ObjectivesAedes-borne viruses (ABV) affect humans on every inhabited continent and frequently cause epidemics. Recent epidemics of chikungunya and Zika viruses (ZIKV) highlight that preparedness for future epidemics requires assessment of susceptibility, particularly among high-risk groups. We sought to determine immunity against the three major circulating ABV among pregnant women in an ABV-endemic area of Colombia.MethodsA cross-sectional seroprevalence study was performed, enrolling women presenting to Labor and Delivery. Cord blood and maternal peripheral blood samples were obtained. IgG seroprevalence to flaviviruses and chikungunya was determined by ELISA. An abbreviated neutralization test was used to estimate the frequency and magnitude of immunity to Zika and four dengue serotypes. Cluster analyses explored epidemiologic factors associated with seroprevalence.ResultsMost women exhibited high levels of neutralizing antibodies to one or more ABV; however, nearly 20% were seronegative for flaviviruses. Our research took place after the epidemic peak of the ZIKV outbreak in Colombia in 2016. However, only 20% of pregnant women had high levels of Zika-neutralizing antibodies consistent with likely protective immunity to ZIKV.ConclusionHence, a high proportion of pregnant women in Risaralda remain susceptible to one or more ABV including the teratogenic ZIKV, indicating a risk for future epidemics in this region.

Background: Antibodies against SARS-CoV-2 can be detected by various testing platforms, but a detailed understanding of assay performance is critical. Methods: We developed and validated a simple enzyme-linked immunosorbent assay (ELISA) to detect IgG binding to the receptor-binding domain (RBD) of SARS-CoV-2, which was then applied for surveillance. ELISA results were compared to a set of complimentary serologic assays using a large panel of clinical research samples. Results: The RBD ELISA exhibited robust performance in ROC curve analysis (AUC> 0.99; Se = 89%, Sp = 99.3%). Antibodies were detected in 23/353 (6.5%) healthcare workers, 6/9 RT-PCR-confirmed mild COVID-19 cases, and 0/30 non-COVID-19 cases from an ambulatory site. RBD ELISA showed a positive correlation with neutralizing activity (p = <0.0001, R2 = 0.26). Conclusions: We applied a validated SARS-CoV-2-specific IgG ELISA in multiple contexts and performed orthogonal testing on samples. This study demonstrates the utility of a simple serologic assay for detecting prior SARS-CoV-2 infection, particularly as a tool for efficiently testing large numbers of samples as in population surveillance. Our work also highlights that precise understanding of SARS-CoV-2 infection and immunity at the individual level, particularly with wide availability of vaccination, may be improved by orthogonal testing and/or more complex assays such as multiplex bead assays.

Abstract Background Sensitive and specific SARS-CoV-2 antibody diagnostics are urgently needed to estimate the seroprevalence of SARS-CoV-2 infection in both the general population and special risk groups. Moreover, validated serologic assays are critical to understanding immunity to SARS-CoV-2 infection over time and identifying correlates of protection. Methods An enzyme-linked immunosorbent assay (ELISA) protocol to detect antibodies (IgG) that bind the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was validated and ROC curve analysis performed by testing a large panel of pre-pandemic sera (n=162) and convalescent sera from RT-PCR-confirmed COVID-19 cases (n=60). We then applied this test in two cohorts: 1) Healthcare personnel (HCP) that were enrolled in a longitudinal surveillance cohort just after peak local transmission and 2) Mildly ill patients being tested for SARS-CoV-2 infection by RT-PCR from NP swabs in an ambulatory testing clinic. Demographics of mildly symptomatic patients tested for SARS-CoV-2 with RT-PCR Results ROC curve analysis yielded an AUC of 0.9953, with a sensitivity and specificity at 91.67% and 99.38% at the optimal OD normalization threshold of 0.20. In 240 HCP surveilled at enrollment, 5.83% had positive IgG results. Of 19 symptomatic patients who presented to the ambulatory clinic, 5/19 had a positive PCR. In convalescence (13–74 days post symptom onset), 3 of those 5 were positive for IgG. Validation of the SARS-CoV-2 RBD ELISA ROC Curve Analysis Conclusion We demonstrated high sensitivity and specificity of the SARS-CoV-2 RBD ELISA. This simple assay is an efficient way to track seroconversion and duration of antibody responses to SARS-CoV-2 for different populations, particularly since RBD-binding antibodies have been shown to correlate with neutralization activity and may be useful to determine protective immunity following natural infection or vaccination. Ongoing work will assess variation in magnitude, character and duration of antibody responses in key populations and seek to maximize deployability of large-scale SARS-CoV-2 serology. Disclosures Jessica Howard-Anderson, MD, MSc, Antibacterial Resistance Leadership Group (ARLG) (Other Financial or Material Support, The ARLG fellowship provides salary support for ID fellowship and mentored research training) Nadine Rouphael, MD, Lilly (Grant/Research Support)Merck (Grant/Research Support)Pfizer (Grant/Research Support)Quidel (Grant/Research Support)Sanofi Pasteur (Grant/Research Support)

