Development and initial characterization of a novel ghrelin receptor CRISPR/Cas9 knockout wistar rat model.

BACKGROUND/OBJECTIVES: Ghrelin, a stomach-derived hormone implicated in numerous behaviors including feeding, reward, stress, and addictive behaviors, acts by binding to the growth hormone secretagogue receptor (GHSR). Here, we present the development, verification, and initial characterization of a novel GHSR knockout (KO) Wistar rat model created with CRISPR genome editing. METHODS: Using CRISPR/Cas9, we developed a GHSR KO in a Wistar background. Loss of GHSR mRNA expression was histologically verified using RNAscope in wild-type (WT; n = 2) and KO (n = 2) rats. We tested the effects of intraperitoneal acyl-ghrelin administration on food consumption and plasma growth hormone (GH) concentrations in WT (n = 8) and KO (n = 8) rats. We also analyzed locomotion, food consumption, and body fat composition in these animals. Body weight was monitored from early development to adulthood. RESULTS: The RNAscope analysis revealed an abundance of GHSR mRNA expression in the hypothalamus, midbrain, and hippocampus in WTs, and no observed probe binding in KOs. Ghrelin administration increased plasma GH levels (p = 0.0067) and food consumption (p = 0.0448) in WT rats but not KOs. KO rats consumed less food overall at basal conditions and weighed significantly less compared with WTs throughout development (p = 0.0001). Compared with WTs, KOs presented higher concentrations of brown adipose tissue (BAT; p = 0.0322). CONCLUSIONS: We have verified GHSR deletion in our KO model using histological, physiological, neuroendocrinological, and behavioral measures. Our findings indicate that GHSR deletion in rats is not only associated with a lack of response to ghrelin, but also associated with decreases in daily food consumption and body growth, and increases in BAT. This GHSR KO Wistar rat model provides a novel tool for studying the role of the ghrelin system in obesity and in a wide range of medical and neuropsychiatric disorders.

Developing effective screening strategies in multiple endocrine neoplasia type 1 (MEN 1) on the basis of clinical and sequencing data of German patients with MEN 1.

BACKGROUND: Multiple-endocrine-neoplasia-type-1 (MEN1) is an autosomal-dominant inherited disorder characterized by the combined occurrence of primary hyperparathyroidism (pHPT), gastroenteropancreatic neuroendocrine tumors (GEP), adenomas of the pituitary gland (APA), adrenal cortical tumors (ADR) and other tumors. As the tumors appear in an unpredictable schedule, uncertainty about screening programs is persisting. OBJECTIVE: To optimize screening and to analyze possible differences in sporadic versus familial cases. METHODS: We analyzed data of 419 individuals including 306 MEN-1 patients (138 isolated and168 familial cases out of 102 unrelated families). RESULTS: A total of 683 tumors occurred consisting of 273 pHPT, 138 APA, 166 GEP, 57 ADR, 24 thymic- and bronchial-carcinoids as well as 25 neoplasms of other tissues. The age-related penetrance was determined as 10%, 35%, 67%, 81% and 100% at 20, 30, 40, 50 and 65 years respectively. Although pHPT being the most frequent first manifestation (41%), also GEP (22%) or APA (21%) were found to be the first presentation. APA occurred significantly more frequent (p<0,05) in isolated (n=138) than in familial (n=168) cases, whereas GEP showed a tendency to occur more often in familial cases. Genotype/phenotype correlation in 140 clinically affected MEN-1 cases showed a tendency for truncating mutations, especially nonsense mutations to be associated to GEP and carcinoids of the lungs and thymus. CONCLUSION: In view of the morbidity and frequency in familial cases an effective screening programme should aim at an early diagnosis of GEP particularly when truncating, especially nonsense mutations are found.

Serum leptin monitoring in anorectic patients during refeeding therapy.

