Characterization of Antibodies against Receptor Activity-Modifying Protein 1 (RAMP1): A Cautionary Tale.

Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field.

Estimating the Burden of Pandemic Infectious Disease: The Case of the Second Wave of Pandemic Influenza H1N1 in Forsyth County, North Carolina.

BACKGROUND: Understanding the burden of influenza A(H1N1)pdm09 virus during the second wave of 2009-2010 is important for future pandemic planning. METHODS: Persons who presented to the emergency department (ED) or were hospitalized with fever and/or acute respiratory symptoms at the academic medical center in Forsyth County, North Carolina were prospectively enrolled and underwent nasal/throat swab testing for influenza A(H1N1)pdm09. Laboratory-confirmed cases of influenza A(H1N1)pdm09 virus identified through active surveillance were compared by capture-recapture analysis to those identified through independent, passive surveillance (physician-ordered influenza testing). This approach estimated the number of total cases, including those not captured by either surveillance method. A second analysis estimated the total number of influenza A(H1N1)pdm09 cases by multiplying weekly influenza percentages determined via active surveillance by weekly counts of influenza-associated discharge diagnoses from administrative data. Market share adjustments were used to estimate influenza A(H1N1)pdm09 virus ED visits or hospitalizations per 1,000 residents. RESULTS: Capture-recapture analysis estimated that 753 residents (95% confidence interval [CI], 424-2,735) with influenza A(H1N1)pdm09 virus were seen in the academic medical center from September 2009 through mid-April 2010; this result yielded an estimated 4.7 (95% CI, 2.6-16.9) influenza A(H1N1)pdm09 virus ED visits or hospitalizations per 1,000 residents. Similarly, 708 visits were estimated using weekly influenza percentages and influenza-associated discharge diagnoses, yielding an estimated 4.4 influenza A(H1N1)pdm09 virus ED visits or hospitalizations per 1,000 residents. CONCLUSION: This study demonstrates that the burden of influenza A(H1N1)pdm09 virus in ED and inpatient settings by capture-recapture analysis was 4-5 per 1,000 residents; this rate was approximately 8-fold higher than that detected by physician-ordered influenza testing.

Adrenomedullin: new inhibitory regulator for cortisol synthesis and secretion.

Preterm birth is associated with immaturity of several crucial physiological functions notably those prevailing in the lung and kidney. Recently, a steroid secretion deficiency was identified in very preterm neonates, associated with a partial yet transient deficiency in 11ß-hydroxylase activity, sustaining cortisol synthesis. However, the P450c11ß enzyme is expressed in preterm adrenal glands, we hypothesized an inhibition of cortisol production by adrenomedullin (ADM), a peptide highly produced in neonates and whose effect on steroidogenesis remains poorly known. We studied the effects of ADM on three models: 104 cord-blood samples of the PREMALDO neonate cohort, genetically targeted mice overexpressing ADM, and two human adrenocortical cell lines (H295R and HAC15 cells). Mid-regional-proADM (MR-proADM) quantification in cord-blood samples showed strong negative correlation with gestational age (P = 0.0004), cortisol production (P < 0.0001), and 11ß-hydroxylase activity index (P < 0.0001). Mean MR-proADM was higher in very preterm than in term neonates (1.12 vs 0.60 nmol/L, P < 0.0001). ADM-overexpression mice revealed a lower 11ß-hydroxylase activity index (P < 0.05). Otherwise, aldosterone levels measured by LC-MS/MS were higher in ADM-overexpression mice (0.83 vs 0.46 ng/mL, P < 0.05). More importantly, the negative relationship between adrenal ADM expression and aldosterone production found in control was lacking in the ADM-overexpression mice. Finally, LC-MS/MS and gene expression studies on H295R and HAC15 cells revealed an ADM-induced inhibition of both cortisol secretion in cell supernatants and CYP11B1 expression. Collectively, our results converge toward an inhibitory effect of ADM on glucocorticoid synthesis in humans and should be considered to explain the steroid secretion deficiency observed at birth in premature newborns.

Calcitonin-Receptor-Like Receptor Signaling Governs Intestinal Lymphatic Innervation and Lipid Uptake.