Background: Healthcare personnel (HCP) may be at increased risk for COVID-19, but differences in risk by work activities are poorly defined Centers for Disease Control and Prevention recommends cohorting hospitalized patients with COVID-19 to reduce in-hospital transmission of SARS-CoV-2, but it is unknown if occupational and non-occupational behaviors differ based on exposure to COVID-19 units Methods: We analyzed a subset of HCP from an ongoing CDC-funded SARSCoV- 2 serosurveillance study HCP were recruited from four Atlanta hospitals of different sizes and patient populations All HCP completed a baseline REDCap survey We used logistic regression to compare occupational activities and infection prevention practices among HCP stratified by exposure to COVID-19 units: low (0% of shifts), medium (1-49% of shifts) or high (≥50% of shifts) Results: Of 211 HCP enrolled (36% emergency department [ED] providers, 35% inpatient RNs, 17% inpatient MDs/APPs, 7% radiology technicians and 6% respiratory therapists [RTs]), the majority (79%) were female and the median age was 35 years Nearly half of the inpatient MD/APPs (46%) and RNs (47%) and over two-thirds of the RTs (67%) worked primarily in the ICU Aerosol generating procedures were common among RNs, MD/APPs, and RTs (26-58% performed ≥1), but rare among ED providers (0-13% performed ≥1) Compared to HCP with low exposure to COVID-19 units, those with medium or high exposure spent a similar proportion of shifts directly at the bedside and were about as likely to practice universal masking Being able to consistently social distance from co-workers was rare (33%);HCP with high exposure to COVID-19 units were less likely to report social distancing in the workplace compared to those with low exposure;however, this was not significantly different (OR 0 6;95% CI: 0 3, 1 1) Concerns about personal protective equipment in COVID-19 units were similar across levels of exposure (Table 1) Conclusion: The proportion of time spent in dedicated COVID-19 units did not appear to influence time HCP spend directly at the bedside or infection prevention practices (social distancing and universal masking) in the workplace Risk for SARSCoV- 2 infection in HCP may depend more on factors acting at the individual level rather than those related to location of work (Table Presented)

Abstract Background The spatial distribution and burden of dengue in sub-Saharan Africa remains highly uncertain, despite high levels of ecological suitability. The goal of this study was to describe the epidemiology of dengue among a cohort of febrile children presenting to outpatient facilities located in areas of western Uganda with differing levels of urbanicity and malaria transmission intensity. Methods Eligible children were first screened for malaria using rapid diagnostic tests. Children with a negative malaria result were tested for dengue using a combination NS1/IgM/IgG rapid test (SD Bioline Dengue Duo). Confirmatory testing by RT-PCR was performed in a subset of participants. Antigen-capture ELISA was performed to estimate seroprevalence. Results Only 6 of 1416 (0.42%) children had a positive dengue rapid test, while none of the RT-PCR results were positive. ELISA testing demonstrated reactive IgG antibodies in 28 (2.2%) participants with the highest prevalence seen at the urban site in Mbarara (19 of 392, 4.9%, p < 0.001). Conclusions Overall, these findings suggest that dengue, while present, is an uncommon cause of non-malarial, pediatric febrile illness in western Uganda. Further investigation into the eocological factors that sustain low-level transmission in urban settings are urgently needed to reduce the risk of epidemics.

