[Mitomycin C associated hemolytic uremic syndrome--a case report and review of the Japanese cases].

A 36-year-old man who developed hemolytic uremic syndrome (HUS) associated with mitomycin C (MMC) after the radical operation for early gastric cancer was reported. He was successfully treated with hemodialysis and transfusion of fresh frozen plasma. However, the pathogenesis and the effective treatment of this syndrome are still undetermined. The literature on MMC-induced HUS in Japan was reviewed, and the relationship between the prognosis and the patients conditions, such as sex, age, site of primary cancer, total dose of MMC, latent period from MMC administration, laboratory findings at the time of diagnosis and treatment with steroids or plasma exchange, were analysed. Patients less than 60 years old or treated with plasma exchange were found to be associated significantly with a favorable outcome. The most frequent cause of death was pulmonary edema or respiratory failure. In conclusion, early treatment with plasma exchange appeared to result in a better prognosis.

Effect of T-cell receptor V beta-specific monoclonal antibodies on cyclophosphamide-induced diabetes mellitus in non-obese diabetic mice.

The expression of specific T-cell receptor gene segments by T lymphocytes appears to be critically important for the induction of several experimental autoimmune diseases mediated by these cells. We examined whether this situation also applied to non-obese diabetic mice by using various T-cell receptor V beta-specific monoclonal antibodies. No significant age- or sex-related differences were observed in V beta usage by peripheral and splenic T lymphocytes. CD8+ T lymphocytes among the islet-derived mononuclear cells isolated from 20-week-old female non-obese diabetic mice showed heterogeneity of their V beta gene usage. In order to examine the role of T lymphocyte subsets expressing specific T-cell receptor V beta segments in the development of diabetes mellitus, T-cell receptor V beta-specific monoclonal antibodies were administered to 10-week-old male non-obese diabetic mice treated with cyclophosphamide. None of the antibodies used could significantly diminish the incidence of cyclophosphamide-induced diabetes and the severity of insulitis [anti-V beta 3 (11 of 22 mice became diabetic, 50%), anti-V beta 5 (9 of 14, 64%), anti-V beta 8 (9 of 21, 43%), anti-V beta 11 (12 of 23, 52%), anti-V beta 14 (7 of 12, 58%), and anti-V beta 5 + anti-V beta 11 (6 of 12, 50%)] when compared with control mice (12 of 21, 57%). In addition, there were no significant differences in T-cell receptor V beta usage between diabetic and non-diabetic cyclophosphamide-treated mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of cyclophosphamide-induced and spontaneous diabetes in NOD/Shi/Kbe mice by anti-MHC class I Kd monoclonal antibody.

The immune mechanisms directly responsible for beta-cell destruction in insulin-dependent diabetes are undefined. We studied the role of MHC class I-restricted T lymphocytes in the development of diabetes in cyclophosphamide (CY)-treated male and untreated female NOD mice (H-2Kd,Db). After administration of CY to 10-wk-old male NOD/Shi/Kbe mice, 37 of 64 (58%) phosphate-buffered saline-injected control mice and 13 of 22 (59%) anti-Kb and 12 of 27 (44%) anti-Db monoclonal antibody (MoAb)-injected mice became diabetic by 14 wk of age, whereas only 3 of 38 (8%) anti-Kd and 2 of 13 (15%) anti-Lyt-2 MoAb-injected mice did. In untreated female NOD/Shi/Kbe mice, 30 of 46 (65%) mice developed spontaneous diabetes by 30 wk of age, whereas none of 9 anti-Kd MoAb-injected mice became diabetic. Immunohistochemical studies showed that islet-infiltrating cells in CY-treated control mice were composed mainly of both L3T4+ and Lyt-2+ T lymphocytes, whereas many L3T4+ and very few Lyt-2+ lymphocytes infiltrated within the islets in anti-Kd MoAb-injected mice. Administration of anti-Lyt-2 MoAb induced the absence of Lyt-2+ T lymphocytes in the islet and spleen. However, anti-Kd MoAb did not change the number of spleen cells or the T-lymphocyte subset and response to concanavalin A. These results suggest that MHC class I Kd-restricted Lyt-2+ T lymphocytes play an important role as direct effector cells in destruction of beta-cells in NOD/Shi/Kbe mice.

