Biomedical subjects
Publications and source records attributed to T Shirai.
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CD5+ B cells have attracted much attention, because of their involvement in both autoimmunity and B cell-type chronic lymphocytic leukemia (B-CLL). B-CLL is a type of leukemia most often occurring among close relatives and is partly associated with the major histocompatibility complex (MHC), a finding relevant to autoimmune disease. We established MHC (H-2)-congenic NZB x NZW (NZB/W) F1 mice (H-2d/z, H-2z/z, and H-2d/d), in that only H-2d/z heterozygotes developed severe SLE, associated with IgG anti-DNA antibodies, as the animals aged. Such age-associated changes occurred in parallel with the decrease in the splenic, but not peritoneal, CD5+ B cells. By contrast, H-2z/z homozygotes did not develop SLE but, in turn, a marked clonal proliferation of CD5+ B cells resembling B-CLL did occur. H-2d/d homozygotes also did not develop the typical SLE, and a moderate CD5+ B frequency persisted. Despite the finding that all the three H-2-congenic NZB/W F1 strains produced IgM anti-DNA antibodies, only the H-2d/z heterozygotes produced IgG antibodies. Whereas the surface phenotype of major IgM producers was CD5+ sIgM+, that of IgG producers was CD5-sIgM-. Genetic and cellular analyses supported our thesis that in the heterozygotes IgM to IgG isotype switching probably emerges in CD5+ B cells and that this event is associated with the loss of CD5 molecules. Because of the lack of genetic elements required for differentiation, only signals for proliferation would be functioning in CD5+ B cells in the H-2z/z homozygotes. These observations infer that certain different, but related, MHC haplotypes may predispose either to B-CLL or to autoimmune disease in close relatives.
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In Experiment 1, groups of thirty 6-wk-old male and female F344 rats were given diets containing 0 (control) or 3% uracil for 104 wk. In the uracil-treated groups, carcinomas, in particular transitional cell carcinomas, developed in the urinary bladder of 90% of the males and 19% of the females. Squamous cell carcinomas also developed in 10% of the males, but not in females. Striking findings were that calculi were present in the urinary bladder of almost all males, but in only 30% of the females, and that induction of urinary bladder carcinomas was related to the presence of calculi. In Experiment 2, groups of thirty 6-wk-old male and female C57BL/6 x C3H F1 mice were given a diet containing 0 (control) or 3% (from Wk 1 to Wk 6) and 2.5% (from Wk 7 to Wk 96) uracil. The total observation period was 96 wk. In the uracil-treated groups, transitional cell carcinomas developed in 76% of the females and 8% of the males. Squamous cell carcinomas developed in only 8% of the males. In Experiment 3, 6-wk-old male F344 rats were given diets containing 3% uracil, 3% uracil plus 5% or 10% NaCl, or 10% NaCl for 36 wk and then diet without chemicals for a recovery period of 4 wk. The incidences of carcinomas and calculi of the urinary bladder were 75% and 81% in the group given uracil alone, 6% and 6% in the group given uracil plus 5% NaCl, and both zero in the group given uracil plus 10% NaCl. Thus, the present study showed that the inductions of urinary bladder carcinomas by uracil, a nongenotoxic compound, in rats and mice showed sex differences and were related to the presence of calculi in the urinary bladder.
Among 15 anti-DNA antibody-producing hybridomas derived from a single NZB X NZW F1 mouse, an IgM and an IgG were shown to use the same VH gene of the Q52 family. Using a combination of two primers both consisting of a mixture of oligonucleotides (one complementary to the 5' end of VH segment and one to the 3' end of VH segment of Q52 family) we determined the sequences of several members of germ-line VH genes in the Q52 family derived from NZB and NZW strains. Comparison of the sequences with those of cloned VH cDNA obtained from the hybridomas revealed that the VH sequence of the IgM anti-DNA antibody was identical to that of a cloned NZW germ-line VH gene, except for the priming sites. In contrast, the VH sequence of the IgG counterpart contained somatically mutated nucleotides. Because the IgG anti-DNA antibody showed a higher DNA binding activity than did the IgM antibody, we conclude that these changes in nucleotide sequences were induced and selected through an antigen-driven mechanism as is the case in a normal immune response. It is tempting to speculate that the germ-line encoded, low-affinity IgM autoantibody undergoes somatic mutations and isotype switching, resulting in generation of pathogenic, high-affinity autoantibodies in autoimmune diseases.