Abstract Background Flavivirus infection represents a major public health problem, with over 2 billion people at risk for one or more infections each year. There are no licensed antiviral treatments for flaviviruses, and effective vaccines are lacking for several. Accurate diagnosis of flaviviruses is also complicated by serologic cross-reactivity. Thus, there is an urgent need to develop public health tools and interventions to reduce the burden of flavivirus infection worldwide. Methods We recently isolated two potently neutralizing human monoclonal antibodies (NmAb) against Zika, which exhibit activity against multiple strains of Zika but do not bind the closely related dengue virus. Mapping studies revealed that the NmAb target distinct epitopes on the envelope protein (E). A competition ELISA (ZE-BOB) was developed with the two NmAb to test the hypotheses that neutralizing Ab responses in the general population would target the same epitopes on Zika E as the novel NmAb. Results We found that competitive Ab responses were detected in 19 of 19 (100%) convalescent sera from travelers with confirmed Zika infection. We tested a panel of 20 sera from individuals with either primary or secondary prior dengue infection and found that 18/20 (90%) contained IgG reactive with Zika virus; however, 0/20 (0%) exhibited ZE-BOB activity. These results indicate that the epitopes targeted by these two Zika NmAb are consistently immunogenic humans infected by Zika. Additionally, these epitopes elicit type-specific Ab to Zika, providing a basis for development of simple serodiagnostic assays with utility in epidemiologic studies as well as in the clinical setting. Because NAb are key determinants of long-term protection against future flavivirus infection, we further hypothesized that the results of the ZE-BOB assay may simultaneously provide a correlate of immunity, which would be a critical tool for vaccine development. In comparing the 50% neutralization titer against Zika with the magnitude of competition by ZE-BOB, there was no correlation in these values, but sera tended to be positive in both or neither assay. Conclusion Therefore, the ZE-BOB assay constitutes a novel tool that is widely deployable for purposes ranging from clinical diagnosis to epidemiologic monitoring to vaccine development for Zika. Disclosures All authors: No reported disclosures.

Muscle atrophy in metabolic conditions like chronic kidney disease (CKD) and diabetes are associated with glucocorticoid production, dysfunctional insulin/Akt/FoxO3 signaling and increased myostatin expression. We recently found that CREB, a transcription factor proposed to regulate myostatin expression, is highly phosphorylated in some wasting conditions. Based on a novel Akt-PDE3/4 signaling paradigm, we hypothesized that reduced Akt signaling contributes to CREB activation and myostatin expression. C2C12 myotubes were incubated with dexamethasone (Dex), an atrophy-inducing synthetic glucocorticoid. Akt/CREB signaling and myostatin expression were evaluated by immunoblot and qPCR analyses. Inhibitors of Akt, phosphodiesterase (PDE)-3/4, and protein kinase A (PKA) signaling were used to test our hypothesis. Incubating myotubes with Dex for 3-24 h inhibited Akt phosphorylation and enhanced CREB phosphorylation as well as myostatin mRNA and protein. Inhibition of PI3K/Akt signaling with LY294002 similarly increased CREB phosphorylation. Isobutyl-methylxanthine (IBMX, a pan PDE inhibitor), milrinone (PDE3 inhibitor) and rolipram (PDE4 inhibitor) augmented CREB phosphorylation and myostatin expression. Inhibition of protein kinase A by PKI reverted Dex- or IBMX-induced CREB phosphorylation and myostatin expression. Our study provides evidence supporting a newly identified mechanism by which a glucocorticoid-related reduction in Akt signaling contributes to myostatin expression via CREB activation. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.

