T cell-extrinsic IL-1 signaling controls long-term gammaherpesvirus infection by suppressing viral reactivation.

Gammaherpesviruses establish life-long infection in over 95% of adults and are associated with several cancers, including B cell lymphomas. Using the murine gammaherpesvirus 68 (MHV68) animal model, we previously showed a pro-viral role of Interleukin-1 (IL-1) signaling that supported viral reactivation during the establishment of chronic infection. Unexpectedly, in this study we found that the proviral effects of IL-1 signaling originally observed during the establishment of chronic gammaherpesvirus infection convert to antiviral effects during the long-term stage of infection. Specifically, IL-1 signaling promoted expansion of antiviral CD8+ T cells and control of viral reactivation in the peritoneal cavity of a long-term infected host. Using a novel mouse model of T cell-specific IL-1 signaling deficiency, we found that the antiviral effects of IL-1 signaling were T cell extrinsic. Our study highlights a dynamic nature of host factors that shape the parameters of chronic gammaherpesvirus infection.

Polymorphism in the TOMM40 gene modifies the risk of developing sporadic inclusion body myositis and the age of onset of symptoms.

A polyT repeat in an intronic polymorphism (rs10524523) in the TOMM40 gene, which encodes an outer mitochondrial membrane translocase involved in the transport of amyloid-ß and other proteins into mitochondria, has been implicated in Alzheimer's disease and APOE-TOMM40 genotypes have been shown to modify disease risk and age at onset of symptoms. Because of the similarities between Alzheimer's disease and sporadic inclusion body myositis (s-IBM), and the importance of amyloid-ß and mitochondrial changes in s-IBM, we investigated whether variation in poly-T repeat lengths in rs10524523 also influence susceptibility and age at onset in a cohort of 90 Caucasian s-IBM patients (55 males; age 69.1 ± 9.6). In carriers of APOE ε3/ε3 or ε3/ε4, genotypes with a very long (VL) poly-T repeat were under-represented in s-IBM compared to controls and were associated with a later age at symptom onset, suggesting that these genotypes may be protective. Our study is the first to suggest that polymorphisms in genes controlling mitochondrial function can influence susceptibility to s-IBM and have disease modifying effects. However, further studies in other s-IBM populations are needed to confirm these findings, as well as expression studies of different TOMM40 alleles in muscle tissue.

Recognition of allo-peptide is governed by novel anchor imposition and limited variations in TCR contact residues.

Immune specificity of a T cell is determined by the TCR contact residues exposed on the antigenic peptide/MHC complex. Naturally processed, biallelic epitopes from H7 minor histocompatibility (mH) antigen vary in position 7 (p7) from aspartic acid (D) to a glutamic acid (E), which differ by an additional methylene (-CH(2)) in the side chain. Here, we show that this variation generates a strong anti-H7a or anti-H7b cytotoxic T cell responses. Further, the H7 allelic peptides use p6 asparagine as their central anchor residue and amino acid variations in either the canonical p5 or the predicted p6 anchor positions in the antigenic epitope were detrimental for TCR recognition. In addition, introduction of any other amino acids, except asparagine, in the polymorphic p7 significantly abolished the ability of anti-H7b TCR recognition. This demonstrates that only an asparagine with an amine group as a side chain instead of a charged oxygen radical could effectively stimulate the anti-H7b specific T cells. Our findings provide evidence that mH antigen-specific TCRs are highly stringent in recognizing their cognate epitopes.

A transgenic model to study the pathogenesis of somatic mtDNA mutation accumulation in beta-cells.

Low levels of somatic mutations accumulate in mitochondrial DNA (mtDNA) as we age; however, the pathogenic nature of these mutations is unknown. In contrast, mutational loads of >30% of mtDNA are associated with electron transport chain defects that result in mitochondrial diseases such as mitochondrial encephalopathy lactic acidosis and stroke-like episodes. Pancreatic beta-cells may be extremely sensitive to the accumulation of mtDNA mutations, as insulin secretion requires the mitochondrial oxidation of glucose to CO(2). Type 2 diabetes arises when beta-cells fail to compensate for the increased demand for insulin, and many type 2 diabetics progress to insulin dependence because of a loss of beta-cell function or beta-cell death. This loss of beta-cell function/beta-cell death has been attributed to the toxic effects of elevated levels of lipids and glucose resulting in the enhanced production of free radicals in beta-cells. mtDNA, localized in close proximity to one of the major cellular sites of free radical production, comprises more than 95% coding sequences such that mutations result in changes in the coding sequence. It has long been known that mtDNA mutations accumulate with age; however, only recently have studies examined the influence of somatic mtDNA mutation accumulation on disease pathogenesis. This article will focus on the effects of low-level somatic mtDNA mutation accumulation on ageing, cardiovascular disease and diabetes.

