Use of a prolactin-Cre/ROSA-YFP transgenic mouse provides no evidence for lactotroph transdifferentiation after weaning, or increase in lactotroph/somatotroph proportion in lactation

In rats, a shift from somatotroph dominance to lactotroph dominance during pregnancy and lactation is well reported. Somatotroph to lactotroph transdifferentiation and increased lactotroph mitotic activity are believed to account for this and associated pituitary hypertrophy. A combination of cell death and transdifferentiation away from the lactotroph phenotype has been reported to restore non-pregnant pituitary proportions after weaning. To attempt to confirm that a similar process occurs in mice, we generated and used a transgenic reporter mouse model (prolactin (PRL)-Cre/ROSA26-expression of yellow fluorescent protein (EYFP)) in which PRL promoter activity at any time resulted in permanent, stable, and highly specific EYFP. Triple immunochemistry for GH, PRL, and EYFP was used to quantify EYFP+ve, PRL−ve, and GH+ve cell populations during pregnancy and lactation, and for up to 3 weeks after weaning, and concurrent changes in cell size were estimated. At all stages, the EYFP reporter was expressed in 80% of the lactotrophs, but in fewer than 1% of other pituitary cell types, indicating that transdifferentiation from those lactotrophs where reporter expression was activated is extremely rare. Contrary to expectations, no increase in the lactotroph/somatotroph ratio was seen during pregnancy and lactation, whether assessed by immunochemistry for the reporter or PRL: findings confirmed by PRL immunochemistry in non-transgenic mice. Mammosomatotrophs were rarely encountered at the age group studied. Individual EYFP+ve cell volumes increased significantly by mid-lactation compared with virgin animals. This, in combination with a modest and non-cell type-specific estrogen-induced increase in mitotic activity, could account for pregnancy-induced changes in overall pituitary size

Chondromodulin-I expression in the growth plate of young uremic rats

Chondromodulin-I expression in the growth plate of young uremic rats.BackgroundGrowth retardation of chronic renal failure is associated with alterations in the growth plate suggestive of a disturbed chondrocyte maturation process and abnormal vascular invasion at the chondro-osseous interphase. Chondromodulin I (ChM-I) is a potent cartilage-specific angiostatic factor. Its pattern of expression in the uremic rat growth plate is unknown. Persistence of ChM-I synthesis and/or imbalance between ChM-I and vascular endothelial growth factor (VEGF) expressions might play a role in the alterations of uremic growth plate.MethodsGrowth cartilage ChM-I expression was investigated by immunohistochemistry, in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) in growth-retarded young uremic rats (UREM), control rats, fed ad libitum (SAL) or pair-fed with the UREM group (SPF), and uremic rats treated with growth hormone (UREM-GH). VEGF expression was analyzed by immunohistochemistry.ResultsChM-I and ChM-I mRNA were confined to the proliferative and early hypertrophic zones of growth cartilage. A similar number of chondrocytes per column was positive for ChM-I in the 4 groups. In accordance with the elongation of the hypertrophic stratum in uremia, the distance (X±SEM, μm) between the extracellular ChM-I signal and the metaphyseal end of growth cartilage was higher (P < 0.003) in UREM (236 ± 40) and UREM-GH (297 ± 17) than in SAL (92 ± 7) and SPF (113 ± 6). No differences in ChM-I expression were appreciated by RT-PCR. Similar VEGF positivity was observed in the hypertrophic chondrocytes of all groups.ConclusionIn experimental uremia, expansion of growth cartilage does not result from increased or persistent expression of ChM-I or from reduced VEGF expression at the cartilage-metaphyseal bone interphase. GH treatment does not modify ChM-I and VEGF expressions

A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient Cavalier King Charles Spaniels is amenable to exon 51 skipping

BACKGROUND Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion "hot spot" is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD "hot spot". METHODOLOGY/PRINCIPAL FINDINGS Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5' donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. CONCLUSIONS/SIGNIFICANCE Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD

In vivo grading of lipids in fatty liver by near-infrared autofluorescence and reflectance

The prevalence of nonalcoholic fatty liver (NAFLD) is rapidly increasing worldwide. When untreated, it may lead to complications such as liver cirrhosis or hepatocarcinoma. The diagnosis of NAFLD is usually obtained by ultrasonography, a technique that can underestimate its prevalence. For this reason, physicians aspire for an accurate, cost-effective, and noninvasive method to determine both the presence and the specific stage of the NAFLD. In this paper, we report an integrated approach for the quantitative estimation of the density of triglycerides in the liver based on the use of autofluorescence and reflectance signals generated by the abdomen of obese C57BL6/J mice. Singular value decomposition is applied to the generated spectra and its corresponding regression model provided a determination coefficient of 0.99 and a root mean square error of 240 mg/dl. This, in turn, enabled the quantitative imaging of triglycerides density in the livers of mice under in vivo conditionsMinisterio de Ciencia e Innovacion, Grant/Award Number: IJC2020-045229-I; Ministerio de Ciencia e Innovacion, Grant/Award Number: NANONERVPID2019-106211RB-I0

Existence of long-lasting experience-dependent plasticity in endocrine cell networks

Experience-dependent plasticity of cell and tissue function is critical for survival by allowing organisms to dynamically adjust physiological processes in response to changing or harsh environmental conditions. Despite the conferred evolutionary advantage, it remains unknown whether emergent experience-dependent properties are present in cell populations organized as networks within endocrine tissues involved in regulating body-wide homeostasis. Here we show, using lactation to repeatedly activate a specific endocrine cell network in situ in the mammalian pituitary, that templates of prior demand are permanently stored through stimulus-evoked alterations to the extent and strength of cell–cell connectivity. Strikingly, following repeat stimulation, evolved population behaviour leads to improved tissue output. As such, long-lasting experience-dependent plasticity is an important feature of endocrine cell networks and underlies functional adaptation of hormone release

Targeting L-type amino acid transporter 1 in innate and adaptive T cells efficiently controls skin inflammation

BACKGROUND: Psoriasis is a frequent inflammatory skin disease that is mainly mediated by IL-23, IL-1β, and IL-17 cytokines. Although psoriasis is a hyperproliferative skin disorder, the possible role of amino acid transporters has remained unexplored. OBJECTIVE: We sought to investigate the role of the essential amino acid transporter L-type amino acid transporter (LAT) 1 (SLC7A5) in psoriasis. METHODS: LAT1 floxed mice were crossed to Cre-expressing mouse strains under the control of keratin 5, CD4, and retinoic acid receptor-related orphan receptor γ. We produced models of skin inflammation induced by imiquimod (IMQ) and IL-23 and tested the effect of inhibiting LAT1 (JPH203) and mammalian target of rapamycin (mTOR [rapamycin]). RESULTS: LAT1 expression is increased in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in human subjects and mice. LAT1 deletion in keratinocytes does not dampen the inflammatory response or their proliferation, which could be maintained by increased expression of the alternative amino acid transporters LAT2 and LAT3. Specific deletion of LAT1 in γδ and CD4 T cells controls the inflammatory response induced by IMQ. LAT1 deletion or inhibition blocks expansion of IL-17-secreting γ4+δ4+ and CD4 T cells and dampens the release of IL-1β, IL-17, and IL-22 in the IMQ-induced model. Moreover, inhibition of LAT1 blocks expansion of human γδ T cells and IL-17 secretion by human CD4 T cells. IL-23 and IL-1β stimulation upregulates LAT1 expression and induces mTOR activation in IL-17+ γδ and TH17 cells. Deletion or inhibition of LAT1 efficiently controls IL-23- and IL-1β-induced phosphatidylinositol 3-kinase/AKT/mTOR activation independent of T-cell receptor signaling. CONCLUSION: Targeting LAT1-mediated amino acid uptake is a potentially useful immunosuppressive strategy to control skin inflammation mediated by the IL-23/IL-1β/IL-17 axis.Funding This manuscript has been funded by grants SAF 2017-82886-R (FS-M) and SAF 2013-42850-R (MF) from the Spanish Ministry of Economy and Competitiveness; CAM (S2017/BMD-3671-INFLAMUNE-CM) from the Comunidad de Madrid (FS-M); CIBERCV, BIOIMID PIE13/041 from Instituto de Salud Carlos III and Fundación La Marató TV3 (20152330 31). The project leading to these results has also received funding from FUNDACIÓN BBVA A EQUIPOS DE INVESTIGACIÓN CIENTÍFICA 2018 and from “la Caixa” Banking Foundation under the project code HR17-00016 (FS-M), and from Agencia Estatal de Investigación, Fondo Europeo de Desarrollo Regional PI17/01972 (E.D).S

