Hans J. Hedrich

Biochemical markers in inbred strains of the rat (Rattus norvegicus).

Klaus Bender 1, M a r k Adams 2, Peter R. Baverstock 2, Maria den Bieman 3, Siegbert Bissbort 1, Rad im Brdi~ka 4, Geoffrey W. Butcher 5, Dona ld V. Cramer 6, Otto yon Deimling 7, Michael F .W. Festing 8, Eberhard Gtinther 9, Rona ld D. G u t t m a n n 1°, Hans J. Hedrich 11, Philip B. Kendall 12, Reinhard Kluge i t , Ren6 Moutier 13, Babette Simon 7, James E. W o m a c k ~4, Junzo Yamada ~5, and Bert van Zutphen 3

Role of intestinal microbiota in transformation of bismuth and other metals and metalloids into volatile methyl and hydride derivatives in humans and mice.

ABSTRACT The present study shows that feces samples of 14 human volunteers and isolated gut segments of mice (small intestine, cecum, and large intestine) are able to transform metals and metalloids into volatile derivatives ex situ during anaerobic incubation at 37°C and neutral pH. Human feces and the gut of mice exhibit highly productive mechanisms for the formation of the toxic volatile derivative trimethylbismuth [(CH3)3Bi] at rather low concentrations of bismuth (0.2 to 1 μmol kg−1 [dry weight]). An increase of bismuth up to 2 to 14 mmol kg−1 (dry weight) upon a single (human volunteers) or continuous (mouse study) administration of colloidal bismuth subcitrate resulted in an average increase of the derivatization rate from approximately 4 pmol h−1 kg−1 (dry weight) to 2,100 pmol h−1 kg−1 (dry weight) in human feces samples and from approximately 5 pmol h−1 kg−1 (dry weight) to 120 pmol h−1 kg−1 (dry weight) in mouse gut samples, respectively. The upshift of the bismuth content also led to an increase of derivatives of other elements (such as arsenic, antimony, and lead in human feces or tellurium and lead in the murine large intestine). The assumption that the gut microbiota plays a dominant role for these transformation processes, as indicated by the production of volatile derivatives of various elements in feces samples, is supported by the observation that the gut segments of germfree mice are unable to transform administered bismuth to (CH3)3Bi.

Homologous collagen-induced arthritis in rats and mice are associated with structurally different major histocompatibility complex DQ-like molecules.

Collagen‐induced arthritis (CIA) in rats, induced with homologous type II collagen (CII), is a genetically more restricted disease and has better resemblance to rheumatoid arthritis by its chronic disease course, than CIA induced with heterologous CII. The DA strain is highly susceptible to CIA induced with homologous CII, while the Lewis strain is resistant. (DAxLew)F1 is susceptible and backcrossing to Lewis reveals a close, but not complete, association of both arthritis and CII responsiveness to the RT1a haplotype. Analyses of congenic strains on DA and Lewis backgrounds suggest that expression of a major histocompatibility complex class II Ba molecule, encoded from the RT1Ba locus, is associated with arthritis susceptibility and CII responsiveness. The second exons coding for the first domains of the α and β chains of both the RT1a and RT11 haplotypes were sequenced and the deduced amino acid sequences compared with the corresponding molecule associated with susceptibility to CIA in the mouse (H‐2 Aq). The sequences of the respective alleles revealed no obvious structural homology explaining the extensive similarities in the development of chronic autoimmune arthritis. Instead, this finding implies that different trimolecular constituents (i.e. class II,T cell receptor, and CII peptides) may yield an antigen presentation event that is able to trigger a similar autoaggressiveness in the two rodent species.

DNA detection in hair of transgenic mice-a simple technique minimizing the distress on the animals

The breeding of transgenic animals requires that each individual offspring be analysed for integration of transgenic deoxyribonucleic acid (DNA), unless exclusively homozygous animals are mated. The standard protocol for identification of transgenic animals (Hogan et al. 1994) is based on tissue samples and preparation of chromosomal DNA including proteinase K digestion and phenol/chloroform extraction. The procedure described here represents a much simpler and faster method to screen offspring for the transgene DNA. It is based on the use of hair bulbs as sample material, which can be directly used for polymerase chain reaction (PCR) after alkaline lysis. This protocol allows large numbers of animals to be easily screened in a minimum amount of time. A unique advantage though, is the reduction of the distress caused to the animals. With respect to the 3Rs (Replacement, Reduction, Refinement), and because of technical advantages this method may replace ear or tail clipping.

