In this paper we report the concentration of terminal complement complexes (TCCs, SC5b-9, an index of complement activation) in newly diagnosed insulindependent diabetes mellitus (IDDM) patient serum and normal human serum. In the nine patients studied, levels of serum soluble TCCs were approximately 1·6-fold higher than in sera obtained from normal control individuals. On incubation of rat islet cells with diluted serum (10%, v/v, concentration), complement activation was increased at a significantly faster rate and the total TCC concentration was significantly higher in culture medium containing IDDM patient serum than in medium containing control serum. The concentration of anti-(glutamic acid decarboxylase) autoantibodies in newly diagnosed IDDM patient serum was on average 60-fold higher than in normal human control serum.IDDM patient serum (10%, v/v) induced apoptosis in islet cells, as determined by islet cell density changes and DNA fragmentation patterns. However, serum from IDDM patients was not able to induce apoptosis of the cells when complement components (C1q and C3) or antibodies were depleted. In addition, glutamine and the potent antioxidant 1-pyrrolidinecarbodithioic acid partially reversed cell death induced by IDDM patient serum in a concentration-dependent manner. The ATP concentration in islet cells incubated for 24 h in the presence of diluted IDDM patient serum was reduced to 4·4% of that observed in islet cells incubated in fetal calf serum or 7·3% of that observed in islet cells incubated in normal human serum. On the basis of these observations, we suggest that the pathway of IDDM patient serum-induced islet cell apoptosis may involve antibody-dependent complement activation, free radical generation and a precipitous fall in ATP levels.

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We have previously reported that newly diagnosed Type-1 diabetic patient sera potently suppressed insulin secretion from a clonal rat pancreatic -cell line (BRIN BD11) but did not alter cell viability. Here, we report that apoptosis in BRIN BD11 cells incubated in various sera types (fetal calf serum (FCS), normal human serum and Type-1 diabetic patient) was virtually undetectable. Although low levels of necrosis were detected, these were not significantly different between cells incubated in sera from different sources. ATP levels were reduced by approximately 30% while nitrite production increased twofold from BRIN BD11 cells incubated for 24 h in the presence of Type-1 diabetic patient sera compared with normal human sera. Additionally, ATP levels were reduced by approximately 40% and DNA fragmentation increased by more than 20-fold in BRIN BD11 cells incubated in FCS in the presence of a pro-inflammatory cytokine cocktail (interleukin-1 , tumour necrosis factor-and interferon-), compared with cells incubated in the absence of cytokines. Nitric oxide production from BRIN BD11 cells was markedly increased (up to 10-fold) irrespective of sera type when the cytokine cocktail was included in the incubation medium.Type-1 diabetic patient sera significantly (P<0·001) raised basal levels of intracellular free Ca 2+ concentration ([Ca 2+ ] i ) in BRIN BD11 cells after a 24-h incubation. The alteration in [Ca 2+ ] i concentration was complement dependent, as removal of the early complement components C1q and C3 resulted in a significant reduction (P<0·01) of sera-induced [Ca 2+ ] i changes. We propose that the mechanism of Type-1 diabetic patient sera-induced inhibition of insulin secretion from clonal -cells may involve complement-stimulated elevation of [Ca 2+ ] i which attenuates the nutrient-induced insulin secretory process possibly by desensitizing the cell to further changes in Ca 2+ .

There are conflicting reports on the effect of serum from patients with insulin-dependent diabetes mellitus (IDDM) or normal human serum on -cell function and insulin secretion. Here, we report that the sera of newly diagnosed IDDM patients potently suppresses insulin secretion from a clonal rat pancreatic -cell line (BRIN-BD11), but do not alter cell viability. Indeed, the viability of the -cells was not significantly different between cells cultured in 10% (v/v) IDDM sera, normal human sera, or fetal calf serum after 24, 48 and 72 h. Alanine-stimulated insulin secretion from cells cultured for 24 h in (10% v/v) IDDM patient sera was reduced to 48% of that secreted from cells cultured in (10% v/v) normal human sera. After depletion of the complement components C1q and C3, the inhibition of insulin secretion induced by IDDM patient sera was significantly reversed (no significant difference was observed between cells cultured in complementdepleted IDDM patient sera and cells cultured in normal human sera or complement-depleted normal human sera).The concentration of glutamic acid decarboxylase (GAD) autoantibodies was markedly increased in the sera of six out of nine newly diagnosed IDDM patients in this study, whereas insulin auto-antibodies (IAA) were detected in the sera of three of the nine patients and islet-cell antibodies (ICA) in the sera of five of them. In addition, the concentration of soluble terminal complement complexes (SC5b-9) was greater in some of the -cell culture media samples after 24 h incubation when the incubation medium was supplemented with IDDM patient sera than when supplementation was with normal human sera.We propose that the mechanism of sera-induced inhibition of insulin secretion from clonal -cells may involve complement-and cytokine-stimulated intracellular events that attenuate the metabolite-induced secretory process.

Drug resistance remains a serious limiting factor in the treatment of acute myeloid leukenua (AML) either at initial presentation or following primary or subsequent relapses. Using specific kinase inhibitors, this study has investigated the conhibution of the Rks/PI3-kinase regulated survival pathways to drug resistance, and resulting suppression of apoptosis. Inhibition of the Raf/MAP-kinase pathway with Apigenin did not m i t i s e promyelocytic HL60 cells to drug-induced apoptosis, suggeshg a la& of involvement for this pathway in drug resistance. In contrast, the use of two speahc PI3-kinaae inhibitors, LY294002and Wortmarnun. ' did cause a significant increase in apoptosis in annbination with cytotoxic drugs. Following this observation, the contribution of two downstream effectom of PISCinaSe, p7OS6-kinase and PKB/Akt were investigated. Inhibition of p7OS6-kinase with rapamcyin did not alter levels of druginduced apoptosis. in contrast PI3-lanase lnhibition led to significant dephosphorylation of downahPam kmase PKB, suggesting that the PI%kinase/PKB survival pathway may play a mapr role in chemoresistance in AML. This pathway has previoudy been associated with modifications of the apoptotic regulator Bad. However we found no widence of Bad hetendher famation with anti-apoptotic regulators Bcl-2, Bd-& or Md-1, nor of alterations in Bax/Bcl-2 or Bax/Mcl-1 Itetemdimers following PI3-kinase inhibition. This suggests that alternative targets of PI3-kinase/PKB, distinct from the BcI-2 family may be responsible for amhibuting to drug resistance in AML. Thts work may represent a novel strategy for treatment of multidrug resistance in myeloid leukemia by combined use of inhibitors with conventional 13 Stress proteins and the regulation of apoptosis in tumour cells.