Morphological analysis of selective destruction of pancreatic beta-cells by cytotoxic T lymphocytes in NOD mice.

Interactions of pancreatic islets and islet-associated mononuclear cells (IAMCs) from the nonobese diabetic (NOD) mouse were morphologically investigated. To obtain IAMCs, pancreatic islets isolated from adult NOD mice were cultured for 7 days with interleukin 2. Noted by light microscopy, interactions between IAMCs and freshly isolated islets from young NOD mice began 30 min after the initiation of the coculture, and 6 h later, normal cellular array of the islets was lost. By electron microscopy, most IAMCs had low nucleus-cytoplasm ratio, the nucleus was notched and exhibited condensed chromatin along the nuclear membrane, and well-developed Golgi complexes and several mitochondria were distributed in the cytoplasm. These IAMCs adhered to beta-cells, but not to alpha- or delta-cells, with their pseudopods and caused cytolysis of beta-cells. Immunohistochemical study with antibodies specific for pancreatic hormones demonstrated that only cells reacting with anti-insulin antibody were selectively lost as the incubation time proceeded. Electron immunohistochemistry by immunogold technique showed that effector cells in IAMCs reacted with anti-CD8 (Lyt-2) antibody, but not anti-CD4 (L3T4) or anti-asialogangliosideM1 antibody. In addition, the concentration of pancreatic hormones in the culture medium, used as a marker of cytolysis, also demonstrated that insulin was significantly increased after 6 h of culture, whereas glucagon and somatostatin were not. These results suggest that CD8+ cytotoxic T lymphocytes are involved in the selective destruction of pancreatic beta-cells in the NOD mouse.

Impaired mitogen-induced expression of high-affinity interleukin 2 receptors on spleen cells from NOD/Shi/Kbe mice.

The immune abnormalities of NOD mice, a model of human type I (insulin-dependent) diabetes, have been postulated to be T-lymphocyte dependent. We measured responsiveness to exogenous interleukin 2 (IL-2) and IL-2 production in spleen mononuclear cells from female NOD/Shi/Kbe mice after stimulating the cells with concanavalin A (ConA blasts) or phytohemagglutinin (PHA blasts). Exogenous IL-2 produced significantly lower proliferative responses in each blast from 3- and 10-wk-old NOD/Shi/Kbe mice than from control strains. IL-2 production in NOD/Shi/Kbe mice was inclined to decrease but not significantly compared with controls. Even sufficient amounts of recombinant IL-2 (rIL-2) or IL-1 (rIL-1), added with mitogens to the preculture medium, failed to provoke normal proliferative responses from NOD/Shi/Kbe mouse cells. To clarify the reason for this defect, we investigated the expression of IL-2 receptors (IL-2Rs) on mitogen-activated cells with anti-IL-2R monoclonal antibody (PC61) and radiolabeled IL-2. Cytofluorometry showed no significant difference between strains in the number of PC61+ ConA and PHA blasts. However, Scatchard analysis with 125I-labeled IL-2 showed that the number of high-affinity IL-2Rs (H-IL-2Rs), the mediators of the biological activity of IL-2, was decreased in NOD/Shi/Kbe mice compared with controls, whereas the number of low-affinity IL-2Rs (L-IL-2Rs) was not different. Separating the L3T4+ and Lyt-2+ populations of T lymphocytes by cell sorting showed both to be deficient in H-IL-2Rs.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-interleukin 2 receptor antibody attenuates low-dose streptozotocin-induced diabetes in mice.

