[Susceptibility to Mycobacterium leprae of congenic hypertensive nude rat (SHR/NCrj-rnu) and production of cytokine from the resident peritoneal macrophages].

We have established a congenic hypertensive nude rat strain, SHR/NCrj-rnu, carrying nude (rnu) and hypertension genes which was produced using females of the SHR/NCrj rat and males of the F344/NJcl nude rat by cross-intercross system for 12 generations. We demonstrated the susceptibility to M. leprae infection of SHR/NCrj-rnu rats as compared with F344/NJcl-rnu rats. SHR/NCrj-rnu rats were highly susceptible to M. leprae, and the SHR/NCrj-rnu rats of both sexes showed massive swelling of legs due to multiplication of M. leprae. However, F344/NJcl-rnu rats of both sexes revealed very poor susceptibility to M. leprae. There was a wide difference in the susceptibility to M. leprae between the SHR/NCrj-rnu and the F344/NJcl-rnu rats. We also examined the cytokine production. The resident peritoneal macrophages of SHR/NCrj-rnu rats produced IL-1 alpha, IL-6, IL-10 and TNF alpha, whereas those of F344/NJcl-rnu rats produced only TNF alpha.

Genetic typing of the mouse and rat nude mutations by PCR and restriction enzyme analysis.

A genetic typing method for the mouse and rat nude mutations by PCR and restriction fragment length polymorphism (RFLP) analysis was developed. Since restriction sites useful for RFLP analysis do not exist in the mouse nu and rat rnu mutations, artificial restriction sites were introduced by PCR with modified primers. Three genotypes in the mouse (nu/nu, nu/+ and +/+) or rat (rnu/rnu, rnu/+ and +/+) are rapidly differentiated with the PCR-RFLP assay. In addition, congenic nude strains can be efficiently established by using this assay. Finally, genetic mapping of the rnu locus was performed with microsatellite markers. The locus order on rat chromosome 10 was D10Mgh14-(2.0cM)-D10Mit2-(1.4cM)-rnu-(0.7cM++ +)-D10Mgh6-(2.7cM)-D10Mit8.

Resistance to chemically-induced mammary tumors in Copenhagen X nude-derived F2 athymic rats: evidence that T-cell immunity is not involved in Copenhagen resistance.

Resistance to chemically-induced mammary tumors in the Copenhagen rat is well defined, but the mechanism of resistance has yet to be determined. We have tested whether or not Copenhagen rat resistance is dependent on T-cells, since several lines of evidence supported an involvement of the immune system. We crossed Copenhagen rats with an athymic nude rat to produce F1s, that were interbred to produce F2 animals, some of which were athymic with partial Copenhagen rat background. A comparison of the mammary tumor incidences between the nude athymic F2 animals and their non-nude littermates allowed us to determine what role, if any, T-cells played in resistance. Following treatment with N-methyl-N-nitrosourea, we observed no difference in the tumor incidences between the two groups. Furthermore, the mammary tumor incidences in the F2 nude and non-nude animals was almost zero. These results indicate that T-cells are not involved in Cop resistance, and that nude rats are resistant to N-methyl-N-nitrosourea-induced mammary tumorigenesis.

Linkage of the athymic nude locus with the myeloperoxidase locus in the rat.

In rats of the BUF/Mna strain epithelial thymoma development is regulated by a single autosomal susceptible gene, Tsr-1. In pre-thymoma stage, BUF/Mna rats have extremely large thymuses, when compared with those of other strains of rats. The large thymus size of this strain is contributed by a thymus-enlargement gene, Ten-1. On the other hand, reduced thymus size and suppression of thymoma development were found in heterozygous BUF/Mna-rnu/+ rats. Linkage studies between RNU and microsatellite and restriction fragment length polymorphism markers in ([BUF/Mna-rnu/rnu x WKY/NCrj] F1 x WKY/NCrj)- and (WKY/NCrj x [BUF/Mna-rnu/rnu x WKY/NCrj] F1)- backcross rats have led to the localization of RNU on chromosome 10. The rat homolog of mouse Mpo (myeloperoxidase) was also assigned to the chromosome 10. The gene order on the chromosome was MYHSE (myosin heavy chain of embryonic skeletal muscle)--(1.0 centimorgan [cM])--SHBG (sex hormone-binding globulin)--(4.0 cM)--RNU (Rowett rat nude)--(10.0 cM)--MPO--(13.0 cM)--AEP (anion exchange protein). Conserved linkage of homologous loci mapped to rat chromosome 10 and mouse chromosome 11 supports the hypothesis that the RNU and MPO loci are rat homologs of the mouse nu and Mpo loci.

