Genetically engineered minipigs model the major clinical features of human neurofibromatosis type 1.

Neurofibromatosis Type 1 (NF1) is a genetic disease caused by mutations in Neurofibromin 1 (NF1). NF1 patients present with a variety of clinical manifestations and are predisposed to cancer development. Many NF1 animal models have been developed, yet none display the spectrum of disease seen in patients and the translational impact of these models has been limited. We describe a minipig model that exhibits clinical hallmarks of NF1, including café au lait macules, neurofibromas, and optic pathway glioma. Spontaneous loss of heterozygosity is observed in this model, a phenomenon also described in NF1 patients. Oral administration of a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor suppresses Ras signaling. To our knowledge, this model provides an unprecedented opportunity to study the complex biology and natural history of NF1 and could prove indispensable for development of imaging methods, biomarkers, and evaluation of safety and efficacy of NF1-targeted therapies

Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure

Background: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. Methods: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. Results: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/invmice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. Conclusions: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis

Phosphatidylserine on viable sperm and phagocytic machinery in oocytes regulate mammalian fertilization

Fertilization is essential for species survival. Although Izumo1 and Juno are critical for initial interaction between gametes, additional molecules necessary for sperm: egg fusion on both the sperm and the oocyte remain to be defined. Here, we show that phosphatidylserine (PtdSer) is exposed on the head region of viable and motile sperm, with PtdSer exposure progressively increasing during sperm transit through the epididymis. Functionally, masking phosphatidylserine on sperm via three different approaches inhibits fertilization. On the oocyte, phosphatidylserine recognition receptors BAl1, CD36, Tim-4, and Mer-TK contribute to fertilization. Further, oocytes lacking the cytoplasmic ELMO1, or functional disruption of RAC1 (both of which signal downstream of BAl1/BAl3), also affect sperm entry into oocytes. Intriguingly, mammalian sperm could fuse with skeletal myoblasts, requiring PtdSer on sperm and BAl1/3, ELMO2, RAC1 in myoblasts. Collectively, these data identify phosphatidylserine on viable sperm and PtdSer recognition receptors on oocytes as key players in sperm: egg fusion

Development of a lung slice preparation for recording ion channel activity in alveolar epithelial type I cells

BACKGROUND: Lung fluid balance in the healthy lung is dependent upon finely regulated vectorial transport of ions across the alveolar epithelium. Classically, the cellular locus of the major ion transport processes has been widely accepted to be the alveolar type II cell. Although evidence is now emerging to suggest that the alveolar type I cell might significantly contribute to the overall ion and fluid homeostasis of the lung, direct assessment of functional ion channels in type I cells has remained elusive. METHODS: Here we describe a development of a lung slice preparation that has allowed positive identification of alveolar type I cells within an intact and viable alveolar epithelium using living cell immunohistochemistry. RESULTS: This technique has allowed, for the first time, single ion channels of identified alveolar type I cells to be recorded using the cell-attached configuration of the patch-clamp technique. CONCLUSION: This exciting new development should facilitate the ascription of function to alveolar type I cells and allow us to integrate this cell type into the general model of alveolar ion and fluid balance in health and disease

Genetic Predisposition for Type 2 Diabetes, but Not for Overweight/Obesity, Is Associated with a Restricted Adipogenesis

BACKGROUND: Development of Type 2 diabetes, like obesity, is promoted by a genetic predisposition. Although several genetic variants have been identified they only account for a small proportion of risk. We have asked if genetic risk is associated with abnormalities in storing excess lipids in the abdominal subcutaneous adipose tissue. METHODOLOGY/PRINCIPAL FINDINGS: We recruited 164 lean and 500 overweight/obese individuals with or without a genetic predisposition for Type 2 diabetes or obesity. Adipose cell size was measured in biopsies from the abdominal adipose tissue as well as insulin sensitivity (HOMA index), HDL-cholesterol and Apo AI and Apo B. 166 additional non-obese individuals with a genetic predisposition for Type 2 diabetes underwent a euglycemic hyperinsulinemic clamp to measure insulin sensitivity. Genetic predisposition for Type 2 diabetes, but not for overweight/obesity, was associated with inappropriate expansion of the adipose cells, reduced insulin sensitivity and a more proatherogenic lipid profile in non-obese individuals. However, obesity per se induced a similar expansion of adipose cells and dysmetabolic state irrespective of genetic predisposition. CONCLUSIONS/SIGNIFICANCE: Genetic predisposition for Type 2 diabetes, but not obesity, is associated with an impaired ability to recruit new adipose cells to store excess lipids in the subcutaneous adipose tissue, thereby promoting ectopic lipid deposition. This becomes particularly evident in non-obese individuals since obesity per se promotes a dysmetabolic state irrespective of genetic predisposition. These results identify a novel susceptibility factor making individuals with a genetic predisposition for Type 2 diabetes particularly sensitive to the environment and caloric excess

Keeping secrets from parents: Longitudinal associations of secrecy in adolescence

