Evidence against a role for NLRP3-driven islet inflammation in db/db mice.

OBJECTIVES: Type 2 diabetes (T2D) is associated with chronic, low grade inflammation. Activation of the NLRP3 inflammasome and secretion of its target interleukin-1ß (IL-1ß) have been implicated in pancreatic ß cell failure in T2D. Specific targeting of the NLRP3 inflammasome to prevent pancreatic ß cell death could allow for selective T2D treatment without compromising all IL-1ß-associated immune responses. We hypothesized that treating a mouse model of T2D with MCC950, a compound that specifically inhibits NLRP3, would prevent pancreatic ß cell death, thereby preventing the onset of T2D. METHODS: Diabetic db/db mice were treated with MCC950 via drinking water for 8 weeks from 6 to 14 weeks of age, a period over which they developed pancreatic ß cell failure. We assessed metabolic parameters such as body composition, glucose tolerance, or insulin secretion over the course of the intervention. RESULTS: MCC950 was a potent inhibitor of NLRP3-induced IL-1ß in vitro and was detected at high levels in the plasma of treated db/db mice. Treatment of pre-diabetic db/db mice with MCC950, however, did not prevent pancreatic dysfunction and full onset of the T2D pathology. When examining the NLRP3 pathway in the pancreas of db/db mice, we could not detect an activation of this pathway nor increased levels of its target IL-1ß. CONCLUSIONS: NLRP3 driven-pancreatic IL-1ß inflammation does not play a key role in the pathogenesis of the db/db murine model of T2D.

Genetic manipulation of cardiac Hsp72 levels does not alter substrate metabolism but reveals insights into high-fat feeding-induced cardiac insulin resistance.

Heat shock protein 72 (Hsp72) protects cells against a variety of stressors, and multiple studies have suggested that Hsp72 plays a cardioprotective role. As skeletal muscle Hsp72 overexpression can protect against high-fat diet (HFD)-induced insulin resistance, alterations in substrate metabolism may be a mechanism by which Hsp72 is cardioprotective. We investigated the impact of transgenically overexpressing (Hsp72 Tg) or deleting Hsp72 (Hsp72 KO) on various aspects of cardiac metabolism. Mice were fed a normal chow (NC) or HFD for 12 weeks from 8 weeks of age to examine the impact of diet-induced obesity on metabolic parameters in the heart. The HFD resulted in an increase in cardiac fatty acid oxidation and a decrease in cardiac glucose oxidation and insulin-stimulated cardiac glucose clearance; however, there was no difference in Hsp72 Tg or Hsp72 KO mice in these rates compared with their respective wild-type control mice. Although HFD-induced cardiac insulin resistance was not rescued in the Hsp72 Tg mice, it was preserved in the skeletal muscle, suggesting tissue-specific effects of Hsp72 overexpression on substrate metabolism. Comparison of two different strains of mice (BALB/c vs. C57BL/6J) also identified strain-specific differences in regard to HFD-induced cardiac lipid accumulation and insulin resistance. These strain differences suggest that cardiac lipid accumulation can be dissociated from cardiac insulin resistance. Our study finds that genetic manipulation of Hsp72 does not lead to alterations in metabolic processes in cardiac tissue under resting conditions, but identifies mouse strain-specific differences in cardiac lipid accumulation and insulin-stimulated glucose clearance.

Targeting gp130 to prevent inflammation and promote insulin action.

Obesity and type 2 diabetes are now the most prevalent metabolic diseases in the Western world and the development of new strategies to treat these metabolic diseases is most warranted. Obesity results in a state of chronic low-grade inflammation in metabolically active tissues such as the liver, adipose tissue, brain and skeletal muscle. Work in our laboratory has focussed on the role of the cytokine interleukin-6 (IL)-6 and other IL-6-like cytokines that signal through the gp130 receptor complex. We have focussed on the role of blocking IL-6 trans-signalling to prevent inflammation on the one hand, and activating membrane-bound signalling to promote insulin sensitivity on the other hand. Since the cloning of the IL-6 gene nearly 30 years ago, a pattern has emerged associating IL-6 with a number of diseases associated with inflammation including rheumatoid arthritis (RA), Crohn's disease and several cancers. Accordingly, tocilizumab, an IL-6 receptor-inhibiting monoclonal antibody, is now useful for the treatment of RA. However, this may not be the most optimal strategy to block inflammation associated with IL-6 and may result in unwanted side effects that, paradoxically, could actually promote metabolic disease.

Magnetoelectric coupling in 4,4'-stilbenedinitrene.

