Interferon γ and glycemic status in diabetes associated with chronic pancreatitis.

BACKGROUND/AIMS: Although the role of cytokines in the etiopathology of chronic pancreatitis (CP) is well recognized, information on pancreatic tissue cytokines in CP with/without associated diabetes is unavailable. The aim of the present study was to identify the differences in pancreatic cytokines and observe their correlations with the glycemic status in CP. METHODS: Pancreata were obtained from CP patients (n = 44), with/without associated diabetes and non-diabetic control subjects (n= 20). Patients with CP were classified into two groups after ascertaining their diabetic status. Pancreatic cytokines (IL 1ß, IL 6, IL 8, IL 10, IL 12P70, TNF α, IFN γ) were analyzed by flow cytometer. The influence of individual and cocktail of cytokines on glucose stimulated insulin release (GSIR) was examined by challenging the islets from control subjects. RESULTS: The pancreatic IFN γ levels in diabetic and non diabetic CP patients were significantly higher in comparison to controls. The glucose stimulated insulin release (GSIR) in response to high glucose concentration in control islets, islets from non-diabetic and diabetic CP patients was 8.2, 5.67 and 3.15 µU × 10(-3)/min/islet equivalent respectively. IFN γ resulted in 82.35% decrease in GSIR from the control islet cells at a concentration of >20 pg/ml which was reversed by epigallocatechin-3-gallate (EGCG). CONCLUSION: These results suggest that IFN γ among other cytokines, play a major role in ß-cell dysfunction associated with CP.

ß-Cell dysfunction in chronic pancreatitis.

Chronic pancreatitis (CP) is a progressive inflammatory disease characterized by irreversible destruction of pancreatic secretory parenchyma, fibrosis, exocrine atrophy, and endocrine insufficiency leading to diabetes. Secondary diabetes occurring in CP subsequent to destruction of pancreatic ß-cells is distinct, since it involves ß-cell dysfunction amidst an inflammatory milieu. Even though considerable knowledge is available on the pathophysiology and clinical management of CP, relatively much less is known about the molecular events leading to ß-cell dysfunction. Investigators have demonstrated that altered morphology, reduced ß-cell mass, and ß-cell numbers result in endocrine insufficiency. However, recent reports and our observations suggest that ß-cell dysfunction develops in the early stages of CP while clinical diabetes manifests later, when there is profound fibrosis. In the early stages, altered internal milieu and physiology arising due to inflammation and release of cytokines might lead to deranged signaling pathways and islet dysfunction. Subsequently, development of fibrosis causes islet destruction. This suggests that endocrine deficiency in CP is multifactorial. Although the role of transcription factors (Pdx-1, MafA, NeuroD) on ß-cell functions is understood, alterations in internal milieu of pancreatic tissue that affects ß-cell functions in CP has not been elucidated. In this review, we summarize the factors that have an effect on islet functions. Understanding molecular events of ß-cell dysfunction in CP can lead to the development of targeted preventive and therapeutic modalities.

Sugar coated ceramic nanocarriers for the oral delivery of hydrophobic drugs: formulation, optimization and evaluation.

In order to enhance the delivery of poorly-soluble drugs, we have explored aquasomes (three-layered, ceramic core based, oligosaccharide coated nanoparticles) as potential carriers for the delivery of model hydrophobic drug piroxicam (log P = 3.1). Ceramic nanoparticles were prepared using two techniques; namely, co-precipitation by refluxing and co-precipitation by sonication. Core preparation was finally done using sonication approach; based on the higher % yield (42.4 ± 0.4%) and shorter duration (1 day) compared to the reflux method (27.4 ± 2.05%, 6 days). Lactose loading onto ceramic core was achieved using adsorption. Colorimetric analysis of lactose coating was done using Anthrone method. Optimization of process variables namely, incubation time and core to coat ratio (for sugar loading) was carried out. Optimum time of incubation was 3 h and the core to coat ratio was 4:1. The drug loading was achieved by incubating the sugar loaded cores in different concentrations of piroxicam solution and it was found that 1.5% w/v piroxicam was optimal. Structural characterization using Fourier-Transform Infra Red Spectroscopy (FTIR) confirmed the presence of sugar coating onto the core. Morphological evaluation using transmission electron microscopy (TEM) revealed spherical nanoparticles (size 56.56 ± 5.93 nm for lactose coated core and 184.75 ± 13.78 nm for piroxicam loaded aquasomes) confirming the nanometric dimensions.