A single nucleotide polymorphism in the Emp3 gene defines the H4 minor histocompatibility antigen.

Minor histocompatibility antigens (minor H antigen) elicit strong T-cell-mediated responses during both graft rejection and graft versus leukemia (GvL) among MHC-matched individuals (where MHC is major histocompatibility complex). Employing expression-cloning methodology, we have identified a cDNA clone, MI-35, encoding the immunodominant H4b minor H antigen within the classical mouse H4 complex. The minimal antigenic epitope derived from H4b presented on Kb class I MHC is SGIVYIHL (SYL8) and the polymorphism is due to C-->T nucleotide modification in p3 resulting in the change of threonine (ACT) to isoleucine (ATT). The results presented here demonstrate that amino acid variation in the allelic epitopes results in the low abundance of H4a peptide. The differential peptide copy number resulted in an immunodominant cytotoxic T cells (CTL) response directed against H4b while the anti-B6 response directed against H4a was easily dominated. These results provide a molecular mechanism for the H4 minor H antigen and suggest a novel mechanism by which alloantigenic disparity caused by conservative amino acid changes can be augmented by posttranslational antigen processing events.

Mechanisms of beta-cell death in response to double-stranded (ds) RNA and interferon-gamma: dsRNA-dependent protein kinase apoptosis and nitric oxide-dependent necrosis.

Viral infection is one environmental factor that has been implicated as a precipitating event that may initiate beta-cell damage during the development of diabetes. This study examines the mechanisms by which the viral replicative intermediate, double-stranded (ds) RNA impairs beta-cell function and induces beta-cell death. The synthetic dsRNA molecule polyinosinic-polycytidylic acid (poly IC) stimulates beta-cell DNA damage and apoptosis without impairing islet secretory function. In contrast, the combination of poly IC and interferon (IFN)-gamma stimulates DNA damage, apoptosis, and necrosis of islet cells, and this damage is associated with the inhibition of glucose-stimulated insulin secretion. Nitric oxide mediates the inhibitory and destructive actions of poly IC + IFN-gamma on insulin secretion and islet cell necrosis. Inhibitors of nitric oxide synthase, aminoguanidine, and N(G)-monomethyl-L-arginine, attenuate poly IC + IFN-gamma-induced DNA damage to levels observed in response to poly IC alone, prevent islet cell necrosis, and prevent the inhibitory actions on glucose-stimulated insulin secretion. N(G)-monomethyl-L-arginine fails to prevent poly IC- and poly IC + IFN-gamma-induced islet cell apoptosis. PKR, the dsRNA-dependent protein kinase that mediates the antiviral response in infected cells, is required for poly IC- and poly IC + IFN-gamma-induced islet cell apoptosis, but not nitric oxide-mediated islet cell necrosis. Alone, poly IC fails to stimulate DNA damage in islets isolated from PKR-deficient mice; however, nitric oxide-dependent DNA damage induced by the combination of poly IC + IFN-gamma is not attenuated by the genetic absence of PKR. These findings indicate that dsRNA stimulates PKR-dependent islet cell apoptosis, an event that is associated with normal islet secretory function. In contrast, poly IC + IFN-gamma-induced inhibition of glucose-stimulated insulin secretion and islet cell necrosis are events that are mediated by islet production of nitric oxide. These findings suggest that at least one IFN-gamma-induced antiviral response (islet cell necrosis) is mediated through a PKR-independent pathway.

Nitric oxide regulates adenylyl cyclase activity in rat striatal membranes.