We investigated the optical properties of 4,4'-stilbenedinitrene at low temperature and in high magnetic fields and compared the results with complementary first principles calculations. Both physical tuning parameters allow us to manipulate the singlet-triplet equilibrium, and by doing so, control the optical contrast (which is on the order of -2.5 × 10(2) cm(-1) at 555 nm and 35 T). Moreover, analysis of the magneto-optical response using a combined population and Beer's law framework reveals the singlet-triplet spin gap and identifies particular features in the absorption difference spectrum as deriving from singlet or triplet state excitations. These findings deepen our understanding of coupling in open shell molecules and show how chemical structure modification can modulate charge-spin interactions in organic biradicals.

Adalimumab induced pulmonary sarcoid reaction.

Sarcoidosis is a multisystem granulomatous inflammatory disease of unknown etiology. There is evidence that Tumor Necrosis Factor alpha (TNF-α) antagonists are useful in the treatment of advanced or refractory disease. However, sarcoidosis-like reaction has been reported with TNF-α blockade in other inflammatory conditions. Here we report a case of sarcoid-like reaction in a patient with psoriatic arthritis shortly after initiation of adalimumab therapy. Stopping adalimumab and systemic anti-inflammatory therapy with corticosteroids resulted in resolution of pulmonary symptoms and chest radiographic findings. Though TNF-α plays a critical role in pathogenesis of sarcoidosis, the development of sarcoid reaction with TNF-α blockade is paradoxical and the mechanism of this response remains unknown. TNF-α induced sarcoid-reaction could involve multiple organs. Its development with one agent does not preclude therapy with other TNF-α blockers.

Skeletal muscle-specific overproduction of constitutively activated c-Jun N-terminal kinase (JNK) induces insulin resistance in mice.

AIMS/HYPOTHESIS: Although skeletal muscle insulin resistance has been associated with activation of c-Jun N-terminal kinase (JNK), whether increased JNK activity causes insulin resistance in this organ is not clear. In this study we examined the metabolic consequences of isolated JNK phosphorylation in muscle tissue. METHODS: Plasmids containing genes encoding a wild-type JNK1 (WT-JNK) or a JNK1/JNKK2 fusion protein (rendering JNK constitutively active; CA-Jnk) were electroporated into one tibialis anterior (TA) muscle of C57Bl/6 mice, with the contralateral TA injected with an empty vector (CON) to serve as a within-animal control. RESULTS: Overproduction of WT-JNK resulted in a modest (~25%) increase in phosphorylation (Thr(183)/Tyr(185)) of JNK, but no differences were observed in Ser(307) phosphorylation of insulin receptor substrate 1 (IRS-1) or total IRS-1 protein, nor in insulin-stimulated glucose clearance into the TA muscle when comparing WT-JNK with CON. By contrast, overexpression of CA-Jnk, which markedly increased the phosphorylation of CA-JNK, also increased serine phosphorylation of IRS-1, markedly decreased total IRS-1 protein, and decreased insulin-stimulated phosphorylation of the insulin receptor (Tyr(1361)) and phosphorylation of Akt at (Ser(473) and Thr(308)) compared with CON. Moreover, overexpression of CA-Jnk decreased insulin-stimulated glucose clearance into the TA muscle compared with CON and these effects were observed without changes in intramuscular lipid species. CONCLUSIONS/INTERPRETATION: Constitutive activation of JNK in skeletal muscle impairs insulin signalling at the level of IRS-1 and Akt, a process which results in the disruption of normal glucose clearance into the muscle.

Communication: Manipulating the singlet-triplet equilibrium in organic biradical materials.

We investigated the tunability of the singlet-triplet equilibrium population in the organic biradical 1,4-phenylenedinitrene via magneto-optical spectroscopy. A rich magnetochromic response occurs because applied field increases the concentration of the triplet state species, which has a unique optical signature by comparison with the singlet biradical and the precursor molecule. A Curie-like analysis of the magneto-optical properties allows us to extract the spin gap, which is smaller than previously supposed. These measurements establish the value of local-probe photophysical techniques for magnetic property determination in open-shell systems such as biradicals where a traditional electron paramagnetic resonance Curie law analysis has intrinsic limitations.

Interleukin-6-deficient mice develop hepatic inflammation and systemic insulin resistance.

