Multi-disciplinary Insights from the First European Forum on Visceral Myopathy 2022 Meeting

Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype–phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first ‘European Forum on Visceral Myopathy’. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy. Graphical Abstract: [Figure not available: see fulltext.

Testosterone production during puberty in two 46,XY patients with disorders of sex development and novel NR5A1 (SF-1) mutations

BACKGROUND: Steroidogenic factor 1 (SF-1, NR5A1) is a key transcriptional regulator of many genes involved in the hypothalamic–pituitary–gonadal axis and mutations in NR5A1 can result in 46,XY disorders of sex development (DSD). Patients with this condition typically present with ambiguous genitalia, partial gonadal dysgenesis, and absent/rudimentary Müllerian structures. In these cases, testosterone is usually low in early infancy, indicating significantly impaired androgen synthesis. Further, Sertoli cell dysfunction is seen (low inhibin B, anti-Müllerian hormone). However, gonadal function at puberty in patients with NR5A1 mutations is unknown. SUBJECTS AND METHODS: Clinical assessment, endocrine evaluation, and genetic analysis were performed in one female and one male with 46,XY DSD who showed spontaneous virilization during puberty. The female patient presented at adolescence with clitoral hypertrophy, whereas the male patient presented at birth with severe hypospadias and entered puberty spontaneously. Molecular analysis of NR5A1 was performed followed by in vitro functional analysis of the two novel mutations detected. RESULTS: Testosterone levels were normal during puberty in both patients. Analysis of NR5A1 revealed two novel heterozygous missense mutations in the ligand-binding domain of SF-1 (patient 1: p.L376F; patient 2: p.G328V). The mutant proteins showed reduced transactivation of the CYP11A promoter in vitro. CONCLUSION: Patients with 46,XY DSD and NR5A1 mutations can produce sufficient testosterone for spontaneous virilization during puberty. Phenotypic females (46,XY) with NR5A1 mutations can present with clitoromegaly at puberty, a phenotype similar to other partial defects of androgen synthesis or action. Testosterone production in 46,XY males with NR5A1 mutations can be sufficient for virilization at puberty. As progressive gonadal dysgenesis is likely, gonadal function should be monitored in adolescence and adulthood, and early sperm cryopreservation considered in male patients if possible

Alterations in Epithelial and Mesenchymal Intestinal Gene Expression During Doxorubicin-Induced Mucositis in Mice

In the current study we aimed to gain insight into epithelial-mesenchymal cross-talk and progenitor compartment modulation during doxorubicin (DOX)-induced mucositis in mice. Intestinal segments were collected on various days after DOX treatment. DOX-induced damage at day 1–2 was characterized by increased epithelial proliferation and apoptosis and a decrease in the expression of epithelial differentiation markers. Concurrently, T-cell factor-4 (TCF4) levels increased and the epithelial differentiation enhancing factor, bone morphogenic protein-4 (BMP4), decreased. During severe damage (day 3), BMP4 levels were significantly increased, which inversely correlated with epithelial proliferation. At the same time, the expression of the epithelial differentiation markers was increasing again. At day 7, BMP4 levels were down-regulated, while the levels of the epithelial differentiation markers and TCF4 were normalized again. These data suggest that in response to DOX-induced damage, BMP4 and TCF4 are modulated in such a way that homeostasis of the progenitor compartment is partly preserved

Mutation Analysis of NR5A1 Encoding Steroidogenic Factor 1 in 77 Patients with 46, XY Disorders of Sex Development (DSD) Including Hypospadias

BACKGROUND: Mutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, n = 11), ambiguous external genitalia without uterus (n = 33) or hypospadias (n = 33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein. CONCLUSIONS/SIGNIFICANCE: Mutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations

Organotypical tissue cultures from adult murine colon as an in vitro model of intestinal mucosa

Together with animal experiments, organotypical cell cultures are important models for analyzing cellular interactions of the mucosal epithelium and pathogenic mechanisms in the gastrointestinal tract. Here, we introduce a three-dimensional culture model from the adult mouse colon for cell biological investigations in an in vivo-like environment. These explant cultures were cultured for up to 2 weeks and maintained typical characteristics of the intestinal mucosa, including a high-prismatic epithelium with specific epithelial cell-to-cell connections, a basal lamina and various connective tissue cell types, as analyzed with immunohistological and electron microscopic methods. The function of the epithelium was tested by treating the cultures with dexamethasone, which resulted in a strong upregulation of the serum- and glucocorticoid-inducible kinase 1 similar to that found in vivo. The culture system was investigated in infection experiments with the fungal pathogen Candida albicans. Wildtype but not Δcph1/Δefg1-knockout Candida adhered to, penetrated and infiltrated the epithelial barrier. The results demonstrate the potential usefulness of this intestinal in vitro model for studying epithelial cell-cell interactions, cellular signaling and microbiological infections in a three-dimensional cell arrangement

Age-Dependent TLR3 Expression of the Intestinal Epithelium Contributes to Rotavirus Susceptibility

