NADPH oxidase inhibition prevents beta cell dysfunction induced by prolonged elevation of oleate in rodents.

AIMS/HYPOTHESIS: The activation of NADPH oxidase has been implicated in NEFA-induced beta cell dysfunction. However, the causal role of this activation in vivo remains unclear. Here, using rodents, we investigated whether pharmacological or genetic inhibition of NADPH oxidase could prevent NEFA-induced beta cell dysfunction in vivo. METHODS: Normal rats were infused for 48 h with saline or oleate with or without the NADPH oxidase inhibitor apocynin. In addition, NADPH oxidase subunit p47(phox)-null mice and wild-type littermate controls were infused with saline or oleate for 48 h. This was followed by measurement of NADPH oxidase activity, reactive oxygen species (ROS) and superoxide imaging and assessment of beta cell function in isolated islets and hyperglycaemic clamps. RESULTS: Oleate infusion in rats increased NADPH oxidase activity, consistent with increased total but not mitochondrial superoxide in islets and impaired beta cell function in isolated islets and during hyperglycaemic clamps. Co-infusion of apocynin with oleate normalised NADPH oxidase activity and total superoxide levels and prevented beta cell dysfunction. Similarly, 48 h NEFA elevation in wild-type mice increased total but not mitochondrial superoxide and impaired beta cell function in isolated islets. p47(phox)-null mice were protected against these effects when subjected to 48 h oleate infusion. Finally, oleate increased the levels of total ROS, in both models, whereas inhibition of NADPH oxidase prevented this increase, suggesting that NADPH oxidase is the main source of ROS in this model. CONCLUSIONS/INTERPRETATION: These data show that NADPH-oxidase-derived cytosolic superoxide is increased in islets upon oleate infusion in vivo; and whole-body NADPH-oxidase inhibition decreases superoxide in concert with restoration of islet function.

Glucose-induced beta cell dysfunction in vivo in rats: link between oxidative stress and endoplasmic reticulum stress.

AIMS/HYPOTHESIS: Endoplasmic reticulum (ER) stress has been implicated in glucose-induced beta cell dysfunction. However, its causal role has not been established in vivo. Our objective was to determine the causal role of ER stress and its link to oxidative stress in glucose-induced beta cell dysfunction in vivo. METHODS: Healthy Wistar rats were infused i.v. with glucose for 48 h to achieve 20 mmol/l hyperglycaemia with or without the co-infusion of the superoxide dismutase mimetic tempol (TPO), or the chemical chaperones 4-phenylbutyrate (PBA) or tauroursodeoxycholic acid (TUDCA). This was followed by assessment of beta cell function and measurement of ER stress markers and superoxide in islets. RESULTS: Glucose infusion for 48 h increased mitochondrial superoxide and ER stress markers and impaired beta cell function. Co-infusion of TPO, which we previously found to reduce mitochondrial superoxide and prevent glucose-induced beta cell dysfunction, reduced ER stress markers. Similar to findings with TPO, co-infusion of PBA, which decreases mitochondrial superoxide, prevented glucose-induced beta cell dysfunction in isolated islets. TUDCA was also effective. Also similar to findings with TPO, PBA prevented beta cell dysfunction during hyperglycaemic clamps in vivo and after hyperglycaemia (15 mmol/l) for 96 h. CONCLUSIONS/INTERPRETATION: Here, we causally implicate ER stress in hyperglycaemia-induced beta cell dysfunction in vivo. We show that: (1) there is a positive feedback cycle between oxidative stress and ER stress in glucose-induced beta cell dysfunction, which involves mitochondrial superoxide; and (2) this cycle can be interrupted by superoxide dismutase mimetics as well as chemical chaperones, which are of potential interest to preserve beta cell function in type 2 diabetes.

An intact kidney slice model to investigate vasa recta properties and function in situ.

BACKGROUND: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. METHODS: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in 'live' kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. RESULTS: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10-30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E(2)) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). CONCLUSIONS: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.

Hyperthermal organic thin film growth on surfaces terminated with self-assembled monolayers. I. The dynamics of trapping.