AIMS/HYPOTHESIS: The role of IL-6 in the development of obesity and hepatic insulin resistance is unclear and still the subject of controversy. We aimed to determine whether global deletion of Il6 in mice (Il6 (-/-)) results in standard chow-induced and high-fat diet (HFD)-induced obesity, hepatosteatosis, inflammation and insulin resistance. METHODS: Male, 8-week-old Il6 (-/-) and littermate control mice were fed a standard chow or HFD for 12 weeks and phenotyped accordingly. RESULTS: Il6 (-/-) mice displayed obesity, hepatosteatosis, liver inflammation and insulin resistance when compared with control mice on a standard chow diet. When fed a HFD, the Il6 (-/-) and control mice had marked, equivalent gains in body weight, fat mass and ectopic lipid deposition in the liver relative to chow-fed animals. Despite this normalisation, the greater liver inflammation, damage and insulin resistance observed in chow-fed Il6 (-/-) mice relative to control persisted when both were fed the HFD. Microarray analysis from livers of mice fed a HFD revealed that genes associated with oxidative phosphorylation, the electron transport chain and tricarboxylic acid cycle were uniformly decreased in Il6 (-/-) relative to control mice. This coincided with reduced maximal activity of the mitochondrial enzyme ß-hydroxyacyl-CoA-dehydrogenase and decreased levels of mitochondrial respiratory chain proteins. CONCLUSIONS/INTERPRETATION: Our data suggest that IL-6 deficiency exacerbates HFD-induced hepatic insulin resistance and inflammation, a process that appears to be related to defects in mitochondrial metabolism.

Intubation success rates by air ambulance personnel during 12-versus 24-hour shifts: does fatigue make a difference?

OBJECTIVES: To determine whether the skill performance and psychomotor agility, as measured by the endotracheal intubation success rate, of air ambulance medical personnel would be affected by the potential fatigue incurred when increasing the length of their shifts from 12 to 24 hours. METHODS: This was a retrospective review of all flight and intubation records from a large air medical transport system from 1997, when 24-hour shifts were in place, and six months (March-August) of 1996, during which 12-hour shifts were scheduled. Records of all intubation efforts during both periods, including multiple attempts per patient, and outcomes of all attempts, were recorded. Results of successes and failures were tabulated for both ultimate intubation outcome per patient and all attempts per patient for each calendar day and for the 12 hours between 19:00 and 07:00 when fatigue might play a role. Results from the two study periods were compared using Fisher's exact test. RESULTS: During the six months of 1996, 190 of 199 (95.5%) patients were ultimately successfully intubated. These patients required 237 attempts (80.1% successful). During 1997, 362 of 376 (96.3%) patients were successfully intubated, and required 438 attempts (82.6% successful). There was no statistically significant difference in the number of ultimately successful intubations (p = 0.66) or total intubation attempts (p = 0.37) between 1996 and 1997. Analysis of intubations between 19:00 and 07:00 revealed 81 of 84 (96.4%) patients successfully intubated in 1996, with 81 of 103 (78.6%) attempts successful. During 1997, 173 of 180 (96.1%) patients were ultimately successfully intubated, with 173 of 212 (81.6%) attempts successful. Again, there was no significant difference in the number of successful intubations (p = 0.99) or intubation attempts (p = 0.55) between 1996 and 1997. CONCLUSION: Psychomotor agility of air ambulance medical personnel, as measured by the success rate of endotracheal intubation, was not affected by the potential additional fatigue incurred as a result of increasing shift length from 12 to 24 hours.

Evaluation of biphasic transthoracic defibrillation in an animal model of prolonged ventricular fibrillation.

OBJECTIVES: To determine whether a biphasic defibrillation waveform (BDW) would produce a superior rate of converting prolonged ventricular fibrillation (VF) into a perfusing rhythm and delay the occurrence of asystole and/or pulseless electrical activity (PEA) during the resuscitation attempt, when compared with a monophasic defibrillation waveform (MDW). METHODS: The authors performed a prospective, randomized, blinded experiment using an established swine model of prolonged VF. Thirty-four mixed-breed domestic swine (mean mass 22.9 kg) were sedated (ketamine/xylazine), anesthetized (isoflurane), and intubated. Aortic and femoral venous catheters were placed. ECG was monitored continuously. The animals were shocked into VF (3-s, 100-mA, 60-Hz shock), and were untreated for 8 minutes. Advanced Cardiac Life Support (ACLS) began with 1 minute of standardized (Thumper) chest compressions and ventilation. The animals were randomized to treatment with either BDW or MDW. Standard ACLS protocols were followed. The energy sequence was 2.5 J/kg first, 3.5 J/kg second, and 4.5 J/kg for all subsequent shocks. Outcome variables were time to event of asystole/PEA, return of spontaneous circulation (ROSC), and one-hour survival. Data were analyzed with two-tailed Fisher's exact test and Kaplan-Meier survival plots (alpha = 0.05). RESULTS: ROSC occurred more frequently in the BDW group (7/17) compared with the MDW group (1/17); p = 0.04. Survival analysis showed that the BDW significantly delayed the occurrence of asystole/PEA during the resuscitation attempt when compared with the MDW; log-ranked p = 0.02. One-hour survival rates (5/17 BDW and 1/17 MDW, p = 0.17) did not differ. CONCLUSIONS: BDW resulted in a superior rate of ROSC and delay in the occurrence of asystole/ PEA during the resuscitation attempt when compared with MDW.

Syncope as an emergency department presentation of pulmonary embolism.

Pulmonary embolism presenting as an isolated syncopal spell can be a difficult clinical correlation to make. We present three cases of pulmonary embolism-induced syncope and review the pathophysiology and diagnostic considerations in this setting. Pulmonary embolism should be considered in the differential diagnosis of every syncopal event that presents to the emergency department, even in the face of cardiac dysrhythmias and normal pulse oximetry values.

Cromolyn sodium for ACE inhibitor-induced cough.

There are several theories on the cause of ACE inhibitor-induced cough, but the exact mechanism is not known. In many patients, if cough develops, the ACE inhibitor can be discontinued and a drug in another therapeutic class used in its place. However, in patients with CHF, diabetic nephropathy, and patients who have experienced a myocardial infarction, discontinuing the ACE inhibitor may not be in the best interest of the patient. In this patient population it would be reasonable to try cromolyn sodium to treat cough, while continuing the ACE inhibitor. Data are not available to support the efficacy of cromolyn sodium to treat cough in patients with diabetic nephropathy, but these patients clearly benefit from the use of an ACE inhibitor. Other factors not addressed in the case reports and the clinical trial such as patient adherence, cost, and quality of life should also play a role in the decision to use cromolyn sodium. Cromolyn sodium has been effective for the treatment of ACE inhibitor-induced cough in many case reports and has had mild success in one small clinical trial. Although none of the reports adequately assessed adverse effects, studies examining cromolyn for other indications have demonstrated a relatively benign adverse effect profile. It is difficult to recommend an exact dose to use because of the dosing variability in the case reports. The majority of the case reports and the one clinical trial used dosages similar to recommendations for bronchial asthma (i.e., 2 puffs [1.6 mg] 4 times daily via MDI or 20-mg capsules 4 times daily via breath-activated inhalation). At this time, the use of cromolyn sodium is a viable option, but more controlled studies are needed to fully elucidate its role in the treatment of ACE inhibitor-induced cough.

Cytogenetic and molecular analysis in trisomy 12p.

We studied a male patient with de novo pure trisomy 12p syndrome by molecular analysis and fluorescence in situ hybridization (FISH) with markers from chromosome 12. G-banding studies demonstrated a 46,XY, 22p+ karyotype and the banding pattern and clinical findings suggested that the extra chromosomal material was derived from 12p. Trisomy 12p was confirmed by dosage analysis with chromosome 12p markers and FISH analysis with a whole chromosome 12 paint. The de novo re-arranged chromosome was of paternal origin. A comparison of the clinical and cytogenetic findings in this patient was made with previously described cases of trisomy 12p. We propose a classification system for 12p trisomy in order to better characterize the correlative relationships between specific cytogenetic constitution and phenotype.

Molecular regulation of transferrin receptor and ferritin expression in the rat gastrointestinal tract.

BACKGROUND & AIMS: The role of the transferrin receptor and ferritin i n the regulation of intestinal dietary iron uptake and excretion is unknown. This study aimed to determine the regulation of transferrin receptor and ferritin messenger RNA (mRNA) in the rat gastrointestinal tract in response to dietary iron changes. METHODS: In situ hybridization studies for transferrin receptor and L-ferritin mRNAs were performed using tissues from normal-iron-deficient, and iron-loaded rats. RESULTS: L-ferritin mRNA was localized to small intestinal crypt and villus epithelial cells and colonic crypt and surface epithelial cells with mRNA levels up-regulated in iron-loaded rats. Transferrin receptor mRNA was detected in crypt epithelial cells of the small and large intestine in iron-deficient and normal rats. In contrast, in iron-loaded rats, transferrin receptor mRNA was also detected in the superficial epithelial cells of the small intestine and colon, which contained increased stores of iron. Transferrin receptor mRNA levels were increased in the colon. CONCLUSIONS: In the iron-deficient and normal rat intestine, transferrin receptor mRNA was expressed only by proliferating crypt epithelial cells. In iron-loaded rats, however, surface enterocytes of the intestine expressed both transferrin receptor mRNA and increased ferritin mRNA levels.

The value of age and severity as predictors of costs in geriatric head trauma patients.

The costs of inpatient hospital care for treating head trauma were examined in 125 patients over the age of 50. Regression analysis indicated that injury severity, as measured by the admitting Glasgow Coma Scale, was a good predictor of hospital cost, while patient age was unrelated to cost. The regression relationship, however, was quadratic (not linear), indicating that least injured and severely injured patients cost less than moderately injured patients. These findings are in direct contrast with the commonly held belief that the elderly consume more hospital resources than younger patients. They suggest the need for a reexamination of the use of age in setting prospective reimbursement amounts in certain diagnoses, and in making resource allocation decisions relative to geriatric programming at the hospital level. Head trauma patients are costly because they are sick, not because they are old.

Bacillus piliformis infection in an adult dog.

Bacillus piliformis infection (Tyzzer's disease) was diagnosed in a 7-year-old spayed dog that had icterus, hepatosplenomegaly, and polyuria. Hematology revealed regenerative anemia, leukocytosis, lymphopenia, and thrombocytopenia. Serum chemical analyses indicated hypocalcemia, high alkaline phosphatase activity, hypoalbuminemia, and hyperglobulinemia. At necropsy, the liver was stippled with gray-white focal lesions. Microscopically, the liver lesions were necrotic and inflammatory. Warthin-Starry-stained sections revealed rod-shaped bacteria in crisscrossing patterns characteristic of B piliformis. This dog was considerably older than dogs previously reported to have Tyzzer's disease and had a concurrent systemic hyphomycosis, suggesting it had been immunocompromised.

Promoter recognition by Escherichia coli RNA polymerase: effects of base substitutions in the -10 and -35 regions.

We have constructed the PRM promoter of phage lambda and eight variants, which represents intermediates in the conversion of this promoter to one that has complete homology to the consensus sequences in the -10 and -35 regions. The in vivo activity of these promoters was determined from the beta-galactosidase or galactokinase activities in cells harboring plasmids, in which the cloned promoters were driving the expression of these genes. Additionally, the kinetics of the interaction of Escherichia coli RNA polymerase with the same series of promoters was measured as a function of RNA polymerase concentration. This allowed the overall rate of functional or open complex formation to be dissected into the equilibrium constant for binding of the polymerase to form a closed promoter complex and the rate of subsequent isomerization to yield the open complex. The following conclusions can be drawn from the data presented: (1) The consensus sequence is optimal for promoter function both in vivo and in vitro. (2) Alterations of the -10 and -35 regions have similar effects on the kinetics of RNA polymerase binding in vitro; with one exception, the same holds for promoter activity in vivo. (3) The in vitro rate of RNA polymerase binding to a promoter is solely determined by the number of positions at which its -10 and -35 regions match the consensus promoter sequence. The functional importance of a match does not appear to be determined by the sequence conservation at the particular position. (4) The extent to which a particular base change affects the kinetic parameters depends on the sequence of the promoter into which it is introduced.

Fractionation of Aspergillus niger cellulases by combined ion exchange affinity chromatography.

Eight chemically modified cellulose supports were tested for their ability to absorb components of the Aspergillus niger cellulase system. At least two of the most effective adsorbents, aminoethyl cellulose and carboxymethyl cellulose, were shown to be useful for the fractionation of cellulases. These supports apparently owe their resolving capacity to both ion exchange and biospecific binding effects; however, the relative importance of each effect is unknown. These observations form the basis for a new cellulase fractionation technique, combined ion exchange-affinity chromatography.

Promoter recognition by Escherichia coli RNA polymerase. Effects of substitutions in the spacer DNA separating the -10 and -35 regions.

A family of variants of the PRM promoter of lambda phage was constructed, bearing nine base pair substitutions in a stretch of the spacer DNA separating the contacted -10 and -35 regions. The substituted sequences were chosen for their potential to adopt structures different from those of average B-form DNA and thus to affect the interaction of RNA polymerase with the two contacted regions. Characterization of the promoters in vitro and in vivo provides additional support for the lack of specific contacts in the substituted spacer region and shows that a small change in the relative rotational orientation of the -10 and -35 regions is inconsequential to promoter function. However, a 2-3-fold reduction in promoter activity is observed with promoters bearing substitutions of nonalternating dG-dC base pairs in either orientation. This corroborates other studies indicating the anomalous behavior of such sequences and suggests that the structure of the spacer DNA can modulate promoter recognition.