MECHANISMS IN ENDOCRINOLOGY: Transient juvenile hypoglycemia in growth hormone receptor deficiency - mechanistic insights from Laron syndrome and tailored animal models.

The aim of the study is to find possible explanations for vanishing juvenile hypoglycemia in growth hormone receptor deficiency (GHRD) in human patients and animal models. We reviewed parameters of glucose metabolism in distinct age groups into two human cohorts (Israeli and Ecuadorian) of Laron syndrome (LS) patients, a mouse model (Ghr-KO mouse) and provided additional data for a porcine model (GHR-KO pig). Juvenile hypoglycemia is a common symptom of GHRD and vanishes in adulthood.

A New Toolbox in Experimental Embryology-Alternative Model Organisms for Studying Preimplantation Development.

Preimplantation development is well conserved across mammalian species, but major differences in developmental kinetics, regulation of early lineage differentiation and implantation require studies in different model organisms, especially to better understand human development. Large domestic species, such as cattle and pig, resemble human development in many different aspects, i.e.

Unbiased analysis of obesity related, fat depot specific changes of adipocyte volumes and numbers using light sheet fluorescence microscopy.

In translational obesity research, objective assessment of adipocyte sizes and numbers is essential to characterize histomorphological alterations linked to obesity, and to evaluate the efficacies of experimental medicinal or dietetic interventions. Design-based quantitative stereological techniques based on the analysis of 2D-histological sections provide unbiased estimates of relevant 3D-parameters of adipocyte morphology, but often involve complex and time-consuming tissue processing and analysis steps. Here we report the application of direct 3D light sheet fluorescence microscopy (LSFM) for effective and accurate analysis of adipocyte volumes and numbers in optically cleared adipose tissue samples from a porcine model of diet-induced obesity (DIO).

Human Engineered Heart Tissue Patches Remuscularize the Injured Heart in a Dose-Dependent Manner.

Background: Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose.

Methods: Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions.

Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos.

Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages.

CFTR Correctors and Antioxidants Partially Normalize Lipid Imbalance but not Abnormal Basal Inflammatory Cytokine Profile in CF Bronchial Epithelial Cells.

A deficiency in cystic fibrosis transmembrane conductance regulator (CFTR) function in CF leads to chronic lung disease. CF is associated with abnormalities in fatty acids, ceramides, and cholesterol, their relationship with CF lung pathology is not completely understood. Therefore, we examined the impact of CFTR deficiency on lipid metabolism and pro-inflammatory signaling in airway epithelium using mass spectrometric, protein array.

Chronic Hyperglycemia Drives Functional Impairment of Lymphocytes in Diabetic Transgenic Pigs.

People with diabetes mellitus have an increased risk for infections, however, there is still a critical gap in precise knowledge about altered immune mechanisms in this disease. Since diabetic transgenic pigs exhibit elevated blood glucose and a stable diabetic phenotype soon after birth, they provide a favorable model to explore functional alterations of immune cells in an early stage of diabetes mellitus . Hence, we investigated peripheral blood mononuclear cells (PBMC) of these diabetic pigs compared to non-diabetic wild-type littermates.

Genome editing for Duchenne muscular dystrophy: a glimpse of the future?

Mutations in Dystrophin, one of the largest proteins in the mammalian body, are causative for a severe form of muscle disease, Duchenne Muscular Dystrophy (DMD), affecting not only skeletal muscle, but also the heart. In particular, exons 45-52 constitute a hotspot for DMD mutations. A variety of molecular therapies have been developed, comprising vectors encoding micro- and minidystrophins as well as utrophin, a protein with partially overlapping functions.

Manipulating the Epigenome in Nuclear Transfer Cloning: Where, When and How.

The nucleus of a differentiated cell can be reprogrammed to a totipotent state by exposure to the cytoplasm of an enucleated oocyte, and the reconstructed nuclear transfer embryo can give rise to an entire organism. Somatic cell nuclear transfer (SCNT) has important implications in animal biotechnology and provides a unique model for studying epigenetic barriers to successful nuclear reprogramming and for testing novel concepts to overcome them. While initial strategies aimed at modulating the global DNA methylation level and states of various histone protein modifications, recent studies use evidence-based approaches to influence specific epigenetic mechanisms in a targeted manner.

Pathways to Clinical Cardiac Xenotransplantation.

Heart transplantation is the only long-lasting lifesaving option for patients with terminal cardiac failure. The number of available human organs is however far below the actual need, resulting in substantial mortality of patients while waiting for a human heart. Mechanical assist devices are used to support cardiac function but are associated with a high risk of severe complications and poor quality of life for the patients.

Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies.

Background: Many genetically multi-modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno-organs may impair their long-term function after orthotopic transplantation in non-human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR-KO) to reduce the size of donor pigs in one step.

A decade of experience with genetically tailored pig models for diabetes and metabolic research.

The global prevalence of diabetes mellitus and other metabolic diseases is rapidly increasing. Animal models play pivotal roles in unravelling disease mechanisms and developing and testing therapeutic strategies. Rodents are the most widely used animal models but may have limitations in their resemblance to human disease mechanisms and phenotypes.

Evidence of early increased sialylation of airway mucins and defective mucociliary clearance in CFTR-deficient piglets.