Calcium Homeostasis in Myogenic Differentiation Factor 1 (MyoD)-Transformed, Virally-Transduced, Skin-Derived Equine Myotubes

Dysfunctional skeletal muscle calcium homeostasis plays a central role in the pathophysiology of several human and animal skeletal muscle disorders, in particular, genetic disorders associated with ryanodine receptor 1 (RYR1) mutations, such as malignant hyperthermia, central core disease, multiminicore disease and certain centronuclear myopathies. In addition, aberrant skeletal muscle calcium handling is believed to play a pivotal role in the highly prevalent disorder of Thoroughbred racehorses, known as Recurrent Exertional Rhabdomyolysis. Traditionally, such defects were studied in human and equine subjects by examining the contractile responses of biopsied muscle strips exposed to caffeine, a potent RYR1 agonist. However, this test is not widely available and, due to its invasive nature, is potentially less suitable for valuable animals in training or in the human paediatric setting. Furthermore, increasingly, RYR1 gene polymorphisms (of unknown pathogenicity and significance) are being identified through next generation sequencing projects. Consequently, we have investigated a less invasive test that can be used to study calcium homeostasis in cultured, skin-derived fibroblasts that are converted to the muscle lineage by viral transduction with a MyoD (myogenic differentiation 1) transgene. Similar models have been utilised to examine calcium homeostasis in human patient cells, however, to date, there has been no detailed assessment of the cells’ calcium homeostasis, and in particular, the responses to agonists and antagonists of RYR1. Here we describe experiments conducted to assess calcium handling of the cells and examine responses to treatment with dantrolene, a drug commonly used for prophylaxis of recurrent exertional rhabdomyolysis in horses and malignant hyperthermia in humans

Combined Immune Defect in B-Cell Lymphoproliferative Disorders Is Associated with Severe Infection and Cancer Progression

This research received no external funding. K.G.-H is supported by The European Social Fund (ESF) through a Río Ortega Grant for Health Research Projects by the Carlos III Health Institute (ISCIII) (CM20/00098).B cell chronic lymphoproliferative diseases (B-CLPD) are associated with secondary antibody deficiency and other innate and adaptive immune defects, whose impact on infectious risk has not been systematically addressed. We performed an immunological analysis of a cohort of 83 B-CLPD patients with recurrent and/or severe infections to ascertain the clinical relevance of the immune deficiency expression. B-cell defects were present in all patients. Patients with combined immune defect had a 3.69-fold higher risk for severe infection (p = 0.001) than those with predominantly antibody defect. Interestingly, by Kaplan–Meier analysis, combined immune defect showed an earlier progression of cancer with a hazard ratio of 3.21, than predominantly antibody defect (p = 0.005). When B-CLPD were classified in low-degree, high-degree, and plasma cell dyscrasias, risk of severe disease and cancer progression significantly diverged in combined immune defect, compared with predominantly antibody defect (p = 0.001). Remarkably, an underlying primary immunodeficiency (PID) was suspected in 12 patients (14%), due to prior history of infections, autoimmune and granulomatous conditions, atypical or variegated course and compatible biological data. This first proposed SID classification might have relevant clinical implications, in terms of predicting severe infections and cancer progression, and might be applied to different B-CLPD entities.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Epidemiology of Exertional Rhabdomyolysis Susceptibility in Standardbred Horses Reveals Associated Risk Factors and Underlying Enhanced Performance

