Efficacy of radiofrequency ablation in the treatment of small functional adrenal neoplasms. To evaluate the use of radiofrequency (RF) ablation as a primary treatment for symptomatic primary functional adrenal neoplasms and determine the efficacy of treatment with use of clinical and biochemical follow-up. After obtaining institutional review board approval, the authors retrospectively evaluated images and medical records from 13 consecutive patients with symptomatic functional adrenal neoplasms (<3.2 cm in diameter) who underwent RF ablation during a 7-year period. There were six men and seven women with a mean age of 54.1 years (range, 42-71 years). Cross-sectional images, findings from clinical examination, and adrenal biochemical markers were available for all patients. Ten of the 13 patients (77%) had an aldosteronoma and one patient each had a cortisol-secreting tumor, testosterone-secreting tumor, and pheochromocytoma. RF ablation was performed by two radiologists using an internally cooled electrode and a pulsed technique according to manufacturer's specifications. Clinical and laboratory follow-up was performed for all patients. Three patients underwent imaging follow-up for other reasons. All patients demonstrated resolution of abnormal biochemical markers after ablation (mean biochemical follow-up, 21.2 months). In addition, all patients experienced resolution of clinical symptoms or syndromes, including hypertension and hypokalemia (in patients with aldosteronoma), Cushing syndrome (in the patient with cortisol-secreting tumor), virilizing symptoms (in the patient with testosterone-secreting tumor), and hypertension (in the patient with pheochromocytoma). For the patients with aldosteronoma, improvements in hypertension management were noted. The mean blood pressure before ablation was 149/90 mm Hg with a mean (±standard deviation) of 3.1 ± 0.6 blood pressure medications, and this decreased to 122/77 mm Hg at a mean of 2.8 months after ablation with 1.3 ± 0.9 medications (P < .001) and 124/75 mm Hg at a mean of 41.4 months. There were two minor complications: one small pneumothorax and one limited hemothorax, neither of which required overnight admission. There were two episodes of transient self-remitting procedural hypertension-one in a patient with aldosteronoma and one in the patient with a cortisol-secreting tumor; however, none of these patients required further therapy during overnight observation. RF ablation may be an effective, minimally invasive method for treating small functional primary adrenal tumors.


Advanced hepatocellular carcinoma: early evaluation of response to bevacizumab therapy at dynamic contrast-enhanced US with quantification--preliminary results. To investigate whether there is any correlation between standard efficacy endpoints-specifically, tumor response, progression-free survival, and overall survival-and tumor perfusion parameters measured by using dynamic contrast material-enhanced ultrasonography (US) in patients with advanced hepatocellular carcinoma (HCC) treated with bevacizumab. The institutional review board approved the study, and all patients provided written informed consent before their enrollment. Between June 3, 2005, and September 28, 2007, 42 patients (33 men, nine women; median age, 62 years; age range, 23-84 years) participated in this phase II study of single-agent bevacizumab treatment. Tumor response (based on RECIST [response evaluation criteria in solid tumors]) at 2 months was assessed in 37 patients, and progression-free survival and overall survival were assessed in all 42 patients. Dynamic contrast-enhanced US (ie, dynamic US) was performed before treatment (day 0); on days 3, 7, 14, and 60 after treatment; and every 2 months thereafter. Tumor perfusion parameters were estimated quantitatively from contrast material uptake curves constructed from raw linear data. The changes in dynamic US functional parameters between day 0 and the later time points were compared between treatment responders and nonresponders by using nonparametric tests. Given multiple comparisons, P < .001 indicated significance. The percentage decrease in several dynamic US parameters between day 0 and day 3 showed trends toward correlation with (a) tumor response in terms of total area under the time-intensity curve (AUC) (P = .02), AUC during wash in (P = .04), AUC during washout (P = .02), and time to peak intensity (P = .03); (b) progression-free survival in terms of time to peak intensity (P = .028); and (c) overall survival in terms of AUC (P = .002) and AUC during washout (P = .003). Dynamic US can be used to quantify dynamic changes in tumor vascularity as early as 3 days after bevacizumab administration in patients with HCC. These early changes in tumor perfusion may be predictive of tumor response at 2 months, progression-free survival, and overall survival, and they may be potential surrogate measures of the effectiveness of antiangiogenic therapy in patients with HCC.


IP(3) receptors: toward understanding their activation. Inositol 1,4,5-trisphosphate receptors (IP(3)R) and their relatives, ryanodine receptors, are the channels that most often mediate Ca(2+) release from intracellular stores. Their regulation by Ca(2+) allows them also to propagate cytosolic Ca(2+) signals regeneratively. This brief review addresses the structural basis of IP(3)R activation by IP(3) and Ca(2+). IP(3) initiates IP(3)R activation by promoting Ca(2+) binding to a stimulatory Ca(2+)-binding site, the identity of which is unresolved. We suggest that interactions of critical phosphate groups in IP(3) with opposite sides of the clam-like IP(3)-binding core cause it to close and propagate a conformational change toward the pore via the adjacent N-terminal suppressor domain. The pore, assembled from the last pair of transmembrane domains and the intervening pore loop from each of the four IP(3)R subunits, forms a structure in which a luminal selectivity filter and a gate at the cytosolic end of the pore control cation fluxes through the IP(3)R.


Acute pancreatitis: imaging utilization practices in an urban teaching hospital--analysis of trends with assessment of independent predictors in correlation with patient outcomes. To evaluate imaging utilization trends in patients with acute pancreatitis (AP) and to assess independent predictors of radiology usage in relation to patient outcomes. Institutional review board approval was obtained for this HIPAA-compliant study; written informed consent was waived. AP-related radiologic studies in 252 patients admitted for AP between June 2005 and December 2007 were collected during and for a 1-year period after hospitalization. Clinical data were collected from patients' medical records, while imaging data were obtained from the radiology information system. Linear regression models were used to investigate predictors and time trends of imaging utilization, after adjustment for confounders. Patient outcomes, measured by using mortality, intensive care unit admission, need for surgical intervention, organ failure, and persistent systemic inflammatory response syndrome, were evaluated by using logistic regression. Mean utilization was 9.9 radiologic studies per patient (95% confidence interval: 7.5, 12.3), with relative value unit (RVU) of 7.8 (95% confidence interval: 6.3, 9.4). Utilization was highest on day 0, declining rapidly by day 4; 53% of imaging occurred during initial hospitalization. Chest radiography (38%) and abdominal computed tomography (CT) (17%) were the most commonly performed studies. Patients with longer hospital stay (P = .001), higher Acute Physiology and Chronic Health Evaluation II score (P = .0012), higher pain levels (P = .003), drug-induced AP (P = .002), and prior episodes of AP (P < .001) underwent significantly more radiologic studies. After adjustment for confounders, a 2.5-fold increase in the use of high-cost (CT and magnetic resonance imaging) examinations and a 1.4-fold increase in RVUs per case-mix-adjusted admissions (P < .05) were observed during the 2.5-year study period. This increased use was not associated with improvement in patient outcomes. AP severity explained substantial variation in imaging utilization. After case-mix adjustment for severity and other patient level factors, there was still increasing use over the course of time without notable improvement in patient outcomes.


Neural correlates of olfactory learning: Critical role of centrifugal neuromodulation. The mammalian olfactory system is well established for its remarkable capability of undergoing experience-dependent plasticity. Although this process involves changes at multiple stages throughout the central olfactory pathway, even the early stages of processing, such as the olfactory bulb and piriform cortex, can display a high degree of plasticity. As in other sensory systems, this plasticity can be controlled by centrifugal inputs from brain regions known to be involved in attention and learning processes. Specifically, both the bulb and cortex receive heavy inputs from cholinergic, noradrenergic, and serotonergic modulatory systems. These neuromodulators are shown to have profound effects on both odor processing and odor memory by acting on both inhibitory local interneurons and output neurons in both regions.


Snapshot inversion recovery: an optimized single-shot T1-weighted inversion-recovery sequence for improved fetal brain anatomic delineation. To prospectively evaluate the clinical effectiveness of snapshot inversion recovery (SNAPIR), which is a dedicated optimized inversion-recovery-prepared single-shot fast spin-echo T1-weighted sequence, in the delineation of normal fetal brain anatomy compared with that of the currently used T1-weighted gradient-echo protocol, which often yields images of poor quality due to motion artifacts and inadequate contrast. This study was approved by the hospital research ethics committee, and informed written consent was obtained from all patients. Forty-one fetuses were examined at 19-37 weeks gestation (mean, 29 weeks gestation) by using both the standard T1-weighted protocol and the optimized T1-weighted SNAPIR protocol with a 1.5-T imager. Two independent blinded observers performed qualitative analysis, evaluating overall diagnostic quality, detailed anatomic delineation, and severity of motion artifacts. Quantitative analysis comprised calculation of contrast ratios (CRs) for the cortical gray matter, subplate, white matter, and cerebrospinal fluid. The Wilcoxon signed rank test was used to compare image rating scores, the paired t test was used to compare CRs, and κ statistics were used to test interobserver agreement. Both overall diagnostic quality (P < .001) and detailed anatomic delineation (P < .001) were enhanced with SNAPIR compared with the standard T1-weighted acquisition. Also, motion artifacts were less severe (P = .008) and less extensive (P < .001) with SNAPIR. Corresponding CRs were increased with SNAPIR in seven of eight examined regions. SNAPIR is a promising robust alternative to the current T1-weighted acquisitions; its role in the detection of disease requires further study.


Renal arteries: isotropic, high-spatial-resolution, unenhanced MR angiography with three-dimensional radial phase contrast. To prospectively compare a new three-dimensional (3D) radial phase-contrast magnetic resonance (MR) angiographic method with contrast material-enhanced MR angiography for anatomic assessment of the renal arteries. An institutional review board approved this prospective HIPAA-compliant study. Informed consent was obtained. Twenty-seven subjects (mean age, 52.6 years ± 20.5 [standard deviation]) were imaged with respiratory-gated phase-contrast vastly undersampled isotropic projection reconstruction (VIPR) prior to contrast-enhanced MR angiographic acquisition with a 3.0-T clinical system. The imaging duration for phase-contrast VIPR was 10 minutes and provided magnitude and complex difference ("angiographic") images with 3D volumetric (320 mm) coverage and isotropic high spatial resolution (1.25 mm(3)). Quantitative analysis consisted of comparing vessel diameters between the two techniques. Qualitative assessment included evaluation of the phase-contrast VIPR and contrast-enhanced MR angiographic techniques for artifacts, noise, and image quality. Bland-Altman analysis was used for comparison of quantitative measurements, and the Wilcoxon signed rank test was used for comparison of qualitative scores. Phase-contrast VIPR images were successfully acquired in all subjects. The vessel diameters measured with phase-contrast VIPR were slightly greater than those measured with contrast-enhanced MR angiography (mean bias = 0.09 mm). Differences in mean artifact, quality scores for the proximal renal arteries, and overall image quality scores between phase-contrast VIPR and contrast-enhanced MR angiographic techniques were not statistically significant (P = .31 and .29, .27 and .39, and .43 and .69 for readers 1 and 2, respectively). The quality scores for the segmental renal arteries were higher for phase-contrast VIPR than for contrast-enhanced MR angiography (P < .05). Although the noise scores were higher with phase-contrast VIPR than with contrast-enhanced MR angiography and were statistically significant (P < .05), the presence of noise did not interfere with the ability to interpret the images. Isotropic, high-spatial-resolution, unenhanced MR angiography of the renal arteries is feasible with 3D radial undersampling.


