John M. Hunt

The 1996 Clinical Research Award: Use of a Pneumatonometer in Burn Scar Assessment

The evaluation of wound outcome after burn injury is a challenging problem in the performance of clinical trials evaluating potential impact on wound healing and scar formation. The purpose of this study was to determine whether an ocular tonometer could be adapted to provide an objective measurement of scar compliance. A pneumatonometer was used to perform measurements of cutaneous compliance at 8 anatomic areas (14 separate sites) on each of 17 normal volunteers and on 59 burn scars. Comparison of different anatomic sites showed there to be significant differences in the cutaneous compliance of different areas. The aggregate compliance of the burn scars in all sites was less than that of the control sites. These results indicate that the pneumatonometer can discern differences in the compliance of normal skin and differences between normal skin and scar and suggest that it may be a useful tool in the objective assessment of scar formation.

ELECTROPHORETIC RESOLUTION OF TWO RAT CLASS I ALLOANTIGENS EXPRESSED ON (WF x F344)F1 LIVER CELLS IN PRIMARY CULTURE

Polyvalent alloantisera, prepared by reciprocal immunization of F344 (RT1lv1 haplotypes) and WF (RT1u haplotype) rats, as well as monoclonal antibodies, were used to immunoprecipitate class I alloantigens from detergent extracts of monolayer cultures of 35S‐methionine‐labelled liver cells. Two‐dimensional IEF/SDS‐PAGE gel analysis resolved the RT1.Alv1 and RT1.Au class I antigens expressed on the liver cells in culture.

Exploitation of Surface Molecules for Separation of Cells from Mosaic Livers

Publisher Summary This chapter focuses on the exploitation of surface molecules for separation of cells from mosaic livers. The liver parenchyma is a biochemically mosaic tissue. This natural mosaicism has been exploited to demonstrate the clonal nature of early hyperplastic liver lesions arising during chemically induced liver carcinogenesis in mice. Natural mosaicism has been employed in an analogous manner in the past to demonstrate clonality of some human tumors. Two immunological approaches to purification of donor-origin hepatocytes from genotypic mosaic livers, differential hepatocyte rosetting, and complement-mediated cytolysis have provided 4- to 11-fold enrichments for donor-origin hepatocytes. A panning technique is suitable for purification of host-origin hepatocytes. A combination of these cell separation strategies should make possible preparative scale purification of donor and host-origin cells from genotypic mosaic rat livers. In principle, the techniques should be applicable to other genotypic mosaic tissues in which developmental or pathogenic cellular lineages must be elucidated. This panning separation of host-origin cells is a positive immunoselective technique in contrast to the two negative selection techniques described as donor-origin cells.

DNA Adduct Formation During Chronic Administration of an Aromatic Amine

DNA adduct formation is considered to play an integral part in the initiation of tumors. Since most human exposure to chemical carcinogens is chronic, it is important to investigate DNA adduct formation in relation to preneoplasia and tumorigenesis in animal models using a similar type of exposure. In this communication, two different experimental systems are described in which rodents were continuously fed 2-acetylaminofluorene (AAF). In both experiments, DNA adducts were detected with antisera specific for the acetylated [N-(deoxyguanosin-8-уl)-2-acetylaminofluorene; dG-C8-AAF] and deacetylated [N-(deoxyguanosin-8-y1)-2-aminofluorene; dG-C8-AF] C8-substituted deoxyguanosine adducts of AAF. The first study compared the tumor incidence in the livers and bladders of mice chronically fed seven different doses of AAF for up to 33 months with the DNA adducts in these tissues after one month of feeding AAF at similar doses. In the second investigation, four different hepatocarcinogenesis protocols were used to induce enzyme-altered, preneoplastic foci in the livers of rats. The animals were then fed AAF for five to six days and the concentration of AAF-DNA adducts was determined in the foci and adjacent tissues. Taken together, these studies suggest that initiating events involving DNA adducts occur relatively early in the carcinogenic process, and that cells progressing to form tumors may not necessarily contain or be able to form DNA adducts. Furthermore, the number of adduct-related events required for tumor initiation appears to differ among tissues of the same animal.

Changes in the expression of class I major histocompatibility complex antigen RNA induced by interferon in rat hepatoma cells

The rat hepatoma cell line 17X was studied to determine if it expressed major histocompatibility complex (MHC) class I antigen RNA and to see if interferon treatment would affect its expression. Under normal cell culture conditions, MHC class I RNA in 17X hepatoma cells is virtually undetectable. Administration of rat interferon at a concentration of 1000 units/ml for 48 h to 17X cells in culture resulted in the appearance of detectable levels of RNA for MHC class I antigens. The interferon-induced increase in class I RNA was accompanied by de novo synthesis of immunoprecipitable, metabolically radiolabeled class I antigens in the 17X hepatoma cells.

In Situ Detection of Hepatic AF-DNA Adduct Formation During Continuous Feeding of 0.02% Acetylaminofluorene and in Enzyme-Altered Foci Induced by Foyr Different Protocols

Formation of carcinogen-DNA adducts is thought to represent the critical event in the initiation of tumorigenesis in many carcinogenesis models. DNA-adduct formation and repair has therefore been profoundly studied, both for human monitoring of carcinogen exposure and in experimental carcinogenesis. Most studies on DNA-adducts are based upon extraction of DNA from whole tissue, with little possibility to distinguish between different cell populations. The ability to differentiate between subpopulations within the tissue is presumably important, both to assess adduct processing within the tumor progenitor cells, and to monitor biological alterations of initiated cell populations.

Detection of epoxide hydrolase in rat hepatoma cell lines and primary rat liver cells by immunoblotting

Rat epoxide hydrolase (EH) (EC 3.3.2.3) is elevated in cells of premalignant liver lesions, and variable EH activity has been reported for hepatocellular carcinomas. To facilitate detection of altered EH levels in liver cells, an immunoblotting method was devised. Rabbit antiserum specific for rat EH was prepared and used to detect EH extracted from suspensions of normal liver cells and from hepatoma cell lines. Compared with normal liver cells, 3 rat hepatoma cell lines, 7777, HTC and 17X, showed virtually undetectable EH levels by immunoblotting. The immunoblotting results rule out the possibility that very low EH enzymatic activity in the hepatoma cells results from production of normal amounts of non-functional enzyme protein.