DNA repair after DNA fragmentation in mouse small intestinal epithelial cells.

In our earlier work, we found that, in mice, i.p. injection of anti-CD3 monoclonal antibody activated intraepithelial lymphocytes (iIEL), leading to DNA fragmentation in villous epithelial cells of the duodenum and jejunum within 30 min. By 2 h after injection, nearly half of the enterocytes had detached from the villi, and DNA fragmentation could barely be detected in the remaining villous epithelium. We hypothesized that DNA had been repaired in enterocytes in which DNA fragmentation had previously been induced. In this study, enterocytes became negative for TUNEL staining at 60 min after anti-CD3 treatment, prior to detachment. The remaining villous epithelial cells, after DNA fragmentation and detachment, were found to be positive for 5-bromo-2-deoxyuridine labeling. To confirm whether fragmented DNA had been repaired in situ, we investigated the appearance and/or mobilization of DNA-repair-related proteins. Focus formation, a typical staining pattern of repair-related proteins including phosphorylated H2AX, phospo-ATM substrate, and Nbs1, was observed 30 min after anti-CD3 injection, with the kinetics virtually identical to that of DNA fragmentation. The co-localization of gamma-H2AX and phospo-ATM substrate was also confirmed. The disappearance of a positive reaction for TUNEL staining in previously fragmented DNA, the appearance of representative DNA-repair-related proteins, the coincidence of the kinetics of DNA fragmentation and this appearance of DNA-repair-related proteins, and the co-localization of two of the repair-related proteins strongly indicated that enterocyte DNA could be repaired after it had been fragmented in vivo. Thus, DNA fragmentation per se may not necessarily be an immediate sign of cell death.

[The safety of docetaxel with cyclophosphamide (TC) therapy as adjuvant chemotherapy for Japanese women with operable breast cancer].

The safety of docetaxel (60 mg/m(2)) plus cyclophosphamide (600 mg/m(2)) every three weeks (TC) as adjuvant therapy for Japanese women with operable breast cancer was evaluated. Ehime TC Study Group initiated the randomized control study,which compared the effects of the TC course number (4 cycles versus 8 cycles) in the adjuvant setting on the treatment outcomes of breast cancer patients. Eight patients were investigated on the side effects of TC therapy, four of them were allocated to 4 cycles of TC, and four to eight cycles from May, 2004 to Feb. 2005. Leukocytopenia and neutropenia of grade 3 or 4 were seen in 50% and 63% of the cases, respectively. No febrile neutropenia was seen. Although the non-hematological side effects of grade 3 or 4 were not observed, alopecia, stomatitis, skin toxicities and edema of grade 2 were seen in 100%, 25%, 25%, 13% of cases, respectively. TC therapy was well tolerated. All anticancer drugs could be administered as scheduled. From these preliminary results, TC therapy seems to be able to be safely prescribed postoperatively for Japanese women operated for breast cancer.

Comparison of CDR3 length among thymocyte subpopulations: impacts of MHC and BV segment on the CDR3 shortening.

Thymocytes are thought to be selected on the basis of antigen specificity between TCR and peptide-MHC (pMHC) ligands. The specificity depends primarily on extensive diversities of complementarity determining region 3 (CDR3), whose specificity is considered to be determined through thymocyte selection. We examined the CDR3 length profiles with 20 BV segments in thymocyte subpopulations from C57BL/6 (H-2(b)), C.B10 (Balb/c congenic, H-2(b)) and Balb/c (H-2(d)) mice. The CDR3 length was shorter in both CD4 single positive (SP) and CD8SP than in double positive (DP), but not altered among DP, double negative (DN) 4 and DN3 subpopulations. The CDR3 shortened more prominently in CD4SP than in CD8SP for C57BL/6 and C.B10, but the shortening was only slight for Balb/c. Although the shortening varied considerably among different BV segments, the greater shortening was observed in most BV segments for CD4SP and in several for CD8SP, in particular, the extent was the greatest in BV1, BV2, BV15, BV16, BV23 and BV26 for CD4SP, and in BV13-1 and BV29 for CD8SP. Moreover, the extent and the pattern of CDR3 shortening were basically the same among highly homologous BV segments (e.g. BV12-1 and 12-2; BV13-1, 13-2 and 13-3). These results taken together indicate that (1) the CDR3 shortening occurred between the DP to the SP stages but never earlier, that (2) there would be the MHC class preference for the CDR3 shortening, that (3) it was in part influenced by MHC haplotype, and finally that (4) the primary structure of particular BV segments would possibly affect the CDR3 length in selected thymocytes. It could be deduced from these results that the CDR3 shortening might play roles in ensuring geometrical disposition of CDRs unique to each BV segment and consequently allow CDRs to intimately interact with pMHC ligands.

Accumulation of intestinal intraepithelial lymphocytes in association with lack of polymeric immunoglobulin receptor.