Resequencing and analysis of variation in the TCF7L2 gene in African Americans suggests that SNP rs7903146 is the causal diabetes susceptibility variant. Variation in the transcription factor 7-like 2 (TCF7L2) locus is associated with type 2 diabetes across multiple ethnicities. The aim of this study was to elucidate which variant in TCF7L2 confers diabetes susceptibility in African Americans. Through the evaluation of tagging single nucleotide polymorphisms (SNPs), type 2 diabetes susceptibility was limited to a 4.3-kb interval, which contains the YRI (African) linkage disequilibrium (LD) block containing rs7903146. To better define the relationship between type 2 diabetes risk and genetic variation we resequenced this 4.3-kb region in 96 African American DNAs. Thirty-three novel and 13 known SNPs were identified: 20 with minor allele frequencies (MAF) >0.05 and 12 with MAF >0.10. These polymorphisms and the previously identified DG10S478 microsatellite were evaluated in African American type 2 diabetic cases (n = 1,033) and controls (n = 1,106). Variants identified from direct sequencing and databases were genotyped or imputed. Fifteen SNPs showed association with type 2 diabetes (P < 0.05) with rs7903146 being the most significant (P = 6.32 × 10(-6)). Results of imputation, haplotype, and conditional analysis of SNPs were consistent with rs7903146 being the trait-defining SNP. Analysis of the DG10S478 microsatellite, which is outside the 4.3-kb LD block, revealed consistent association of risk allele 8 with type 2 diabetes (odds ratio [OR] = 1.33; P = 0.022) as reported in European populations; however, allele 16 (MAF = 0.016 cases and 0.032 controls) was strongly associated with reduced risk (OR = 0.39; P = 5.02 × 10(-5)) in contrast with previous studies. In African Americans, these observations suggest that rs7903146 is the trait-defining polymorphism associated with type 2 diabetes risk. Collectively, these results support ethnic differences in type 2 diabetes associations.


Urinary 8-hydroxy-2'-deoxyguanosine as a biomarker of oxidative DNA damage induced by ambient pollution in the Normative Aging Study. Studies show that exposure to air pollution damages human health, but the mechanisms are not fully understood. One suggested pathway is via oxidative stress. This study examines associations between exposure to air pollution and oxidative DNA damage, as indicated by urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations in ageing participants during 2006-2008. We fit linear regression models to examine associations between air pollutants and 8-OHdG adjusting for potential confounders. 8-OHdG was significantly associated with ambient particulate matter ≤2.5 μm in aerodynamic diameter (PM(2.5)), nitrogen dioxide (NO(2)), maximal 1 h ozone (O(3)), sulphate (SO(4)(2-)) and organic carbon (OC), but not with black carbon (BC), carbon monoxide (CO), the number of particles (PN) or elemental carbon (EC). Effects were more apparent with multi-week averages of exposures. Per IQR increases in 21-day averages of PM(2.5), PN, BC, EC, OC, CO, SO(4)(2-), NO(2) and maximal 1 h O(3) were associated with 30.8% (95% CI 9.3% to 52.2%), -13.1% (95% CI -41.7% to 15.5%), 3.0% (95% CI -19.8% to 25.8%), 5.3% (95% CI -23.6% to 34.2%), 24.4% (95% CI 1.8% to 47.1%), -2.0% (95% CI -12.4% to 8.3%), 29.8% (95% CI 6.3% to 53.3%), 32.2% (95% CI 7.4% to 56.9%) and 47.7% (95% CI 3.6% to 91.7%) changes in 8-OHdG, respectively. This study suggests that ageing participants experienced an increased risk of developing oxidative DNA injury after exposure to secondary, but not primary, ambient pollutants.


Congenital hyperinsulinism and glucose hypersensitivity in homozygous and heterozygous carriers of Kir6.2 (KCNJ11) mutation V290M mutation: K(ATP) channel inactivation mechanism and clinical management. The ATP-sensitive K(+) channel (K(ATP)) controls insulin secretion from the islet. Gain- or loss-of-function mutations in channel subunits underlie human neonatal diabetes and congenital hyperinsulinism (HI), respectively. In this study, we sought to identify the mechanistic basis of K(ATP)-induced HI in two probands and to characterize the clinical course. We analyzed HI in two probands and characterized the course of clinical treatment in each, as well as properties of mutant K(ATP) channels expressed in COSm6 cells using Rb efflux and patch-clamp methods. We identified mutation V290M in the pore-forming Kir6.2 subunit in each proband. In vitro expression in COSm6 cells supports the mutation resulting in an inactivating phenotype, which leads to significantly reduced activity in intact cells when expressed homomerically, and to a lesser extent when expressed heteromerically with wild-type subunits. In one heterozygous proband, a fluoro-DOPA scan revealed a causal focal lesion, indicating uniparental disomy with loss of heterozygosity. In a second family, the proband, homozygous for the mutation, was diagnosed with severe diazoxide-unresponsive hypersinsulinism at 2 weeks of age. The patient continues to be treated successfully with octreotide and amlodipine. The parents and a male sibling are heterozygous carriers without overt clinical HI. Interestingly, both the mother and the sibling exhibit evidence of abnormally enhanced glucose tolerance. V290M results in inactivating K(ATP) channels that underlie HI. Homozygous individuals may be managed medically, without pancreatectomy. Heterozygous carriers also show evidence of enhanced glucose sensitivity, consistent with incomplete loss of K(ATP) channel activity.


High connectivity between reduced cortical thickness and disrupted white matter tracts in long-standing type 1 diabetes. Previous studies have observed disruptions in brain white and gray matter structure in individuals with type 1 diabetes, and these structural differences have been associated with neurocognitive testing deficiencies. This study investigated the relationship between cerebral cortical thickness reductions and white matter microstructural integrity loss in a group of patients with type 1 diabetes and in healthy control subjects using diffusion tensor imaging (DTI). Twenty-five subjects with type 1 diabetes for at least 15 years and 25 age- and sex-matched control subjects underwent structural T1 and proton-density and DTI on a 3.0 Tesla scanner. Fractional anisotropy measurements were made on major cerebral white matter tracts, and DTI tractography was performed to identify cortical regions with high connectivity to these tracts. Posterior white matter tracts with reduced fractional anisotropy (optic radiations, posterior corona radiata, and the splenium region of the corpus callosum) were found to have high connectivity with a number of posterior cortical regions, including the cuneus, precuneus, fusiform, and posterior parietal cortical regions. A significant reduction in cortical thickness in the diabetic group was observed in the regions with high connectivity to the optic radiations and posterior corona radiata tracts (P < 0.05). The direct relationship between white and gray matter structural pathology has not been previously demonstrated in subjects with long-standing type 1 diabetes. The relationship between posterior white matter microstructural integrity disruption and lower cortical thickness demonstrated using a novel DTI connectivity technique suggests a common or interrelated pathophysiological mechanism in type 1 diabetes.


Levels of oxidized LDL and advanced glycation end products-modified LDL in circulating immune complexes are strongly associated with increased levels of carotid intima-media thickness and its progression in type 1 diabetes. High cholesterol levels in circulating immune complexes (IC), surrogate markers of modified LDL, are associated with increased carotid intima-media thickness (IMT) and cardiovascular events in type 1 diabetes. Different modifications of LDL are involved in IC formation, but which of these are predictive of vascular events is not known. Therefore, we measured oxidized LDL (oxLDL), advanced glycation end products-modified LDL (AGE-LDL), and malondialdehyde-modified LDL (MDA-LDL) in IC and determined their relationship with increased carotid IMT and compared the strength of the association with that observed with conventional risk factors. Levels of oxLDL, AGE-LDL, and MDA-LDL were measured in circulating IC isolated from sera of 479 patients of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) cohort, collected at baseline. Internal and common carotid IMT were measured 8 and 14 years later by DCCT/EDIC. OxLDL, AGE-LDL, and MDA-LDL levels in circulating IC were significantly correlated with diabetes duration, BMI, and lipid and blood pressure, but not with age. Multivariate logistic regression models indicated that individuals in the highest versus lowest quartile of oxLDL and AGE-LDL in IC had a 6.11-fold [confidence interval (CI) 2.51-14.8] and a 6.4-fold (CI 2.53-16.2) increase in the odds of having high carotid IMT, respectively, after adjusting for conventional risk factors. Parallel analyses resulted in odds ratios of 2.62 (CI 1.24, 5.55) for LDL-C, 1.45 (CI 0.69, 3.03) for diastolic blood pressure, and 2.33 (CI 1.09, 4.99) for A1C. OxLDL and AGE-LDL in circulating IC were significantly associated with progression and increased levels of carotid IMT in type 1 diabetes.


mt-Nd2(a) Modifies resistance against autoimmune type 1 diabetes in NOD mice at the level of the pancreatic β-cell. To investigate whether a single nucleotide polymorphism (SNP) in the mitochondrial gene for NADH dehydrogenase 2 (mt-Nd2) can modulate susceptibility to type 1 diabetes in NOD mice. NOD/ShiLtJ mice conplastic for the alloxan resistant (ALR)/Lt-derived mt-Nd2(a) allele (NOD.mt(ALR)) were created and compared with standard NOD (carrying the mt-Nd2(c) allele) for susceptibility to spontaneous autoimmune diabetes, or to diabetes elicited by reciprocal adoptive splenic leukocyte transfers, as well as by adoptive transfer of diabetogenic T-cell clones. β-Cell lines derived from either the NOD (NIT-1) or the NOD.mt(ALR) (NIT-4) were also created to compare their susceptibility to cytolysis by diabetogenic CD8(+) T-cells in vitro. NOD mice differing at this single SNP developed spontaneous or adoptively transferred diabetes at comparable rates and percentages. However, conplastic mice with the mt-Nd2(a) allele exhibited resistance to transfer of diabetes by the CD4(+) T-cell clone BDC 2.5 as well as the CD8(+) AI4 T-cell clones from T-cell receptor transgenic animals. NIT-4 cells with mt-Nd2(a) were also more resistant to AI4-mediated destruction in vitro than NIT-1 cells. Conplastic introduction into NOD mice of a variant mt-Nd2 allele alone was not sufficient to prevent spontaneous autoimmune diabetes. Subtle nonhematopoietic type 1 diabetes resistance was observed during adoptive transfer experiments with T-cell clones. This study confirms that genetic polymorphisms in mitochondria can modulate β-cell sensitivity to autoimmune T-cell effectors.