The effects of cholesterol on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced rat prostate carcinogenesis were investigated in male F344 rats. Animals were given 10 subcutaneous (s.c.) injections of 50 mg/kg body weight of DMAB at 2-week intervals, each time 2 days after transfer from 1 week on 0.75 ppm of an ethinyl estradiol (EE)-supplemented diet to a basal diet. From week 20, the animals received treatment with cholesterol (2 or 1%, in the diet), saponin (1 or 0.2 mg/rat/week, s.c.), or clofibrate (0.3%, in the diet) for 40 weeks. Although serum cholesterol levels were significantly increased by administration of cholesterol itself, and were decreased by clofibrate, the incidences of atypical hyperplasias and carcinomas of the ventral prostate were very similar with all treatments. Thus, under the present conditions, high or low cholesteremia did not affect rat prostate lesion development.
The present experiment was undertaken to investigate possible synergism in induction of rat prostate tumor. F344 rats were repeatedly administered the prostate carcinogens 3,2'-dimethyl-4-aminobiphenyl (DMAB),N-nitrosobis(2-oxopropyl)amine(BOP), and N-methylnitrosourea (MNU) sequentially or together at times of hormonal castration induced regenerative cell proliferation of prostate epithelial cells. Group 1 animals received two 100 mg/kg doses of DMAB, two 20 mg/kg applications of BOP, and two times 15 mg/kg of MNU. Groups 2, 3, and 4, respectively, received each of the carcinogen treatment alone. Group 5 was given 6 applications of all three in combination, each at one-third the dose administered to the other groups. The results showed a clear synergism in enhanced tumor development in the colon but not in the prostate, in which the incidence of lesions induced by the three carcinogens was similar to that with DMAB alone.
Eight pesticides were tested in a medium-term bioassay based upon the induction of preneoplastic lesions in the liver. Rats were initially given diethylnitrosamine intraperitoneally at a dose of 200 mg/kg body weight and 2 weeks later were treated with the pesticides for 6 weeks and then killed; all rats had a partial hepatectomy at week 3. Hepatocarcinogenic potential was assessed by comparing the number and area of glutathione S-transferase placental form positive foci in the liver with those of controls given diethylnitrosamine (DEN) alone. Positive results were seen with p,p-DDT and Triadimefon. Permethrin (mixture of 39% cis form and 61% trans form) showed borderline results. Permethrin (25/75), Deltamethrin, Cypermethrin (52/48), while Trimorphamide and Propineb gave negative results. Our findings provide experimental evidence to indicate that compounds active in this assay have a potential for liver carcinogenicity in rodents.
To examine the induction and repair of UV-induced DNA damage, indirect immunofluorescence was performed on UVB-irradiated organ-cultured normal human skin using monoclonal antibodies specific for either cyclobutane pyrimidine dimers or (6-4) photoproducts. Nuclear immunofluorescence of cyclobutane pyrimidine dimers and (6-4) photoproducts were observed in a dose-dependent manner after UVB irradiation. The intensity of nuclear immunofluorescence of the upper epidermal layers was stronger and clearer than that of the lower epidermal layers. DNA repair time-course studies showed that both types of DNA damage could be repaired within 24 h after UVB irradiation.