The regulation of adenylyl cyclase activity by nitric oxide (NO) was studied in rat (Sprague-Dawley) striatal membranes. Three chemically distinct NO donors attenuated forskolin-stimulated activity but did not alter basal activity. Maximum inhibition resulted in a 50% decrease in forskolin-stimulated activity, consistent with the presence of multiple isoforms of adenylyl cyclase and our previous findings that only the forskolin-stimulated activity of the type-5 and -6 isoform family of enzymes is inhibited by NO. To monitor primarily the type-5 isoform, we examined the ability of NO donors to attenuate D(1)-agonist-stimulated adenylyl cyclase activity. Under those conditions, complete inhibition was observed. The data indicate that NO attenuates neuromodulator-stimulated cAMP signaling in the striatum.

K cells: a novel target for insulin gene therapy for the prevention of diabetes.

Recently, gut K cells have been shown to express glucokinase, the glucose sensor of pancreatic beta cells, and transgenic mice expressing human insulin under the control of a K cell-specific promoter are resistant to diabetes development induced by the beta-cell toxin streptozotocin. These novel findings suggest that gut K cells might be a suitable target for gene therapeutic treatment of type 1 diabetes mellitus.

Double-stranded RNA-dependent protein kinase is not required for double-stranded RNA-induced nitric oxide synthase expression or nuclear factor-kappaB activation by islets.

Environmental factors, such as viral infection, have been implicated in the destruction of beta-cells during the development of autoimmune diabetes. Double-stranded RNA (dsRNA), produced during viral replication, is an active component of a viral infection that stimulates antiviral responses in infected cells. Previous studies have shown that treatment of rat islets with dsRNA in combination with gamma-interferon (IFN-gamma) results in a nitric oxide-dependent inhibition of glucose-stimulated insulin secretion. This study examines the role of nuclear factor-kappaB (NF-kappaB) and the dsRNA-dependent protein kinase (PKR) in dsRNA + IFN-gamma-induced nitric oxide synthase (iNOS) expression and nitric oxide production by rat, mouse, and human islets. Treatment of rat and human islets with dsRNA in the form of polyinosinic-polycytidylic acid (poly IC) and IFN-gamma resulted in iNOS expression and nitric oxide production. Inhibitors of NF-kappaB activation-the proteasome inhibitor MG-132 and the antioxidant pyrrolidine-dithiocarbamate (PDTC)-prevented poly IC + IFN-gamma-induced iNOS expression and nitric oxide production. Incubation of rat islets for 3 h or human islets for 2 h with poly IC alone or poly IC + IFN-gamma resulted in NF-kappaB nuclear translocation and degradation of the NF-kappaB inhibitor protein, IkappaB, events that are prevented by MG-132. PKR has been shown to participate in dsRNA-induced NF-kappaB activation in a number of cell types, including mouse embryonic fibroblasts. However, poly IC stimulated NF-kappaB nuclear translocation and IkappaB degradation to similar levels in islets isolated from mice devoid of PKR (PKR-/-) and wild-type mice (PKR+/+). Furthermore, the genetic absence of PKR did not affect dsRNA + IFN-gamma-induced iNOS expression, nitric oxide production, or the inhibitory actions of these agents on glucose-stimulated insulin secretion. These results suggest that 1) NF-KB activation is required for dsRNA + IFN-gamma-induced iNOS expression, 2) PKR is not required for either dsRNA-induced NF-kappaB activation or dsRNA + IFN-y-induced iNOS expression by islets, and 3) PKR is not required for dsRNA + IFN-gamma-induced inhibition of glucose-stimulated insulin secretion by islets.

Pancreatic beta-cell damage mediated by beta-cell production of interleukin-1. A novel mechanism for virus-induced diabetes.

