A novel home-based method for preparing suspensions of anti-TB drugs.

BACKGROUND: Tablets are the most widely available dosage form for the treatment of TB; however, adult tablets fail to meet the needs of young children who cannot swallow these tablets or require dose titration. We tested a new, simple device (XTEMP-R®) and the methodology for converting tablets of TB drugs into a homogeneous suspension for home use by children and caregivers.METHODS: XTEMP-R is a new device used for converting tablets into liquid preparations. Four TB drugs - pretomanid, delamanid, clofazimine and bedaquiline - were dispersed in the device utilizing water and simple syrup. The reproducibility of accurately delivering aliquots from the suspension upon preparation and upon redispersion after storing for 2 days was studied.RESULTS: Suspensions of each of the drugs tested were easily prepared in about 10 min and were visually uniform in consistency. Dosages in 2 and 5 mL were assessed in suspension, and those in 5 mL tested upon redispersion after 2 days. The observed range for these dosages spanned from 94.6% to 101.1% of the theoretical concentration for the suspensions under examination. The cleaned device had no detectable residual drug.CONCLUSION: XTEMP-R can be used at home by caregivers to prepare doses of suspensions accurately for children and patients who cannot swallow tablets.

Stable, compounded bedaquiline suspensions to support practical implementation of pediatric dosing in the field.

BACKGROUND: Bedaquiline (BDQ) tablets are indicated as part of a combination regimen for the treatment of multidrug-resistant TB in adults, adolescents and children. A dispersible tablet formulation is now approved but is not currently available in all settings. The aim of this study was to develop stable extemporaneous liquid formulations of BDQ that can be stored at room temperature or 30°C for several weeks, to support pragmatic pediatric dosing in the field and reduce wastage.METHODS: BDQ tablets were suspended in simple syrup and a sugar-free vehicle. Each 20 mg/mL formulation was stored at room temperature or 30°C for 30 days in amber dispensing bottles. Appearance, BDQ potency, pH and microbial counts were determined on Days 0, 15 and 30.RESULTS: The BDQ potency in both formulations remained at 98-101% of the theoretical concentration for 30 days. The appearance, pH and microbial count of sugar-free formulation did not change during the 30-day storage. The simple syrup formulation was stable for 15 days as microbial growth was observed on Day 30.CONCLUSIONS: BDQ may be prepared in syrup or sugar-free suspensions: syrup suspensions can be stored for 15 days at room temperature and 30C, whereas sugar-free suspensions can be stored for 30 days at room temperature and 30C. This information will support practical BDQ dosing for children in the field.

Extemporaneously compounded liquid formulations of clofazimine.

BACKGROUND: Clofazimine (CFZ) is routinely used worldwide for the treatment of leprosy and TB. However, no liquid or dispersible tablet formulations of CFZ are currently available commercially for patients with challenges ingesting soft gelatin capsules or solid formulations. The aim of this research was to develop stable extemporaneous liquid formulations of CFZ that can be stored at room temperature for several weeks to enable practical dosing in the field. METHODS: Two formulations were prepared in syrup and sugar-free vehicles with CFZ tablets using a simple method that can be used in a routine pharmacy. Suspensions were stored at room temperature and at 30°C for 30 days. Formulation aliquots were tested on Days 0, 15 and 30 for appearance, pH, potency and microbial counts. RESULTS: Appearance remained unchanged during storage. The pH of both formulations was between 4.0 and 6.0. Potency was between 90% and 110% for 30 days in the syrup formulation and for 15 days in the sugar-free formulation. Microbial counts met United States Pharmacopeia 1111 limits for oral aqueous liquids and specific organisms were absent. CONCLUSIONS: A simple field-friendly method was successfully developed for the preparation of CFZ liquid formulations using commonly available ingredients. This will permit practical dosing and titration for children and other patients with swallowing challenges.

Sugar and sugar-free liquid formulations of delamanid for patients with rifampicin-resistant TB.