Although murine peritoneal B cells were homogenously positive for an epitope Lp-2, coded for by the alternative exon 4 of the CD45 gene, they were heterogenous with respect to the expression of another CD45R epitope, 6B2, of unknown exon dependency. While the majority of 6B2-high peritoneal B cells was composed of CD5- B cells, those with low or negative 6B2 were CD5+ B cells. Both 6B2+ and 6B2- peritoneal B cells expressed mainly the same largest CD45R transcripts, with all three alternative exon (4, 5, and 6) sequences. Further, a CD5+ B lymphoma cell line, BCL-1, which was found to be Lp-2+6B2- also had the largest isoform of CD45R molecules with all three alternate structures. Although enzyme digestion studies suggested that the 6B2 epitope resides in protein, not in sugar structures, it is likely that a post-translational modification of CD45R molecules is responsible for the presence or absence of 6B2 epitope expression on peritoneal CD5+ B cells. This event may be related to the differential role of CD45R molecules in regulating lymphocyte function.
The alterations of second-messenger ligand binding and cerebral blood flow (CBF) were evaluated in the gerbil brain after 2-h unilateral common carotid artery occlusion. [3H]Forskolin (FK) and [3H]phorbol-12,13-dibutyrate (PDBu) were used as specific ligands for adenylate cyclase (AC) and protein kinase C (PKC) activity estimation, respectively. CBF was determined at the end of the experiment by the [14C]iodoantipyrine method. A quantitative autoradiographic method permitted simultaneous measurement of the three parameters in the same brain. The levels in the caudate-putamen, globus pallidus, and hippocampus were analyzed. The animals were divided into three groups: Group 1 with severe ischemia (CBF in the lateral nuclei of the thalamus (CBFt) less than 50 ml/100 g/min), Group 2 with mild ischemia (CBFt greater than or equal to 50 ml/100 g/min), and the Sham Group. The PDBu binding revealed a statistically significant increase in the caudate-putamen, lateral nuclei of the thalamus and hippocampus (CA1 and CA3 regions and dentate gyrus) on the ischemic side in Group 1 as compared to that in Group 2 and the Sham Group. In contrast, the FK binding did not show any significant changes in any of the regions. These data and our previous findings for 6-h ischemia suggest that (1) PKC translocation to the cell membrane may occur at the early ischemic phase in particular regions including the caudate-putamen, lateral nuclei of the thalamus and hippocampus, with the translocated PKC gradually diminishing during the subsequent ischemic period; and (2) the suppression of the AC system observed in 6-h ischemia may not appear in the early ischemic phase.
In the F1 hybrid of phenotypically normal NZW (H-2z) and systemic lupus erythematosus (SLE)-prone BXSB mice (H-2b), features of the disease became more severe than those seen in the BXSB mice, regardless of the presence or absence of the Yaa (Y-chromosome-linked autoimmune acceleration) mutant gene. To determine whether the gene(s) linked to the major histocompatibility complex (MHC) of NZW mice is involved in this event, we developed the H-2-congenic NZW.H-2d strain and compared the severity of autoimmune disease between (NZW x BXSB) F1 (H-2z/b) and (NZW.H-2d x BXSB) F1 mice (H-2d/b). The H-2z/b, but not H-2d/b, heterozygous F1 mice of both sexes showed an accelerated, higher incidence of proteinuria and a more severe thrombocytopenia than did the BXSB mice. In NZW x (NZW x BXSB) F1 backcross mice, the H-2z/b heterozygous progeny showed more severe disease than did the H-2z/z homozygotes. Thus, disease-accelerating events in (NZW x BXSB) F1 mice are linked to the H-2z/b heterozygosity. Because H-2d/z heterozygosity plays a crucial role for SLE in (NZB x NZW) F1 mice, in which SLE features differ from those in (NZW x BXSB) F1 mice, the present observations may imply that the different but related MHC heterozygosity acts as a predisposing genetic element in these different SLE syndromes.
Tragacanth gum was administered at dietary levels of 0 (control), 1.25 and 5.0% to groups of 50 male and 50 female B6C3F1 mice for 96 wk after which all animals were maintained on a basal diet without tragacanth gum for a further 10 wk. Mean body weights of females in the 5.0% and 1.25% groups were lower than those of the controls after 11 and 16 wk, respectively. However, there were no treatment-related clinical signs or adverse effects on survival rate, urinalysis, haematology, blood biochemistry and organ weight. While detailed histopathology revealed the development of squamous cell hyperplasias, papillomas and one carcinoma in the forestomach, there was no significant treatment-related increase in the incidence of any preneoplastic or neoplastic lesion. Thus, under the experimental conditions used, tragacanth gum was not carcinogenic in B6C3F1 mice of either sex.
The correlation between urolithiasis due to uracil administration and epithelial proliferation in the urinary bladder was investigated in male F344 rats. Animals, 6 weeks old at the start, which ingested 3% uracil alone in the diet for 4 weeks developed urolithiasis and papillomatosis of the urinary bladder, whereas addition of NaCl, particularly at high concentration (10%), to the 3% uracil in the diet blocked production of both. In contrast, NaHCO3 was without influence on either calculi or proliferative changes. These results indicate that uracil-induced epithelial proliferation is due to direct mechanical irritation by calculi, and not to any chemical stimulus presented by uracil itself.
A variety of positive or negative enzyme altered foci have been proposed as preneoplastic marker lesions in the rat liver. Frozen sections are required in some cases. We have compared the suitability of various histochemically or immunohistochemically demonstrated markers and concluded that immunohistochemically-stained glutathione S-transferase placental form (GST-P) positive foci are particularly useful for practical application in risk assessment. Advantages include ease of quantitative foci analysis on a number of samples since acetone or formalin-fixed paraffin blocks can be used and clear contrast of foci against the surrounding liver tissue facilitates recognition. We have established a liver medium-term bioassay model of 8 weeks' duration using diethylnitrosamine as an initiator and GST-P positive foci as the endpoint lesions. At present, 58 non-genotoxic chemicals for which carcinogenicity data are available have been examined and many carcinogenic agents, mostly liver carcinogens, have been satisfactorily detected as having carcinogenic potential. Exceptional examples are two peroxisome proliferators, clofibrate and DEHP. For these chemical, several peroxisomal enzymes such as catalase and enoyl CoA hydratase have been tested as markers.
Autoimmune-prone New Zealand Black mice produce a large amount of autoantibodies cytotoxic for thymocytes (natural thymocytotoxic autoantibodies, NTA). A monoclonal NTA (NTA260) has been found to react with brain tissues as well as the cell surface of thymocytes. We investigated the expression of NTA 260 antigen in the primary culture of fetal brain cells. NTA260 labeled strongly the cytoplasm of nerve cells after fixation, but failed to stain the living cells. Western blot analysis revealed that NTA260 recognized predominantly a band at approximately 53 kDa in brain and thymic extracts. These findings indicate that neuronal NTA260 antigen, which has a molecular mass similar to that of thymocytes, is likely an intracellular component.
To investigate the association of glomerular involvement with biliary atresia, we carried out the following studies: (1) review of the clinical records of 120 patients, (2) histologic study of the kidneys obtained at autopsy from 28 patients, and (3) measurements of circulating immune complexes. Of 90 patients with adequate follow-up information, 40 (44.4%) had hematuria, proteinuria, or both. All the kidney specimens showed a wide variety of mesangial proliferation, and immunoglobulins were present in 23 of 26 cases. There was a good correlation between the glomerular alterations and the period of reduced hepatic function. When IgA was present in the mesangium, IgA2 and secretory components were detected. Elevated serum IgA and circulating IgA-containing immune complex levels were found in patients with prolonged obstructive jaundice. These findings indicate that glomerular alterations may occur subsequently in patients with biliary atresia, and that IgA of intestinal mucosal origin plays some role in the development of these lesions.