Viral infection is one environmental factor that may initiate beta-cell damage during the development of autoimmune diabetes. Formed during viral replication, double-stranded RNA (dsRNA) activates the antiviral response in infected cells. In combination, synthetic dsRNA (polyinosinic-polycytidylic acid, poly(I-C)) and interferon (IFN)-gamma stimulate inducible nitric-oxide synthase (iNOS) expression, inhibit insulin secretion, and induce islet degeneration. Interleukin-1 (IL-1) appears to mediate dsRNA + IFN-gamma-induced islet damage in a nitric oxide-dependent manner, as the interleukin-1 receptor antagonist protein prevents dsRNA + IFN-gamma-induced iNOS expression, inhibition of insulin secretion, and islet degeneration. IL-1beta is synthesized as an inactive precursor protein that requires cleavage by the IL-1beta-converting enzyme (ICE) for activation. dsRNA and IFN-gamma stimulate IL-1beta expression and ICE activation in primary beta-cells, respectively. Selective ICE inhibition attenuates dsRNA + IFN-gamma-induced iNOS expression by primary beta-cells. In addition, poly(I-C) + IFN-gamma-induced iNOS expression and nitric oxide production by human islets are prevented by interleukin-1 receptor antagonist protein, indicating that human islets respond to dsRNA and IFN-gamma in a manner similar to rat islets. These studies provide biochemical evidence for a novel mechanism by which viral infection may initiate beta-cell damage during the development of autoimmune diabetes. The viral replicative intermediate dsRNA stimulates beta-cell production of pro-IL-1beta, and following cleavage to its mature form by IFN-gamma-activated ICE, IL-1 then initiates beta-cell damage in a nitric oxide-dependent fashion.

Adoptive transfer of acute lung injury.

In this study, we describe a novel adoptive transfer protocol to study acute lung injury in the rat. We show that bronchoalveolar lavage (BAL) cells isolated from rats 5 h after intratracheal administration of lipopolysaccharide (LPS) induce a lung injury when transferred to normal control recipient rats. This lung injury is characterized by increased alveolar-arterial oxygen difference and extravasation of Evans blue dye (EBD) into lungs of recipient rats. Recipient rats receiving similar numbers of donor cells isolated from healthy rats do not show adverse changes in the alveolar-arterial oxygen difference or in extravasation of EBD. The adoptive transfer-induced lung injury is associated with increased numbers of neutrophils in the BAL, the levels of which are similar to the numbers observed in BAL cells isolated from rats treated for 5 h with LPS. As an indicator of BAL cell activation, donor BAL cell inducible nitric oxide synthase (iNOS) expression was compared with BAL cell iNOS expression 48 h after adoptive transfer. BAL cells isolated 5 h after LPS administration expressed iNOS immediately after isolation. In contrast, BAL cells isolated 48 h after adoptive transfer did not express iNOS immediately after isolation but expressed iNOS following a 24-h ex vivo culture. These findings indicate that the activation state of donor BAL cells differs from BAL cells isolated 48 h after adoptive transfer, suggesting that donor BAL cells may stimulate migration of new inflammatory cells into the recipient rats lungs.

Potential role of PKR in double-stranded RNA-induced macrophage activation.

In this study, the role of the double-stranded (ds) RNA-dependent protein kinase (PKR) in macrophage activation was examined. dsRNA [polyinosinic:polycytidylic acid (poly IC)]-stimulated inducible nitric oxide synthase, interleukin (IL)-1alpha and IL-1beta mRNA expression, nitrite formation and IL-1 release are attenuated in RAW264.7 cells stably expressing dominant negative (dn) mutants of PKR. The transcriptional regulator nuclear factor (NF)-kappaB is activated by dsRNA, and appears to be required for dsRNA-induced macrophage activation. While dnPKR mutants prevent macrophage activation, they fail to attenuate dsRNA-induced IkappaB degradation or NF-kappaB nuclear localization. The inhibitory actions of dnPKR on dsRNA-induced macrophage activation can be overcome by treatment with interferon (IFN)-gamma, an event associated with PKR degradation. Furthermore, dsRNA + IFN-gamma stimulate inducible nitric oxide synthase expression, IkappaB degradation and NF-kappaB nuclear localization to similar levels in macrophages isolated from PKR(-/-) and PKR(+/+) mice. These findings indicate that both NF-kappaB and PKR are required for dsRNA-induced macrophage activation; however, dsRNA-induced NF-kappaB activation occurs by PKR-independent mechanisms in macrophages. In addition, the PKR dependence of dsRNA-induced macrophage activation can be overcome by IFN-gamma.

Anti-inflammatory actions of 15-deoxy-delta 12,14-prostaglandin J2 and troglitazone: evidence for heat shock-dependent and -independent inhibition of cytokine-induced inducible nitric oxide synthase expression.