Muscle atrophy in diseases like chronic kidney disease and diabetes is associated with dysfunctional regulation of insulin/Akt/FoxO3 and myostatin‐SMAD3 signaling pathways. We recently found CREB, a transcription factor that regulates maintenance and regeneration of skeletal muscle, is phosphorylated in wasting conditions. In silico analysis identifies a CREB binding site in the myostatin promoter. This study investigated the mechanism by which atrophy‐inducing conditions regulate CREB phosphorylation and its impact on myostatin expression. Differentiated C2C12 myotubes were incubated with combinations of dexamethasone (Dex), LY294002 (PI‐3 kinase inhibitor), H89 (protein kinase A inhibitor), isobutylmethyl xanthine (IBMX, a general phosphodiesterase (PDE) inhibitor), milrinone (PDE3 inhibitor), or rolipram (PDE4 inhibitor) for up to 24 h. Cells were harvested for analysis of proteins or mRNAs. Treatment of myotubes with Dex for 6–24h increased CREB phosphorylation (pCREB) within 3 h and sustained the increase over 24h. Dex also inhibited Akt phosphorylation (pAkt) over a similar time frame. Inhibition of pAkt by LY294002 mimicked Dex by increasing pCREB; the actions of LY294002 and Dex on pCREB were not additive. Since Akt can activate PDE3/PDE4 and reduce cAMP/PKA signaling in non‐muscle cells, we tested whether Dex‐induced inhibition of PDE3/4 increases pCREB. Without Dex, IBMX, milrinone, and rolipram increased pCREB. The Dex‐induced increase in pCREB was attenuated by the PKA inhibitor H89. When added individually, Dex, milrinone, rolipram and IBMX increase myostatin mRNA and protein significantly. Our study identifies a new mechanism by which atrophy‐inducing conditions activate CREB and contribute to myostatin expression. Signals that attenuate insulin/Akt signaling reduce the PDE3/4 activity. This leads to an increase in cAMP, PKA activity, CREB phosphorylation and myostatin expression.Support or Funding InformationFunded by NIH RO1 DK95610 and VA Merit X01BX001456

Type 2 diabetes mellitus is a complex metabolic disease, and cardiovascular disease is a leading complication of diabetes. Epicardial adipose tissue surrounding the heart displays biochemical, thermogenic, and cardioprotective properties. However, the metabolic cross-talk between epicardial fat and the myocardium is largely unknown. This study sought to understand epicardial adipose tissue metabolism from heart failure patients with or without diabetes. We aimed to unravel possible differences in glucose and lipid metabolism between human epicardial and subcutaneous adipocytes and elucidate the potential underlying mechanisms involved in heart failure. Insulin-stimulated [(14)C]glucose uptake and isoproterenol-stimulated lipolysis were measured in isolated epicardial and subcutaneous adipocytes. The expression of genes involved in glucose and lipid metabolism was analyzed by reverse transcription-polymerase chain reaction in adipocytes. In addition, epicardial and subcutaneous fatty acid composition was analyzed by high-resolution proton nuclear magnetic resonance spectroscopy. The difference between basal and insulin conditions in glucose uptake was significantly decreased (P= 0.006) in epicardial compared with subcutaneous adipocytes. Moreover, a significant (P< 0.001) decrease in the isoproterenol-stimulated lipolysis was also observed when the two fat depots were compared, and it was strongly correlated with lipolysis, lipid storage, and inflammation-related gene expression. Moreover, the fatty acid composition of these tissues was significantly altered by diabetes. These results emphasize potential metabolic differences between both fat depots in the presence of heart failure and highlight epicardial fat as a possible therapeutic target in situ in the cardiac microenvironment.