The protective role of Smad7 in diabetic kidney disease: mechanism and therapeutic potential. Although Smad3 has been considered as a downstream mediator of transforming growth factor-β (TGF-β) signaling in diabetes complications, the role of Smad7 in diabetes remains largely unclear. The current study tests the hypothesis that Smad7 may play a protective role and has therapeutic potential for diabetic kidney disease. Protective role of Smad7 in diabetic kidney disease was examined in streptozotocin-induced diabetic mice that have Smad7 gene knockout (KO) and in diabetic rats given Smad7 gene transfer using an ultrasound-microbubble-mediated technique. We found that mice deficient for Smad7 developed more severe diabetic kidney injury than wild-type mice as evidenced by a significant increase in microalbuminuria, renal fibrosis (collagen I, IV, and fibronectin), and renal inflammation (interleukin-1β [IL-1β], tumor necrosis factor-α [TNF-α], monocyte chemoattractant protein-1 [MCP-1], intracellular adhesion molecule-1 [ICAM-1], and macrophages). Further studies revealed that enhanced renal fibrosis and inflammation in Smad7 KO mice with diabetes were associated with increased activation of both TGF-β/Smad2/3 and nuclear factor-κB (NF-κB) signaling pathways. To develop a therapeutic potential for diabetic kidney disease, Smad7 gene was transferred into the kidney in diabetic rats by an ultrasound-microbubble-mediated technique. Although overexpression of renal Smad7 had no effect on levels of blood glucose, it significantly attenuated the development of microalbuminuria, TGF-β/Smad3-mediated renal fibrosis such as collagen I and IV and fibronectin accumulation and NF-κB/p65-driven renal inflammation including IL-1β, TNF-α, MCP-1, and ICAM-1 expression and macrophage infiltration in diabetic rats. Smad7 plays a protective role in diabetic renal injury. Overexpression of Smad7 may represent a novel therapy for the diabetic kidney complication.


Effects of acute hyperglucagonemia on hepatic and intestinal lipoprotein production and clearance in healthy humans. The metabolism of hepatic- and intestinally derived lipoproteins is regulated in a complex fashion by nutrients, hormones, and neurologic and other factors. Recent studies in animal models suggest an important role for glucagon acting via the glucagon receptor in regulating hepatic triglyceride (TG) secretion. Here we examined the direct effects of glucagon on regulation of hepatic and intestinal lipoprotein metabolism in humans. Eight healthy men underwent two studies each, in random order, 4-6 weeks apart in which de novo lipogenesis, kinetics of larger VLDL1 TG, and kinetics of VLDL1 and smaller VLDL2 apolipoprotein (apo)B100 and B48 were studied using established stable isotope enrichment methods. Subjects were studied in the constant fed state under conditions of a pancreatic clamp (with infusion of somatostatin, insulin, and growth hormone) at either basal glucagon (BG study, 64.5 ± 2.1 pg/mL) or hyperglucagonemia (high glucagon [HG] study, 183.2 ± 5.1 pg/mL). There were no significant differences in plasma concentration of VLDL1 or VLDL2 TG, apoB100 or apoB48 between BG and HG studies. There was, however, lower (P < 0.05) VLDL1 apoB100 fractional catabolic rate (-39%) and production rate (-30%) in HG versus BG, but no difference in de novo lipogenesis or TG turnover, and glucagon had no effect on intestinal (B48-containing) lipoprotein metabolism. Glucagon acutely regulates hepatic but not intestinal lipoprotein particle metabolism in humans both by decreasing hepatic lipoprotein particle production as well as by inhibiting particle clearance, with no net effect on particle concentration.


Disruption of hepatocyte growth factor/c-Met signaling enhances pancreatic beta-cell death and accelerates the onset of diabetes. To determine the role of hepatocyte growth factor (HGF)/c-Met on β-cell survival in diabetogenic conditions in vivo and in response to cytokines in vitro. We generated pancreas-specific c-Met-null (PancMet KO) mice and characterized their response to diabetes induced by multiple low-dose streptozotocin (MLDS) administration. We also analyzed the effect of HGF/c-Met signaling in vitro on cytokine-induced β-cell death in mouse and human islets, specifically examining the role of nuclear factor (NF)-κB. Islets exposed in vitro to cytokines or from MLDS-treated mice displayed significantly increased HGF and c-Met levels, suggesting a potential role for HGF/c-Met in β-cell survival against diabetogenic agents. Adult PancMet KO mice displayed normal glucose and β-cell homeostasis, indicating that pancreatic c-Met loss is not detrimental for β-cell growth and function under basal conditions. However, PancMet KO mice were more susceptible to MLDS-induced diabetes. They displayed higher blood glucose levels, marked hypoinsulinemia, and reduced β-cell mass compared with wild-type littermates. PancMet KO mice showed enhanced intraislet infiltration, islet nitric oxide (NO) and chemokine production, and β-cell apoptosis. c-Met-null β-cells were more sensitive to cytokine-induced cell death in vitro, an effect mediated by NF-κB activation and NO production. Conversely, HGF treatment decreased p65/NF-κB activation and fully protected mouse and, more important, human β-cells against cytokines. These results show that HGF/c-Met is critical for β-cell survival by attenuating NF-κB signaling and suggest that activation of the HGF/c-Met signaling pathway represents a novel strategy for enhancing β-cell protection.


Molecular mechanisms for activation of the agouti-related protein and stimulation of appetite. The agouti-related protein (Agrp) is a powerful orexigenic peptide, but little is known about its transcriptional regulation. The objective of this study was to determine molecular mechanisms for the activation of hypothalamic Agrp and identify compounds that stimulate appetite. We used promoter analyses methods, hypothalamic cell culture and transfection, immunohistochemistry, luciferase-expressing transgenic mice, in vivo bioluminescence, anitisense RNA, mouse feeding studies, indirect calorimetry, real-time PCR, and Western blots. We found that the Krüppel-like factor 4 (Klf4) is a potent activator of Agrp by binding to a specific CACCC-box in its minimal promoter. We also found that an extract of tarragon, termed PMI-5011, activated hypothalamic Klf4 and Agrp. In vivo, PMI-5011 increased Agrp promoter activity in luciferase-expressing transgenic mice, increased hypothalamic Klf4 and Agrp expression, increased hypothalamic Orexin and melanin-concentrating hormone, increased food intake, reduced circulating insulin and leptin levels, attenuated energy expenditure, and enhanced body weight but only when using a high-fat diet. These data show that Klf4 augmented hypothalamic Agrp by binding to a specific CACCC-box onto its minimal promoter. In addition, the tarragon extract PMI-5011 activated Klf4 and orexigenic neuropeptides and reduced peripheral insulin and leptin levels leading to positive energy balance.


β-cell dysfunctional ERAD/ubiquitin/proteasome system in type 2 diabetes mediated by islet amyloid polypeptide-induced UCH-L1 deficiency. The islet in type 2 diabetes is characterized by β-cell apoptosis, β-cell endoplasmic reticulum stress, and islet amyloid deposits derived from islet amyloid polypeptide (IAPP). Toxic oligomers of IAPP form intracellularly in β-cells in humans with type 2 diabetes, suggesting impaired clearance of misfolded proteins. In this study, we investigated whether human-IAPP (h-IAPP) disrupts the endoplasmic reticulum-associated degradation/ubiquitin/proteasome system. We used pancreatic tissue from humans with and without type 2 diabetes, isolated islets from h-IAPP transgenic rats, isolated human islets, and INS 832/13 cells transduced with adenoviruses expressing either h-IAPP or a comparable expression of rodent-IAPP. Immunofluorescence and Western blotting were used to detect polyubiquitinated proteins and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) protein levels. Proteasome activity was measured in isolated rat and human islets. UCH-L1 was knocked down by small-interfering RNA in INS 832/13 cells and apoptosis was evaluated. We report accumulation of polyubiquinated proteins and UCH-L1 deficiency in β-cells of humans with type 2 diabetes. These findings were reproduced by expression of oligomeric h-IAPP but not soluble rat-IAPP. Downregulation of UCH-L1 expression and activity to reproduce that caused by h-IAPP in β-cells induced endoplasmic reticulum stress leading to apoptosis. Our results indicate that defective protein degradation in β-cells in type 2 diabetes can, at least in part, be attributed to misfolded h-IAPP leading to UCH-L1 deficiency, which in turn further compromises β-cell viability.


Genital ulceration does not increase HIV-1 shedding in cervical or vaginal secretions of women taking antiretroviral therapy. Genital ulcer disease (GUD) is associated with increased HIV-1-RNA shedding in antiretroviral therapy (ART)-naive women. The effect of GUD on HIV-1 shedding among ART-treated women is not known. The objective of this study was to test the hypothesis that genital ulcerations increase genital HIV-1-RNA shedding in women receiving ART. Eligible women initiated ART and attended monthly visits with inspection for genital lesions and collection of genital swabs. GUD cases diagnosed after 2 months or more on ART were included for analysis and served as their own controls. HIV-1 RNA was quantitated in specimens collected before, during and after GUD for all cases. The lower limit of quantitation was 100 HIV-1-RNA copies/swab. Using the pre-GUD visit as the reference, the detection of genital HIV-1 RNA before versus during and after GUD episodes was compared. 36 women had GUD episodes after ART initiation. HIV-1 RNA was detected before, during and after GUD in cervical secretions from four (11%), one (3%) and six (17%) women, respectively, and in vaginal secretions from three (8%), four (11%) and four (11%) women, respectively. After adjustment for time on ART, there was no difference in the detection of cervical HIV-1 RNA before versus during GUD (adjusted OR 0.22, 95% CI 0.04 to 1.23). Likewise, GUD did not increase HIV-1 detection in vaginal secretions (adjusted OR 1.32, 95% CI 0.29 to 5.92). GUD did not significantly increase cervical or vaginal HIV-1 shedding. The results suggest that ART maintains its effectiveness for genital HIV-1 suppression despite GUD episodes.