In this study, the anti-inflammatory actions of the peroxisome proliferator-activated receptor (PPAR)-gamma agonists 15-deoxy-delta 12,14-prostaglandin J2 (15-d-delta 12,14-PGJ2) and troglitazone have been examined. Treatment of RAW 264.7 cells and CD-1 mouse peritoneal macrophages with lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) results in inducible nitric oxide synthase (iNOS), inducible cyclooxygenase (COX-2) and interleukin-1 (IL-1) expression, increased production of nitric oxide, and the release of IL-1. In a concentration-dependent manner, 15-d-delta 12,14-PGJ2 inhibits each of these proinflammatory actions of LPS + IFN-gamma, with half-maximal inhibition at approximately 0.5 microg/ml and complete inhibition at 1-5 microg/ml. The inhibitory actions of 15-d-delta 12,14-PGJ2 on LPS + IFN-gamma-induced inflammatory events are not associated with the inhibition of iNOS enzymatic activity or macrophage cell death, but appear to result from an inhibition of iNOS and IL-1 transcription. In addition, the anti-inflammatory actions of 15-d-delta 12,14-PGJ2 are not limited to peritoneal macrophages, as 15-d-delta 12,14-PGJ2 prevents TNF-alpha + LPS-induced resident islet macrophage expression of IL-1beta and beta-cell expression of iNOS stimulated by the local release of IL-1 in rat islets. 15-d-delta 12,14-PGJ2 appears to be approximately 10-fold more effective at inhibiting resident islet macrophage activation (in response to TNF + LPS) than IL-1-induced nitrite production by beta-cells. Two mechanisms appear to be associated with the antiinflammatory actions of both 15-d-delta 12,14-PGJ2 and troglitazone: 1) the direct inhibition of cytokine- and endotoxin-stimulated iNOS and IL-1 transcription; and 2) the inhibition of IL-1 signaling, an event associated with PPAR-gamma agonist-induced activation of the heat shock response (as assayed by heat shock protein 70 expression). These findings indicate that the PPAR-gamma agonists, troglitazone and the J series of prostaglandins, are potent anti-inflammatory agents that prevent cytokine- and endotoxin-stimulated activation of peripheral and resident tissue macrophages and cytokine-induced iNOS expression by beta-cells by the inhibition of transcriptional activation and induction of the heat shock response.

Targeted overactivity of beta cell K(ATP) channels induces profound neonatal diabetes.

A paradigm for control of insulin secretion is that glucose metabolism elevates cytoplasmic [ATP]/[ADP] in beta cells, closing K(ATP) channels and causing depolarization, Ca2+ entry, and insulin release. Decreased responsiveness of K(ATP) channels to elevated [ATP]/[ADP] should therefore lead to decreased insulin secretion and diabetes. To test this critical prediction, we generated transgenic mice expressing beta cell K(ATP) channels with reduced ATP sensitivity. Animals develop severe hyperglycemia, hypoinsulinemia, and ketoacidosis within 2 days and typically die within 5. Nevertheless, islet morphology, insulin localization, and alpha and beta cell distributions were normal (before day 3), pointing to reduced insulin secretion as causal. The data indicate that normal K(ATP) channel activity is critical for maintenance of euglycemia and that overactivity can cause diabetes by inhibiting insulin secretion.

Prolonged STAT1 activation is associated with interferon-gamma priming for interleukin-1-induced inducible nitric-oxide synthase expression by islets of Langerhans.