Circulating concentrations of leptin are exceedingly low in severe malnutrition as seen in the acute state of anorexia nervosa (AN). During refeeding therapy plasma leptin levels increase to normal and in some cases peak at values in excess of the BMI of matched controls even before a normal body weight has been achieved. Peak leptin levels are possibly the cause of an increased energy expenditure during this stage of the disorder and might predispose to renewed weight loss (rebound phenomenon). In this study we investigated the role of leptin fluctuations as a prognostic factor of therapeutic success in AN. In 11 anorectic female patients serum leptin levels, BMI and body fat percentage were evaluated in four-week intervals during a conventional refeeding program over three months (group 1). The results of the first two measurements were used to determine a range of increases in leptin levels in relation to increases in BMI. The values between the 25th and 75th percentiles determined the reference range. In a second group of 9 anorectic female patients serum leptin levels, BMI, body fat percentage and the increase in the leptin level in relation to the BMI of each subject were investigated for three months every two weeks. These patients were also treated according to the same conventional refeeding program, but the caloric intake was reduced or increased (+/-250 kcal/d) if the increase in the leptin level, in relation to the increase in the BMI, had exceeded or fallen short of the reference range. During the refeeding therapy every subject of each group experienced increases in serum leptin levels, BMI and body fat percentage. Six subjects of group 1 and six subjects of the second group had an increase in leptin levels in relation to the increase of the BMI out of the reference range at least once. To investigate the therapeutic outcome of leptin monitoring and the following alteration of caloric intake, weight gain of the patients of both groups during the whole treatment was compared. No significant difference was found. Our results probably do not support the findings that high leptin levels predispose to a renewed loss of weight. The outcome in our patients whose caloric intake was modified due to their serum leptin levels was not significantly improved.

Sequential actions of BMP receptors control neural precursor cell production and fate.

Bone morphogenetic proteins (BMPs) have diverse and sometimes paradoxical effects during embryonic development. To determine the mechanisms underlying BMP actions, we analyzed the expression and function of two BMP receptors, BMPR-IA and BMPR-IB, in neural precursor cells in vitro and in vivo. Neural precursor cells always express Bmpr-1a, but Bmpr-1b is not expressed until embryonic day 9 and is restricted to the dorsal neural tube surrounding the source of BMP ligands. BMPR-IA activation induces (and Sonic hedgehog prevents) expression of Bmpr-1b along with dorsal identity genes in precursor cells and promotes their proliferation. When BMPR-IB is activated, it limits precursor cell numbers by causing mitotic arrest. This results in apoptosis in early gestation embryos and terminal differentiation in mid-gestation embryos. Thus, BMP actions are first inducing (through BMPR-IA) and then terminating (through BMPR-IB), based on the accumulation of BMPR-IB relative to BMPR-IA. We describe a feed-forward mechanism to explain how the sequential actions of these receptors control the production and fate of dorsal precursor cells from neural stem cells.

Bone marrow cells regenerate infarcted myocardium.

Myocardial infarction leads to loss of tissue and impairment of cardiac performance. The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time. Injury to a target organ is sensed by distant stem cells, which migrate to the site of damage and undergo alternate stem cell differentiation; these events promote structural and functional repair. This high degree of stem cell plasticity prompted us to test whether dead myocardium could be restored by transplanting bone marrow cells in infarcted mice. We sorted lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein by fluorescence-activated cell sorting on the basis of c-kit expression. Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct. Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells. The developing tissue comprised proliferating myocytes and vascular structures. Our studies indicate that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease.

POU transcription factors control expression of CNS stem cell-specific genes.

Multipotential stem cells throughout the developing central nervous system have common properties. Among these is expression of the intermediate filament protein nestin and the brain fatty acid binding protein (B-FABP). To determine if common mechanisms control transcription in CNS stem cells, the regulatory elements of these two genes were mapped in transgenic mice. A 257 basepair enhancer of the rat nestin gene is sufficient for expression throughout the embryonic neuroepithelium. This enhancer contains two sites bound by the class III POU proteins Brn-1, Brn-2, Brn-4, and Tst-1. Only one of the two POU sites is required for CNS expression. An adjacent hormone response element is necessary for expression in the dorsal midbrain and forebrain. The regulatory sites of the B-FABP gene are strikingly similar to those of the nestin gene. A hybrid POU/Pbx binding site is recognized in vitro by Pbx-1, Brn-1 and Brn-2. This site is essential for expression in most of the CNS. In addition, a hormone response element is necessary for forebrain expression. Both the nestin and B-FABP genes therefore depend on POU binding sites for general CNS expression, with hormone response elements additionally required for activity in the anterior CNS. These data indicate that regulation by POU proteins and hormone receptors is a general mechanism for CNS stem cell-specific transcription.

Chicken B cells undergo discrete developmental changes in surface carbohydrate structure that appear to play a role in directing lymphocyte migration during embryogenesis.

The migration of progenitor cells to specific microenvironments is essential for the development of complex organisms. Avian species possess a unique primary lymphoid organ, the bursa of Fabricius, that plays a central role in the development of B cells. B cell progenitors, however, arise outside the bursa of Fabricius and, during embryonic development, must migrate through the vasculature to the bursa of Fabricius. In this report, we demonstrate that these progenitor B cells express the sialyl Lewis x carbohydrate structure previously shown to be a ligand for the selectin family of vascular adhesion receptors. Soon after migration to the bursa of Fabricius, B cell progenitors are induced to undergo a developmental switch and terminate the expression of sialyl Lewis x in a temporal pattern that correlates with the developmental decline in the ability of these cells to home to the bursa of Fabricius upon transplantation. The induction of the developmental switch in the glycosylation pattern of developing B cells requires the bursal environment. In addition, sialyl Lewis x carbohydrate determinants or structurally similar determinants on the surface of immortalized bursal lymphoid stem cells participate in the adherence of these cells to the vascular regions of the bursal microenvironment. These data demonstrate that the carbohydrate structure sialyl Lewis x is developmentally regulated during chicken B cell development and may facilitate the migration of B cell progenitors to the bursal microenvironment by serving as a ligand for a lectin-like adhesion receptor.

Differential regulation of V(D)J recombination during development of avian B and T cells.

The lymphoid immune system is comprised of two major cell types, B cells and T cells, originally identified in avian species. Although both lineages arise from hematopoietic stem cells, avian B cells require a period of development in the bursa of Fabricius while T cells undergo development in the thymus. Each cell type expresses a lineage-specific antigen receptor encoded by genes created by the rearrangement of individual members of variable (V), diversity (D), and joining (J) gene segment families during embryonic development. In this report, we demonstrate that productive rearrangement of the TCR beta gene occurs exclusively in the thymus during normal development. TCR beta rearrangements involving gene segments from the V beta 1 gene family can be detected beginning on day 12 of development, while rearrangements involving the other family of V beta gene segments, V beta 2, were first detected on day 14 of embryogenesis. In contrast, productive rearrangements of Ig light (IgL) and heavy (IgH) chain genes were not restricted to the bursa of Fabricius. Instead, VH-DJH heavy chain rearrangements and VL-JL light chain rearrangements were detected primarily in the embryonic spleen, beginning as early as embryonic day 10, even in birds bursectomized at 60 h of development. Within the spleen, Ig rearrangement was confined to the subset of cells that express the chB6 surface protein. Unlike bursal lymphocytes, which express the recombinase activating gene (RAG)-2 but not RAG-1, splenic B cell precursors also express RAG-1. The data indicate that, while B cell precursors initiate recombination prior to migration of the bursa of Fabricius, T cell precursors undergo V(D)J recombination following migration to the thymus. Thus, distinct developmental mechanisms appear to regulate the process of receptor rearrangement during avian B and T cell development.