Contraceptive practices, sexual and reproductive health needs of HIV-positive and negative female sex workers in Goa, India. In India, female sex workers (FSWs), suffer from high HIV prevalence and abortions. Contraceptive use among general population women is well understood. However, FSWs contraceptives practices and reproductive health needs are under-researched. We investigated contraceptive practices among HIV-positive and negative FSWs in Goa, India and explored its association with socio-demographic and sex work related factors. Cross-sectional study using respondent driven sampling recruited 326 FSWs. They completed an interviewer-administered questionnaire and were screened for STI/HIV. Multivariable logistic regression was used to explore factors associated with sterilisation relative to no contraception. HIV prevalence was high (26%). Of the 59 FSWs planning pregnancy, 33% were HIV-positive and 5-7% had Gonorrhoea, Chlamydia and Trichomonas. 25% and 65% of FSWs screened-positive for Syphilis and Herpes simplex virus type 2 antibodies respectively. Among the 260 FSWs analysed for contraceptive use, 39% did not use contraceptives, and 26% had experienced abortion. Half the FSWs had undergone sterilisation, and only 5% used condoms for contraception. Among HIV-positive FSWs, 45% did not use contraceptives. Sterilisation was independently associated with older age, illiteracy, having an intimate non-paying male partner, having children and financial autonomy. Exposure to National AIDS Control Organisation's HIV-prevention interventions was reported by 34% FSWs and was not significantly associated with contraceptive use (adjusted odds ratio 1.4, 95% CI 0.7 to 2.9). HIV-prevention interventions should promote contraception, especially among young and HIV-positive FSWs. Integrating HIV treatment and care services with HIV-prevention interventions is vital to avert HIV-positive births.


Interleukin-1β produced in response to islet autoantigen presentation differentiates T-helper 17 cells at the expense of regulatory T-cells: Implications for the timing of tolerizing immunotherapy. The effectiveness of tolerizing immunotherapeutic strategies, such as anti-CD40L or dendritic cells (DCs), is greater when administered to young nonobese diabetic (NOD) mice than at peak insulitis. RelB(lo) DCs, generated in the presence of an nuclear factor-κB inhibitor, induce T-regulatory (Treg) cells and suppress inflammation in a model of rheumatoid arthritis. Interleukin (IL)-1β is overexpressed in humans and mice at risk of type 1 diabetes, dysregulates Treg cells, and accelerates diabetes in NOD mice. We investigated the relationship between IL-1β production and the response to RelB(lo) DCs in the prediabetic period. We injected RelB(lo) DCs subcutaneously into 4- or 14-week-old NOD mice and tracked the incidence of diabetes and effect on Treg cell function. We measured the expression of proinflammatory cytokines by stimulated splenocytes and unstimulated islets from mice of different ages and strains and proliferative and cytokine responses of T effectors to Treg in vitro. Tolerizing RelB(lo) DCs significantly inhibited diabetes progression when administered to 4-week-old but not 14-week-old mice. IL-1β production by NOD splenocytes and mRNA expression by islets increased from 6 to 16 weeks of age when major histocompatibility complex (MHC)-restricted islet antigen presentation to autoreactive T-cells occurred. IL-1 reduced the capacity of Treg cells to suppress effector cells and promoted their conversion to Th17 cells. RelB(lo) DCs exacerbated the IL-1-dependent decline in Treg function and promoted Th17 conversion. IL-1β, generated by islet-autoreactive cells in MHC-susceptible mice, accelerates diabetes by differentiating Th17 at the expense of Treg. Tolerizing DC therapies can regulate islet autoantigen priming and prevent diabetes, but progression past the IL-1β/IL-17 checkpoint signals the need for other strategies.


Comment on: Yang et al. (2010) Associations of hyperglycemia and insulin usage with the risk of cancer in type 2 diabetes: the Hong Kong Diabetes Registry. Diabetes;59:1254-1260.


Outstanding Scientific Achievement Award Lecture 2010: deconstructing leptin: from signals to circuits. Martin G. Myers Jr., MD, PhD, received the American Diabetes Association's prestigious 2010 Outstanding Scientific Achievement Award at the Association's 70th Scientific Sessions in Orlando, Florida, on 28 June 2010. The Outstanding Scientific Achievement Award recognizes outstanding scientific achievement in the field of diabetes, taking into consideration independence of thought and originality. Currently the Marilyn H. Vincent Professor of Diabetes Research at the University of Michigan, Ann Arbor, and Associate Professor in internal medicine and in molecular and integrative physiology at the University of Michigan Medical School, Dr. Myers began his impressive track record in diabetes research as a graduate student in the laboratory of Dr. Morris White at the Joslin Diabetes Center/Harvard Medical School. There, Dr. Myers deciphered many of the insulin signaling pathways engaged by insulin receptor substrate proteins. Following his graduation from the Harvard MD-PhD Program in 1997, Dr. Myers was promoted to instructor in medicine at the Joslin Diabetes Center/Harvard Medical School. He began his independent work by building a molecular framework for understanding the mechanisms of leptin signaling, including how individual phosphorylation sites on the leptin receptor recruit distinct signaling molecules. He was promoted to assistant professor at Harvard in 1999. In 2004, Dr. Myers moved to the University of Michigan, where he built upon the molecular framework of leptin signaling to probe the regulation of metabolism by individual leptin signals. Dr. Myers' laboratory revealed the specificity of leptin signals in metabolic control, including the role for leptin-STAT3 signaling in the regulation of energy balance and glucose homeostasis. His group also defined roles for leptin receptor feedback inhibition and hypothalamic mTor signaling in metabolism. Dr. Myers' laboratory has recently developed novel molecular approaches to elucidate the leptin-regulated brain circuits that contribute to metabolic control, enabling the discovery of novel brain systems and their functions. In 1998, Dr. Myers received the American Diabetes Association's Career Development Award for his scientific abilities. Dr. Myers' current support includes the National Institute of Diabetes and Digestive and Kidney Diseases MERIT Award.


Health-chair reform: your chair: comfortable but deadly.


Physical activity, sedentary behavior, and health: paradigm paralysis or paradigm shift?


Final answer: ghrelin can suppress insulin secretion in humans, but is it clinically relevant?


Building genetic scores to predict risk of complex diseases in humans: is it possible?


Diabetes family history: a metabolic storm you should not sit out.


The welcome resurgence of the α-cell: a pro glucagon commentary.


Marine pollution.

Marine pollution.


Mass balances and life-cycle inventory for a garden waste windrow composting plant (Aarhus, Denmark). A comprehensive life-cycle inventory of all consumptions and emissions of environmental relevance was made for the windrow composting plant treating garden waste in Aarhus (Denmark). The flows of materials and substances within the facility were balanced using the mass-balance model STAN. The overall fuel and electricity use at the facility (3.04 L diesel Mg(-1) wet waste (ww) and 0.2 k Wh Mg(-1) ww) was low whereas the emissions of CH( 4) and N(2)O from the windrows (2.4 ± 0.5 kg CH( 4)-C Mg(-1) ww and 0.06 ± 0.03 kg N(2)O-N Mg(-1) ww) were relatively high compared to data reported in similar studies. The loss of carbon during the 14-month-long composting was 56%. CH(4) made up 2.1% of the C lost. Loss of nitrogen-containing compounds was identified as the most sensible and uncertain parameter and could be relevant for global warming (N(2)O emissions), acidification (NH(3) emissions), and eutrophication (NH(3) and NO(3) emissions). The compost produced had a very low content of heavy metals and was suitable for use in gardens and/or agriculture.


Nobuhiko Katunuma: an outstanding scientist in the field of proteolysis and warm-hearted 'Kendo Fighter' biochemist. Professor Nobuhiko Katunuma is well known for his outstanding contribution to the understanding of proteolysis in general and cysteine proteinases and their inhibitors in mammals. In fact, he is a world pioneer in the field. In 1963, he started his highly successful scientific career as a Professor at the Institute for Enzyme Research, the University of Tokushima. During the initial 30 years of his career, he was interested in vitamin B6 metabolism and discovered the acceleration of turnover rates of pyridoxal enzyme in apoprotein formation. After this period, his interest expanded to lysosomal cystein proteinases and their endogenous inhibitors. After determining the crystal structure of human cathepsin B, he generated a series of chemically synthesized specific inhibitors of cathepsins. These inhibitors are currently used throughout the world and some of them have been applied therapeutically in various diseases. During his career and even at present, Professor Katunuma has been studying Biochemistry in Medicine and also practicing to become a 'Kendo sword fencing Fighter'.


Independent and combined association of physical activity and cardiac disease on mortality risk in the very old. This study investigated physical activity as a predictor of all-cause mortality among 75- and 80-year-old people with and without chronic cardiac disease over a 10-year follow-up period. Using the Evergreen Project data, four study groups were formed according to the respondent's self-reported level of physical activity as well as chronic cardiac diseases: active without cardiac disease (control group = ANCD), active with cardiac disease (ACD), sedentary without cardiac disease (SNCD), and sedentary with cardiac disease (SCD). In the analyses, the ACD (HR 1.69, 95% CI 1.02-2.81) and the SNCD (1.76, 1.14-2.73) groups had almost one and a half times greater risk of dying than the control group, while the SCD group had almost three times (2.77, 1.80-4.26) greater risk of dying than the control group. Among the older people with cardiac disease, a physically active lifestyle was associated with lower mortality.


Field application of the H9M2e enzyme-linked immunosorbent assay for differentiation of H9N2 avian influenza virus-infected chickens from vaccinated chickens. Vaccination for control of H9N2 low-pathogenicity avian influenza (LPAI) in chickens began in 2007 in South Korea where the H9N2 virus is prevalent. Recently, an enzyme-linked immunosorbent assay (ELISA) using the extracellular domain of the M2 protein (M2e ELISA) was developed as another strategy to differentiate between vaccinated and infected chickens. Here, an ELISA using the extracellular domain of the M2 protein of H9N2 LPAI virus (H9M2e ELISA) was applied to differentiate infected from vaccinated chickens using the H9N2 LPAI virus M2 peptide. The specificity and sensitivity of the optimized H9M2e ELISA were 96.1% and 83.8% (the absorbance of the sample to the absorbance for the positive control [S/P ratio] ≥ 0.6), respectively, with the cutoff value (S/P ratio = 0.6), and the criterion of avian influenza (AI) infection in a chicken house was established as >20% reactivity of anti-M2e antibody per house with this cutoff value. After infection in naïve chickens and once-vaccinated chickens with a hemagglutination inhibition (HI) assay titer of 9.25 ± 0.75 log(2) units, the sera from infected chickens were confirmed as AI infected when the chickens were 1 week old in both groups, and AI infection lasted for 24 weeks and 9 weeks in naïve and once-vaccinated chickens, respectively, although in twice-vaccinated chickens with a higher HI titer of 11.17 ± 0.37 log(2) units, anti-M2e antibody in infected sera did not reach a level indicating AI infection. In field application, anti-M2e antibody produced in infected chickens after vaccination or in reinfected chickens could be identified as AI infection, although HI test could not distinguish infected from vaccinated sera. These results indicate the utility of H9M2e ELISA as a surveillance tool in control of H9N2 LPAI infections.