Contains fulltext : 55705.pdf (publisher's version ) (Closed access)A 2-wave survey study among 1173 10-14-year-olds tested the longitudinal contribution of secrecy from parents to psychosocial and behavioral problems in adolescence. Additionally, it investigated a hypothesized contribution of secrecy from parents to adolescent development by examining its relation with self-control. Results showed that keeping secrets from parents is associated with substantial psychosocial and behavioral disadvantages in adolescence even after controlling for possible confounding variables, including communication with parents, trust in parents, and perceived parental supportiveness. Contrary to prediction, secrecy was also negatively associated with feelings of self-control. Secrecy from parents thus appears to be an important risk factor for adolescent psychosocial well-being and behavioral adjustment.12 p

Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis

Apoptotic cells release ‘find-me’ signals at the earliest stages of death to recruit phagocytes1. The nucleotides ATP and UTP represent one class of find-me signals2, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 over-expression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the ‘selective’ plasma membrane permeability of early apoptotic cells to specific dyes3. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases

Role of the Gut Endoderm in Relaying Left-Right Patterning in Mice

Analysis of Sox17 mutant mice reveals that gap junction coupling across the gut endoderm of the embryo transmits the left-right asymmetric signal from the node to the site of asymmetric organogenesis in mice

Novel Pathway of Adipogenesis through Cross-Talk between Adipose Tissue Macrophages, Adipose Stem Cells and Adipocytes: Evidence of Cell Plasticity

INTRODUCTION: Previous studies highlight a complex relationship between lineage and phenotype for adipose tissue macrophages (ATMs), adipose stem cells (ASCs), and adipocytes, suggesting a high degree of plasticity of these cells. In the present study, using a novel co-culture system, we further characterized the interaction between ATMs, ASCs and adipocytes. RESEARCH DESIGN AND METHODS: Human adipocytes and the stromal vascular fraction containing ATMs and ASCs were isolated from human adipose tissue and co-cultured for 24 hours. FACS was used to characterize ATMs and ASCs before and after co-culture. Preadipocytes generated after co-culture were characterized by immunostaining for DLK (preadipocytes), CD14 and CD68 (ATMs), CD34 (ASCs), and Nile Red staining for lipid drops. qRT-PCR was used to quantify adipogenic markers such as C/EBPα and PPARγ. A novel fluorescent nanobead lineage tracing method was utilized before co-culture where fluorescent nanobeads were internalized by CD68 (+) ATMs. RESULTS: Co-culture of adipocytes with ATMs and ASCs increased the formation of new preadipocytes, thereby increasing lipid accumulation and C/EBPα and PPARγ gene expression. Preadipocytes originating after co-culture were positive for markers of preadipocytes, ATMs and ASCs. Moreover, fluorescent nanobeads were internalized by ATMs before co-culture and the new preadipocytes formed after co-culture also contained fluorescent nanobeads, suggesting that new preadipocytes originated in part from ATMs. The formation of CD34(+)/CD68(+)/DLK (+) cell spheres supported the interaction of ATMs, ASCs and preadipocytes. CONCLUSIONS: Cross-talk between adipocytes, ATMs and ASCs promotes preadipocyte formation. The regulation of this novel adipogenic pathway involves differentiation of ATMs to preadipocytes. The presence of CD34(+)/CD68(+)/DLK(+) cells grouped in spheres suggest that paracrine interactions between these cell types plays an important role in the generation and proliferation of new preadipocytes. This phenomenon may reflect the in vivo plasticity of adipose tissue in which ATMs play an additional role during inflammation and other disease states. Understanding this novel pathway could influence adipogenesis, leading to new treatments for obesity, inflammation, and type 2 diabetes

Immunodominant T Cell Determinants of Aquaporin-4, the Autoantigen Associated with Neuromyelitis Optica

Autoantibodies that target the water channel aquaporin-4 (AQP4) in neuromyelitis optica (NMO) are IgG1, a T cell-dependent Ig subclass. However, a role for AQP4-specific T cells in this CNS inflammatory disease is not known. To evaluate their potential role in CNS autoimmunity, we have identified and characterized T cells that respond to AQP4 in C57BL/6 and SJL/J mice, two strains that are commonly studied in models of CNS inflammatory diseases. Mice were immunized with either overlapping peptides or intact hAQP4 protein encompassing the entire 323 amino acid sequence. T cell determinants identified from examination of the AQP4 peptide (p) library were located within AQP4 p21-40, p91-110, p101-120, p166-180, p231-250 and p261-280 in C57BL/6 mice, and within p11-30, p21-40, p101-120, p126-140 and p261-280 in SJL/J mice. AQP4-specific T cells were CD4+ and MHC II-restricted. In recall responses to immunization with intact AQP4, T cells responded primarily to p21-40, indicating this region contains the immunodominant T cell epitope(s) for both strains. AQP4 p21-40-primed T cells secreted both IFN-γ and IL-17. The core immunodominant AQP4 21-40 T cell determinant was mapped to residues 24-35 in C57BL/6 mice and 23-35 in SJL/J mice. Our identification of the AQP4 T cell determinants and characterization of its immunodominant determinant should permit investigators to evaluate the role of AQP4-specific T cells in vivo and to develop AQP4-targeted murine NMO models