Two distinct alpha beta T-cell lineages can be distinguished by the differential usage of T-cell receptor V beta gene segments.

Avian T cells can be divided into three subpopulations based on their expression of distinct T-cell receptors (TCR1, TCR2, and TCR3), ontogeny, and tissue distribution. The TCR1 cells appear to be the equivalent of mammalian gamma delta cells, but the derivation of cells expressing TCR2 and TCR3 has been unclear. Here we report that chickens contain two families of TCR beta variable (V) gene segments, V beta 1 and V beta 2. Furthermore, TCR2 and TCR3 represent subsets of alpha beta cells that are defined by mutually exclusive usage of these two families of V beta gene segments. Sequence comparisons of V beta 1 and V beta 2 with mammalian TCR beta V segments reveal that V beta 1 gene segments encode the conserved amino acids used to define the mammalian V beta consensus subgroup I, while V beta 2 encodes the amino acids used to define the mammalian V beta subgroup II. Although the beta chains of TCR2 and TCR3 cells are encoded by the same diversity (D), joining (J), and constant (C) region segments, V beta 1 gene segments undergo rearrangement before V beta 2 gene segments during T-cell development. This may result from the fact that TCR2 cells undergo V-DJ joining by deletional rearrangement, whereas TCR3 cells undergo V-DJ joining by inversional rearrangement. These data suggest that the TCR alpha beta cells can be divided into two distinct and evolutionarily conserved lineages based on V beta gene segment usage. The clear-cut separation of these lineages in the chicken may help to define their immunologic role.

An avian B-lymphocyte protein associated with beta 2-microglobulin.

A member of the family of beta 2-microglobulin (beta 2m)-associated cell surface glycoproteins was identified by the CB3 monoclonal antibody. The Mr 50,000 heavy chain of the CB3 antigen differs from conventional class I heavy chains (Mr 45,000) in the extent of glycosylation, charge, and peptide composition. Because of its selective expression on avian B cells and its similarity to mammalian class I-like molecules, we speculate that the CB3 antigen may play a role in T- and B-cell interactions,

Molecular cloning of the Mason-Pfizer monkey virus genome: biological characterization of genome length clones and molecular comparisons to other retroviruses.

The molecular cloning of the DNA provirus of Mason-Pfizer monkey virus (M-PMV) is described. Fourteen independent clones of integrated M-PMV proviruses were isolated from a human embryo kidney cell line that had been previously derived from a single cell clone infected with M-PMV. Characterization of these clones for size of insert, restriction pattern of flanking DNA, and presence of repetitive DNA in the flanking sequences revealed that 10 of the isolates were identical while the four remaining clones were unique. Three independent clones of unintegrated M-PMV proviruses containing a single copy of the long terminal repeat (LTR) were cloned from acutely infected human embryo kidney cells, Transfection assays revealed that 13 of 14 integrated proviruses and 2 of 3 unintegrated proviruses were capable of producing infectious virus. One of the integrated provirus clones (clone 6A) produced consistently higher titers of virus than all of the other clones in all assays used and in two different cell lines, indicating that it contained a mutation that enhances virus replication. The virus recovered after transfection was shown to be capable of inducing cell fusion in nontransformed cell lines, confirming that this property is associated with M-PMV. One of the clones was hybridized under conditions of varying stringency, to molecular clones of type B, C, and D retroviruses. These studies revealed M-PMV to be most closely related to squirrel monkey retrovirus (D-type virus) and more distantly related to mouse mammary tumor virus (B-type virus). Hybridization was also detected with clones from the pol gene region of a family of human endogenous sequences. No homology was detected with Rous sarcoma virus or most mammalian C-type viruses tested. The exceptions were baboon endogenous virus and RD114 in which previously identified homology in the env gene was confirmed. These results suggest that the type D and type B viruses can be linked together in a group of viruses of similar ancestral origin analogous to that recently proposed for the human T-cell leukemia viruses and bovine leukemia virus.