Analysis of genetic changes in rat endometrial carcinomas by means of comparative genomic hybridization

Animals of the BDII inbred rat strain are known to be genetically predisposed to endometrial adenocarcinoma (EAC). Using them as models of human EACs, we studied tumors arising in F1 and F2 progeny from BDII animals crossed to animals from two other inbred strains, in which EACs were quite rare. In order to identify chromosomal regions exhibiting DNA copy number changes, comparative genomic hybridization (CGH) was applied in a series corresponding to 27 different solid tumors, most of which were classified as EACs, from these animals. The main findings from the study were that, although many different chromosomes were involved in copy number variation, some of the changes detected were recurrent and quite specific. Among specific changes found were gains in rat chromosome (RNO) regions 4q12 approximately q22, 6q14 approximately q16, and whole chromosome arms in some of the small metacentric chromosomes (e.g., RNO14, 16, and 18). RNO10 was involved in gain in the terminal and proximal regions. Each of these regions contains previously identified cancer-related genes representing possible candidates to be involved in the development of EAC. Furthermore, it was observed that there were clear differences in the pattern of copy number changes between tumors occurring in the two different crosses, and also between solid tumors and cell cultures. Endometrial cancer is the most common human gynecological cancer, but not much is known about specific genetic changes influencing this disease. Two genetic alterations that have been reported from human endometrial cancer are amplification of the ERBB2 gene and mutations in the 12 codon of the KRAS gene. One case of Erbb2 amplification was found but there were no Kras mutations in the rat material studied. We conclude that molecular genetic analysis of the rat BDII model will provide important new information about EAC in mammals.

Genetic identification of multiple susceptibility genes involved in the development of endometrial carcinoma in a rat model

There are clear indications that inheritance plays an essential role in certain cases of human endometrial cancer, and there are at least 2 forms of early‐onset heritable endometrial adenocarcinomas (EACs). Females of the BDII inbred rat strain are known to be genetically predisposed to endometrial carcinoma, and we have performed a genetic analysis of susceptibility to endometrial cancer in this strain. F2 populations were generated by crossing BDII females with males from 2 different strains with a low incidence of EAC, and the occurrence of endometrial cancer was studied. Three chromosome regions associated to EAC susceptibility were identified, and the susceptibility genes in these regions were designated Ecs1, Ecs2 and Ecs3. Our results indicate that the genes affecting susceptibility to EAC are different in the 2 crosses, suggesting that the genes behind the susceptibility in BDII animals may interact with different genes in different genetic backgrounds.

Amplification of Mycn, Ddx1, Rrm2, and Odc1 in rat uterine endometrial carcinomas.

The BDII rat is genetically predisposed to estrogen‐dependent endometrial adenocarcinoma and represents a valuable model for this type of tumor. Tumors arising in strain crosses involving the BDII rats had previously been screened for DNA copy number changes using comparative genome hybridization (CGH). It was found that extra copies of the proximal region of rat chromosome (RNO) 6 commonly could be detected in these tumors. Based on RH‐mapping data and comparative mapping with mouse and human, seven cancer‐related genes were predicted to be situated in RNO6q14–q16. Rat PACs were isolated for the N‐myc proto‐oncogene (Mycn), apolipoprotein B (Apob), the DEAD box gene 1 (Ddx1), ornithine decarboxylase 1 (Odc1), proopiomelanocortin (Pomc1), ribonucleotide reductase, M2 polypeptide (Rrm2), and syndecan 1 (Sdc1). The localization of the genes to the region was verified by FISH (fluorescence in situ hybridization) mapping, and the detailed order among them was determined by dual‐color FISH. By Southern blot analysis, it was found that the Mycn locus was highly amplified in two out of 10 cell cultures derived from the tumors. In one of them (designated RUT30), the amplification level of Mycn was estimated at 140×. Two other genes were coamplified (Ddx1 and Rrm2) at much lower levels. Similarly, in another culture (designated RUT2), Mycn was amplified more than 40×, whereas three of the other genes (Ddx1, Rrm2, and Odc1) were coamplified at lower levels. Using FISH on metaphase chromosomes from the cell cultures analyzed, the amplified sequences were shown to be located in typical HSRs. With competitive RT‐PCR, distinct overexpression of Mycn and Ddx1 could be demonstrated in both RUT2 and RUT30. In addition, Mycn was overexpressed in two other tumors not exhibiting Mycn amplification. Taken together, our results suggest that overexpression of Mycn plays an important role in the development of endometrial cancer in the BDII rat. In humans, Mycn amplification has been reported mainly from tumors of neuronal origin. To our knowledge, this is the first report of Mycn amplification and overexpression in hormone‐dependent tumors.