Predictors of mortality in rhinocerebral mycosis.

INTRODUCTION: Rhinocerebral mycosis is a rapidly progressive fatal opportunistic infection, predominantly affecting people in an immunocompromised state. Aggressive surgical therapy, with repeated debridement in combination with intravenous amphotericin B can lead to a high rate of cure. AIM: To determine the predictors of mortality in rhinocerebral mycosis. MATERIALS AND METHODS: The demographic data, clinical features, radiological (MRI/CT) findings, treatment details of patients with a diagnosis of rhinocerebral mycosis confirmed on histopathology were analyzed retrospectively. The outcome was assessed as alive and dead. Univariate analysis with odds ratio (OR) was employed in data analysis. Chi-square test was used for P value. RESULTS: There were 38 patients. The age range was 7-82 (mean 48.68) years; 30 (79%) were males. Craniofacial pain was the most common initial presenting symptom, noted in 29 (76.3%). Rhino-orbital involvement was noted in 24 (63.2%) and 12 (31.6%) had associated focal neurological deficits. Immunocompromised state was noted in 24 (63.2%). Eighteen (47.4%) patients died. The predictors for mortality: odds ratio (95% CI) were 2.45 (1.01-3.89) for elderly age, 5.67 (4.13-7.21) for intracranial extension, 2.6 (1.26-3.94) for immunocompromised state, 2.62 (1.25-3.99) for infection with zygomycosis and 2.33 (1.01-3.65) for anemia. CONCLUSION: Rhinocerebral mycosis is associated with high mortality in spite of aggressive therapy. Intracranial extension with focal neurological deficits is a major predictor of mortality in rhinocerebral mycosis.

Influence of extremely low frequency magnetic fields on Ca2+ signaling and NMDA receptor functions in rat hippocampus.

Extremely low frequency (ELF<300Hz) electromagnetic fields affect several neuronal activities including memory. Because ELF magnetic fields cause altered Ca(2+) homeostasis in neural tissues, we examined their influence on Ca(2+) signaling enzymes in hippocampus and related them with NMDA receptor functions. Hippocampal regions were obtained from brains of 21-day-old rats that were exposed for 90 days to 50Hz magnetic fields at 50 and 100 microT intensities. In comparison to controls, ELF exposure caused increased intracellular Ca(2+) levels concomitant with increased activities of Ca(2+)-dependent protein kinase C (PKC), cAMP-dependent protein kinase and calcineurin as well as decreased activity of Ca(2+)-calmodulin-dependent protein kinase in hippocampal regions. Simultaneous ligand-binding studies revealed decreased binding to N-methyl-D-aspartic acid (NMDA) receptors. The combined results suggest that perturbed neuronal functions caused by ELF exposure may involve altered Ca(2+) signaling events contributing to aberrant NMDA receptor activities.

Biochemical analysis of oxidative stress in the production of aflatoxin and its precursor intermediates.