We have examined the initial stages of growth of a crystalline small molecule organic thin film, diindenoperylene (DIP), on SiO(2) surfaces terminated with a series of self-assembled monolayers (SAMs). In this study we make use of supersonic molecular beam techniques to vary the incident kinetic energy of the DIP molecules, and we use in situ, real time synchrotron x-ray scattering to monitor the buildup of each molecular layer in the growing thin film. We find that the effects of the SAMs are most apparent concerning growth in the sub-monolayer regime, before the substrate is entirely covered by the DIP thin film. In this coverage regime on bare SiO(2), and SiO(2) terminated with either hexamethyldisilazane or perflurooctyltrichlorosilane the adsorption dynamics are consistent with trapping-mediated adsorption as observed in more simple systems, where the probability of adsorption decreases significantly with increasing kinetic energy. Once these surfaces are covered with DIP, however, the adsorption probability increases, particularly at the highest incident kinetic energy, and the probability of adsorption exhibits only a weak dependence on the incident kinetic energy. In contrast, on surfaces terminated by octyl- (OTS) and octadecyltrichlorosilane (ODTS) the trapping probability is high and exhibits little dependence on the incident kinetic energy, essentially the same as what is observed on these same surfaces covered by DIP. We postulate, which is backed by the results of molecular dynamics simulations, that direct molecular insertion into the OTS and ODTS layers is a primary explanation for efficient trapping on these surfaces.

Coverage dependent adsorption dynamics in hyperthermal organic thin film growth.

We have examined the dynamics of adsorption of diindenoperylene (DIP) on SiO(2) and SiO(2) modified with an interfacial organic layer using in situ real time synchrotron x-ray scattering, focusing on the effects of coverage. On both surfaces we observe a substantial increase in the probability of adsorption with increasing coverage, which is most dramatic at the highest incident kinetic energies. On the initially uncovered surfaces, we observe a smooth decrease in the probability of adsorption with increasing incident kinetic energy, indicative of trapping-mediated adsorption. Once both surfaces are covered by DIP, the effects of incident kinetic energy are greatly reduced, and trapping is very efficient over the range of kinetic energies examined. Possible reasons for efficient trapping at high coverage and at high incident kinetic energy include more efficient momentum transfer due to mass matching, and possibly direct molecular insertion. Comparison to results on another small-molecule, pentacene, suggests that this behavior should be common to hyperthermal growth of a variety of other small-molecule thin films.

Factors associated with imaging in patients with early breast cancer after initial treatment.

Background: Overuse of surveillance imaging in patients after curative treatment for early breast cancer (ebc) was recently identified as one of the Choosing Wisely Canada initiatives to improve the quality of cancer care. We undertook a population-level examination of imaging practices in Ontario as they existed before the launch of that initiative. Methods: Patients diagnosed with ebc between 2006 and 2010 in Ontario were identified from the Ontario Cancer Registry. Records were linked deterministically to provincial health care databases to obtain comprehensive follow-up. We identified all advanced imaging exams [aies: computed tomography (ct), bone scan, positron-emission tomography] and basic imaging exams (bies: ultrasonography, chest radiography) occurring within the first 2 years after curative treatment. Poisson regression was used to assess associations between patient or provider characteristics and the rate of aies. Results: Of 30,006 women with ebc, 58.6% received at least 1 bie, and 30.6% received at least 1 aie in year 1 after treatment. In year 2, 52.7% received at least 1 bie, and 25.7% received at least 1 aie. The most common aies were chest cts and bone scans. The rate of aies increased with older age, higher disease stage, comorbidity, chemotherapy exposure, and prior staging investigations (p < 0.001). Imaging was ordered mainly by medical oncologists (38%), followed by primary care physicians (23%), surgeons (13%), and emergency room physicians (7%). Conclusions: Despite recommendations against its use, imaging is common in ebc survivors. Understanding the factors associated with aie use helps to identify areas for further research and is required to lower imaging rates and to improve survivorship care.

Sympathetic and baroreceptor reflex function in neurally mediated syncope evoked by tilt.

The pathophysiology of neurally mediated syncope is poorly understood. It has been widely assumed that excessive sympathetic activation in a setting of left ventricular hypovolemia stimulates ventricular afferents that trigger hypotension and bradycardia. We tested this hypothesis by determining if excessive sympathetic activation precedes development of neurally mediated syncope, and if this correlates with alterations in baroreflex function. We studied the changes in intraarterial blood pressure (BP), heart rate (HR), central venous pressure (CVP), muscle sympathetic nerve activity (MSNA), and plasma catecholamines evoked by upright tilt in recurrent neurally mediated syncope patients (SYN, 5+/-1 episodes/mo, n = 14), age- and sex-matched controls (CON, n = 23), and in healthy subjects who consistently experienced syncope during tilt (FS+, n = 20). Baroreflex responses were evaluated from changes in HR, BP, and MSNA that were obtained after infusions of phenylephrine and sodium nitroprusside. Compared to CON, patients with SYN had blunted increases in MSNA at low tilt levels, followed by a progressive decrease and ultimately complete disappearance of MSNA with syncope. SYN patients also had attenuation of norepinephrine increases and lower baroreflex slope sensitivity, both during tilt and after pharmacologic testing. FS+ subjects had the largest decrease in CVP with tilt and had significant increases in MSNA and heart rate baroreflex slopes. These data challenge the view that excessive generalized sympathetic activation is the precursor of the hemodynamic abnormality underlying recurrent neurally mediated syncope.