BACKGROUND: Delamanid (DLM) tablets are recommended for the treatment of rifampicin-resistant TB. However, no liquid or dispersible tablet formulation of DLM is currently commercially available for patients with challenges ingesting these tablets. The aim of this study was to develop stable extemporaneous liquid formulations of DLM that can be stored at room temperature for several weeks.METHODS: DLM tablets were suspended in 1) simple syrup and 2) a specially formulated sugar-free vehicle. These suspensions containing DLM 5 mg/mL were stored in plastic prescription bottles at room temperature or 30°C for 30 days. These suspensions were evaluated for appearance, potency, pH, and microbial counts at Days 0, 15, and 30.RESULTS: The potency of DLM in each formulation remained at 98-104% of the theoretical concentration for 30 days. The appearance, pH, and microbial count did not change for the sugar-free formulation during the 30-day storage period. Microbial growth, however, was observed in the simple syrup formulation on Day 30 but not on Day 15.CONCLUSION: DLM can be formulated in sugar or sugar-free suspensions and stored at room temperature or 30°C for at least 15 and 30 days, respectively.

Stable sugar and sugar-free suspensions of pretomanid.

BACKGROUND: Pretomanid (PMD) tablets are indicated as part of a combination regimen for the treatment of adults with pulmonary extensively drug-resistant, treatment-intolerant or non-responsive multidrug-resistant TB. No commercial liquid formulation is currently available for patients unable to swallow these tablets.OBJECTIVE: To develop stable extemporaneous liquid formulations of PMD that can be stored at room temperature or 30°C for at least 4 weeks.METHODS: Crushed PMD tablets were formulated into 20 mg/mL suspensions in a simple syrup and sugar-free formulation. The PMD formulations were stored at room temperature and at 30°C for 30 days in dispensing bottles. Appearance, pH, potency and microbial counts of the suspensions were determined on Days 0, 15 and 30.RESULTS: The potency of PMD remained at 99.7-103.4% of the theoretical concentration in each formulation. The appearance, pH and microbial count did not change during the 30-day storage period. Simple syrup formulations did not require preservatives for microbial stability.CONCLUSIONS: PMD oral suspension formulations in simple syrup or in sugar-free vehicle were easily prepared by utilising commonly available equipment and ingredients and were stable for 30 days. These formulations are appropriate alternatives for patients with swallowing difficulties.

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.

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.

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.

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.

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.

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.

Expression of carbonic anhydrase V in pancreatic beta cells suggests role for mitochondrial carbonic anhydrase in insulin secretion.

Carbonic anhydrase V (CA-V) is a mitochondrial enzyme that provides bicarbonate for pyruvate carboxylase in liver and kidney. In the course of a survey of the tissue distribution of CA-V, we detected intense immunostaining in pancreatic islets when sections from rat and mouse pancreases were reacted with a polyclonal antibody to recombinant mouse CA-V. The distribution and large number of CA-V-positive cells in each islet suggested that they represented beta cells. Double immunofluorescence staining of tissue sections and isolated islet cells showed cellular colocalization of CA-V and insulin, confirming that beta cells contain CA-V. Western blotting of rat islets of Langerhans and primary beta cells showed 33- and 30-kDa polypeptides of precursor and mature CA-V, respectively. The CA-V expression was beta cell-specific since no CA-V immunoreaction was detected in the primary alpha cells. Immunohistochemical staining for CA-I, CA-II, CA-IV, CA-VI, and CA-IX was negative in beta cells, and Western blotting of beta cells also failed to identify any CA in beta cells except CA-V. The specific localization of CA-V in beta cells led us to hypothesize that CA-V may be functionally linked to the regulation of insulin secretion. Consistent with this hypothesis, the CA inhibitor acetazolamide was found to be a strong inhibitor of glucose-stimulated insulin secretion by isolated rat pancreatic islets.

IL-1 produced and released endogenously within human islets inhibits beta cell function.