BACKGROUND: Exertional rhabdomyolysis syndrome is recognised in many athletic horse breeds and in recent years specific forms of the syndrome have been identified. However, although Standardbred horses are used worldwide for racing, there is a paucity of information about the epidemiological and performance-related aspects of the syndrome in this breed. The objectives of this study therefore were to determine the incidence, risk factors and performance effects of exertional rhabdomyolysis syndrome in Standardbred trotters and to compare the epidemiology and genetics of the syndrome with that in other breeds. METHODOLOGY/PRINCIPAL FINDINGS: A questionnaire-based case-control study (with analysis of online race records) was conducted following identification of horses that were determined susceptible to exertional rhabdomyolysis (based on serum biochemistry) from a total of 683 horses in 22 yards. Thirty six exertional rhabdomyolysis-susceptible horses were subsequently genotyped for the skeletal muscle glycogen synthase (GYS1) mutation responsible for type 1 polysaccharide storage myopathy. A total of 44 susceptible horses was reported, resulting in an annual incidence of 6.4 (95% CI 4.6-8.2%) per 100 horses. Female horses were at significantly greater risk than males (odds ratio 7.1; 95% CI 2.1-23.4; p = 0.001) and nervous horses were at a greater risk than horses with calm or average temperaments (odds ratio 7.9; 95% CI 2.3-27.0; p = 0.001). Rhabdomyolysis-susceptible cases performed better from standstill starts (p = 0.04) than controls and had a higher percentage of wins (p = 0.006). All exertional rhabdomyolysis-susceptible horses tested were negative for the R309H GYS1 mutation. CONCLUSIONS/SIGNIFICANCE: Exertional rhabdomyolysis syndrome in Standardbred horses has a similar incidence and risk factors to the syndrome in Thoroughbred horses. If the disorder has a genetic basis in Standardbreds, improved performance in susceptible animals may be responsible for maintenance of the disorder in the population

Pleiotropic effects of trisomy and pharmacologic modulation on structural, functional, molecular, and genetic systems in a Down syndrome mouse model.

Down syndrome (DS) is characterized by skeletal and brain structural malformations, cognitive impairment, altered hippocampal metabolite concentration and gene expression imbalance. These alterations were usually investigated separately, and the potential rescuing effects of green tea extracts enriched in epigallocatechin-3-gallate (GTE-EGCG) provided disparate results due to different experimental conditions. We overcame these limitations by conducting the first longitudinal controlled experiment evaluating genotype and GTE-EGCG prenatal chronic treatment effects before and after treatment discontinuation. Our findings revealed that the Ts65Dn mouse model reflected the pleiotropic nature of DS, exhibiting brachycephalic skull, ventriculomegaly, neurodevelopmental delay, hyperactivity, and impaired memory robustness with altered hippocampal metabolite concentration and gene expression. GTE-EGCG treatment modulated most systems simultaneously but did not rescue DS phenotypes. On the contrary, the treatment exacerbated trisomic phenotypes including body weight, tibia microarchitecture, neurodevelopment, adult cognition, and metabolite concentration, not supporting the therapeutic use of GTE-EGCG as a prenatal chronic treatment. Our results highlight the importance of longitudinal experiments assessing the co-modulation of multiple systems throughout development when characterizing preclinical models in complex disorders and evaluating the pleiotropic effects and general safety of pharmacological treatments