Recent evidence indicates that activated T cells and macrophages play an important role in the induction of insulitis and diabetes in certain strains of mice treated with multiple subdiabetogenic doses of streptozotocin. In the present study, we treated C57BL/6J mice with five daily doses of 40 mg/ml streptozotocin and examined the prophylactic effect of an anti-interleukin 2 receptor monoclonal antibody (PC61). In mice treated with streptozotocin, interleukin 2 receptor-positive mononuclear cells were shown to infiltrate into the islets and soluble interleukin 2 receptors in the sera were significantly increased compared with control mice. The administration of PC61 to the mice attenuated the insulitis, and diminished interleukin 2 receptor-positive cells from islets and soluble interleukin 2 receptors in the sera. Moreover, the administration of PC61 significantly reduced the development of hyperglycaemia shown in these mice (12.8 +/- 1.1 mmol/l vs 18.5 +/- 0.7 mmol/l, p less than 0.005). As judged by flow cytometric analysis, this antibody did not cause any changes in either spleen cell counts or T cell subsets. Interleukin 2 receptors were expressed on a minor population of spleen cells regardless of treatment with PC61 (STZ + normal rat IgG: 2.1 +/- 0.3%, STZ + PC61: 2.4 +/- 0.3%). Even after stimulation of spleen cells with concanavalin A or alloantigen, interleukin 2 receptor expression was not significantly different between the two groups. Our studies suggest that interleukin 2 receptor-positive activated T cells or macrophages are important in the development of multi-low-dose streptozotocin diabetes and that an anti-interleukin 2 receptor antibody can attenuate this process.

The presence of splenic T cells specific for islet cell antigens in nonobese diabetic mice.

Nonobese diabetic (NOD) mice display a syndrome with dramatic clinical and pathological features similar to Type 1 diabetes in man. Mononuclear cells intensively infiltrate the pancreas (insulitis), mostly T cells taking part. However, the functional role and specificity of these cells are currently uncertain. We investigated the response of splenic T cells from NOD mice to islet cells, using interleukin 2 (IL-2) production and cell proliferation. Splenic T cells from NOD mice responded with IL-2 production and proliferation when both islet cell antigens from NOD mice and mitomycin C-treated spleen cells (source of antigen-presenting cells) from NOD mice or major histocompatibility complex (MHC)-compatible ILI mice were present. Splenic T cells could produce IL-2 in response to islet cells from sources other than the NOD mouse, but could not produce significantly this lymphokine in response to submandibular gland, gastric mucosal, liver, spleen, and ovarian cells from NOD mice. NOD T cells produced this antigen-specific response first when the mice were 8 weeks old, the response grew stronger until 20 weeks of age and then tapered off. The present study indicates the presence of T cells specific for islet cell antigens in the spleen of NOD mice and suggests that the antigen-specific T cell response increases in parallel with the development of insulitis.

Destruction of pancreatic islet cells by cytotoxic T lymphocytes in nonobese diabetic mice.

Proliferation of islet-associated leukocytes occurred when isolated islets from 20-wk-old female nonobese diabetic (NOD) mice were cultured with 10 U/ml rIL-2 for 7 days. Co-culture of these leukocytes with freshly isolated islets from 6- to 8-wk-old NOD donors in the presence of 1 U/ml rIL-2 produced islet structural deformation within 24 h and islet cytolysis within 48 h. Three lines of evidence suggest that these leukocytes were composed mainly of CTL specific for islet cells. First, morphologically, these proliferating cells adhered to NOD islets at 6 h and killed islets within 48 h of culture, but these phenomena could not be observed in the other tissues from NOD mice. These islet-derived cells were cytotoxic to NOD islet cells in a 51Cr-release assay, whereas no appreciable cytotoxicity was observed when NOD Con A-induced splenic blasts or fibroblasts were used as targets. Second, a flow cytometric analysis showed that these cells consisted of 97% Thy-1.2, 69% Lyt-2, 8% L3T4, and 4% asialo-GM1-positive cells, whereas Mac-1-positive cells could not be seen in these assays. After treatment with anti-Thy-1.2 or Lyt-2 mAb and C, these cells lost their activity to lyse NOD islet cells. However, these cells still had a full killing activity after the depletion of L3T4 or asialo GM1-positive cells. Third, islet cells from BALB/c, DBA/2, and B10.GD mice which share the same H-2K Ag with NOD mice were susceptible to cytolytic activity of these cells, whereas islet cells from NON, C57BL/6, C57BL/10, and C3H mice remained intact. Furthermore, anti-Kd antibody was capable of blocking this cytolysis. These results suggest that CTL expressing Thy-1.2 and Lyt-2 phenotypes appear to recognize the islet cell Ag with the restriction of MHC class I Kd, and then destroy NOD islet cells.