Background: Bacterial colonization in cystic fibrosis (CF) lungs has been directly associated to the loss of CFTR function, and/or secondarily linked to repetitive cycles of chronic inflammation/infection. We hypothesized that altered molecular properties of mucins could contribute to this process.

Methods: Newborn CFTR and CFTR were sacrificed before and 6 h after inoculation with luminescent Pseudomonas aeruginosa into the tracheal carina.

Progressive Proteome Changes in the Myocardium of a Pig Model for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is characterized by progressive muscle weakness. Even though DMD manifests first in skeletal muscle, heart failure is a major cause of death in late-stage DMD. To get insights into DMD-associated cardiomyopathy, we performed a proteome analysis of myocardium from a genetically engineered porcine DMD model resembling clinical and pathological hallmarks of human DMD.

The impact of transcription inhibition during in vitro maturation on the proteome of bovine oocytes†.

Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC-MS/MS analysis of germinal vesicle oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out.

Xeno-organ donor pigs with multiple genetic modifications - the more the better?

The number of donated human organs and tissues for patients with terminal organ failure falls far short of the need. Alternative sources, such as organs and tissues from animals, are therefore urgently required. During the past few years, major progress has been made in the development of genetically multi-modified donor pigs, and their organs have been shown to be safe and efficacious in life-supporting transplantation models into non-human primates, paving the way to clinical xenotransplantation studies.

Proteome profile of neutrophils from a transgenic diabetic pig model shows distinct changes.

INS transgenic pigs develop a stable diabetic phenotype early after birth and therefore allow studying the influence of hyperglycemia on primary immune cells in an early stage of diabetes mellitus in vivo. Since immune response is altered in diabetes mellitus, with deviant neutrophil function discussed as one of the possible causes in humans and mouse models, we investigated these immune cells in INS transgenic pigs and wild type controls at protein level. A total of 2371 proteins were quantified by label-free LC-MS/MS.

Intrinsic alterations in peripheral neutrophils from cystic fibrosis newborn piglets.

Background: The hallmark of the cystic fibrosis (CF) lung disease is a neutrophil dominated lung environment that is associated to chronic lung tissue destruction and ultimately the patient's death. It is unclear whether the exacerbated neutrophil response is primary related to a defective CFTR or rather secondary to chronic bacterial colonization and inflammation. Here, we hypothesized that CF peripheral blood neutrophils present intrinsic alteration at birth before the start of an inflammatory process.

Cellular and Molecular Probing of Intact Human Organs.

Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs.

Porcine models for studying complications and organ crosstalk in diabetes mellitus.

The worldwide prevalence of diabetes mellitus and obesity is rapidly increasing not only in adults but also in children and adolescents. Diabetes is associated with macrovascular complications increasing the risk for cardiovascular disease and stroke, as well as microvascular complications leading to diabetic nephropathy, retinopathy and neuropathy. Animal models are essential for studying disease mechanisms and for developing and testing diagnostic procedures and therapeutic strategies.

Multi-omics insights into functional alterations of the liver in insulin-deficient diabetes mellitus.

Objective: The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically.

Early weaning completely eliminates porcine cytomegalovirus from a newly established pig donor facility for xenotransplantation.

For clinical xenotransplantation, transplants must be free of porcine cytomegalovirus (PCMV). Piglets become infected primarily in the perinatal period by the mother sow. While individual donor animals can be protected from infection by isolation husbandry, success is not guaranteed and this strategy poses the risk of undetected infections and raises animal welfare questions.

Recent progress in porcine islet isolation, culture and engraftment strategies for xenotransplantation.

Purpose Of Review: Xenotransplantation of porcine islets is a realistic option to restore β-cell function in type 1 diabetic patients. Among other factors, such as islet donor age (fetal, neonatal and adult) and genotype (wild type and genetically modified), choice of the transplantation site, and immune protection of the islets, efficient strategies for islet isolation, culture and engraftment are critical for the success of islet xenotransplantation.

Recent Findings: Neonatal porcine islets (NPIs) are immature at isolation and need to be matured in vitro or in vivo before they become fully functional.

Will Genetic Engineering Carry Xenotransplantation of Pig Islets to the Clinic?

Purpose Of Review: Porcine islets represent a potentially attractive beta-cell source for xenotransplantation into patients with type 1 diabetes, who are not eligible to islet allo-transplantation due to a lack of suitable human donor organs. Recent progress in genetic engineering/gene editing of donor pigs provides new opportunities to overcome rejection of xeno-islets, to improve their engraftment and insulin secretion capacity, and to reduce the risk for transmission of porcine endogenous retroviruses. This review summarizes the current issues and progress in islet xenotransplantation with special emphasis on genetically modified/gene edited donor pigs.

Dilution correction for dynamically influenced urinary analyte data.

Urinary analyte data has to be corrected for the sample specific dilution as the dilution varies intra- and interpersonally dramatically, leading to non-comparable concentration measures. Most methods of dilution correction utilized nowadays like probabilistic quotient normalization or total spectra normalization result in a division of the raw data by a dilution correction factor. Here, however, we show that the implicit assumption behind the application of division, log-linearity between the urinary flow rate and the raw urinary concentration, does not hold for analytes which are not in steady state in blood.