Resident macrophages have been suggested to participate in the initiation of beta cell damage during the development of autoimmune diabetes. The purpose of this study was to determine if the endogenous production and release of interleukin 1 (IL-1) in human islets of Langerhans by resident macrophages results in the inhibition of beta cell function. Treatment of human islets with a combination of tumor necrosis factor (TNF) + lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) stimulates inducible nitric oxide synthase (iNOS) expression, nitric oxide production, and inhibits glucose-stimulated insulin secretion. The IL-1 receptor antagonist protein (IRAP) prevents TNF + LPS + IFN-gamma-induced iNOS expression and nitrite production, and attenuates the inhibitory effects on glucose-stimulated insulin secretion by human islets. Inhibition of iNOS activity by aminoguanidine also attenuates TNF + LPS + IFN-gamma-induced inhibition of insulin secretion by human islets. These results indicate that the inhibitory effects of TNF + LPS + IFN-gamma are mediated by nitric oxide, produced by the actions of IL-1 released endogenously within human islets. Reverse transcriptase polymerase chain reaction was used to confirm that TNF + LPS + IFN-gamma stimulates the expression of both IL-1alpha and IL-1beta in human islets. Two forms of evidence indicate that resident macrophages are the human islet cellular source of IL-1: culture conditions that deplete islet lymphoid cells prevent TNF + LPS + IFN-gamma-induced iNOS expression, nitric oxide production, and IL-1 mRNA expression by human islets; and IL-1 and the macrophage surface marker CD69 colocalize in human islets treated with TNF + LPS + IFN-gamma as determined by immunohistochemical analysis. Lastly, nitric oxide production is not required for TNF + LPS + IFN-gamma-induced IL-1 release in human islets. However, cellular damage stimulates IL-1 release by islet macrophages. These findings support the hypothesis that activated islet macrophages may mediate beta cell damage during the development of insulin-dependent diabetes by releasing IL-1 in human islets followed by cytokine-induced iNOS expression by beta cells.

Double-stranded RNA-induced inducible nitric-oxide synthase expression and interleukin-1 release by murine macrophages requires NF-kappaB activation.

The effects of double-stranded RNA (synthetic polyinosinic-polycytidylic acid; poly(I-C)) on macrophage expression of inducible nitric-oxide synthase (iNOS), production of nitric oxide, and release of interleukin-1 (IL-1) were investigated. Individually, poly(I-C), interferon-gamma (IFN-gamma), and lipopolysaccharide (LPS) stimulate nitrite production and iNOS expression by RAW 264.7 cells. In combination, the effects of poly(I-C) + IFN-gamma are additive, while poly(I-C) does not further potentiate LPS-induced nitrite production. These results suggest that poly(I-C) and LPS may stimulate iNOS expression by similar signaling pathways, which may be independent of pathways activated by IFN-gamma. LPS-induced iNOS expression is associated with the activation of NF-kappaB. We show that inhibition of NF-kappaB by pyrrolidinedithiocarbamate prevents poly(I-C) + IFN-gamma-, poly(I-C) + LPS-, and LPS-induced iNOS expression, nitrite production and IkappaB degradation by RAW 264.7 cells. The effects of poly(I-C) on iNOS expression appear to be cell-type specific. Poly(I-C), alone or in combination with IFN-gamma, does not stimulate, nor does poly(I-C) potentiate, IL-1-induced nitrite production by rat insulinoma RINm5F cells. In addition, we show that the combination of poly(I-C) + IFN-gamma stimulates iNOS expression, nitrite production, IkappaB degradation, and the release of IL-1 by primary mouse macrophages, and these effects are prevented by pyrrolidinedithiocarbamate. These findings indicate that double-stranded RNA, in the presence of IFN-gamma, is a potent activator of macrophages, stimulating iNOS expression, nitrite production, and IL-1 release by a mechanism which requires the activation of NF-kappaB.

Potential role of resident islet macrophage activation in the initiation of autoimmune diabetes.