Correlation of immune activation during late pregnancy and early postpartum with increases in plasma HIV RNA, CD4/CD8 T cells, and serum activation markers. A previously observed rise in the plasma viral load postpartum in both treated and untreated HIV-positive women remains unexplained. Virological and immunological markers were evaluated in HIV-negative controls and HIV-positive pregnant women with and without antiretroviral treatment. Plasma HIV RNA, CD4/CD8 T cells, and serum activation markers were sequentially measured during the third trimester, at delivery, and 2 to 8 weeks postpartum in a cohort of HIV-positive pregnant women (n = 96) enrolled in a maternal-fetal HIV transmission study and a control group of HIV-negative pregnant women (n = 28). Mean plasma HIV RNA (P = 0.003) increased from delivery to postpartum, and mean CD4 T cells (P = 0.002) and serum β2-microglobulin (P < 0.0001) increased from the third trimester through postpartum among the HIV-positive women. Mean CD8 T cells increased from the third trimester through postpartum in women receiving zidovudine (ZDV) and in those not treated (P < 0.05) but remained stable in those on highly active antiretroviral therapy (HAART) and the HIV-negative controls. Increases in serum β2-microglobulin were correlated with increases in HIV RNA (P = 0.01). HIV-positive pregnant women showed postpartum increases in plasma HIV RNA, CD4 T cells, and serum β2-microglobulin regardless of the treatment regimen. The rise in CD4 T cells and β2-microglobulin was also observed in HIV-negative pregnant women, suggesting hormonal changes and/or labor-induced cytokines may contribute to immune activation. Immune activation correlated with increased plasma HIV RNA in postpartum women despite treatment, although HAART appeared to blunt the effect. The observed rise in plasma HIV RNA postpartum, which correlated with markers of immune activation, may have implications for enhanced transmission to infants through early breast-feeding and to sexual partners.


Evaluation of Rickettsia japonica pathogenesis and reservoir potential in dogs by experimental inoculation and epidemiologic survey. Rickettsia japonica pathogenesis and reservoir potential in dogs were evaluated by both experimental inoculation and epidemiologic survey. In the experimental inoculation study, dogs 1 and 2 were pretreated with an immunosuppressive dose of cyclosporine 14 days before inoculation and became ill after exposure to R. japonica. Dogs exhibited clinical signs, including fever, anorexia, depression, and decreased water consumption, between 36 and 96 h after inoculation, but these signs disappeared spontaneously by 5 days after inoculation. Dogs 3 and 4 were not pretreated with cyclosporine, and no clinical signs were detected in them throughout the 14-day observation period. The control dog was clinically normal and had a normal rectal temperature throughout the study period. We attempted to detect rickettsial DNA from peripheral blood and aspiration samples from kidney and spleen by nested PCR, but all samples examined were negative. The control dog lacked detectable titers to R. japonica antigen on day 14, while positive antibodies to R. japonica were detected in all four experimentally infected dogs, with titers of 1:160 to 1:80. In the epidemiologic survey, 24 (1.8%) of the 1,363 dogs examined throughout Japan had antibodies against R. japonica, with titers of 1:40 or more. However, we observed neither clinical signs at the time of sample collection nor nested PCR results indicative of rickettsial infection in these dogs. In conclusion, dogs in Japan can be exposed to R. japonica, and infected dogs with immunosuppressive conditions can temporarily develop clinical symptoms, including fever, anorexia, depression, and decreased water consumption.


No association of abnormal cranial venous drainage with multiple sclerosis: a magnetic resonance venography and flow-quantification study. Recent studies using colour-coded Doppler sonography showed that chronic impaired venous drainage from the central nervous system is almost exclusively found in multiple sclerosis (MS) patients. This study aimed to investigate the intracranial and extracranial venous anatomy and the intracerebral venous flow profile in patients with MS and healthy controls using magnetic resonance venography (MRV). Twenty patients with definite MS and 20 age- and gender-matched healthy controls were examined. MR imaging was performed on a whole-body 3T MR system including both 3D phase-contrast and dynamic 3D contrast-enhanced MRV as well as flow quantification of the internal cerebral veins and the straight sinus. Image analysis was performed by two experienced interventional neuroradiologists blinded to clinical data and structural brain imaging. The intracranial and extracranial neck veins were analysed for stenosis/occlusion and alternative venous drainage pattern. A completely normal venous anatomy was observed in 10 MS patients and 12 controls. Anomalies of the venous system (venous stenosis/occlusions) were found in 10 MS patients and eight healthy controls. An anomalous venous system in combination with associated alternative venous drainage was observed in six MS patients and five healthy controls. Flow quantification showed no venous backflow in any MS patient or control. Findings suggestive of anomalies of the cranial venous outflow anatomy were frequently observed in both MS patients and healthy controls. Given the normal intracranial venous flow quantification results, it is likely that these findings reflect anatomical variants of venous drainage rather than clinically relevant venous outflow obstructions.


Escherichia coli K88ac fimbriae expressing heat-labile and heat-stable (STa) toxin epitopes elicit antibodies that neutralize cholera toxin and STa toxin and inhibit adherence of K88ac fimbrial E. coli. Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, (8)LCSEYRNTQIYTIN(21)) and an STa toxoid epitope ((5)CCELCCNPQCAGCY(18)) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC.


Functional role of ipsilateral motor areas in multiple sclerosis. In patients with multiple sclerosis (MS), motor tasks are associated with increased activation of ipsilateral motor cortical areas. The authors examined the role of two ipsilateral motor areas during performance of a simple motor task in MS patients in relation to their motor impairment and CNS injury. Single pulses of transcranial magnetic stimulation (TMS) were used to interfere transiently with neuronal processing in the contralateral (M1(CONTRA)) or ipsilateral (M1(IPSI)) primary motor cortex or ipsilateral dorsal premotor cortex (PMd(IPSI)) during a simple reaction time (RT) task in 26 right-handed patients with moderately severe stable MS and matched healthy controls. Subjects responded to an auditorily presented Go signal as quickly as possible by performing isometric right-thumb abductions. TMS was applied 100 ms after the Go signal. Motor impairment was evaluated by hand function tests. CNS injury was assessed by magnetic resonance spectroscopy (normalised N-acetyl-aspartate spectra, NAA/Cr), by the total cerebral T2-weighted MRI hyperintense lesion load, and by corticomuscular latency (CML) to the abductor pollicis brevis muscle. TMS applied to M1(CONTRA) slowed RT in patients and controls. In contrast, stimulation of M1(IPSI) or PMd(IPSI) increased RT only in MS patients. In patients, the relative RT changes following TMS over M1(IPSI) or PMd(IPSI) did not correlate with any of the motor function tests or with NAA/Cr or total cerebral lesion load. However, RT changes following TMS over M1(IPSI) correlated inversely with CML. Recruitment of ipsilateral motor areas may be a functionally relevant, yet limited adaptive response to chronic brain injury in MS patients.


Efficacy of Bifidobacterium breve and Lactobacillus casei oral supplementation on necrotizing enterocolitis in very-low-birth-weight preterm infants: a double-blind, randomized, controlled trial. Probiotics are used for the prevention of necrotizing enterocolitis (NEC) because of their positive effects on intestinal motor function, modulation of inflammatory response, and mucosal barrier function. The objective was to assess whether the combined use of Lactobacillus casei and Bifidobacterium breve may prevent the occurrence of NEC stage ≥ 2 by the criteria of Bell in very-low-birth-weight preterm infants. A double-blind, randomized, controlled clinical trial was conducted in 231 preterm infants weighing from 750 to 1499 g at birth. The intervention group was composed of 119 infants who received human milk with probiotic supplementation (B. breve and L. casei) and a control group of 112 infants who received human milk containing no probiotics. The primary outcome was the occurrence of NEC stage ≥ 2 as defined by Bell's modified criteria. Four confirmed cases of NEC stage ≥ 2 by Bell's criteria occurred only in the control group. Oral supplementation of B. breve and L. casei reduced the occurrence of NEC (Bell's stage ≥ 2). It was considered that an improvement in intestinal motility might have contributed to this result. This trial was registered at www.isrctin.org as number 67165178 (International Standard Randomized Controlled Trial).


Vitamin D and breast cancer recurrence in the Women's Healthy Eating and Living (WHEL) Study. There is a paucity of research evaluating the relation between vitamin D and recurrence of breast cancer after treatment. This study was designed to evaluate the associations between circulating concentrations of 25-hydroxyvitamin D [25(OH)D] and dietary, supplemental, and total intake of vitamin D and recurrent or new breast cancer events within the Women's Healthy Eating and Living (WHEL) Study. A prospective cohort study design (n = 3085) was used to evaluate the relation between dietary, supplemental, and total vitamin D intake and recurrent breast cancer, and a nested case-control study with 512 matched pairs was used for analysis of the association between 25(OH)D and breast cancer recurrence. No relation between 25(OH)D and breast cancer recurrence was observed. Compared with women with serum concentrations of 25(OH)D ≥ 30 ng/mL, adjusted odds ratios (95% CI) for breast cancer recurrence were 1.14 (0.57, 2.31) for those with concentrations < 10 ng/mL, 1.00 (0.68-1.48) for concentrations ≥ 10 and < 20 ng/mL, and 1.05 (0.76, 1.47) for concentrations ≥ 20 and < 30 ng/mL. No significant associations were observed when analyses were stratified by pre- and postmenopausal status or for local, regional, or distant recurrence or death. Vitamin D intake was not related to breast cancer recurrence overall, although for premenopausal women there was a significant inverse association between dietary vitamin D intake and recurrence (P for trend = 0.02). These results do not provide support for a relation between concentrations of 25(OH)D after treatment and the recurrence of breast cancer. This trial is registered at clinicaltrials.gov for the WHEL Study as NCT00003787.