In this study, the ability of interferon-gamma (IFN-gamma) to prime rat and nonobese diabetic (NOD) mouse islets for interleukin-1 (IL-1)-stimulated expression of inducible nitric-oxide synthase (iNOS) has been examined. IL-1-induced iNOS expression by rat islets is concentration-dependent with maximal expression occurring in response to 1.0 unit/ml. Individually, neither 0.1 unit/ml IL-1 nor 150 units/ml IFN-gamma stimulates iNOS expression or nitrite production by rat islets. However, a 30-60-min pulse of rat islets with IFN-gamma, followed by washing to remove the cytokine and continued culture with 0.1 unit/ml IL-1 for 40 h, results in iNOS expression and nitrite production to levels similar in magnitude to the individual effects of 1.0 unit/ml IL-1. A 1-h pulse with IFN-gamma primes for IL-1-induced islet degeneration that is mediated by the expression of iNOS and increased production of nitric oxide. IFN-gamma also primes for IL-1-induced iNOS expression and nitrite formation by NOD mouse islets. The priming actions of IFN-gamma appear to be selective for beta-cells, as IFN-gamma primes for IL-1-induced nitrite formation by primary beta-cells and RINm5F insulinoma cells, but not primary alpha-cells. The priming actions of IFN-gamma for IL-1-induced iNOS expression do not require de novo protein synthesis as preincubation of RINm5F cells with cycloheximide does not inhibit iNOS mRNA accumulation under priming conditions. The priming actions of IFN-gamma on IL-1-induced iNOS expression persists for extended periods of up to 7 days and are associated with persistent signal transducers and activators of transcription (STAT)-1 activation. A 30-min pulse of rat islets with IFN-gamma stimulates STAT1 phosphorylation, and STAT1 remains phosphorylated for up to 7 days following IFN-gamma removal. In addition, STAT1 remains nuclear for up to 7 days after IFN-gamma removal. These results indicate that IFN-gamma primes for IL-1-induced islet degeneration via a nitric oxide-dependent mechanism. These findings also provide evidence that the priming actions of IFN-gamma for IL-1-induced iNOS expression by islets are associated with the prolonged phosphorylation and activation of STAT1.

Evidence that beta cell death in the nonobese diabetic mouse is Fas independent.

Recent studies suggest that Fas expression on pancreatic beta cells may be important in the development of autoimmune diabetes in the nonobese diabetic (NOD) mouse. To address this, pancreatic islets from NOD mice were analyzed by flow cytometry to directly identify which cells express Fas and Fas ligand (FasL) ex vivo and after in vitro culture with cytokines. Fas expression was not detected on beta cells isolated from young (35 days) NOD mice. In vitro, incubation of NOD mouse islets with both IL-1 and IFN-gamma was required to achieve sufficient Fas expression and sensitivity for islets to be susceptible to lysis by soluble FasL. In islets isolated from older (>/=125 days) NOD mice, Fas expression was detected on a limited number of beta cells (1-5%). FasL was not detected on beta cells from either NOD or Fas-deficient MRLlpr/lpr islets. Also, both NOD and MRLlpr/lpr islets were equally susceptible to cytokine-induced cell death. This eliminates the possibility that cytokine-treated murine islet cells commit "suicide" due to simultaneous expression of Fas and FasL. Last, we show that NO is not required for cytokine-induced Fas expression and Fas-mediated apoptosis of islet cells. These findings indicate that beta cells can be killed by Fas-dependent cytotoxicity; however, our results raise further doubts about the clinical significance of Fas-mediated beta cell destruction because few Fas-positive cells were isolated immediately before the development of diabetes.

Double-stranded RNA inhibits beta-cell function and induces islet damage by stimulating beta-cell production of nitric oxide.

Viral infection has been implicated as a triggering event that may initiate beta-cell damage during the development of autoimmune diabetes. In this study, the effects of the viral replicative intermediate, double-stranded RNA (dsRNA) (in the form of synthetic polyinosinic-polycytidylic acid (poly IC)) on islet expression of inducible nitric oxide synthase (iNOS), production of nitric oxide, and islet function and viability were investigated. Treatment of rat islets with poly(IC) + interferon-gamma (IFN-gamma) stimulates the time- and concentration-dependent expression of iNOS and production of nitrite by rat islets. iNOS expression and nitrite production by rat islets in response to poly(IC) + IFN-gamma correlate with an inhibition of insulin secretion and islet degeneration, effects that are prevented by the iNOS inhibitor aminoguanidine (AG). We have previously shown that poly(IC) + IFN-gamma activates resident macrophages, stimulating iNOS expression, nitric oxide production and interleukin-1 (IL-1) release. In addition, in response to tumor necrosis factor-alpha (TNF-alpha) + lipopolysaccharide, activated resident macrophages mediate beta-cell damage via intraislet IL-1 release followed by IL-1-induced iNOS expression by beta-cells. The inhibitory and destructive effects of poly(IC) + IFN-gamma, however, do not appear to require resident macrophages. Treatment of macrophage-depleted rat islets for 40 h with poly(IC) + IFN-gamma results in the expression of iNOS, production of nitrite, and inhibition of insulin secretion. The destructive effects of dsRNA + IFN-gamma on islets appear to be mediated by a direct interaction with beta-cells. Poly IC + IFN-gamma stimulates iNOS expression and inhibits insulin secretion by primary beta-cells purified by fluorescence-activated cell sorting. In addition, AG prevents the inhibitory effects of poly(IC) + IFN-gamma on glucose-stimulated insulin secretion by beta-cells. These results indicate that dsRNA + IFN-gamma interacts directly with beta-cells stimulating iNOS expression and inhibiting insulin secretion in a nitric oxide-dependent manner. These findings provide biochemical evidence for a novel mechanism by which viral infection may directly mediate the initial destruction of beta-cells during the development of autoimmune diabetes.