The purpose of this study was to evaluate the effects of resident islet macrophage activation on beta cell function. Treatment of freshly isolated rat islets with TNF-alpha and LPS results in a potent inhibition of glucose-stimulated insulin secretion. The inhibitory actions of TNF + LPS are mediated by the intraislet production and release of IL-1 followed by IL-1-induced inducible nitric oxide synthase (iNOS) expression by beta cells. The IL-1R antagonist protein completely prevents TNF + LPS-induced nitrite production, iNOS expression and the inhibitory effects on glucose-stimulated insulin secretion by rat islets. Resident macrophages appear to be the source of IL-1, as a 7-day culture of rat islets at 24 degrees C (conditions known to deplete islets of lymphoid cells) prevents TNF + LPS-induced iNOS expression, nitrite production, and the inhibitory effects on insulin secretion. In addition, macrophage depletion also inhibits TNF + LPS-induced IL-1alpha and IL-1beta mRNA expression in rat islets. Immunocytochemical colocalization of IL-1beta with the macrophage-specific marker ED1 was used to provide direct support for resident macrophages as the islet cellular source of IL-1. IL-1beta appears to mediate the inhibitory actions of TNF + LPS on beta cell function as TNF + LPS-induced expression of IL-1beta is fourfold higher than IL-1alpha, and Ab neutralization of IL-1beta prevents TNF + LPS-induced nitrite production by rat islets. These findings support a mechanism by which the activation of resident islet macrophages and the intraislet release of IL-1 may mediate the initial dysfunction and destruction of beta cells during the development of autoimmune diabetes.

Evidence for the presence of type I IL-1 receptors on beta-cells of islets of Langerhans.

The cytokine interleukin-1beta (IL-1beta) has been shown to inhibit insulin secretion and destroy pancreatic islets by a mechanism that involves the expression of inducible nitric oxide synthase (iNOS), and the production of nitric oxide (NO). Insulin containing beta-cells, selectively destroyed during the development of autoimmune diabetes, appear to be the islet cellular source of iNOS following treatment with IL-1beta. In this study we have evaluated the presence of type I IL-1 signaling receptors on purified pancreatic beta-cells. We show that the interleukin-1 receptor antagonist protein (IRAP) prevents IL-1beta-induced nitrite formation and IL-1beta-induced inhibition of insulin secretion by isolated islets and primary beta-cells purified by fluorescence-activated cell sorting (FACS). The protective effects of IRAP correlate with an inhibition of IL-1beta-induced iNOS expression by islets and FACS purified beta-cells. To provide direct evidence to support beta-cell expression of IL-1 type I signaling receptors, we show that antiserum specific for the type I IL-1 receptor neutralizes IL-1beta-induced nitrite formation by RINm5F cells, and that RINm5F cells express the type I IL-1 receptor at the protein level. Using reverse transcriptase-polymerase chain reaction (RT-PCR), the expression of type I IL-1 signaling receptors by FACS purified beta-cells and not alpha-cells is demonstrated. These results provide direct support for the expression of type I IL-1 receptors by primary pancreatic beta-cells, the cell type selectively destroyed during the development of autoimmune diabetes.

Characteristics of the myocardial PM-FABP: effect of diabetes mellitus.

Properties of the myocardial PM-FABP were studied in normal and STZ-diabetic rats. The fluorescent fatty acids trans-parinaric and cis-parinaric acids were used as analogs of straight-chain (saturated) and kinked-chain (unsaturated) fatty acids respectively. Parinaric acid binding was sensitive to trypsin. Trans-parinaric acid binding was more sensitive to this protease than the binding of cis-parinaric acid. Based on the difference in sensitivity of parinaric acid binding we believe that there are two separate binding sites associated with myocardial PM-FABP; one for unsaturated fats and the other for saturated fats. Diabetes enhanced both cis- and trans-parinaric acid binding capacity in cardiomyocytes; cis-parinaric acid by 2 fold and trans-parinaric acid by 2.6 fold. In addition, there was a concomitant accumulation of free fatty acids and triglycerides in the hearts of the diabetic animals. There was a 2.2 fold increase for fatty acids and a 1.6 fold increase for trigylcerides. This association between myocardial fatty acid build-up and enhanced myocardial PM-FABP during diabetes suggest that this carrier protein might have contributed to lipid accumulation in the hearts of the diabetic rats.

Irreversible inhibition of metabolic function and islet destruction after a 36-hour exposure to interleukin-1beta.