Mapping of H2-M homolog and MOG genes in the rat MHC

Class I genes of the rat major histocompatibility complex (MHC; RTI system) are encoded by two subregions, which are separated by regions coding for class II (RT1.B/D) and class III molecules. By convention, the RT1.A region is left of the class II region; it codes for one or very few classical class I molecules. The RT1. C/E region, located on the other side of the RT1 complex, contains a large number of class I genes (Jameson et al. 1992). The internal organization of this extended class I region has not been defined in detail. It is therefore of interest that genes similar to H2-M genes, which are the most distal group of class I genes in the mouse, have been demonstrated in the rat (Wang et al. 1991, 1995). The rat genes similar to H2-M genes are now called RT1.M genes, and they are different from the previously defined oligomorphic class I gene RT1.M (Wonigeit and Hfinisch 1991), which has been renamed RT1.R (Gill et al. 1995). A homolog of the H2-M3 gene has been isolated, and the sequence of RT1.M3 cDNA is 88% identical to H2-M3 (Wang et al. 1995). RT1.M3 as well as homologs of H2-M2 and H2-M4 have been mapped with established recombinant haplotypes to the extended class I region on the RT1. C/E side of the class II/III region (Wang et al. 1995). We now report on a new recombinant haplotype, r38, derived from the parental strains LEW (RTIO and BN (RTIn), in which the rat homologs of H2-M2 and H2M4 are separated from other class I genes that differ

A multiple transgenic mouse model with a partially humanized activation pathway for helper T cell responses.

Mice expressing human CD4 and human MHC II molecules provide a valuable model both for the investigation of the immunopathogenetic role of human autoantigens and for the development of therapeutic strategies based on modulating helper T cell activation in vivo. Here we present a novel mouse model expressing HLA-DR17 (a split antigen of HLA-DR3) together with human CD4 in the absence of murine cd4 (CD4/DR3 mice). Human CD4 accurately replaces murine cd4 within T cells. In particular, the preservation of cd8(+) and CD4(+) T cell subsets distinguishes CD4/DR3 mice from other multiple transgenic models in which the alternative T cell subsets are fundamentally disturbed. Moreover, human CD4 is also faithfully expressed on antigen presenting cells such as dendritic cells and monocyte/macrophages, so that the overall transgenic CD4 expression pattern resembles very closely that of humans. HLA-DR3 expression in the thymus correlates very closely to that of mouse MHC II. In contrast, only 70% of mouse MHC II positive cells in spleen, lymph node, and peripheral blood coexpress HLA-DR3. No significant bias was found with regard to particular leucocytes in this respect. The stimulation of helper T cells clearly depends on the interaction between the human transgene products, since mAbs to HLA-DR and/or CD4 completely blocked in vitro recall responses to tetanus toxoid. CD4/DR3 mice represent a partially humanized animal model which will facilitate studies of DR3-associated autoimmune responses and the in vivo determination of the therapeutic potential of mAbs to human CD4.

High-density marker loss of heterozygosity analysis of rat chromosome 10 in endometrial adenocarcinoma.

Endometrial cancer is a disease with serious impact on the human population, but not much is known about genetic factors involved in this complex disease. Female BDII rats are genetically predisposed to spontaneous endometrial carcinoma, and the BDII inbred strain provides an experimental animal model for endometrial carcinoma development. In the present study, BDII females were crossed with males from two nonsusceptible inbred rat strains. Endometrial adenocarcinomas (EACs) developed in a proportion of the F1 and F2 progeny. We screened 18 EAC solid tumors and 9 EAC cell cultures for loss of heterozygosity (LOH) using fluorescent‐PCR‐based marker allelotyping methodology with 47 microsatellite markers covering the proximal part of rat chromosome 10 (RNO10). Conclusive evidence was obtained for LOH/deletion involving about 56 cM in the proximal part of RNO10 in DNA from six out of seven informative tumor cell cultures. Analysis of the solid tumors confirmed the presence of LOH in this part of RNO10 in 14 of 17 informative tumors. However, from the studies in the solid tumors it appeared that in fact three separate segments in the proximal part of RNO10 were affected. These three LOH/deletion regions were located approximately in cytogenetic bands 10q11‐12, 10q22, and 10q24.