The relevance of oxidative stress in the production of aflatoxin and its precursors was examined in different mutants of Aspergillus parasiticus, which produce aflatoxin or its precursor intermediates, and compared with results obtained from a non-toxigenic strain. In comparison to the non-toxigenic strain (SRRC 255), an aflatoxin producing strain (NRRL 2999) or mutants that accumulate aflatoxin precursors such as norsolorinic acid (by SRRC 162) or versicolorin (by NRRL 6196) or O-methyl sterigmatocystin (by SRRC 2043) had greater oxygen requirements and higher contents of reactive oxygen species. These changes were in the graded order of NRRL 2999 > SRRC 2043 > NRRL 6196 > SRRC 162 > SRRC 255, indicating incremental accumulation of reactive oxygen species, being least in the non-toxigenic strain and increasing progressively during the ternary steps of aflatoxin formation. Oxidative stress in these strains was evident by increased activities of xanthine oxidase and free radical scavenging enzymes (superoxide dismutase and glutathione peroxidase) as compared to the non-toxigenic strain (SRRC 255). Culturing the toxigenic strain in presence of 0.1-10 muM H(2)O(2 )in the medium resulted in enhanced aflatoxin production, which could be related to dose-dependent increase in [(14)C]-acetate incorporation into aflatoxin B(1) and increased acetyl CoA carboxylase activity. The combined results suggest that formation of secondary metabolites such as aflatoxin and its precursors by A. parasiticus may occur as a compensatory response to reactive oxygen species accumulation.

Prevention of isoproterenol-induced cardiac hypertrophy by eugenol, an antioxidant.

Recent reports on the involvement of calcineurin in cardiac hypertrophy and its susceptibility to free radicals, prompted us to examine possible beneficial effects of dietary antioxidants in this regard. In continuation of initialin vitro studies revealing eugenol to be a potent calcineurin inhibitor, we investigated its ability to reverse isoproterenol-induced cardiac hypertrophy in rats. Intraperitoneal administration of isoproterenol (1 mg/kg body wt/day for 10 days) induced cardiac hypertrophy with increased heart weight and enhanced apoptosis of myocytes concomitant with accumulation of reactive oxygen species, decreased glutathione contents, increased activities of calcineurin and protein kinase C in ventricular tissue. Administering eugenol for 3 days (1 mg/kg body wt/twice a day), followed by combined administration of isoproterenol and eugenol resulted in significant reversal of cardiac hypertrophy and restoration of above changes. These results suggest that eugenol, a natural antioxidant of dietary origin, may offer potential benefits in the management of cardiac hypertrophy.

Serum calcineurin activity in relation to oxidative stress and glycemic control in type II diabetes mellitus.

OBJECTIVES: In view of the well-recognized prevalence of oxidative stress in diabetes mellitus and the susceptibility of calcineurin (Ca(2+)-calmodulin dependent protein phosphatase 2 B) to free radicals, calcineurin was assayed in the sera of type II diabetic patients. DESIGN AND METHODS: Serum contents of thiobarbituric acid reactive substances, calcineurin and calmodulin, as well as activities of calcineurin and superoxide dismutase were measured in 81 diabetic patients and compared with age-matched controls. RESULTS: Oxidative stress in diabetic subjects was evidenced by increased thiobarbituric acid reactive substances, decreased superoxide dismutase activity concomitant with decreased calcineurin activity in sera. The observed decrease in calcineurin activity had a reciprocal correlation with fasting blood sugar, thiobarbituric acid reactive substances, and glycosylated hemoglobin. CONCLUSION: The inverse correlation observed between serum calcineurin activity and glycosylated hemoglobin levels suggests that an assay of serum calcineurin activity may be useful in simultaneous assessment of oxidative stress and glycemic control in type II diabetes mellitus.

Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa.

Two metal response elements, flanking an antioxidant response element, were identified in regions upstream (-3730 bp) to copper metallothionein (CuMT) gene of Neurospora crassa. Presence of copper in culture media, but not of pro-oxidants like H2O2 or menadione, induced CuMT gene expression that could not be completely abolished by antioxidants such as N-acetyl cysteine and ascorbic acid. Gel shift assays revealed the ability of nuclear extracts from copper induced cultures to bind PCR-amplified metal response or antioxidant response elements. Similar observations could not be made with cultures exposed either to pro-oxidants or antioxidants. These results differentiate between CuMT gene induction by copper from antioxidant functions associated with the identified upstream elements.