Evaluation of iron chelating and antioxidant potential of Epilobium hirsutum for the management of iron overload disease.

The present study deals with the investigation of iron chelating and antioxidant potential of Epilobium hirsutum in iron-overloaded rats. Iron overload was induced by 6 IP injections of Iron dextran (12.5mg/100g) administered uniformly over a period of 30 days. Different fractions of E. hirsutum were given orally and deferoxamine (DFO) subcutaneously for 30 days. The extent of iron chelation and various biochemical parameters were estimated on 15th and 30th day of treatment. In-vitro study was assessed by EDTA and DFO method; the results exhibited a dose-dependant iron chelation. The methanolic fraction of methanolic extract (MFME) and methanolic fraction of aqueous extract (MFAE) of E. hirsutum showed significant (p<0.01) iron chelating and antioxidant potential as compared to disease control (DC) rats. The animals treated with MFME and MFAE of E. hirsutum showed significant (p<0.01) vital organ protection as compared to DC rats. The animals treated for longer duration (30th day) reveals better iron chelation potential than shorter ones (15th day). Superior iron chelation was seen at higher dose (300mg/kg) as compared to lower dose (150mg/kg). Taken into an account, our result reveals the reversible iron chelating and antioxidant ability of E. hirsutum and gives some evidence for its possible mechanism via excretion of iron in urine and feces.

Investigation into Iron Chelating and Antioxidant Potential of Melilotus officinalis in Iron Dextran Induced Iron Overloaded Sprague Dawley Rat Model.

Excess of iron leads to generates free radicals, causes organ damage. Melilotus officinalis (Fabaceae) reported to have various pharmacological activities. It contains flavonoids and phenolic compounds which have iron chelating and antioxidant property. Hence, present study was designed to investigate the beneficial effects of different fractions of M. officinalis for the management of iron overload disease and its complications. Iron overload was induced by 6 IP injections of iron dextran (12.5 mg/100 g) uniformly distributed over the period of 30 days. The different fractions of M. officinalis were given orally and Deferoxamine (DFO) subcutaneously for 30 days. The iron chelating and various biochemical parameters were estimated on 15th and 30th day. The different fractions of M. officinalis demonstrated dose dependant in-vitro iron chelating ability. There were significant (P<0.01) iron chelating potential shows in rats treated with methanolic fraction of methanolic extract (MFME) and methanolic fraction of aqueous extract (MFAE) of M. officinalis as compared to disease control (DC) rats. The rats treated with MFME and MFAE of M. officinalis shows significant (P<0.01) antioxidant and vital organ protective effect as compared to DC rats. Better iron chelation was observed on 30th day and at higher dose (300 mg/kg) as compared to 15th day and at lower dose (150 mg/kg). The present study concludes that MFME and MFAE of M. officinalis have reversible iron chelating and antioxidant potential in rats. The study also proves the possible mechanism of action, as an iron chelator by increasing the excretion of iron in urine and feces.

Characterization of the low temperature thermoluminescence band Zv in leaf: an explanation for its variable nature.

Out of the six thermoluminescence bands reported for a mature leaf, one band (Zv) appearing at the lowest temperatures is dependent on the temperature of illumination. The characteristics of this band in fresh leaf are compared with those is a leaf heated to 60 degrees C for 5 min. It is concluded here that this band, following illumination at temperatures lower than 173 K, is part of Arnold and Azzi's Z band (Arnold, W. and Azzi, J.R. (1971) Photochem. Photobiol. 14, 233--240). However, it is part of peak I when observed subsequent to illumination beyond 173 K. An explanation for the appearance of this band at different temperatures is proposed.

Collaborative study for the establishment of the WHO 3(rd) International Standard for Endotoxin, the Ph. Eur. endotoxin biological reference preparation batch 5 and the USP Reference Standard for Endotoxin Lot H0K354.