Objective. Cyclosporine A (CsA) and sirolimus (SRL) are immunosuppressive agents (IA) associated with new onset diabetes after transplantation and dyslipidemia. We aim to evaluate the molecular effects of CsA (5 mg/kg/day) and SRL (1 mg/kg/day) treatment for 3 and 9 weeks on lipid metabolism, in Wistar rats.Materials/Methods. Lipolysis was evaluated in isolated adipocytes, while triglycerides (TG) and non-esterified fatty acid (NEFA) were measured in serum. Gene and protein expression involved in lipid metabolism was assessed in adipose tissue and liver.Results. CsA and SRL treatments of rats for 3 and 9 weeks increased isoproterenol-stimulated lipolysis by 5-9 fold and 4-6 fold in isolated adipocytes, respectively. While CsA increased adipocyte weight and diameter, as well as NEFA and TG levels in circulation after 9 weeks, SRL treatment caused ectopic deposition of TG in the liver after 3 weeks. Moreover, ACC1 and FAS protein expression was increased after 3 weeks (>100%, p < 0.01), while HSL was increased after 9 weeks of CsA treatment. On the other hand, SRL decreased the expression of lipogenic genes, including ACC1 (50%, p < 0.05), lipin1 (25%, p < 0.05), PPAR-gamma (42%, p < 0.05) and SCD1 (80%, p < 0.001) in adipose tissue, after 3 weeks of treatment.Conclusion. The effects of both IAs on expression of lipolytic and lipogenic genes suggest that these agents influence lipid metabolism, thus contributing to the dyslipidemia observed during immunosuppressive therapy. (C) 2014 Elsevier Inc. All rights reserved.

Cyclosporin A (CsA) and sirolimus (SRL) are immunosuppressive agents (IAs) associated with dyslipidemia, insulin resistance and new onset diabetes after transplantation (NODAT). However, the molecular mechanisms involved are not fully understood. We investigated the effects of six-week treatment of either CsA or SRL on glucose and lipid metabolism in Wistar rats. The results show that, compared with vehicle-treated rats, SRL-treated rats were significantly lighter starting at week 5. CsA or SRL caused glucose intolerance, increased storage of lipids in the liver and skeletal muscle, and decreased the insulin-stimulated glucose uptake in isolated adipocytes. Furthermore, these agents significantly decreased genes involved in insulin action and glucose uptake, such as, IRS-1, Glut4 and Glut1, and increased genes and/or proteins involved in hepatic lipogenesis and gluconeogenesis, while decreasing them in adipose tissue. After either treatment PGC1α gene expression was down regulated in skeletal muscle, an important player in fatty acid oxidation. Moreover, there was an increase in IL-6 gene expression in adipose tissue in the SRL-treated rats, suggesting stimulation of lipolysis. The results of the present study suggest that CsA and SRL lead to metabolic alterations in liver, muscle and adipose tissue, which may contribute to the development of dyslipidemia and insulin resistance associated with immunosuppressive therapy.

Body weight is regulated by coordinating energy intake and energy expenditure. Transforming growth factor beta (TGF beta)/bone morphogenetic protein (BMP) signaling has been shown to regulate energy balance in lower organisms, but whether a similar pathway exists in mammals is unknown. We have previously demonstrated that BMP7 can regulate brown adipogenesis and energy expenditure. In the current study, we have uncovered a novel role for BMP7 in appetite regulation. Systemic treatment of diet-induced obese mice with BMP7 resulted in increased energy expenditure and decreased food intake, leading to a significant reduction in body weight and improvement of metabolic syndrome. Similar degrees of weight loss with reduced appetite were also observed in BMP7-treated ob/ob mice, suggesting a leptin-independent mechanism utilized by BMP7. Intracerebroventricular administration of BMP7 to mice led to an acute decrease in food intake, which was mediated, at least in part, by a central rapamycin-sensitive mTOR-p70S6 kinase pathway. Together, these results underscore the importance of BMP7 in regulating both food intake and energy expenditure, and suggest new therapeutic approaches for obesity and its comorbidities.-Townsend, K. L., Suzuki, R., Huang, T. L., Jing, E., Schulz, T. J., Lee, K., Taniguchi, C. M., Espinoza, D. O., McDougall, L. E., Zhang, H., He, T.-C., Kokkotou, E., Tseng, Y.-H. Bone morphogenetic protein 7 (BMP7) reverses obesity and regulates appetite through a central mTOR pathway. FASEB J. 26, 2187-2196 (2012). www.fasebj.org

