Gain-of-function polymorphism in mouse and human Ltk: implications for the pathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE), a complex multigenic disease, is a typical antibody-mediated autoimmune disease characterized by production of autoantibodies against a variety of autoantigens and immune complex-type tissue inflammation, most prominently in the kidney. Evidence suggests that genetic factors predisposing to aberrant proliferation/maturation of self-reactive B cells initiate and propagate the disease. In SLE-prone New Zealand Black (NZB) mice and their F1 cross with New Zealand White (NZW) mice, B cell abnormalities can be ascribed mainly to self-reactive CD5+ B1 cells. Our genome-wide scans to search for susceptibility genes for aberrant activation of B1 cells in these mice showed evidence that the gene, Ltk, encoding leukocyte tyrosine kinase (LTK), is a possible candidate. LTK is a receptor-type protein tyrosine kinase, belonging to the insulin receptor superfamily, and is mainly expressed in B lymphocyte precursors and neuronal tissues. Sequence and functional analyses of the gene revealed that NZB has a gain-of-function polymorphism in the LTK kinase domain near YXXM, a binding motif of the p85 subunit of phosphatidylinositol 3-kinase (PI3K). SLE patients also had this type of Ltk polymorphism with a significantly higher frequency compared with the healthy controls. Our findings suggest that these polymorphic LTKs cause up-regulation of the PI3K pathway and possibly form one genetic component of susceptibility to abnormal proliferation of self-reactive B cells in SLE.     

Immunohistochemical analysis of thymidylate synthase, p16(INK4a), cyclin-dependent kinase 4 and cyclin D1 in colorectal cancers receiving preoperative chemotherapy: significance of p16(INK4a)-mediated cellular arrest as an indicator of chemosensitivity to 5-fluorouracil

High expression of thymidylate synthase (TS) is allegedly associated with the chemoresistance to 5-fluorouracil (5-FU) in colorectal cancers. However, low TS expression does not necessarily imply chemosensitivity. Inactivation of p16(INK4a) correlates with poor prognosis in various cancers. We immunohistochemically evaluated the relationship between the expression of TS, p16(INK4a), CDK4 and cyclin D1 and the effect of 5-FU-based chemotherapy in colorectal cancers. After antigen retrieval, immunoperoxidase staining was performed on the paraffin-embedded, biopsy and surgical specimens of 37 advanced colorectal cancers preoperatively treated with peroral administration of 5-FU derivatives. As a control group, 31 colorectal cancers without preoperative treatment were analyzed. High TS expression was found in 23 (74%) of 31 tumors resected from histological non-responders and in 19 (61%) of 31 controls but in none of six responders. High p16(INK4a) expression was seen in 83% of the responders, 52% of the non-responders and 32% of the controls. The TS-low/p16(INK4a)-high phenotype was noted in 83% of the responders, but only in 3% of the non-responders (P = 0.0001). Induction of p16(INK4a) expression after chemotherapy was predominantly seen in the responders. Neither CDK4 nor cyclin D1 expression was related to the chemotherapeutic effects. In conclusion, the combination of low expression of TS and induction of p16(INK4a) after chemotherapy can be important indicators of the sensitivity to 5-FU-based chemotherapy in colorectal cancers.     

Survey of atopic dermatitis and skin barrier functions in Japanese and Chinese school students]

BACKGROUND: The comparative studies of the prevalence of atopic dermatitis and skin barrier functions in Japanese and Chinese were performed. METHODS: Clinical investigations were performed in 68 elementary school students in Lhasa, Tibet Autonomous Region and 67 students in Yixing, Jiangsu Province in China, and 99 students in Nishinomiya, Hyogo in Japan. Transepidermal water loss (TEWL) and capacitance were measured. Questionary survey about bathing frequency was also performed for students in Lhasa, Yixing and Osaka. RESULT: The prevalence rate of atopic dermatitis was 0% in Lhasa, 2.63% in Yixing, 4.26% in Nishinomiya. TEWL of students in Nishinomiya was higher than that in Yixing and Lhasa. Capacitance of students in Lhasa was lower than that in Nishinomiya and Yixing. The frequency of taking a bath in Lhasa was about 2.2 times per month and fewer than that in Nishinomiya and Yixing. CONCLUSION: There was tendency that the prevalence of atopic dermatitis increased according to increase of TEWL. It was thought that more investigations are necessary whether the development of habitat and environment influence the prevalence of atopic dermatitis and skin barrier function.     

Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-{gamma} production by anti-tumor T cells

Unfractionated spleen cells taken from tumor-bearing mice 2weeks after tumor implantation contained tumor-primed T cellswhich produced cytokines including IL-2 and IFN- when culturedin vitro. With progressive tumor growth this initial lymphokine-producingcapacity decreased. Here, we investigated the ability of IL-12to (I) restore suppressed IFN- production, (II) cause tumorregression and (II) induce anti-tumor protective immunity. Additionof rIL-12 to spleen cell cultures from 4- to 10-week-old tumor-bearingmice resulted in a striking enhancement in the production ofIFN- compared with cultures of these cells in the absence ofrIL-12 or of normal spleen cells in the presence of rIL-12.Five I.p. injections of rIL-12 into mice bearing s.c. tumorsinduced complete tumor regression. This was found when rIL-12was given at early (1–2 weeks), intermediate (4–5weeks) or even late (7 weeks) stages of tumor growth. Furthermore,IL-12-treated mice which rejected the primary tumor exhibitedcomplete resistance to a rechallenge with the same tumor butdid not reject a second syngenetic tumor. Immunohistochemicalanalyses following IL-12 treatment revealed that CD4⁺ and CD8⁺T cells infiltrate the tumor. More importantly, IFN- mRNA expressionwas observed in fresh tumor masses from tumor-bearing mice receivingIL-12 treatment The importance of IFN- was further demonstratedby the observation that the systemic administration of anti-IFN-mAb prior to IL-12 treatment completely abrogated the anti-tumoreffect of IL-12. Thus, these results indicate that administrationof modest levels of rIL-12 to tumor-bearing mice results intumor regression through mechanisms involving reversal of suppressedIFN- production by anti-tumor T cells and the establishmentof a tumor-specific protective immune response.     

Lack of intermediate-affinity interleukin-2 receptor in mice leads to dependence on interleukin-2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependant mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind ¹²⁵I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2Rα gene is therefore required for the formation of the functional IL-2R in mice.     

Possible role of hepatocyte growth factor in regeneration of human peritoneal mesothelial cells

Human peritoneal mesothelial cells (HPMCs) play an important role in peritoneal functions. During long term peritoneal dialysis, it has been reported that HPMCs are damaged by high glucose solution via the signal of transforming growth factor (TGF)-beta1 produced by HPMCs. In this study, we focused on the effect of hepatocyte growth factor (HGF), known as an anti-fibrotic and anti-TGF-beta1 agent, on HPMCs damaged by high glucose solution. HPMCs were isolated from specimens of the omentum from nonuremic patients after informed consent had been obtained. After confirming adhesion for 6 hours, 100 microL of DMEM with 0.5%FCS were added at different concentrations (D-glucose; 6, 30 mM) with or without HGF (10, 30, 100 ng/mL) for 48 hours. We examined the effects of a high concentration of glucose and then focused on following four critical points: 1) the production of HGF from HPMCs exposed to a high concentration of glucose, 2) the expression of c-Met on HPMCs, 3) the viability of those cells, and 4) matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase-2 (TIMP-2). The following significant changes are described herein: high glucose solution and TGF-beta1 i) decreased HGF production from HPMCs and ii) up-regulated expression of c-Met on HPMCs, and addition of HGF iii) restored viability of HPMCs damaged by glucose, iv) suppressed TGF-beta1 production by HGF, and v) induced up-regulation of MMP-2 and decreased TIMP-2 production by HPMCs. Levels of HGF decreased by high concentrations of glucose in the peritoneal cavity may induce the loss of HPMCs and thereby result in peritoneal fibrosis. These results suggest that HGF is an effective agent in the regeneration of peritoneal membrane damaged by high glucose solution.     

Modulation of myocardial reactive hyperemia

Pflügers Archiv - European Journal of Physiology -