Polyglutamine aggregates stimulate ER stress signals and caspase-12 activation

Accumulation of unfolded and malfolded proteins causes endoplasmic reticulum (ER) stress, stimulating unfolded protein response (UPR) and c-Jun N-terminal kinase (JNK) activation and activating caspase-12 located on the ER. Little is known about the relationship between the ER stress and polyglutamine [poly(Q)] aggregates. Poly(Q)72 repeats [poly(Q)(72)] induced the stimulation of ER stress signals such as JNK activation, upregulation of Grp78/Bip and caspase-12 activation in C2C5 cells. We prepared antiserum against the cleavage site of mouse caspase-12 at D(318) (anti-m12D318), and showed that poly(Q)(72) with perinuclear aggregates, cytoplasmic inclusions and nuclear inclusions stimulated JNK activation and anti-m12D318 immunoreactivity, but poly(Q)(72) with dispersed aggregates and small nuclear aggregates showed a significantly less effect. Poly(Q)(72) and poly(Q)(11) dispersed in cytoplasm did not. Anti-m12D318-positive cells showed apoptotic features. Unlike anti-m8D387 immunoreactivity, the anti-m12D318 immunoreactivity was not coaggregated with poly(Q). Ac-IETD-fmk (caspase-8 inhibitor) and Ac-DEVD-CHO (caspase-3 inhibitor) did not prevent the anti-m12D318 immunoreactivity induced by poly(Q)(72) aggregates. Anti-m12D318 immunoreactivity was detected in caspase-8(-/-) and caspase-3(-/-) mouse embryonic fibroblasts expressing poly(Q)(72) aggregates. Thus, caspase-12 was activated by poly(Q)(72) aggregates via a pathway independent of caspase-8 and caspase-3 activation, and caspase-12 activation was closely associated with poly(Q) aggregate-mediated cell death. Stimulation of ER stress signals may be involved in the pathogenesis of neurodegenerative disorders with poly(Q) expansion.     

Undifferentiated (embryonal) sarcoma of the liver in middle-aged adults: smooth muscle differentiation determined by immunohistochemistry and electron microscopy

Undifferentiated (embryonal) sarcoma of the liver (UESL) is a rare pediatric liver malignancy that is extremely uncommon in middle-aged individuals. We studied 2 cases of UESL in middle-aged adults (1 case in a 49-year-old woman and the other in a 62-year-old man) by histology, immunohistochemistry, and electron microscopy to clarify the cellular characteristics of this peculiar tumor. One tumor showed a mixture of spindle cells, polygonal cells, and multinucleated giant cells within a myxoid matrix and also revealed focal areas of a storiform pattern in a metastatic lesion. The other tumor was composed mainly of anaplastic large cells admixed with few fibrous or spindle-shaped components and many multinucleated giant cells. In both cases, some tumor cells contained eosinophilic hyaline globules that were diastase resistant and periodic acid-Schiff positive. Immunohistochemically, the tumor cells showed positive staining for smooth muscle markers, such as desmin, alpha-smooth muscle actin, and muscle-specific actin, and also for histiocytic markers, such as alpha-1-antitrypsin, alpha-1-antichymotrypsin, and CD68. Electron microscope examination revealed thin myofilaments with focal densities and intermediate filaments in the cytoplasm of tumor cells. Our studies suggest that UESL exhibits at least a partial smooth muscle phenotype in middle-aged adults, and this specific differentiation may be more common in this age group than in children. Tumor cells of UESL with smooth muscle differentiation in middle-aged adults show phenotypic diversity comparable to those of malignant fibrous histiocytoma with myofibroblastic differentiation.     

Catalase, a Specific Antigen in the Feces of Human Subjects Infected with Helicobacter pylori

Recently, we reported the production of three new monoclonal antibodies with high specificity for a Helicobacter pylori antigen suitable for diagnosis of H. pylori infection. The aim of the present study was to identify the antigen recognized by these monoclonal antibodies concerning both H. pylori and the feces of human subjects infected with H. pylori. The cellular antigen was purified from an H. pylori cell extract by immunoaffinity column chromatography with the monoclonal antibody as a ligand. The amino-terminal amino acid sequences (eight residues) of the purified antigen and H. pylori catalase were the same. The molecular weights of native and subunit, specific catalase activity, and UV and visible spectra of the purified antigen were in good agreement with those of H. pylori catalase. The human fecal antigens were purified from two fecal samples of two H. pylori-positive subjects by ammonium sulfate precipitation, CM-Sephadex C₅₀ chromatography, and the same immunoaffinity chromatography used for the H. pylori cellular antigen. The fecal antigens had catalase activity. The amino-terminal amino acid sequences (five residues) of the human fecal antigen and H. pylori catalase were the same. The monoclonal antibodies reacted with the native cellular antigen, but did not react with the denatured antigen, human catalase, and bovine catalase. The results show that the target antigen of the monoclonal antibodies is native H. pylori catalase and that the monoclonal antibodies are able to specifically detect the antigen, which exists in an intact form, retaining the catalase activity in human feces.     