Biological effects of power frequency magnetic fields: Neurochemical and toxicological changes in developing chick embryos.

BACKGROUND: There are several reports that indicate a linkage between exposure to power frequency (50 - 60 Hz) magnetic fields with abnormalities in the early embryonic development of the chicken. The present study was designed to understand whether power frequency electromagnetic fields could act as an environmental insult and invoke any neurochemical or toxicological changes in developing chick embryo model. METHODS: Fertilized chicken eggs were subjected to continuous exposure to magnetic fields (50 Hz) of varying intensities (5, 50 or 100 microT) for a period of up to 15 days. The embryos were taken out of the eggs on day 5, day 10 and day 15. Neurochemical (norepinephrine and 5-hydroxytryptamine) and amino acid (tyrosine, glutamine and tryptophan) contents were measured, along with an assay of the enzyme glutamine synthetase in the brain. Preliminary toxicological investigations were carried out based on aminotransferases (AST and ALT) and lactate dehydrogenase activities in the whole embryo as well as in the liver. RESULTS: The study revealed that there was a significant increase (p < 0.01 and p < 0.001) in the level of norepinephrine accompanied by a significant decrease (p < 0.01 and p < 0.001) in the tyrosine content in the brain on day 15 following exposure to 5, 50 and 100 microT magnetic fields. There was a significant increase (p < 0.001) in glutamine synthetase activity resulting in the significantly enhanced (p < 0.001) level of glutamine in the brain on day 15 (for 100 microT only). The possible mechanisms for these alterations are discussed. Further, magnetic fields had no effect on the levels of tryptophan and 5-hydroxytryptamine in the brain. Similarly, there was no effect on the activity of either aminotransferases or lactate dehydrogenase in the whole embryo or liver due to magnetic field exposure. CONCLUSIONS: Based on these studies we conclude that magnetic field-induced changes in norepinephrine levels might help explain alterations in the circadian rhythm, observed during magnetic field stress. Also, the enhanced level of glutamine can act as a contributing factor for developmental abnormalities.

Calcineurin activity as an indicator of oxidative stress in normal islet cells and insulinoma cells.

A comparative study was conducted to evaluate calcineurin activity in normal pancreatic beta cells and insulinoma cells in relation to their oxidative state. In comparison to normal islets, insulinoma cells had enhanced oxidative stress as evidenced by increased content of thiobarbituric acid reactive substances. In addition, diminished activity of calcineurin in insulinoma cells was concomitant with decreased content of reduced glutathione and glutathione peroxidase activity signifying diminished antioxidant status in these cells. Culturing insulinoma cells in presence of the calcineurin inhibitor cyclosporin A resulted in further decrease of calcineurin activity with restoration of glutathione peroxidase but without restoration of reduced glutathione levels. These results indicate that an estimate of oxidative stress in pancreatic islets and insulinoma cells can be obtained by assaying calcineurin activity.

Clinical significance of serum calcineurin in acute leukemia.

BACKGROUND: Calcineurin is involved in T-lymphocyte activation as well as in the maturation of hematopoietic cells. Identification of this predominantly intracellular phosphatase and of calmodulin (CaM) in human sera warranted their assessment in different types of acute leukemias. METHODS: Phosphatase activity of calcineurin (CaN) was assayed, involving the measurement of trifluoperazine-sensitive neutral phosphatase, in sera of leukemic patients before and after treatment. Calcineurin and calmodulin contents were also determined by ELISA employing monoclonal antibodies specific to the proteins. RESULTS: The activity of calcineurin was decreased by 75% and 85% in sera of patients diagnosed either for acute lymphoid leukemia and acute myeloid leukemia, respectively, without apparent changes in calmodulin or calcineurin contents under both these conditions. In addition, the decreased calcineurin activity in acute myeloid leukemia was restored to levels comparable to non-leukemic individuals upon treatment. This was not observed in cases of acute lymphoid leukemia. CONCLUSIONS: These results suggest diagnostic utility in the measurement of serum calcineurin activity in acute leukemia. Restoration of normal calcineurin activity in patients undergoing treatment for acute myeloid leukemia may provide a means to monitor patient response to the prescribed therapeutic regimen.