Erythrocyte membrane phospholipid fatty acids, desaturase activity, and dietary fatty acids in relation to risk of type 2 diabetes in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study. The long-term role of fatty acids (FAs) in the cause of diabetes remains largely unclear. We aimed to investigate erythrocyte membrane FAs, desaturase activity, and dietary FAs in relation to the incidence of type 2 diabetes. We applied a nested case-cohort design (n = 2724, including 673 incident diabetes cases) within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study, which involves 27,548 middle-aged subjects. Thirty erythrocyte membrane FAs (percentage of total FAs) and FA intake (percentage of total fat) were measured at baseline, and physician-confirmed incident diabetes was assessed during a mean follow-up of 7.0 y. We evaluated Δ⁵ desaturase (D5D) and Δ⁶ desaturase (D6D) activity by using FA product-to-precursor ratios (traditional approach) and by investigating variants in FADS1 and FADS2 genes that encode these desaturases (Mendelian randomization approach). As a main finding, erythrocyte 16:1n-7 and 18:3n-6 and FA ratios, which reflect stearoyl coenzyme A desaturase (SCD) and D6D activity, were directly related to diabetes risk in multivariable-adjusted models [relative risks (95% CIs) comparing extreme quintiles: 16:1n-7, 2.11 (1.46, 3.05); 18:3n-6, 2.00 (1.38, 2.88); SCD, 2.61 (1.75, 3.89); and D6D, 2.46 (1.67, 3.63)], whereas the FA ratio that reflects D5D activity was inversely associated with risk [0.46 (0.31, 0.70)]. The Mendelian randomization approach corroborated the direct relation for D6D activity and tended to support the inverse relation for D5D activity. Proportions of dietary FAs showed only modest to low correlations with erythrocyte FAs and were not significantly associated with risk. The FA profile of erythrocyte membrane phospholipids and activity of desaturase enzymes are strongly linked to the incidence of type 2 diabetes.


Fatty acid consumption and risk of fracture in the Women's Health Initiative. Fatty acids (FAs) may be important dietary components that modulate osteoporotic fracture risk. The objective was to examine FA intake in relation to osteoporotic fractures. The participants were postmenopausal women enrolled in the Women's Health Initiative (n = 137,486). Total fractures were identified by self-report; hip fractures were confirmed by medical record review. FA intake was estimated from baseline food-frequency questionnaires and standardized to total caloric intake. No data on omega-3 (n-3) FA supplements were available. Cox proportional hazard models were constructed to estimate risk of fracture. Higher saturated FA consumption was associated with higher hip fracture risk [quartile 4 multivariate-adjusted hazard ratio (HR): 1.31; 95% CI: 1.11, 1.55; P for trend = 0.001]. Lower total fracture risk was associated with a higher monounsaturated FA intake (quartile 3 HR: 0.94; 95% CI: 0.89, 0.98; P for trend = 0.050) and polyunsaturated FA intake (quartile 4 HR: 0.95; 95% CI: 0.90, 0.99; P for trend = 0.019). Unexpectedly, higher consumption of marine n-3 FAs was associated with greater total fracture risk (quartile 4 HR: 1.07; 95% CI: 1.02, 1.12; P for trend = 0.010), whereas a higher n-6 FA intake was associated with a lower total fracture risk (quartile 4 HR: 0.94; 95% CI: 0.89, 0.98; P for trend 0.009). These results suggest that saturated FA intake may significantly increase hip fracture risk, whereas monounsaturated and polyunsaturated FA intakes may decrease total fracture risk. In postmenopausal women with a low intake of marine n-3 FAs, a higher intake of n-6 FAs may modestly decrease total fracture risk. This trial was registered at clinicaltrials.gov as NCT00000611.


Intake of probiotic food and risk of spontaneous preterm delivery. Preterm delivery represents a substantial problem in perinatal medicine worldwide. Current knowledge on potential influences of probiotics in food on pregnancy complications caused by microbes is limited. We hypothesized that intake of food with probiotics might reduce pregnancy complications caused by pathogenic microorganisms and, through this, reduce the risk of spontaneous preterm delivery. This study was performed in the Norwegian Mother and Child Cohort on the basis of answers to a food-frequency questionnaire. We studied intake of milk-based products containing probiotic lactobacilli and spontaneous preterm delivery by using a prospective cohort study design (n = 950 cases and 17,938 controls) for the pregnancy outcome of spontaneous preterm delivery (< 37 gestational weeks). Analyses were adjusted for the covariates of parity, maternal educational level, and physical activity. Pregnancies that resulted in spontaneous preterm delivery were associated with any intake of milk-based probiotic products in an adjusted model [odds ratio (OR): 0.857; 95% CI: 0.741, 0.992]. By categorizing intake into none, low, and high intakes of the milk-based probiotic products, a significant association was observed for high intake (OR: 0.820; 95% CI: 0.681, 0.986). Women who reported habitual intake of probiotic dairy products had a reduced risk of spontaneous preterm delivery.


Rice intake and type 2 diabetes in Japanese men and women: the Japan Public Health Center-based Prospective Study. Refined carbohydrates have been suggested to deteriorate glucose metabolism; however, whether persons with elevated intakes of white rice, which is a major staple food for the Japanese, experience increased risk of developing type 2 diabetes remains unclear. We prospectively investigated the association between white rice intake and risk of type 2 diabetes. Participants were 25,666 men and 33,622 women aged 45-75 y who participated in the second survey of the Japan Public Health Center-based Prospective Study and who had no prior history of diabetes. We ascertained food intake by using a validated 147-item food-frequency questionnaire. Odds ratios of self-reported, physician-diagnosed type 2 diabetes over 5 y were estimated by using logistic regressions. A total of 1103 new cases of type 2 diabetes were self-reported. There was a significant association between rice intake and an increased risk of type 2 diabetes in women; the multivariate-adjusted odds ratio for the highest compared with lowest quartiles of rice intake was 1.65 (95% CI: 1.06, 2.57; P for trend = 0.005). In men, the association was unclear, although there was a suggestion of a positive association in persons who were not engaged in strenuous physical activity (P for trend = 0.08). Elevated intake of white rice is associated with an increased risk of type 2 diabetes in Japanese women. The finding that is suggestive of a positive association of rice intake in physically inactive men deserves further investigation.


Dietary omega-3 fatty acids and fish consumption and risk of type 2 diabetes. Although dietary omega-3 (n-3) fatty acids may confer some cardiovascular benefits, it is unclear whether these nutrients may also unfavorably affect risk of type 2 diabetes (T2D). We evaluated whether dietary omega-3 fatty acids and fish consumption were associated with increased risk of T2D. This was a prospective study of 36,328 women (mean age: 54.6 y) who participated in the Women's Health Study and who were followed from 1992 to 2008. Incident T2D was self-reported and validated primarily through the collection of supplementary information from participants. Information on omega-3 and fish intakes was obtained by using a validated food-frequency questionnaire. We used Cox proportional hazard models to estimate adjusted relative risks. During an average follow-up of 12.4 y, 2370 women developed T2D. Marine but not plant-based omega-3 fatty acids were positively associated with incident T2D. From the lowest to highest quintiles of marine omega-3 intake, the multivariable-adjusted hazard ratios (95% CIs) for T2D were 1.0 (referent), 1.17 (1.03, 1.33), 1.20 (1.05, 1.38), 1.46 (1.28, 1.66), and 1.44 (1.25, 1.65), respectively (P for trend < 0.0001). A similar association was observed with fish intake, but additional adjustment for docosahexaenoic acid led to the elimination of the association. The relation between marine omega-3 fatty acids and T2D was observed in hypertensive and nonhypertensive subjects and in women who reported infrequent fish consumption. Our data suggest an increased risk of T2D with the intake of long-chain omega-3 fatty acids, especially with higher intakes (≥ 0.20 g omega-3/d or ≥ 2 servings of fish/d). The Women's Health Study was registered at clinicaltrials.gov as NCT00000479.


The identification of children with adverse risk factor levels by body mass index cutoffs from 2 classification systems: the Bogalusa Heart Study. The cutoffs from the Centers for Disease Control and Prevention (CDC) growth charts and from the Cooper Institute (FitnessGram) are widely used to identify children who have a high body mass index (BMI). We compared the abilities of these 2 systems to identify children who have adverse lipid concentrations and blood pressure measurements and the reliability (consistency) of each classification system over time (mean follow-up: 7 y). A cross-sectional analysis based on data from 22,896 examinations of 5- to 17-y-olds was conducted. Principal components analyses were used to summarize levels of the 5 risk factors, and likelihood ratios and the κ statistic were used to compare the screening abilities of the 2 systems. Of these children, 3972 were included in longitudinal analyses. There were marked differences in the prevalence of a high FitnessGram BMI by age, with the prevalence among boys increasing from 2.5% to 21% between the ages of 5 and 11 y. The identification of adverse risk factors by the 2 systems was only fair (κ = 0.25), but there was little difference in the abilities of the CDC and FitnessGram cutoffs to identify high-risk children. Longitudinal analyses, however, indicated that the agreement between initial and follow-up FitnessGram classifications was substantially lower than that based on CDC cutoffs (κ = 0.28 compared with 0.49). The FitnessGram and CDC cutoffs have similar abilities to identify high-risk children. However, a high FitnessGram BMI is difficult to interpret because the reliability over time is low, and the prevalence increases markedly with age.


A dose-finding trial of the effect of long-term folic acid intervention: implications for food fortification policy. The lowest dose of folic acid required to achieve effective reductions in homocysteine is controversial but important for food fortification policy given recent concerns about the potential adverse effects of overexposure to this vitamin. We compared the effectiveness of 0.2 mg folic acid/d with that of 0.4 and 0.8 mg/d at lowering homocysteine concentrations over a 6-mo period. A randomized dose-finding trial with folic acid was conducted. Of 203 participants screened, 101 patients with ischemic heart disease and 71 healthy volunteers completed the study. Participants were randomly assigned to receive placebo or folic acid at doses of 0.2, 0.4, or 0.8 mg/d for 26 wk; subsamples of patients with ischemic heart disease were also examined at 6 or 12 wk. Participants with higher baseline homocysteine concentrations had the greatest reductions in homocysteine in response to folic acid doses of 0.2 mg (-20.6%), 0.4 mg (-20.7%), and 0.8 mg (-27.8%); in those with lower baseline homocysteine concentrations, the responses were -8.2%, -8.9%, and -8.3%, respectively. No significant differences in homocysteine responses to the different doses were observed. In the patient group sampled at intervals during the intervention, the maximal homocysteine response appeared to be achieved by 6 wk in the 0.8-mg/d group and by 12 wk in the 0.4-mg/d group. However, the homocysteine response was suboptimal in the 0.2-mg/d group at both 6 and 12 wk compared with that at 26 wk. A folic acid dose as low as 0.2 mg/d can, if administered for 6 mo, effectively lower homocysteine concentrations. Higher doses may not be necessary because they result in no further significant lowering, whereas doses even lower than 0.2 mg/d may be effective in the longer term. Previous trials probably overestimated the folic acid dose required because of a treatment duration that was too short. This trial was registered at clinicaltrials.gov as ISRCTN45296887.