[Cellular immune dysfunction in the NOD mouse: suppression of concanavalin A-induced responses in spleen cells by activated macrophages].

It is generally accepted that T lymphocyte-mediated autoimmunity contributes to the pathogenesis of Type 1 diabetes in humans and animals. Using spleen cells from nonobese diabetic (NOD) mice, a model of human Type 1 diabetes, we have analyzed the subset of T lymphocytes by flow cytometry and investigated concanavalin A (Con A)-induced interleukin 2 (IL-2) production and cell proliferation. NOD mice showed a higher percentage of Thy1.2+, L3T4+, and Lyt2+ T lymphocytes than did control ICR mice through the whole age examined. Spleen cells from a large majority of NOD mice were found to generate very low IL-2 production and cell proliferation in response to Con A. However, a few mice preserved their responsiveness to Con A. The following reasons may indicate that macrophage-mediated suppression participates in the deficient function of NOD spleen cells. (a) Macrophage depletion from NOD spleen cells retrieved Con A-induced IL-2 production. (b) Thioglycollate-induced peritoneal exudate cells containing many activated macrophages could completely suppress cell proliferation. (c) Prostaglandin synthetase inhibitor indomethacin reversed the suppression of IL-2 production by macrophages. (d) Conversely, exogenous prostaglandins could show the partial suppression of IL-2 production. These results suggest that activated macrophages suppress the response of NOD spleen cells to Con A mostly through prostaglandins. This impairment may contribute to the pathogenesis of Type 1 diabetes in NOD mice.

Suppressor T-cell abnormality in NOD mice before onset of diabetes.

To investigate the pathological role of suppressor T-cells in non-obese diabetic (NOD) mice, we stimulated splenic T-lymphocytes from diabetes-prone NOD mice with concanavalin A (ConA) and then evaluated their ability to suppress the lymphocyte-proliferative responses to mitogen and allogenic cells. Lymphocytes from NOD mice showed significantly less suppressor ability than did those from BALB/c mice and non-obese non-diabetic (NON) mice, the corresponding non-diabetic sister strain of the NOD mouse, both in the mitogen response and in the mixed lymphocyte reaction (MLR). We used monoclonal antibodies and flow cytometry to analyze the lymphocytic surface phenotypes, and found markedly fewer Lyt2+ T-lymphocytes (suppressor/cytotoxic T-lymphocyte) in the NOD mice than in both controls after exposure to ConA. These results suggest that suppressor T-cell activity is already depressed in NOD mice before diabetes begins and that a substantial decrease in the number of suppressor T-cells induced by ConA may explain this depressed suppressor activity. This impairment may contribute to the pathogenesis of type 1 diabetes in NOD mice.

Suppression of concanavalin A-induced responses in splenic lymphocytes by activated macrophages in the non-obese diabetic mouse.

Spleen cells from non-obese diabetic mice were found to generate low interleukin 2 production and cell proliferation in response to concanavalin A. However, some of non-obese diabetic mice maintained in the same environment preserved their responsiveness to this T cell mitogen. Non-obese diabetic mice at every age had a higher percentage of Thyl.2, L3T4, and Lyt2-positive spleen cells than did control mice, suggesting that the dysfunction of spleen cells did not depend on the number of T cells or the ratio of these subpopulations. Evidence for macrophage-mediated suppression participating in the deficient function of splenic lymphocytes in this mouse model of insulin-dependent diabetes includes: 1) the restoration of mitogen-induced interleukin 2 production after the macrophages have been depleted by silica absorption form spleen cells; 2) the complete suppression of the cell proliferation by thioglycollate-stimulated peritoneal exudate cells from non-obese diabetic and control mice, and the partial suppression by spleen macrophages from non-obese diabetic mice; 3) the reversal of the suppression of interleukin 2 production by the prostaglandin synthetase inhibitor indomethacin (0.1-1 microgram/ml); 4) the partial suppression of interleukin 2 production, conversely, by the exogenous prostaglandins E1 and E2 (2.5 x 10(-6) mol/l). These results indicate that the activated macrophages existing among the spleen cells suppress the response of splenic T cells to concanavalin A. This impairment may contribute to the pathogenesis of insulin-dependent diabetes in non-obese diabetic mice.