Oxidative stress as a prerequisite for aflatoxin production by Aspergillus parasiticus.

The relevance of free radical generation and oxidative stress with regard to aflatoxin production was examined by comparing the oxygen requirement and antioxidant status of a toxigenic strain of Aspergillus parasiticus with that of a nontoxigenic strain at early (trophophase) and late logarithmic (idiophase) growth phases. In comparison to the nontoxigenic strain, wherein the oxygen requirements were relatively unaltered at various growth phases, the toxigenic strain exhibited greater oxygen requirements at trophophase coinciding with onset of aflatoxin production. The activities of antioxidant enzymes such as xanthine oxidase, superoxide dismutase, and glutathione peroxidase and the mycelial contents of thiobarbituric acid-reactive substances as well as of reduced glutathione were all enhanced during the progression of toxigenic strain from trophophase to idiophase. The combined results suggest that aflatoxin production by the toxigenic strain may be a consequence of increased oxidative stress leading to enhanced lipid peroxidation and free radical generation.

Calmodulin mediated activation of acetyl-CoA carboxylase during aflatoxin production by Aspergillus parasiticus.

The relevance of Ca2+-calmodulin-mediated processes in channelling acetate for aflatoxin formation was investigated by studying the influence of trifluoperazine (an anticalmodulin agent) on [14C]-acetate incorporation and activity of acetyl-CoA carboxylase in Aspergillus parasiticus NRRL 2999. Culturing the organism in presence of 0.14 mmol l-1 trifluoperazine resulted in 55% decrease of [14C]-acetate incorporation into aflatoxin B1, along with an 80% decrease in acetyl-CoA carboxylase activity at periods corresponding to maximal aflatoxin production. Concomitant decrement (35%) in the activity of glucose-6-phosphate dehydrogenase indicated decreased availability of reduction potential (NADPH) required for aflatoxin biosynthesis. The ability of calmodulin to activate and trifluoperazine to inhibit acetyl-CoA carboxylase activity in a dose-dependent manner was also noted under in vitro conditions. The combined results suggest calmodulin-mediated activation of acetyl-CoA carboxylase as an important event for aflatoxin production.

Regulation of aflatoxin production by Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation.

To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.

Antiaflatoxigenic activity of eugenol is due to inhibition of lipid peroxidation.

Eugenol inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999 in a dose-dependent manner up to a concentration of 0.75 mmol l-1 without inhibiting growth. When the mould was grown for 3 d in the presence of 0.45 mmol l-1 eugenol (concentration inhibiting aflatoxin production by 50%), in vivo activities of components of polysubstrate monooxygenase were decreased at idiophase, concomitant with decreased activities of enzymes involved in free radical scavenging, lipid peroxidation and maintenance of redox potential. These results indicate that antiaflatoxigenic actions of eugenol may be related to inhibition of the ternary steps of aflatoxin biosynthesis involving lipid peroxidation and oxygenation.

Requirement of Ca2+ for aflatoxin production: inhibitory effect of Ca2+ channel blockers on aflatoxin production by Aspergillus parasiticus NRRL 2999.

Aflatoxin production by Aspergillus parasiticus NRRL 2999 was inhibited when Ca2+ channel blockers, i.e., verapamil and diltiazem (> 1 mmol 1(-1)), were included in the culture medium. Inhibition was not accompanied by growth inhibition, nor was the [14C]-glucose uptake by the organism altered. However, both the compounds inhibited [14C]-acetate incorporation into aflatoxin B1 in a dose-dependent manner and decreased sporulation of the organism. Even though a nutritional role for Ca2+ has not been demonstrated unequivocally in fungi, the present study suggests the importance of Ca2+ in the production of these secondary metabolites.