An international collaborative study was organised jointly by the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC), the United States Pharmacopeia (USP) and the European Directorate for the Quality of Medicines & HealthCare (EDQM/Council of Europe) for the establishment of harmonised replacement endotoxin standards for these 3 organisations. Thirty-five laboratories worldwide, including Official Medicines Control Laboratories (OMCLs) and manufacturers enrolled in the study. Three candidate preparations (10/178, 10/190 and 10/196) were produced with the same material and same formulation as the current reference standards with the objective of generating a new (3(rd)) International Standard (IS) with the same potency (10 000 IU/vial) as the current (2(nd)) IS, as well as new European Pharmacopoeia (Ph. Eur.). and USP standards. The suitability of the candidate preparations to act as the reference standard in assays for endotoxin performed according to compendial methods was evaluated. Their potency was calibrated against the WHO 2(nd) IS for Endotoxin (94/580). Gelation and photometric methods produced similar results for each of the candidate preparations. The overall potency estimates for the 3 batches were comparable. Given the intrinsic assay precision, the observed differences between the batches may be considered unimportant for the intended use of these materials. Overall, these results were in line with those generated for the establishment of the current preparations of reference standards. Accelerated degradation testing of vials stored at elevated temperatures supported the long-term stability of the 3 candidate preparations. It was agreed between the 3 organisations that batch 10/178 be shared between WHO and EDQM and that batches 10/190 and 10/196 be allocated to USP, with a common assigned value of 10 000 IU/vial. This value maintains the continuity of the global harmonisation of reference materials and unitage for the testing of endotoxins in parenteral pharmaceutical products. Based on the results of the collaborative study, batch 10/178 was established by the European Pharmacopoeia Commission as the Ph. Eur. Endotoxin Biological Reference Preparation (BRP) batch 5. The same batch was also established by the Expert Committee on Biological Standardisation (ECBS) of WHO as the WHO 3(rd) IS for Endotoxin. Batch 10/190 was adopted as the USP Endotoxin Reference Standard, lot H0K354 and vials from this same batch (10/190) will serve as the United States Food and Drug Administration (USFDA) Endotoxin Standard, EC-7.

Taurine deficiency after intensive chemotherapy and/or radiation.

Taurine, a nonessential amino acid (AA), is the most abundant free AA in the intracellular space. We measured plasma AA concentrations in 36 patients 7-28 d after intensive chemotherapy and/or radiation. Plasma taurine concentrations were uniformly low in all patients (20.0 +/- 6.4 mumol/L, mean +/- SD). Plasma taurine in 11 healthy volunteer control subjects was 45.0 +/- 20.3 mumol/L (P less than 0.001). Other AA concentrations, specifically those of precursor AAs methionine and cystine, were normal. We prospectively measured plasma AA concentrations in 12 patients before starting and 6-10 d after completing intensive cytotoxic treatment. Values before treatment were 37.2 +/- 11.6, 109.6 +/- 30.7, and 18.5 +/- 4.8 for taurine, cystine, and methionine, respectively, and were 24.3 +/- 6.0, 111.2 +/- 23.8, and 24.0 +/- 14.5 after treatment. Pretreatment plasma taurine correlated directly with the magnitude of decrease in plasma taurine during cytotoxic treatment (n = 12, r = 0.85, P less than 0.01). Intensive cytotoxic chemotherapy and/or radiation leads to a reduction in plasma taurine concentrations without any change in its precursor AAs, methionine and cystine. The clinical relevance of plasma taurine depletion will need further study.

Aminoglycoside nephrotoxicity in obstructive jaundice.

PURPOSE: Although it is known that liver disease predisposes to aminoglycoside nephrotoxicity, specific features of such disease that may predispose to aminoglycoside-induced renal injury have not been identified. We sought to identify such features. PATIENTS AND METHODS: We undertook a retrospective review of the charts of 42 consecutive patients with biliary obstruction and/or cholangitis who had received more than three doses of an aminoglycoside. RESULTS: Comparison of patients in whom aminoglycoside nephrotoxicity did and did not develop revealed no differences in age, race, sex, dose, and duration of aminoglycoside therapy; mean peak and trough aminoglycoside levels; initial pre-treatment levels of serum creatinine, aspartate transaminase, alkaline phosphatase, or albumin; or prothrombin time. The initial pre-treatment serum bilirubin level was higher in the patients in whom aminoglycoside nephrotoxicity developed (12.2 +/- 8.8 mg/dl versus 3.4 +/- 3.2 mg/dl, p less than 0.01). Aminoglycoside nephrotoxicity occurred in eight patients (19 percent): in seven of 15 patients (47 percent) with an initial bilirubin value greater than 5.0 mg/dl, but in only one of 27 patients (4 percent) with an initial bilirubin value below 5.0 mg/dl (p less than 0.01). The pre-treatment bilirubin level correlated with the change in creatinine during aminoglycoside therapy (n = 42, r = 0.66, p less than 0.01). CONCLUSION: Aminoglycosides should probably be avoided in patients with biliary obstruction and a high serum bilirubin level.