The purpose of this study was to identify the duration of exposure of islets to interleukin 1beta (IL-1beta) that results in irreversible damage. Treatment of rat islets for 18 h with IL-1beta results in an inhibition of glucose-stimulated insulin secretion, mitochondrial aconitase activity, and total protein synthesis. The addition of N(G)-monomethyl-L-arginine (NMMA) or aminoguanidine to islets preincubated for 18 h with IL-1beta, followed by continued culture for 8 h (with both NMMA and IL-1beta), results in the recovery of islet secretory function, aconitase activity, and protein synthesis. However, islet metabolic function is irreversibly inhibited after a 36-h incubation with IL-1beta, as an additional 8-h incubation with NMMA or aminoguanidine does not stimulate the recovery of insulin secretion, aconitase activity, or protein synthesis. The irreversible inhibition of metabolic function correlates with the commitment of islets to destruction. Treatment of islets for 96 h with IL-1beta results in islet degeneration. NMMA, added to islets 24 h after the addition of IL-1beta, followed by continued culture for 72 h (with NMMA and IL-1beta), prevents islet degeneration. However, NMMA added to islets 36 h or 48 h after the addition of IL-1beta, followed by continued culture for a total of 96 h, does not prevent islet degeneration. New messenger RNA expression appears to be required for islet recovery from IL-1beta-induced damage as actinomycin D prevents the recovery of islet aconitase activity. Lastly, treatment of human islets with a combination of IL-1beta and interferon-gamma (IFNgamma) results in a potent inhibition of mitochondrial aconitase activity. NMMA, when cocultured with IL-1beta + IFNgamma, completely prevents cytokine-induced inhibition of human islet aconitase activity. NMMA, when added to human islets pretreated for 18 h with IL-1beta + IFNgamma, stimulates the recovery of mitochondrial aconitase activity after an additional 8 h incubation. These findings indicate that nitric oxide-induced islet damage is reversible; however, prolonged production of nitric oxide (after a 36-h exposure to IL-1beta) results in the irreversible inhibition of islet metabolic and secretory function.

Interferon-gamma increases the sensitivity of islets of Langerhans for inducible nitric-oxide synthase expression induced by interleukin 1.

The purpose of this study was to evaluate the effects of interferon-gamma (IFN-gamma) alone and in combination with interleukin 1beta (IL-1beta) on inducible nitric-oxide synthase (iNOS) mRNA and protein expression, nitrite production, and insulin secretion by islets of Langerhans. Treatment of rat islets with IL-1beta results in a concentration-dependent increase in the production of nitrite that is maximal at 5 units/ml. Individually, 0. 1 unit/ml IL-1beta or 150 units/ml rat IFN-gamma do not stimulate iNOS expression or nitrite production by rat islets; however, in combination, these cytokines induce the expression of iNOS and the production of nitrite to levels similar in magnitude to the individual effects of 5 units/ml IL-1beta. The islet beta-cell, selectively destroyed during insulin-dependent diabetes mellitus, appears to be one islet cellular source of iNOS as 150 units/ml rat IFN-gamma and 0.1 unit/ml IL-1beta induced similar effects in primary beta-cells purified by fluorescence-activated cell sorting and in the rat insulinoma cell line, RINm5F. iNOS expression and nitrite production by rat islets in response to 150 units/ml rat IFN-gamma and 0.1 unit/ml IL-1beta are correlated with an inhibition of insulin secretion and islet degeneration that are prevented by the iNOS inhibitor aminoguanidine. The mechanism by which IFN-gamma increases the sensitivity of beta-cells for IL-1-induced iNOS expression appears to be associated with an increase in the stability of iNOS mRNA. Last, cellular damage during physical dispersion of islets results in the release of sufficient amounts of IL-1beta to induce iNOS expression and nitrite production in the presence of exogenously added rat IFN-gamma. The cellular source of IL-1beta under these conditions is believed to be resident islet macrophages as depletion of macrophages prior to dispersion prevents IFN-gamma-induced iNOS expression and nitrite formation by dispersed islet cells. These studies show that the T-lymphocyte cytokine, IFN-gamma, increases the sensitivity of rat islets to the effects of IL-1beta on iNOS expression and nitrite production by 10-fold, in part, through the stabilization of iNOS mRNA. Our studies also support an effector role for IFN-gamma, in concert with resident islet macrophage release of IL-1beta, in mediating beta-cell destruction during the development of autoimmune diabetes.