New member of the winged-helix protein family disrupted in mouse and rat nude mutations.

Mutations at the nude locus of mice and rats disrupt normal hair growth and thymus development, causing nude mice and rats to be immune-deficient. The mouse nude locus has been localized on chromosome 11 (refs 3, 4) within a region of < 1 megabase. Here we show that one of the genes from this critical region, designated whn, encodes a new member of the winged-helix domain family of transcription factors, and that it is disrupted on mouse nu and rat rnuN alleles. Mutant transcripts do not encode the characteristic DNA-binding domain, strongly suggesting that the whn gene is the nude gene. Mutations in winged-helix domain genes cause homeotic transformations in Drosophila and distort cell-fate decisions during vulval development in Caenorhabditis elegans. The whn gene is thus the first member of this class of genes to be implicated in a specific developmental defect in vertebrates.

Regional mapping of the Rowett nude gene (RONU) to rat chromosome 10q24-->q32 by localizing linked SYB2 and GH loci.

The Rowett nude gene (RONU) has been mapped on rat chromosome (Chr) 10 by linkage analysis using (ACI x F344/N-RONU/RONU)F1 x F344/N-RONU/RONU backcross progeny. The gene order on the chromosome was RR92- (16.1 cM) - RR24 - (17.9 cM) - MYHSE (myosin heavy chain, embryonic) - (1.0 cM) - SYB2 (synaptobrevin 2) - (1.0 cM) - SHBG (sex hormone-binding globulin) - (4.0 cM) - RONU (Rowett nude) - (29.0 cM) - AEP (anion exchange protein), PPY (pancreatic polypeptide) - (3.0 cM) - ACE (angiotensin I converting enzyme), GH (growth hormone). The RONU locus was localized to 10q24-->q32 by fluorescence in situ hybridization of the closely linked SYB2 and loosely linked GH loci on the opposite side. Conserved linkage of homologous loci mapped to rat Chr 10 and mouse Chr 11 supports the hypothesis that the RONU locus is a rat homolog of the mouse nu locus.

Genetic mapping of the athymic nude (RNU) locus in the rat to a region on chromosome 10.

The nude trait in the rat is transmitted in an autosomal recessive manner and is associated with thymic aplasia, T-cell deficiency, and hairlessness. Congenic rats homozygous for the RNU (Rowett nude) locus are important models in the study of inflammatory disease, tumor growth, and transplant rejection. The RNU locus has not been previously mapped, and the nature of the gene product is unknown. To determine the map location of this gene, a single F344.rnu/rnu (athymic nude congenic Fischer rat) male congenic rat was bred with 3 LEW/N (NIH stock Lewis rat) female rats to produce F1 progeny. Twelve F1 brother-sister breeding pairs were established. Forty-nine phenotypically nude F2 offspring (198 total) were obtained. Linkage analysis done on F2 DNA revealed highly significant cosegregation between the nude phenotype and eight polymorphic markers located on Chromosome (Chr) 10. The tightest linkages were with: MYH3 (embryonic, skeletal myosin heavy chain) and SHBG (sex hormone-binding globulin), giving 2 point lod scores of 20.2, and 20.0, respectively. The map order and map distances, determined by multipoint linkage calculations, were: RR24-(16.1 cM)-MYH3-(3.5 cM)-SHBG-(4.7 cM)-RNU-(11.9 cM)-F16F2-(24.1 cM)-CLATP (citrate lyase ATPase)-(2.4 cM)-ACE (angiotensin converting enzyme)/PPY (pancreatic polypeptide)-(14.1 cM)-RR1023. The position of the RNU locus in the rat corresponds closely with that of the recently reported nu locus in the mouse. This finding suggests that the nude phenotype in the rat and the mouse arise from defects in homologous genes.

Alloreactive lymphokine-activated killer cells from athymic nude rats do not express CD3-associated alpha/beta or gamma/delta T cell receptors.