Prevalence and clinical implications of hypocalcemia in acutely ill patients in a medical intensive care setting.

The incidence and the clinical implications of hypocalcemia were evaluated in acutely ill patients admitted to the Medical Intensive Care Unit of the Detroit Receiving Hospital. Total and ionized calcium levels were prospectively evaluated upon admission for all patients over a three-month interval. A high proportion of patients (62 of 88, 70 percent) were found to have decreased levels of both total and ionized calcium. Known causes of hypocalcemia could be identified in only 28 patients (45 percent). These included hypomagnesemia (17, 28 percent), renal insufficiency (five, 8 percent), alkalosis (four, 6 percent), and acute pancreatitis (two, 3 percent). In the remaining 34 patients (55 percent), no readily identifiable cause could be found. These 34 patients had a lower mean albumin level than did the 23 normocalcemic patients (p less than 0.01), but there were no differences in age, pH, serum creatinine, magnesium, or phosphate between the two groups. Serum albumin correlated directly with ionized calcium levels (n = 82, r = 0.33, p less than 0.01), as well as with total calcium levels (n = 76, r = 0.70, p less than 0.01). There was a strong association between sepsis and hypocalcemia. Patients who survived the hospitalization had higher mean ionized calcium, total calcium, and albumin values than did nonsurvivors, but there were no differences in age, serum creatinine, magnesium, and phosphate between the two groups. The mortality of the hypocalcemic patients (44 percent) was significantly greater (p less than 0.05) than the mortality of the normocalcemic patients (17 percent). These findings suggest that hypocalcemia is a very common abnormality in acutely ill patients and is associated with a poor prognosis.

Potentiation of butyrate-induced differentiation in human colon tumor cells by deoxycholate.

Human colon adenocarcinoma cells, treated with deoxycholate for 24 h prior to exposure to 1 mM butyrate, exhibited dose-dependent increases in the activities of three markers of colonic differentiation (alkaline phosphatase, lactase and CEA). Treatment with deoxycholate alone, for 24 h or longer, did not increase the secretion of CEA or the activities of either of the brush border-associated enzyme activities. Increases in differentiation markers were found to be bile acid-specific. Pretreatment with either dehydrocholic acid or cholic acid, even at cytotoxic concentrations, led to no significant butyrate-induced increases in brush-border associated hydrolase activities. The addition of a bacterial superoxide dismutase decreased the short-term cytotoxicity of deoxycholate and increased the maturation-potentiating effects of the bile acid in HCT-116 DO cells. The results of these studies demonstrate that bile acids, which are commonly thought to have tumor promoting activities in vivo, may also have physiological effects which serve to limit carcinogenic processes in the human colon by potentiating tumor cell differentiation.

Frequency and clinical significance of the S1235R mutation in the cystic fibrosis transmembrane conductance regulator gene: results from a collaborative study.

More than 900 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been reported to the cystic fibrosis (CF) consortium. A missense mutation, S1235R, was originally reported in a CF patient with a second mutation (G628R) on the same chromosome. The clinical significance of S1235R was not clear. S1235R is not among the commonly reported mutations, and it is not routinely screened for in most laboratories. However, we have detected the S1235R allele at a frequency that is significantly higher than that of many other CF mutations. Among more than 3,000 patients tested for either a possible diagnosis of CF or to determine CF carrier status, we identified 51 patients heterozygous for S1235R. No patients were homozygous for S1235R. Five patients were compound heterozygotes for a second CFTR mutation: two cases (one family) were N1303K/S1235R and three unrelated cases were deltaF508/S1235R. Our data suggest that S1235R, when combined with a second CF mutation, may be pathogenic, although phenotypic manifestations appear to be variable. The possibility that this represents a rare polymorphism cannot be discounted completely. Genetic counseling is difficult when S1235R is identified, even in the presence of a second known mutation, especially in prenatal cases.

Proteins and cells on PEG immobilized silicon surfaces.

Silicon surfaces were modified by covalent attachment of a self-assembled (SA) polyethylene glycol (PEG) film. Adsorption of albumin, fibrinogen, and IgG to PEG immobilized silicon surfaces was studied by ellipsometry to evaluate the non-fouling and non-immunogenic properties of the surfaces. The adhesion and proliferation of human fibroblast and Hela cells onto the modified surfaces were investigated to examine their tissue biocompatibility. Coated PEG chains showed the effective depression of both plasma protein adsorption and cell attachment to the modified surfaces. The mechanisms accounting for the reduction of protein adsorption and cell adhesion on modified surfaces were discussed.