture tumor cell spheroids. The quality and performance of various hepatocyte systems will be assessed by comparing hepatocyte fine structure (via transmission electron microscopy), extracellular matrix and organization of cytoskeletal elements (via immunofluorescence), responsiveness to growth factor stimulation (determined by DNA synthesis) and cell cycle status and presence of normal liver-specific functions, as measured by Northern and Western blot analysis and nuclear run-on assays.
Cytokine Gene Expression and Allergic Contact Dermatitis
Craig A. Elmets, MD
Case Western Reserve University, Cleveland, Ohio
Three haptens which cause allergic contact dermatitis (ACD) in humans will be applied to organ cultured human skin and freshly prepared Langerhans cells in order to examine the role that cytokines play in the expression of ACD. Increases in selected cytokines will be examined in cultures exposed to urushiol (poison ivy), nickel, and dinitrochlorobenzene, as well as sodium lauryl sulfate irritant. When an optimal in vitro assay has been established, its validity as a predictor of contact allergy in humans will be tested by comparing it to the clinical reaction in vivo.
Epidermal Cell Expression of Co-Stimulatory Activity in the Induction of Allergic Contact Dermatitis
Anthony A. Gaspari, MD
University of Rochester, Rochester, New York
Gaspari and colleagues are attempting to clarify the relationship between allergic contact dermatitis and the regulation of antigen presenting functions by epidermal cells. Previous studies have demonstrated that Langerhans cells regulate antigen presenting functions, changing their phenotype, morphology and immunostimulatory functions in response to keratinocyte derived cytokines. The goal of this project is to characterize biochemically the B7/BB-1 antigen that is expressed by Langerhans cells and keratinocytes, in order to discover whether allergens act on Langerhans cells to induce allergic contact dermatitis. These studies use human cells.
Development of In Vitro Monitoring Techniques Based on Genetically Engineered Bioluminescence
David S. Holmes, PhD
Clarkson University, Potsdam, New York
The firefly luciferase gene (luc) will be used as a reporter to evaluate the effect of animoglycoside drugs on the cellular metabolism of a well characterized epithelial cell line derived from the pig kidney. The objective of this study is to isolate a clonal line that expresses light consistently under defined conditions of growth.
Since cellular light expression is a function of ATP concentration and aminoglycoside drugs are known to reduce ATP levels, the researchers believe that light output should correlate with drug activity. The research will also attempt to determine whether the development of aminoglycoside drugs retaining antimicrobial activity but exhibiting reduced toxicity is feasible.
Development of a QSAR Database for Skin Contact Allergens
Howard I. Maibach, MD
University of California, San Francisco, California
Maibach and colleagues continue to characterize allergenics and non-allergenics, using descriptors related to mechanisms of transport and tissue binding, to construct QSAR (quantitative structure activity relationship) computer database for skin contact allergens. The development of a mathematical model for allergenicity will decrease the need for animal testing. Two-valued regression analysis has proven the most precise statistical technique utilized thus far and will be applied to all future modeling studies. The model currently includes 36 allergens and 36 non-allergens with high predictivity over a broad range of molecular structures.
Oncogenes in Transgenic Mice: Applications for Generating Novel Immortalized Cell Lines from the Nervous System
Albee Messing, V.MD, PhD
University of Wisconsin-Madison, Madison, Wisconsin
This investigator is exploring two strategies for generating novel cell lines chowing properties of differentiated cells. In the first, he will place a potent transforming gene from SV40 under control of regulatory sequences from a neural specific gene, which will then be introduced into the germ line of mice. The resulting tumors will be used for subsequent derivation of immortalized cell lines. The second strategy combines the same SV40 T antigen gene with regulatory sequences from an interferon-inducible mouse gene. The Mx1-SV40 mice will be used to establish immortalized cell lines in which expression of the oncogene is controlled by levels of interferon in the culture medium. The cell lines produced by these strategies should reduce the need for in vivo models in neurotoxicology and in other types of experimental research.
Phototoxicity of Solar Irradiation of Cultured Human Keratinocytes
Lester Packer, PhD
University of California, Berkeley, California
Changes in antioxidant status reflect changes in free radical production rates and can thus be used as biomarkers of oxidative stress. In these studies, cultured human skin cells and excised human dermis and epidermis will be irradiated with simulated solar light, with samples tested for photooxidative damage as indicated by accumulation of lipid peroxidation products and protein carbonyls. Responses in the whole skin will be compared to those of the cultured cells and indices of photooxidative stress will be related to indices of photooxidative damage in order to find sensitive predictors of phototoxicity. The experiment will then be repeated in vivo.
Standardization and Simplification of an In Vitro Assay to Screen the Efficacy of Anti-Metastatic Agents
Margaret Penno, PhD
Johns Hopkins School of Medicine, Baltimore, Maryland
Tumor metastasis is difficult to study in both humans and animals. Earlier studies have demonstrated that tumor cell invasion is potentiated by proteins involved in adhesion, selective proteolysis and cellular movement across subendothelial basement membranes. Human venule endothelial cells will be used by the investigator to create a screening assay to measure the invasive capacity of tumor cells and the effects of potential anti-metastatic agents. Invasion assays will test the ability of human monocytes and lung carcinomas to cross a 100 µg barrier of Matrigel in a two-chamber well. A non-invasive line of normal fibroblasts will serve as a control.
Specific goals are to determine the reproducibility and quality of the simplified chamber assay, to determine the former with the addition of endothelial cells and to determine the utility of the chamber assay to study the effect of selected antibodies to act as anti-metastatic agents.