Nitric oxide participates in early events associated with NNMU-induced acute lung injury in rats.

In this study, the biochemical mechanisms by which N-nitroso-N-methylurethane (NNMU) induces acute lung injury are examined. Polymorphonuclear neutrophil infiltration into the lungs first appears in the bronchoalveolar lavage (BAL) fluid 24 h after NNMU injection (10.58 +/- 3.00% of total cells; P < 0.05 vs. control animals). However, NNMU-induced elevation of the alveolar-arterial O2 difference requires 72 h to develop. Daily intraperitoneal injections of the inducible nitric oxide (. NO) synthase (iNOS)-selective inhibitor aminoguanidine (AG) initiated 24 h after NNMU administration improve the survival of NNMU-treated animals. However, AG administration initiated 48 or 72 h after NNMU injection does not significantly improve the survival of NNMU-treated animals. These results suggest that. NO participates in events that occur early in NNMU-induced acute lung injury. BAL cells isolated from rats 24 and 48 h after NNMU injection produce elevated. NO and express iNOS during a 24-h ex vivo culture. AG attenuates. NO production but does not affect iNOS expression, whereas actinomycin D prevents iNOS expression and attenuates. NO production by BAL cells during this ex vivo culture. These results suggest that NNMU-derived BAL cells can stimulate iNOS expression and. NO production during culture. In 48-h NNMU-exposed rats, iNOS expression is elevated in homogenates of whole lavaged lungs but not in BAL cells derived from the same lung. These findings suggest that the pathogenic mechanism by which NNMU induces acute lung injury involves BAL cell stimulation of iNOS expression and. NO production in lung tissue.

Heat shock inhibits cytokine-induced nitric oxide synthase expression by rat and human islets.

In this study the effects of heat shock on interleukin-1beta (IL-1)-induced inhibition of islet metabolic function were examined. Treatment of rat islets for 18 h with IL-1 results in a potent inhibition of glucose-stimulated insulin secretion. The inhibitory effects of IL-1 on insulin secretion are completely prevented if islets are pretreated for 60 min at 42 C before cytokine stimulation. Heat shock also prevents IL-1-induced inhibition of insulinoma RINm5F cell mitochondrial aconitase activity. The protective effects of heat shock on islet metabolic function are associated with the inhibition of IL-1-stimulated inducible nitric oxide synthase (iNOS or NOS II) expression. Islets heat shocked for 60 min at 42 C fail to express iNOS (messenger RNA or protein) or produce nitrite in response to IL-1. IL-1-induced iNOS expression by rat islets requires activation of the transcriptional regulator nuclear factor kappaB (NF-kappaB). Heat shock prevents IL-1-induced NF-kappaB nuclear localization by inhibiting inhibitory protein kappaB (IkappaB) degradation in rat islets. Similar to rat islets, heat shock (stimulated by 90 min incubation at 42 C) prevents IL-1 + interferon gamma-induced iNOS expression and NF-kappaB nuclear localization in human islets. IL-1 also stimulates heat-shock protein 70 (hsp 70) expression by rat islets, and hsp 70 expression is dependent on islet production of nitric oxide. Last, evidence is presented that implicates nitric oxide as a stimulus for the expression of proteins that participate in islet recovery from nitric oxide-mediated damage. These studies indicate that heat shock prevents cytokine-induced islet damage by inhibiting iNOS expression, and suggest that nitric oxide is one effector molecule that stimulates the expression of factors involved in beta-cell recovery from nitric oxide-mediated damage.