Immunoglobulin A (IgA) is transported by the polymeric immunoglobulin receptor (pIgR) through epithelial cells of the gut, the airways, the tear and salivary glands, and the lactating mammary gland, and IgA accumulates in serum and the intestinal lamina propria of pIgR-deficient (pIgR(-/-)) mice. Intraepithelial lymphocytes (IEL) increased in number and Thy-1(+)CD8alphabeta(+)TCRalphabeta(+) IEL preferentially expanded in the small intestine (SI) of pIgR(-/-) mice. Cytotoxic activity of SI-IEL was comparable in pIgR(+/+) and pIgR(-/-) mice. Accumulation and cytotoxic activity of SI-IEL was attenuated in germ-free pIgR(-/-) mice. Furthermore, Thy-1(+)CD8alphabeta(+) IEL did not expand in pIgR(-/-)TCRbetadelta(-/-) mice compared with TCRbetadelta(-/-) mice, and SI-IEL from pIgR(-/-)TCRbetadelta(-/-) mice as well as TCRbetadelta(-/-) mice expressed perforin and granzyme B mRNA and serine esterase. The proliferative status of SI-IEL from pIgR(+/+) and pIgR(-/-) mice was similar, but adoptive transfer experiment showed that SI-IEL from pIgR(-/-) mice might have a stronger tendency to migrate into the intestinal epithelia than those from pIgR(+/+) mice. These results demonstrate that the accumulation of Thy-1(+)CD8alphabeta(+)TCRalphabeta(+) IEL in pIgR(-/-) mice triggered by intestinal microorganisms needed the expression of functional TCR and might be caused by lymphocyte migration into the intestinal epithelia.

Disruption of the WFS1 gene in mice causes progressive beta-cell loss and impaired stimulus-secretion coupling in insulin secretion.

Wolfram syndrome, an autosomal recessive disorder characterized by juvenile-onset diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 gene. In order to gain insight into the pathophysiology of this disease, we disrupted the wfs1 gene in mice. The mutant mice developed glucose intolerance or overt diabetes due to insufficient insulin secretion in vivo. Islets isolated from mutant mice exhibited a decrease in insulin secretion in response to glucose. The defective insulin secretion was accompanied by reduced cellular calcium responses to the secretagogue. Immunohistochemical analyses with morphometry and measurement of whole-pancreas insulin content demonstrated progressive beta-cell loss in mutant mice, while the alpha-cell, which barely expresses WFS1 protein, was preserved. Furthermore, isolated islets from mutant mice exhibited increased apoptosis, as assessed by DNA fragment formation, at high concentration of glucose or with exposure to endoplasmic reticulum-stress inducers. These results strongly suggest that WFS1 protein plays an important role in both stimulus-secretion coupling for insulin exocytosis and maintenance of beta-cell mass, deterioration of which leads to impaired glucose homeostasis. These WFS1 mutant mice provide a valuable tool for understanding better the pathophysiology of Wolfram syndrome as well as WFS1 function.

DNA fragmentation and detachment of enterocytes induced by anti-CD3 mAb-activated intraepithelial lymphocytes.

To elucidate the role of intraepithelial lymphocytes (IEL) and enterocytes in the defense mechanism of the small intestine, we designed experiments to stimulate the IEL by anti-CD3epsilon, anti-TCRalphabeta, or anti-TCRgammadelta monoclonal antibodies (mAbs), and to examine the subsequent changes to the enterocytes. The enterocytes of the duodenum and jejunum, but not of the ileum, showed massive DNA fragmentation 30 min after intraperitoneal injection of anti-CD3 mAb. These responses were also induced by anti-TCRgammadelta mAb, but not by anti-TCRalphabeta mAb, and were completely inhibited by cyclosporin A. Nearly half of the enterocytes of the villi in the duodenum and jejunum were exfoliated into the lumen 4 h after the injection of the mAb. Administration of anti-CD3 mAb also induced DNA fragmentation in Fas-deficient MRL/lpr mice, indicating that the Fas-Fas ligand system was not involved in these events. The anti-CD3 mAb treatment also induced massive DNA fragmentation in the intestinal epithelium of the duodenum and jejunum in TNF-receptor-1-deficient mice, whereas TNF-alpha induced only the detachment of intestinal epithelium of wild-type mice, implying the dissociation of two independent factors and/or mechanisms for DNA fragmentation and the subsequent epithelial cell detachment in the murine duodenum and jejunum. The mAb failed to exfoliate the epithelium in TNF-R1-deficient mice. Thus, TCRgammadelta(+) IEL, when treated with anti-CD3 or anti-TCRgammadelta mAbs, induced rapid DNA fragmentation and subsequent detachment of the duodenal and jejunal epithelia, but not in the ileum ("the silent ileum"), partly because of the paucity of TCRgammadelta(+) IELs in the ileum.

Distribution of two types of lymphocytes (intraepithelial and lamina-propria-associated) in the murine small intestine.

The intestine, which is exposed to nutrition and to food-derived antigens and microbes including viruses and bacteria, might be an important site for the immune response. Crucial structural and functional differences exist between the small and large intestine, regional differences even having been demonstrated within the small intestine. Accordingly, intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) might be heterogeneous among the different intestinal regions. The aim of this study has been to describe, as accurately as possible, the numbers and T-cell receptor (TCR) phenotypes of IELs and LPLs present in distinct regions of the murine small intestine under physiological conditions. Using an immunohistological technique to differentiate IELs from LPLs, the differential enumeration of IELs and LPLs in distinct regions of the murine small intestine, based upon their definition originally determined by their location, has been performed for the first time and has demonstrated that (1) there are more IELs than LPLs in the duodenum and jejunum, but more LPLs than IELs in the ileum, (2) in the duodenum and jejunum, TCRgammadelta IELs account for 70%-75% of the total CD3(+) IELs, a much greater percentage than previously reported, (3) the ratio of TCRgammadelta to TCRalphabeta IELs is inverted in the ileum, with more than 75% IELs being TCRalphabeta-positive, (4) the lamina propria forms one functional unit throughout the small intestine in terms of the TCR subset components (TCRalphabeta:TCRgammadelta=3:1), and (5) the ileum is entirely different from other regions of the small intestine. To deepen our understanding of the functional significance of the small intestine as an immunologically competent organ, the precise distributions of IELs and LPLs, the ratio of their various subsets, and the strict distinction of IELs and LPLs, as described in this study, is indispensable.