Development of a Characterized Human Proximal Tubule Cell Line
Lorraine C. Racusen, MD
Johns Hopkins School of Medicine, Baltimore, Maryland
Racusen and colleagues have characterized clones of human renal proximal tubular cells which were immortalized by exposure to an adeno 12/SV40 virus. Clone 8 has been selected for further propagation and characterization and has been purified by immunodissection using a monoclonal antibody. The investigator will assess ultrastructural, biochemical, and transport properties of this clone under optimal growth conditions. Toxic effects on morphology and function will be assessed in the transformed cells and in non-transformed cultured human proximal tubule cells to validate the immortalized cell line as an in vitro model for toxicity testing.
An In Vitro Model for Human Peripheral Nerve Demyelination
J. Lynn Rutkowski, PhD
University of Michigan, Ann Arbor, Michigan
In the second year of this research, Rutkowski and colleagues will generate neuronal cell lines from rat embryonic progenitor cells and assess the feasibility of an in vitro myelinating system using the rodent cells. The validity of this model will then be examined by treating the myelinated co-cultures with neurotoxins which affect either neurons or Schwann cells. The objective is to develop an in vitro model for neurotoxicity testing using human cell lines.
Hepatocyte-Kupffer Cell Co-Cultures for Evaluation of Hepatotoxicity Due to Hepatotoxicant Interactions
I. Glenn Sipes, PhD
University of Arizona, Tucson, Arizona
The investigators has developed procedures to isolate hepatocytes and Kupffer cells (KC) from rat livers, to maintain them in culture, to monitor various drug biotransformation activities in hepatocytes, to assess chemical-induced injury to the hepatocyte and to monitor the release of cytotoxic factors from the KC. The co-culture of these cell types is being used to investigate how KC and hepatocytes interact to enhance killing of hepatocytes. In future studies, investigators hope to establish co-culture systems with human liver cells.
Development of an In Vitro Model for Human Response to Dioxin
Thomas A. Sutter, PhD
Johns Hopkins School of Public Health, Baltimore, Maryland
The investigator's objective is to develope an epithelial cell culture system for measurements of human response to TCDD. Sutter and colleagues will investigate the application of human epithelial cell culture systems in assessments of high to low dose extrapolations for human biological response and inter-individual variation.
Specific objectives include defining culture conditions that permit both the efficient growth of normal human epidermal keratinocytes and their response to treatment with TCDD and determining the relationship between administered dose of TCDD in culture medium to dose of TCDD in cell culture. The researchers will determine the TCDD concentration-response relationships for increases in levels of TCDD-inducible mRNA species and corresponding proteins in a single strain of human epidermal keratinocytes.
The Development of Immortalized Rabbit Kidney Proximal Tubule Cell Culture System in Serum Free Medium
Mary L. Taub, PhD
State university of New York at Buffalo, Buffalo, New York
Taub and colleagues are developing an immortalized rabbit kidney proximal tubule cell culture system for nephrotoxicity studies. The investigators have transformed primary rabbit kidney proximal tubule cells with pRSVT plasmid containing SV40 genes and have maintained the transformants in culture for over 20 passages.
They will now examine the cytotoxic effects of nephrotoxins on pRSVT transformed rabbit kidney cells and attempt to identify the mechanisms by which the nephrotoxins mercuric chloride and cephaloridine exert their cytotoxic effects. Glutathione levels, oxidative metabolism and the activity of the p-aminohippurate transport system will be examined in the pRSVT transformed cells following treatment with the toxins.
A Human In Vitro Skin Model for Toxicity Screening
Benjamin F. Trump, MD
University of Maryland at Baltimore, Baltimore, Maryland
The objective of this third year research project is to refine earlier studies which have identified early indicators of cutaneous toxicity using muring keratinocyte JB6 cells. These early indicators include measurement of intracellular ionized calcium levels and measurement of immediate early gene induction. Genes under study include c-fos, c-myc, and hsp70. Confocal laser scanning microscopy will be coupled with newly developed fluorescent probes to permit more rapid assessment of ion changes. The investigators will also use genetically engineered JB6 cell lines to assess the significance of these assays, using both primary and immortalized human cell lines.
Biochemical Characterization of ML-1 Cell Differentiation for Toxicological Studies
Michael A. Trush, PhD
Johns Hopkins School of Public Health, Baltimore, Maryland
Trush and colleagues will use the human myeloblastic leukemia cell, ML-1, as a model of monocytic differentiation to evaluate the effects of xenobiotics on cell differentiation. They will develop a battery of assays which assess cell function and biochemistry and investigate the manner in which culture conditions may alter the differentiation and biochemistry of these cells
During the first year of the grant they plan to complete characterization of the ML-1 cell line, to validate the role of mitochondrial development in mononuclear cell function, to study the effects of modification of culture conditions and to examine the effect of the addition of cytokines on cell differentiation. They will also study the effects of hydroquinone on ML-1 cell differentiation and function.
Development of In Vitro Bioassay for Diagnosis of Insecticide-Induced Delayed Neurotoxicity
Uri Wormser, PhD
The Hebrew Univeristy, Jerusalem, Israel
Wormser and colleagues will develop an in vitro bioassay to detect exposure to delayed neurotoxicity-inducing agents. Previous studies have established the ability of sera from humans and animals suffering from certain types of neuronal damage to destroy cultured nervous tissue. Preliminary findings by these investigators have confirmed that sera of paralyzed chickens and of patients with peripheral neuropathy are cytotoxic in an in vitro pheochromocytoma (PC12) cell system.
During the first year of this project, sera of acutely, subacutely and subchronically TOCP-intoxicated hens will be checked for toxic effects in the PC12 system as the investigators attempt to determine the lowest dose of TOCP which produces sear toxicity, the longest post-exposure interval in which intoxication can be detected, and the relative efficiency of the proposed assay in comparison to known parameters for diagnosis of delayed neurotoxicity.