The absorption of dietary fat requires complex neuroendocrine-mediated regulation of chylomicron trafficking through enterocytes and intestinal lymphatic vessels. Calcitonin-receptor-like receptor (Calcrl) is a G protein-coupled receptor that can bind either a lymphangiogenic ligand adrenomedullin, with coreceptor RAMP2, or the neuropeptide CGRP, with coreceptor RAMP1. The extent to which this common GPCR controls lipid absorption via lymphatics or enteric innervation remains unclear. We used conditional and inducible genetic deletion of Calcrl in lymphatics to elucidate the pathophysiological consequences of this receptor pathway under conditions of high-fat diet. Inefficient absorption of dietary fat coupled with altered lymphatic endothelial junctions in Calcrl fl/fl /Prox1-CreER T2 mice results in excessive, transcellular lipid accumulation and abnormal enterocyte chylomicron processing and failure to gain weight. Interestingly, Calcrl fl/fl /Prox1-CreER T2 animals show reduced and disorganized mucosal and submucosal innervation. Consistently, mice with genetic loss of the CGRP coreceptor RAMP1 also displayed mucosal and submucosal innervation deficits, substantiating the CGRP-biased function of Calcrl in the neurolymphocrine axis. Thus, the common Calcrl receptor is a critical regulator of lipid absorption through its cell-specific functions in neurolymphocrine crosstalk.

Genetic loss of proadrenomedullin N-terminal 20 peptide (PAMP) in mice is compatible with survival.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are small peptides derived from a common precursor, pre-proadrenomedullin. Although AM and PAMP share hypotensive effects in the cardiovascular system, the peptides also exert diverse and distinct effects on endocrine physiology, innate immunity, cytoskeletal biology and receptor signaling pathways. Tremendous knowledge has been gleaned from the study of several genetic animal models of AM deletion or overexpression, some of which also simultaneously delete the coding region for PAMP peptide. However, deletion of PAMP without concurrent deletion of AM in an animal model is not currently available for the study of PAMP function. Here, we present the generation of AdmΔPAMP/ΔPAMP and AdmΔPAMP/- mice, which lack the coding sequence for PAMP while preserving the coding sequence for AM. AdmΔPAMP/ΔPAMP mice survive to adulthood without any obvious abnormalities and are fertile, though AdmΔPAMP/- females have small litters. Interestingly, these animals express lower levels of Adm mRNA and AM peptide than wild type animals, but these levels are still compatible with survival. Importantly, despite reduced levels, the spatiotemporal expression of AM peptide within the hearts of AdmΔPAMP/- mice remains similar to wild type animals. AdmΔPAMP/ΔPAMP mice are now a publicly available tool for future investigations of PAMP function.

Lymphatic deletion of calcitonin receptor-like receptor exacerbates intestinal inflammation.

Lymphatics play a critical role in maintaining gastrointestinal homeostasis and in the absorption of dietary lipids, yet their roles in intestinal inflammation remain elusive. Given the increasing prevalence of inflammatory bowel disease, we investigated whether lymphatic vessels contribute to, or may be causative of, disease progression. We generated a mouse model with temporal and spatial deletion of the key lymphangiogenic receptor for the adrenomedullin peptide, calcitonin receptor-like receptor (Calcrl), and found that the loss of lymphatic Calcrl was sufficient to induce intestinal lymphangiectasia, characterized by dilated lacteals and protein-losing enteropathy. Upon indomethacin challenge, Calcrlfl/fl/Prox1-CreERT2 mice demonstrated persistent inflammation and failure to recover and thrive. The epithelium and crypts of Calcrlfl/fl/Prox1-CreERT2 mice exhibited exacerbated hallmarks of disease progression, and the lacteals demonstrated an inability to absorb lipids. Furthermore, we identified Calcrl/adrenomedullin signaling as an essential upstream regulator of the Notch pathway, previously shown to be critical for intestinal lacteal maintenance and junctional integrity. In conclusion, lymphatic insufficiency and lymphangiectasia caused by loss of lymphatic Calcrl exacerbates intestinal recovery following mucosal injury and underscores the importance of lymphatic function in promoting recovery from intestinal inflammation.

Relative timing of influenza disease by age group.

A detailed understanding of influenza movement in communities during yearly epidemics is needed to inform improved influenza control programs. We sought to determine the relative timing of influenza presentation and symptom onset by age group and influenza strain. Prospective, laboratory-confirmed surveillance was performed over three moderate influenza seasons in emergency departments and inpatient settings of both medical centers in Winston-Salem, NC. Influenza disease presented first in school age children through community epidemics of influenza A(H1N1)pdm09 and influenza B, and first in persons 5-49 years old for influenza A(H3N2). This finding indicates that influenza prevention in persons 5-49 years of age may be particularly important in influenza epidemic control.

Evaluation of the limit of detection of the BD Veritor™ system flu A+B test and two rapid influenza detection tests for influenza virus.

We evaluated the limits of detection of 3 rapid influenza diagnostic tests-BD Veritor™ System for Flu A+B, Binax NOW® Influenza A+B, and QuickVue® Influenza-for influenza strains circulating in 2010-2012. Limits of detection varied by influenza strain, with Veritor™ Flu A+B test showing the lowest limit of detection for all strains.