Direct in vivo protein transduction into a specific restricted brain area in rats

Attempts at protein transduction into specific restricted brain areas have remained unsuccessful. We attempted targeted, direct in vivo protein transduction by microinjecting beta-galactosidase (beta-gal) with hemagglutinating virus of Japan envelope (HVJ-E) vector into the rat nucleus tractus solitarius (NTS). The medulla oblongata including the NTS was removed 6h post-injection and cryostat sections were histochemically stained to detect beta-gal enzymatic activity. beta-gal-positive cells were present in these sections as was beta-gal activity determined by colorimetric analysis. beta-gal-positive cells were not present in the rats microinjected only beta-gal protein without HVJ-E vector. Our findings suggest that direct in vivo protein transduction into specific restricted brain areas is possible. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the central nervous system.     

Histiocytic necrotizing lymphadenitis without granulocytic infiltration

Summary Twenty-seven cases of an unusual necrotizing lymphadenitis previously described only in Japan are reported as occurring in West Germany (23 cases), Iran (1 case), Italy (1 case), Korea (1 case) and Spain (1 case). The lesion frequently develops in the cervical lymph nodes of young women. It is characterized by infiltration of the cortex and/or paracortex by large collections of proliferating histiocytes and is devoid of granulocytes. Complete or, more often, incomplete necrosis of lymphoid tissue is seen in all cases. In cases with incomplete necrosis, the histiocytes are interspersed with pyknotic cells and nuclear debris. Based on the histological findings, the term histiocytic necrotizing lymphadenitis without granulocytic infiltration is proposed. Lesions to be considered in a differential diagnosis are malignant histiocytic neoplasms and necrotizing lymphadenitis with granulocytic infiltration, which is seen in lupus erythematosus and bacterial infections. The aetiology of histiocytic necrotizing lymphadenitis without granulocytic infiltration is still unclear. Some clinical and histological features indicate the possibility of an underlying viral infection.This study was supported by the Kind-Philipp-Stiftung     

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.     

Flow cytometric and functional analysis of mononuclear cells infiltrating the liver in experimental autoimmune hepatitis.

Experimental autoimmune hepatitis was produced by immunizing Wistar rats with syngeneic liver proteins. Mononuclear cells infiltrating the liver tissue were identified by immunohistochemical techniques using monoclonal antibodies specific for subpopulations of rat lymphocytes. The strong infiltration of CD8+ cytotoxic T lymphocytes (CTL) were found in the portal areas. Subpopulations of mononuclear cells infiltrating the liver, spleen cells and peripheral blood lymphocytes were identified by flow cytometry. Flow cytometric analysis revealed the presence of CD5- and CD8+ lymphocytes in the liver tissues. Mononuclear cells infiltrating the liver were isolated from Wistar rats having autoimmune hepatitis to determine whether those exhibit cytotoxicity against syngeneic hepatocytes; they exhibited cytotoxicity against isolated syngeneic hepatocytes, but failed to lyse K562 cells, syngeneic concanavalin A-activated splenocytes and allogeneic hepatocytes. Depletion of CD8+ T cells significantly reduced the cytotoxic ability of mononuclear cells infiltrating into the liver against syngeneic hepatocytes. These findings support the idea that liver cell injury in experimental autoimmune hepatitis may at least in part be mediated by CTL.     

Primary cutaneous B-cell lymphoma in Japanese patients

Primary cutaneous B-cell lymphoma (PCBCL) is a rare group of lymphoproliferative disorders. There have been few reports of Japanese patients with PCBCL, so the present study investigated the clinicopathological and immunological features and Bcl-2 gene rearrangement and protein expression in 28 Japanese patients with PCBCL. According to the Revised European-American Lymphoma (REAL) classification, there were 25 diffuse large B-cell lymphomas (DLBCL), one Burkitt type lymphoma, one lymphoblastic lymphoma and one marginal zone cell lymphoma. Of the 25 DLBCL, 17 were in males and eight in females, with an average age of 69.4 years. Follow-up data were available in 19 cases of DLBCL of which seven died and 12 were alive. The overall 5-year survival rate was 61%. Cases of DLBCL involving the legs were found to have poorer clinical outcomes; two of four cases with leg lesions died, with a mean survival of 13 months. Of 14 cases with non-leg lesions, four died, and the mean survival was 38.9 months. Only one case of Burkitt type lymphoma was CD10 positive. Bcl-2 rearrangement was not observed in 13 cases studied by polymerase chain reaction. Bcl-2 expression was observed in nine of 13 cases studied. All five cases with leg lesions exhibited Bcl-2 expression, but four of six cases with non-leg lesions also expressed the protein. These results show that DLBCL is the most frequent subtype of PCBCL in Japanese patients and that the prognosis of Japanese patients with DLBCL is worse than that of reported European cases. The study also found that PCBCL was frequently associated with Bcl-2 expression, which was not site-confined, and that there was no evidence for a follicular center origin of PCBCL.