Brown adipose tissue plays an important role in obesity, insulin resistance, and diabetes. We have previously shown that the transition from brown preadipocytes to mature adipocytes is mediated in part by insulin receptor substrate (IRS)-1 and the cell cycle regulator protein necdin. In this study, we used pharmacological inhibitors and adenoviral dominant negative constructs to demonstrate that this transition involves IRS-1 activation of Ras and ERK1/2, resulting in phosphorylation of cAMP response element-binding protein (CREB) and suppression of necdin expression. This signaling did not include an elevation of intracellular calcium. A constitutively active form of CREB expressed in IRS-1 knockout cells decreased necdin promoter activity, necdin mRNA, and necdin protein levels, leading to a partial restoration of differentiation. By contrast, forkhead box protein (Fox)O1, which is regulated by the phosphoinositide 3 kinase-Akt pathway, increased necdin promoter activity. Based on reporter gene assays using truncations of the necdin promoter and chromatin immunoprecipitation studies, we demonstrated that CREB and FoxO1 are recruited to the necdin promoter, likely interacting with specific consensus sequences in the proximal region. Based on these results, we propose that insulin/IGF-I act through IRS-1 phosphorylation to stimulate differentiation of brown preadipocytes via two complementary pathways: 1) the Ras-ERK1/2 pathway to activate CREB and 2) the phosphoinositide 3 kinase-Akt pathway to deactivate FoxO1. These two pathways combine to decrease necdin levels and permit the clonal expansion and coordinated gene expression necessary to complete brown adipocyte differentiation.

Environmental factors, such as the macronutrient composition of the diet, can have a profound impact on risk of diabetes and metabolic syndrome. In the present study we demonstrate how a single, simple dietary factor—leucine—can modify insulin resistance by acting on multiple tissues and at multiple levels of metabolism. Mice were placed on a normal or high fat diet (HFD). Dietary leucine was doubled by addition to the drinking water. mRNA, protein and complete metabolomic profiles were assessed in the major insulin sensitive tissues and serum, and correlated with changes in glucose homeostasis and insulin signaling. After 8 weeks on HFD, mice developed obesity, fatty liver, inflammatory changes in adipose tissue and insulin resistance at the level of IRS-1 phosphorylation, as well as alterations in metabolomic profile of amino acid metabolites, TCA cycle intermediates, glucose and cholesterol metabolites, and fatty acids in liver, muscle, fat and serum. Doubling dietary leucine reversed many of the metabolite abnormalities and caused a marked improvement in glucose tolerance and insulin signaling without altering food intake or weight gain. Increased dietary leucine was also associated with a decrease in hepatic steatosis and a decrease in inflammation in adipose tissue. These changes occurred despite an increase in insulin-stimulated phosphorylation of p70S6 kinase indicating enhanced activation of mTOR, a phenomenon normally associated with insulin resistance. These data indicate that modest changes in a single environmental/nutrient factor can modify multiple metabolic and signaling pathways and modify HFD induced metabolic syndrome by acting at a systemic level on multiple tissues. These data also suggest that increasing dietary leucine may provide an adjunct in the management of obesity-related insulin resistance.

Background Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca2+. Methodology Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme. Principal Findings NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI. Conclusions/Significance We propose that W542 and Ca2+ are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca2+ is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis.