Immunohistochemical expression of MAM-3 and MAM-6 antigens in salivary gland tumours

Summary MAM-3 and MAM-6 antigens of human milk fat globule membrane were detected immunohistochemically in 93 cases of salivary gland tumours as well as in normal glands. The antigens were visualized in 10% formalin-fixed paraffin sections. MAM-3 (MoAbs 115G3, 67D11) antigen was distributed in intercalated and striated duct cells of the normal salivary glands, and in luminal tumour cells and squamous metaplastic cells of pleomorphic adenomas. In pleomorphic adenomas the frequency of positive staining with MoAb 67D11 (54/67; 80.6%) was higher than that with MoAb 115G3 (36/67; 53.7%). MAM-6 (MoAbs 115D8, 115F5) antigen was expressed in luminal and lateral borders of serous acinar cells and ductal of the normal glands, and also in luminal borders of tubulo-ductal and glandular structures of salivary gland tumours. Ductal basal cells were characterized by existence of positive staining for MAM-6 antigen, in adenolymphomas MAM-6 antigen was restricted to the basal tumour cells. Some mucous cells of mucoepidermoid tumours were stained specifically with MoAb 115G3, and epidermoid cells of mucoepidermoid carcinomas manifested MAM-6 antigen staining. Immunohistochemical localization of MAM-6 antigen resembled that of epithelial membrane antigen (EMA) detected with MoAb.     

Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice

Missense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3beta and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD).     

Clonal expansion of multiphenotypic Epstein-Barr virus-infected lymphocytes in chronic active Epstein-Barr virus infection

Chronic active Epstein-Barr virus (EBV) infection has been recognized as clonal non-neoplastic lymphoproliferative diseases. However, some reports of cases with a multiphenotypic expansion of EBV-infected lymphocytes give rise to questions of how EBV infects multiphenotypic lymphocytes and whether chronic active EBV infection is a truly monoclonal lymphoproliferative disease. We report two patients with chronic active EBV infection who showed expansion of multiphenotypic EBV-infected lymphocytes. EBV DNA was detected in CD4+ and CD8+ T cells and in B cells from pleural fluid of one patient and in T and B cells from a cervical lymph node of the other patient by polymerase chain reaction (PCR). Although real-time PCR showed that there were equally high loads of EBV genomes in CD4+ and CD8+ T cells from the pleural fluid, Southern blot hybridization with terminal repeats of the EBV genome showed a single band of the same molecular weight in three tissue samples from the patient. The results indicated biphenotypic expansions of CD4+ and CD8+ T cells infected with the same clone of EBV. Furthermore, bisulfite PCR analysis showed hypermethylated status in the Cp region in the two patients regardless of their cell populations. There has been a discrepancy between clonality and expansion of multiphenotypic EBV-infected lymphocytes. We speculate that lymphoid progenitor cells that have not differentiated into T and B cell progenitors are infected with EBV, resulting in clonal expansion of EBV-infected multiphenotypic cells.     

Changes in T,B, and NK Lymphocyte Subsets During and After Normal Pregnancy

PROBLEM: Pregnancy affects the maternal immune system and the clinical course of maternal diseases. Here we report the changes in the detailed lymphocyte subsets of helper T cells, suppressor T cells, CD5⁺ B cells, T cell receptor (TCR) αβ-positive T cells (Tαβ cells), TCRαβ-negative T cell (Tγδ cells), and others during and after pregnancy through to one year postpartum, and discuss the significance of the changes. METHOD: The absolute numbers of helper T cells, suppressor T cells, cytotoxic T cells, TCRαβ-negative T cells (Tγδ cells), CD5^— B cells, CD5⁺ B cells, and NK cell subsets were examined by two-color flow cytometry in peripheral blood from 51 healthy non-pregnant women, 106 healthy pregnant women, and 148 healthy postpartum women. RESULTS: In early pregnancy, the numbers of suppressor T cells and NK cells with strong cytotoxicity (NK⁺⁺⁺ cells) increased, and the number of cytotoxic T cells decreased. In late pregnancy, the helper T cell and NK⁺⁺⁺ cell numbers decreased. Tαβ, CD5^— B and CD5⁺ B cells decreased during pregnancy. After delivery, helper T cells and cytotoxic T cells increased from 1 to 4 months postpartum, and suppressor T cells increased at 7 months postpartum. TCRαβ-negative T cells increased at 4 to 10 months postpartum. Both CD5^— and CD5⁺ B cells decreased further at 1 month postpartum, but CD5⁺ B cells increased markedly at 7 to 10 months postpartum. CONCLUSIONS: These data indicate that 1) early increases of suppressor T cells and NK⁺⁺⁺ cells during pregnancy may be related to the mechanism to accept or reject the fetus in early pregnancy, respectively; 2) late decreases of helper T cells and NK⁺⁺⁺ cells may be related to the maintenance of pregnancy: 3) postpartum increases of helper T cells, cytotoxic T cells, TCRαβ-negative T cells (Tγδ cells), and CD5⁺ B cells may be related to the postpartum aggravation of autoimmune diseases; and 4) the immunological effects of pregnancy remains until about 1 year after delivery.     

Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test

In order to evaluate the utility of the mouse lymphoma assay(MLA) for detecting in vitro clastogens and spindle poisonsand to compare it with the in vitro chromosomal aberration test(CA), we conducted an international collaborative study of theMLA that included 45 Japanese laboratories and seven overseaslaboratories under the cooperation of the Ministry of Healthand Welfare of Japan and the Japanese Pharmaceutical Manufacturer'sAssociation. We examined 40 chemicals; 33 were reportedly positivein the CA but negative in the bacterial reverse mutation assay,six were negative in both assays and one was positive in both.We assayed mutations of the thymidine kinase (TK) locus (tk)of L5178Y tk^+/– mouse lymphoma cells using the microwellmethod. According to our standard protocol, cells were exposedto the chemical for 3 h, cultured for 2 days and TK-deficientmutants were expressed in 96-well plates under trifluorothymidine.Each chemical was coded and tested by two or three laboratories.Among the 34 CA-positive chemicals, positive MLA results wereobtained for 20 and negative results were obtained for nine.The remaining five chemicals were inconclusive or equivocalbecause of discrepant inter-laboratory results or reproduceddiscrepant results, respectively. Among the six CA-negativechemicals, one was negative in the MLA, two were positive andthree were inconclusive. Thus, the MLA could detect only 59%(20/34) of CA-positive chemicals. We concluded that the MLAwas not as sensitive as the CA. Some MLA-negative chemicalsevoked positive responses in the CA only after long continuoustreatment. These might also be genotoxic in the MLA with longcontinuous treatment. Improvement of the MLA protocol, includingalteration of the duration of the treatment, might render theMLA as sensitive as the CA. ⁸ To whom correspondence should be addressed. Tel: +81 3 37009847; Fax: +81 3 3700 2348; Email: sofuni{at}nihs.go.jp     

Histochemical studies on glycosaminoglycans in Bruch's membrane of postnatal rat eyes

Localization of glycosaminoglycans (GAG) in Bruch's membrane of postnatal rat eyeballs was examined histochemically. Fixed eyeballs from postnatal rats (ages 5 days and 8 weeks) were routinely processed and embedded in paraffin wax or Quetol 651 resin. Paraffin-embedded tissue sections were stained with hematoxylin and eosin or sensitized high iron diamine procedure in combination with selective methods such as GAG-degrading enzyme digestions and/or a chemical modification, and examined by light microscopy. Quetol 651-embedded ultrathin sections were stained with heavy metals and examined by electron microscopy. In rats at postnatal day 5, Bruch's membrane contained mainly chondroitin sulfate (CS) and heparan sulfate (HS). In contrast, at 8 weeks after birth the membrane included a large amount of dermatan sulfate (DS) and HS. According to electron microscopic findings, Bruch's membrane on day 5 consisted of only 3 layers without a central elastic layer. However, at 8 weeks after birth the membrane was constructed of 5 layers. These findings suggested that the difference in GAG molecular species in the membranes at 5 days and at 8 weeks after birth could be correlated with the development and maturation of the collagenous layer in Bruch's membrane. Moreover, maturation of Bruch's membrane may contributes to the architectural stabilization of the outer portions of the photoreceptor cells.     

Pathogenic role of polyclonal and polymeric IgA in a murine model of mesangial proliferative glomerulonephritis with IgA deposition.

Molecular size and charge distribution of IgA of sera and glomerular eluates were investigated in ddY mice, spontaneously developing mesangial proliferative glomerulonephritis (GN) with IgA deposition after 40 weeks of age. Serum IgA levels were increased in aged ddY mice more than 40 weeks old with a significant increase (P less than 0.01) at the age of 60 weeks, comparing with those of BALB/c mice. The isoelectric focusing (IEF) spectrotype of pooled serum IgA in 60-week-old mice ranged from 4.2 to 5.5, being similar to those in younger ddY (16 weeks old) and control BALB/c mice (12 weeks old) without enhanced expression of specific IgA peaks. However, IgA in the glomerular eluate from the 60-week-old mice showed limited anionic spectrotypes from pH 4.2 to 4.8. HPLC of IgA in pooled sera and glomerular eluates of 16-, 40- and 60-week-old ddY mice, revealed markedly increased ratios of the dimeric IgA (dIgA) and polymeric IgA (pIgA) in the total IgA with age. In the contrast to serum profiles, monomeric IgA (mIgA) was always detected as the smallest peak of the IgA fractions in glomerular eluates. Furthermore, aged mice with severe GN showed a higher percentage of dIgA and pIgA in total IgA (80%) in the sera than that of the mice with mild GN (64%). HPLC analysis under acid condition of glomerular IgA from 40-week-old ddY mice showed a similar pattern of dIgA and pIgA peaks in neutral buffer without the appearance of mIgA. These findings suggest that there is a selective mechanism for glomerular accumulation of more acid IgA among the polyclonally expanded IgA in old ddY mice, and that the polymeric form of IgA plays a pathogenic role in the development of mesangial proliferative GN in these mice.     

Evaluation of DNA probes for specific detection of Vibrio cholerae O139 Bengal.

Two DNA probes, 2R1 and 2R3, prepared from a region in the chromosome specific for the lipopolysaccharide O side chains of Vibrio cholerae O139 (M.K. Waldor and J.J. Mekalanos, Lancet 343:1366, 1994) were examined for their specificity and sensitivity. Both probes did not hybridize with any strain of V. cholerae belonging to serogroups other than O139 and to any of the other species examined belonging to the family Vibrionaceae. Among the 126 strains of V. cholerae O139 examined, probe 2R1 hybridized with 125 strains while probe 2R3 hybridized with all 126 strains. Both probes were found to be highly specific and sensitive and can be used for the specific identification of V. cholerae O139.     

Tumor necrosis factor reduces lifespan of human endothelial cells in vitro

Tumor necrosis factor (TNF) is known to regulate the proliferation and function of vascular endothelial cells (ECs). We have examined the effects of TNF on the growth and aging of human ECs of different origins and compared them with those in human normal diploid fibroblasts. The results obtained were as follows: (1) TNF reduces the growth rate and in vitro life span of ECs in both dose- and treatment length-dependent fashions; (2) ECs are significantly more sensitive to TNF than fibroblasts; and (3) the life span shortening effect of TNF on ECs increases as a function of in vitro cell age. These results suggest that the aging of ECs is modified by TNF exposure.