A low-glycemic index diet combined with exercise reduces insulin resistance, postprandial hyperinsulinemia, and glucose-dependent insulinotropic polypeptide responses in obese, prediabetic humans. The optimal lifestyle intervention that reverses diabetes risk factors is not known. We examined the effect of a low-glycemic index (GI) diet and exercise intervention on glucose metabolism and insulin secretion in obese, prediabetic individuals. Twenty-two participants [mean ± SEM age: 66 ± 1 y; body mass index (in kg/m(2)): 34.4 ± 0.8] underwent a 12-wk exercise-training intervention (1 h/d for 5 d/wk at ≈ 85% of maximum heart rate) while randomly assigned to receive either a low-GI diet (LoGIX; 40 ± 0.3 units) or a high-GI diet (HiGIX; 80 ± 0.6 units). Body composition (measured by using dual-energy X-ray absorptiometry and computed tomography), insulin sensitivity (measured with a hyperinsulinemic euglycemic clamp with [6,6-(2)H(2)]-glucose), and oral glucose-induced insulin and incretin hormone secretion were examined. Both groups lost equal amounts of body weight (-8.8 ± 0.9%) and adiposity and showed similar improvements in peripheral tissue (+76.2 ± 14.9%) and hepatic insulin sensitivity (+27.1 ± 7.1%) (all P < 0.05). However, oral glucose-induced insulin secretion was reduced only in the LoGIX group (6.59 ± 0.86 nmol in the prestudy compared with 4.70 ± 0.67 nmol in the poststudy, P < 0.05), which was a change related to the suppressed postprandial response of glucose-dependent insulinotropic polypeptide. When corrected for changes in β cell glucose exposure, changes in insulin secretion were attenuated in the LoGIX group but became significantly elevated in the HiGIX group. Although lifestyle-induced weight loss improves insulin resistance in prediabetic individuals, postprandial hyperinsulinemia is reduced only when a low-GI diet is consumed. In contrast, a high-GI diet impairs pancreatic β cell and intestinal K cell function despite significant weight loss. These findings highlight the important role of the gut in mediating the effects of a low-GI diet on type 2 diabetes risk reduction.


The B cell response is redundant and highly focused on V1V2 during early subtype C infection in a Zambian seroconverter. High-titer autologous neutralizing antibody responses have been demonstrated during early subtype C human immunodeficiency virus type 1 (HIV-1) infection. However, characterization of this response against autologous virus at the monoclonal antibody (MAb) level has only recently begun to be elucidated. Here we describe five monoclonal antibodies derived from a subtype C-infected seroconverter and their neutralizing activities against pseudoviruses that carry envelope glycoproteins from 48 days (0 month), 2 months, and 8 months after the estimated time of infection. Sequence analysis indicated that the MAbs arose from three distinct B cell clones, and their pattern of neutralization compared to that in patient plasma suggested that they circulated between 2 and 8 months after infection. Neutralization by MAbs representative of each B cell clone was mapped to two residues: position 134 in V1 and position 189 in V2. Mutational analysis revealed cooperative effects between glycans and residues at these two positions, arguing that they contribute to a single epitope. Analysis of the cognate gp120 sequence through homology modeling places this potential epitope near the interface between the V1 and V2 loops. Additionally, the escape mutation R189S in V2, which conferred resistance against all three MAbs, had no detrimental effect on virus replication in vitro. Taken together, our data demonstrate that independent B cells repeatedly targeted a single structure in V1V2 during early infection. Despite this assault, a single amino acid change was sufficient to confer complete escape with minimal impact on replication fitness.


A vaccinia virus deletion mutant reveals the presence of additional inhibitors of NF-kappaB. The classical nuclear factor kappa B (NF-κB) signaling pathway is an important regulator of inflammation and innate immunity that is activated by a wide variety of stimuli, including virus infection, tumor necrosis factor alpha (TNF-α), and interleukin 1β (IL-1β). Poxviruses, including vaccinia virus (VV) and ectromelia virus, encode multiple proteins that function in immune evasion. Recently, a growing number of genes encoded by poxviruses have been shown to target and disrupt the NF-κB signaling pathway. To determine if additional gene products that interfere with NF-κB signaling existed, we used a vaccinia virus deletion mutant, VV811, which is missing 55 open reading frames lacking all known inhibitors of TNF-α-induced NF-κB activation. Immunofluorescence analysis of HeLa cells treated with TNF-α and IL-1β revealed that NF-κB translocation to the nucleus was inhibited in VV811-infected cells. This was further confirmed through Western blotting of cytoplasmic and nuclear extracts for NF-κB. Additionally, VV811 infection inhibited TNF-α-induced IκBα degradation. In contrast to vaccinia virus strain Copenhagen (VVCop)-infected cells, VV811 infection resulted in the dramatic accumulation of phosphorylated IκBα. Correspondingly, coimmunoprecipitation assays demonstrated that the NF-κB-inhibitory IκBα-p65-p50 complex was intact in VV811-infected cells. Significantly, cells treated with 1-β-d-arabinofuranosylcytosine, an inhibitor of poxvirus late gene expression, demonstrated that an additional vaccinia virus late gene was involved in the stabilization of IκBα. Overall, this work indicates that unidentified inhibitors of NF-κB exist in vaccinia virus. The complex inhibition of NF-κB by vaccinia virus illustrates the importance of NF-κB activation in the antiviral response.


Structure-based analysis of the interaction between the simian virus 40 T-antigen origin binding domain and single-stranded DNA. The origin-binding domain (OBD) of simian virus 40 (SV40) large T-antigen (T-Ag) is essential for many of T-Ag's interactions with DNA. Nevertheless, many important issues related to DNA binding, for example, how single-stranded DNA (ssDNA) transits along the T-Ag OBD, have yet to be established. Therefore, X-ray crystallography was used to determine the costructure of the T-Ag OBD bound to DNA substrates such as the single-stranded region of a forked oligonucleotide. A second structure of the T-Ag OBD crystallized in the presence of poly(dT)(12) is also reported. To test the conclusions derived from these structures, residues identified as being involved in binding to ssDNA by crystallography or by an earlier nuclear magnetic resonance study were mutated, and their binding to DNA was characterized via fluorescence anisotropy. In addition, these mutations were introduced into full-length T-Ag, and these mutants were tested for their ability to support replication. When considered in terms of additional homology-based sequence alignments, our studies refine our understanding of how the T-Ag OBDs encoded by the polyomavirus family interact with ssDNA, a critical step during the initiation of DNA replication.


Poliovirus RNA is released from the capsid near a twofold symmetry axis. After recognizing and binding to its host cell, poliovirus (like other nonenveloped viruses) faces the challenge of translocating its genome across a cellular membrane and into the cytoplasm. To avoid entanglement with the capsid, the RNA must exit via a single site on the virion surface. However, the mechanism by which a single site is selected (from among 60 equivalents) is unknown; and until now, even its location on the virion surface has been controversial. To help to elucidate the mechanism of infection, we have used single-particle cryo-electron microscopy and tomography to reconstruct conformationally altered intermediates that are formed by the poliovirion at various stages of the poliovirus infection process. Recently, we reported icosahedrally symmetric structures for two forms of the end-state 80S empty capsid particle. Surprisingly, RNA was frequently visible near the capsid; and in a subset of the virions, RNA was seen on both the inside and outside of the capsid, caught in the act of exiting. To visualize RNA exiting, we have now determined asymmetric reconstructions from that subset, using both single-particle cryo-electron microscopy and cryo-electron tomographic methods, producing independent reconstructions at ∼50-Å resolution. Contrary to predictions in the literature, the footprint of RNA on the capsid surface is located close to a viral 2-fold axis, covering a slot-shaped area of reduced density that is present in both of the symmetrized 80S reconstructions and which extends by about 20 Å away from the 2-fold axis toward each neighboring 5-fold axis.


Template recognition mechanisms by replicase proteins differ between bipartite positive-strand genomic RNAs of a plant virus. Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3' untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism.


A nuclear inhibitor of NF-kappaB encoded by a poxvirus. Poxviruses have evolved various strategies to inhibit cytoplasmic events leading to activation of the nuclear factor κB (NF-κB) signaling pathway, with individual viruses often encoding multiple NF-κB inhibitors. Here, the novel orf virus (ORFV)-encoded protein ORFV002 was shown to inhibit nuclear events regulating NF-κB transcriptional activity. ORFV002 expression in cell cultures significantly decreased wild-type-virus-, tumor necrosis factor alpha (TNF-α)-, and lipopolysaccharide (LPS)-induced NF-κB-mediated gene expression. Expression of ORFV002 in cells, while not affecting phosphorylation or nuclear translocation of NF-κB-p65, markedly decreased TNF-α- and wild-type-virus-induced acetylation of NF-κB-p65, a p300-mediated nuclear modification of NF-κB-p65 that regulates its transactivating activity. ORFV002 was shown to colocalize and interact with NF-κB-p65, and expression of ORFV002 in cell cultures resulted in a reduced interaction of NF-κB-p65 with p300, suggesting that ORFV002 interferes with NF-κB-p65/p300 association. Deletion of ORFV002 from the OV-IA82 genome had no significant effect on ORFV pathogenesis in sheep, indicating that ORFV002 is nonessential for virus virulence in the natural host. This represents the first description of a nuclear inhibitor of NF-κB encoded by a poxvirus.


Cytomegalovirus microRNA expression is tissue specific and is associated with persistence. MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in posttranscriptional regulation. miRNAs are utilized in organisms ranging from plants to higher mammals, and data have shown that DNA viruses also use this method for host and viral gene regulation. Here, we report the sequencing of the small RNAs in rat cytomegalovirus (RCMV)-infected fibroblasts and persistently infected salivary glands. We identified 24 unique miRNAs that mapped to hairpin structures found within the viral genome. While most miRNAs were detected in both samples, four were detected exclusively in the infected fibroblasts and two were specific for the infected salivary glands. The RCMV miRNAs are distributed across the viral genome on both the positive and negative strands, with clusters of miRNAs at a number of locations, including near viral genes r1 and r111. The RCMV miRNAs have a genomic positional orientation similar to that of the miRNAs described for mouse cytomegalovirus, but they do not share any substantial sequence conservation. Similar to other reported miRNAs, the RCMV miRNAs had considerable variation at their 3' and 5' ends. Interestingly, we found a number of specific examples of differential isoform usage between the fibroblast and salivary gland samples. We determined by real-time PCR that expression of the RCMV miRNA miR-r111.1-2 is highly expressed in the salivary glands and that miR-R87-1 is expressed in most tissues during the acute infection phase. Our study identified the miRNAs expressed by RCMV in vitro and in vivo and demonstrated that expression is tissue specific and associated with a stage of viral infection.


Differential regulation of human papillomavirus type 8 by interferon regulatory factors 3 and 7. The genus β human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patients with epidermodysplasia verruciformis, and evidence for its protumorigenic potential in the general population increases. To date, strategies to suppress genus β HPV infections are limited. Interferon regulatory factors IRF-3 and IRF-7 play key roles in the activation of the innate immune response to viral infections. In this study, we show for the first time that both IRF-3 and IRF-7 regulate transcription of a papillomavirus, but with opposing effects. IRF-7, expressed in the suprabasal layers of human epidermis, increased HPV8 late promoter activity via direct binding to viral DNA. UV-B light-induced activation of the HPV8 promoter involved IRF-7 as a downstream effector. In contrast, IRF-3, expressed in all layers of human epidermis, induced strong HPV8 suppression in primary keratinocytes. IRF-3-mediated suppression prevailed over IRF-7-induced HPV8 transcription. Unlike the E6 oncoprotein of the mucosal high-risk HPV16, the HPV8 E6 protein did not bind to IRF-3 and only weakly antagonized its activity. Strong antiviral activity was also observed, when keratinocytes were treated with potent IRF-3 activators, poly(I:C) or RNA bearing 5' phosphates. In conclusion, we show that IRF-3 activation induces a state of cell-autonomous immunity against HPV in primary human keratinocytes. Our study suggests that local application of IRF-3-activating compounds might constitute an attractive novel therapeutic strategy against HPV8-associated diseases, particularly in epidermodysplasia verruciformis patients.


Function of the small hydrophobic protein of J paramyxovirus. At 18,954 nucleotides, the J paramyxovirus (JPV) genome is one of the largest in the family Paramyxoviridae, consisting of eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. To study the function of novel paramyxovirus genes in JPV, a plasmid containing a full-length cDNA clone of the genome of JPV was constructed. In this study, the function of the small hydrophobic (SH) protein of JPV was examined by generating a recombinant JPV lacking the coding sequence of the SH protein (rJPVΔSH). rJPVΔSH was viable and had no growth defect in tissue culture cells. However, more tumor necrosis factor alpha (TNF-α) was produced during rJPVΔSH infection, suggesting that SH plays a role in inhibiting TNF-α production. rJPVΔSH induced more apoptosis in tissue culture cells than rJPV. Virus-induced apoptosis was inhibited by neutralizing antibody against TNF-α, suggesting that TNF-α contributes to JPV-induced apoptosis in vitro. The expression of JPV SH protein inhibited TNF-α-induced NF-κB activation in a reporter gene assay, suggesting that JPV SH protein can inhibit TNF-α signaling in vitro. Furthermore, infection of mice with rJPVΔSH induced more TNF-α expression, indicating that SH plays a role in blocking TNF-α expression in vivo.


Adenovirus membrane penetration activates the NLRP3 inflammasome. Adenovirus type 5 (Ad5) infection of macrophages results in rapid secretion of interleukin-1β (IL-1β) and is dependent on the inflammasome components NLRP3 and ASC and the catalytic activity of caspase-1. Using lentivirus-expressed short hairpin RNA (shRNA) and competitive inhibitors, we show that Ad-induced IL-1β release is dependent upon Toll-like receptor 9 (TLR9) sensing of the Ad5 double-stranded DNA (dsDNA) genome in human cell lines and primary monocyte-derived macrophages but not in mouse macrophages. Additionally, a temperature-sensitive mutant of Ad5 unable to penetrate endosomal membranes, ts1, is unable to induce IL-1β release in TLR2-primed THP-1 cells, suggesting that penetration of endosomal membranes is required for IL-1β release. Disruption of lysosomal membranes and the release of cathepsin B into the cytoplasm are required for Ad-induced NLRP3 activation. Ad5 cell entry also induces reactive oxygen species (ROS) production, and inhibitors of ROS prevent Ad-induced IL-1β release. Ad5 activation of NLRP3 also induces necrotic cell death, resulting in the release of the proinflammatory molecule HMGB1. This work further defines the mechanisms of virally induced inflammasome activation.


Translational control of the abundance of cytoplasmic poly(A) binding protein in human cytomegalovirus-infected cells. Irrespective of their effects on ongoing host protein synthesis, productive replication of the representative alphaherpesvirus herpes simplex virus type 1, the representative gammaherpesvirus Kaposi's sarcoma herpesvirus, and the representative betaherpesvirus human cytomegalovirus [HCMV] stimulates the assembly of the multisubunit, cap-binding translation factor eIF4F. However, only HCMV replication is associated with an increased abundance of eIF4F core components (eIF4E, eIF4G, eIF4A) and the eIF4F-associated factor poly(A) binding protein (PABP). Here, we demonstrate that the increase in translation factor concentration was readily detected in an asynchronous population of HCMV-infected primary human fibroblasts, abolished by prior UV inactivation of virus, and genetically dependent upon viral immediate-early genes. Strikingly, while increased mRNA steady-state levels accompanied the rise in eIF4E and eIF4G protein levels, the overall abundance of PABP mRNA, together with the half-life of the polypeptide it encodes, remained relatively unchanged by HCMV infection. Instead, HCMV-induced PABP accumulation resulted from new protein synthesis and was sensitive to the mTORC1-selective inhibitor rapamycin, which interferes with phosphorylation of the mTORC1 substrate p70 S6K and the translational repressor 4E-BP1. While virus-induced PABP accumulation did not require p70 S6K, it was inhibited by the expression of a dominant-acting 4E-BP1 variant unable to be inactivated by mTORC1. Finally, unlike the situation in alpha- or gammaherpesvirus-infected cells, where PABP is redistributed to nuclei, PABP accumulated in the cytoplasm of HCMV-infected cells. Thus, cytoplasmic PABP accumulation is translationally controlled in HCMV-infected cells via a mechanism requiring mTORC1-mediated inhibition of the cellular 4E-BP1 translational repressor.


A new model of Epstein-Barr virus infection reveals an important role for early lytic viral protein expression in the development of lymphomas. Epstein-Barr virus (EBV) infects cells in latent or lytic forms, but the role of lytic infection in EBV-induced lymphomas is unclear. Here, we have used a new humanized mouse model, in which both human fetal CD34(+) hematopoietic stem cells and thymus/liver tissue are transplanted, to compare EBV pathogenesis and lymphoma formation following infection with a lytic replication-defective BZLF1-deleted (Z-KO) virus or a lytically active BZLF1(+) control. Both the control and Z-KO viruses established long-term viral latency in all infected animals. The infection appeared well controlled in some animals, but others eventually developed CD20(+) diffuse large B cell lymphomas (DLBCL). Animals infected with the control virus developed tumors more frequently than Z-KO virus-infected animals. Specific immune responses against EBV-infected B cells were generated in mice infected with either the control virus or the Z-KO virus. In both cases, forms of viral latency (type I and type IIB) were observed that are less immunogenic than the highly transforming form (type III) commonly found in tumors of immunocompromised hosts, suggesting that immune pressure contributed to the outcome of the infection. These results point to an important role for lytic EBV infection in the development of B cell lymphomas in the context of an active host immune response.


Reovirus infection or ectopic expression of outer capsid protein micro1 induces apoptosis independently of the cellular proapoptotic proteins Bax and Bak. Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein μ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed μ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of μ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for μ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of μ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing μ1, indicating that caspases were not required. Additionally, μ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax(-/-)Bak(-/-) MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak.


Norovirus GII.4 strain antigenic variation. Noroviruses are the principal cause of epidemic gastroenteritis worldwide. Multiple reports have concluded that the major capsid proteins of GII.4 strains, which cause 80% of norovirus infections worldwide, are evolving rapidly, resulting in new epidemic strains. Surrogate neutralization assays using sera from outbreaks and from immunized mice suggest that, as with influenza virus, antigenic variation maintains GII.4 persistence in the face of human population herd immunity. To test this hypothesis, mice were hyperimmunized with virus-like particles (VLPs) representing an early (GII.4-1987) and a contemporary (GII.4-2006) GII.4 strain. Anti-GII.4-1987 IgG monoclonal antibodies (MAbs) strongly reacted with GII.4 VLPs derived between only 1987 and 2002. Ligand binding blockade was more efficient with GII.4-1987 and GII.4-1997 VLPs than with GII.4-2002. Anti-GII.4-2006 IgG MAbs recognized either a broad panel of GII.4 VLPs (1987 to 2006) or a subset of contemporary (2004 to 2006) VLPs. Most 2006 antibodies did not recognize or only poorly recognized GII.4 VLPs of 2007 or 2008, documenting rapid antigenic evolution of GII.4 capsids. Generally, 2006 MAbs blocked homotypic VLP-ligand binding but were unable to block VLPs representing strains primarily circulating during or earlier than 2002. These analyses demonstrate that both subtle and significant evolutionary change has occurred within antibody epitopes between epidemic strains, providing direct evidence that the GII.4 noroviruses are undergoing antigenic variation, likely in response to herd immunity. As with influenza virus, HIV, and hepatitis C virus, norovirus antigenic variation will significantly influence the design of efficacious vaccines and immunotherapeutics against these important human pathogens.


Successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus. Newly emerging viruses often circulate as a heterogeneous swarm in wild animal reservoirs prior to their emergence in humans, and their antigenic identities are often unknown until an outbreak situation. The newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV) and reemerging influenza virus cause disproportionate disease in the aged, who are also notoriously difficult to successfully vaccinate, likely due to immunosenescence. To protect against future emerging strains, vaccine platforms should induce broad cross-reactive immunity that is sufficient to protect from homologous and heterologous challenge in all ages. From initial studies, we hypothesized that attenuated Venezuelan equine encephalitis virus (VEE) replicon particle (VRP) vaccine glycoproteins mediated vaccine failure in the aged. We then compared the efficacies of vaccines bearing attenuated (VRP(3014)) or wild-type VEE glycoproteins (VRP(3000)) in young and aged mice within novel models of severe SARS-CoV pathogenesis. Aged animals receiving VRP(3000)-based vaccines were protected from SARS-CoV disease, while animals receiving the VRP(3014)-based vaccines were not. The superior protection for the aged observed with VRP(3000)-based vaccines was confirmed in a lethal influenza virus challenge model. While the VRP(3000) vaccine's immune responses in the aged were sufficient to protect against lethal homologous and heterologous challenge, our data suggest that innate defects within the VRP(3014) platform mediate vaccine failure. Exploration into the mechanism(s) of successful vaccination in the immunosenescent should aid in the development of successful vaccine strategies for other viral diseases disproportionately affecting the elderly, like West Nile virus, influenza virus, norovirus, or other emerging viruses of the future.