Lymphokine activated killer (LAK) cells from athymic nude rats, previously shown to selectively kill MHC-incompatible small lymphocytes in vitro, were examined for rearrangement and expression of genes for the T cell antigen receptors. Southern blots showed no rearrangement of the TCR beta-chain genes, and Northern blots showed transcription only of truncated 1.0 kb beta-chain messages, but not of full-length, 1.3 kb beta-chain mRNA. Transcription of the alpha-chain of the TCR could not be detected, and surface staining with the mAb R73 showed no expression of the rat TCR alpha/beta heterodimer. Transcripts hybridizing with a rat TCR C gamma probe were detected on Northern blots, but probes for all presently characterized mouse V gamma genes failed to hybridize to the same filters, indicating that the C gamma-containing transcripts probably were from non-rearrangement genes. CD3 delta- and epsilon-chain transcripts could not be detected by Northern blot analysis. Less than 2% of the cells stained with the anti-rat CD3 monoclonal antibody 1F4, and incubation with 1F4 had no effect on the alloreactivity of nude rat LAK cells. We have previously shown that immunoglobulin is not involved in the killing of the allogeneic lymphocytes. The most likely interpretation of these results is therefore that nude rat LAK cells express a novel receptor structure involved in allorecognition.

Histochemical heterogeneity of dermal mast cells in athymic and normal rats.

Mucosal mast cells (MMC) and connective tissue mast cells (CTMC) of the rat contain different proteoglycans, which can be distinguished using histochemical methods. The chondroitin sulphate proteoglycan of the MMC, unlike the heparin of the CTMC, does not show fluorescent berberine binding, is susceptible to aldehyde fixatives and stains preferentially with Alcian Blue in a staining sequence with Safranin. The majority of the dermal mast cells are typical CTMC and are located in the deep part of the dermis. Subepidermal mast cells are comparatively few in normal rats but numerous in athymic rats and mice. These cells differ from other dermal mast cells in that they stain preferentially with Alcian Blue and they appear to contain little histamine. We examined some of the histochemical properties of the skin mast cells of female PVG-rnu/rnu rats and their heterozygous littermates aged from 5 to 29 weeks. The thiazine dye-binding of the subepidermal mast cells was partially blocked by formaldehyde fixation and only about half of them showed a weakly fluorescent berberine binding. The critical electrolyte concentration of the Alcian Blue staining of the subepidermal mast cells was between that of CTMC and MMC. Deaminative cleavage with nitrous acid abolished the staining of all skin mast cells, while that of the MMC was unaffected. There were no statistically significant differences in the staining patterns of the dermal mast cells between different ages or groups of rat. These results indicate that the subepidermal mast cells contain a heparin proteoglycan which is, however, different from that of the typical CTMC of other sites. They thus appear to represent a second example of a mast cell within a defined anatomical location exhibiting a distinct proteoglycan expression.

Possible single dosage effects of the nude gene: suppression of spontaneous development of thymoma and nephropathy in BUF/Mna-rnu/+ rats.

Single dosage effects of the rat nude gene (rnu) on spontaneous development of epithelial thymoma, muscle atrophy and nephrotic syndrome were studied by comparing littermates of rnu /+ and +/+ rats on a high thymoma strain, BUF/Mna, background. Heterozygous rnu/+ rats had a significantly smaller thymus than the +/+ littermates at 6 weeks of age. The incidence of thymoma at 12 months of age was extremely low in the female rnu/+ rats (3%) as compared with that of the +/+ rats (94%). Development of the nephrotic syndrome but not of the muscle atrophy was also suppressed in the heterozygotes. The results suggest that a recessive mutant gene, rnu, in a single dosage, interfered with critical steps of the disease processes of the thymoma and nephrotic syndrome in BUF/Mna-background rats.

Effects of homozygosity of the nude (rnu) gene in an inbred strain of rats: studies of lymphoid and non--lymphoid organs in different age groups of nude rats of LEW background at a stage in the gene transfer.

Several age groups of nude homozygous rnu/rnu and heterozygous rnu/+ rats of the same genetic background at an early stage of back-crossing (LEW/Mol) were compared as to body and organ weights, histological appearance and cell density of lymphoid organs, haematological values and differential counts of bone marrow and peripheral blood. No thymic tissue was found in the nude animals. 7-week-old nudes were smaller than control animals and had relatively larger non-lymphoid organs and cell-depleted peripheral lymphoid organs. Other age groups showed little difference. Peripheral blood of nude rats showed no signs of lymphopaenia in contrast with the findings in nude mice. The number of thoracic duct lymphocytes was, however, significantly smaller in all age groups of the nude rats, and the bone marrow tended to contain fewer lymphocytes.