Rotavirus is a major cause of diarrhea worldwide and exhibits a pronounced small intestinal epithelial cell (IEC) tropism. Both human infants and neonatal mice are highly susceptible, whereas adult individuals remain asymptomatic and shed only low numbers of viral particles. Here we investigated age-dependent mechanisms of the intestinal epithelial innate immune response to rotavirus infection in an oral mouse infection model. Expression of the innate immune receptor for viral dsRNA, Toll-like receptor (Tlr) 3 was low in the epithelium of suckling mice but strongly increased during the postnatal period inversely correlating with rotavirus susceptibility, viral shedding and histological damage. Adult mice deficient in Tlr3 (Tlr3−/−) or the adaptor molecule Trif (TrifLps2/Lps2) exerted significantly higher viral shedding and decreased epithelial expression of proinflammatory and antiviral genes as compared to wild-type animals. In contrast, neonatal mice deficient in Tlr3 or Trif did not display impaired cell stimulation or enhanced rotavirus susceptibility. Using chimeric mice, a major contribution of the non-hematopoietic cell compartment in the Trif-mediated antiviral host response was detected in adult animals. Finally, a significant age-dependent increase of TLR3 expression was also detected in human small intestinal biopsies. Thus, upregulation of epithelial TLR3 expression during infancy might contribute to the age-dependent susceptibility to rotavirus infection

Differential gene expression in male and female rainbow trout embryos prior to the onset of gross morphological differentiation of the gonads

<p>Abstract</p> <p>Background</p> <p>There are large differences between the sexes at the genetic level; these differences include heterogametic sex chromosomes and/or differences in expression of genes between the sexes. In rainbow trout (<it>Oncorhynchus mykiss</it>) qRT-PCR studies have found significant differences in expression of several candidate sex determining genes. However, these genes represent a very small fraction of the genome and research in other species suggests there are large portions of the transcriptome that are differentially expressed between the sexes. These differences are especially noticeable once gonad differentiation and maturation has occurred, but less is known at earlier stages of development. Here we use data from a microarray and qRT-PCR to identify genes differentially expressed between the sexes at three time points in pre-hatch embryos, prior to the known timing of sexual differentiation in this species.</p> <p>Results</p> <p>The microarray study revealed 883 differentially expressed features between the sexes with roughly equal numbers of male and female upregulated features across time points. Most of the differentially expressed genes on the microarray were not related to sex function, suggesting large scale differences in gene expression between the sexes are present early in development. Candidate gene analysis revealed <it>sox9</it>, <it>DMRT1</it>, <it>Nr5a1 </it>and <it>wt1 </it>were upregulated in males at some time points and <it>foxl2</it>, <it>ovol1</it>, <it>fst </it>and <it>cyp19a1a </it>were upregulated in females at some time points.</p> <p>Conclusion</p> <p>This is the first study to identify sexual dimorphism in expression of the genome during embryogenesis in any fish and demonstrates that transcriptional differences are present before the completion of gonadogenesis.</p

The association between insight and depressive symptoms in schizophrenia: Undirected and Bayesian network analyses

Background. Greater levels of insight may be linked with depressive symptoms among patients with schizophrenia, however, it would be useful to characterize this association at symptom-level, in order to inform research on interventions. Methods. Data on depressive symptoms (Calgary Depression Scale for Schizophrenia) and insight (G12 item from the Positive and Negative Syndrome Scale) were obtained from 921 community-dwelling, clinically-stable individuals with a DSM-IV diagnosis of schizophrenia, recruited in a nationwide multicenter study. Network analysis was used to explore the most relevant connections between insight and depressive symptoms, including potential confounders in the model (neurocognitive and social-cognitive functioning, positive, negative and disorganization symptoms, extrapyramidal symptoms, hostility, internalized stigma, and perceived discrimination). Bayesian network analysis was used to estimate a directed acyclic graph (DAG) while investigating the most likely direction of the putative causal association between insight and depression. Results. After adjusting for confounders, better levels of insight were associated with greater self-depreciation, pathological guilt, morning depression and suicidal ideation. No difference in global network structure was detected for socioeconomic status, service engagement or illness severity. The DAG confirmed the presence of an association between greater insight and self-depreciation, suggesting the more probable causal direction was from insight to depressive symptoms. Conclusions. In schizophrenia, better levels of insight may cause self-depreciation and, possibly, other depressive symptoms. Person-centered and narrative psychotherapeutic approaches may be particularly fit to improve patient insight without dampening self-esteem

Oestrogen blocks the nuclear entry of SOX9 in the developing gonad of a marsupial mammal

<p>Abstract</p> <p>Background</p> <p>Hormones are critical for early gonadal development in nonmammalian vertebrates, and oestrogen is required for normal ovarian development. In contrast, mammals determine sex by the presence or absence of the <it>SRY </it>gene, and hormones are not thought to play a role in early gonadal development. Despite an XY sex-determining system in marsupial mammals, exposure to oestrogen can override <it>SRY </it>and induce ovarian development of XY gonads if administered early enough. Here we assess the effect of exogenous oestrogen on the molecular pathways of mammalian gonadal development.</p> <p>Results</p> <p>We examined the expression of key testicular (<it>SRY</it>, <it>SOX9</it>, <it>AMH </it>and <it>FGF9</it>) and ovarian (<it>WNT4</it>, <it>RSPO1</it>, <it>FOXL2 </it>and <it>FST</it>) markers during gonadal development in the marsupial tammar wallaby (<it>Macropus eugenii</it>) and used these data to determine the effect of oestrogen exposure on gonadal fate. During normal development, we observed male specific upregulation of <it>AMH </it>and <it>SOX9 </it>as in the mouse and human testis, but this upregulation was initiated before the peak in <it>SRY </it>expression and 4 days before testicular cord formation. Similarly, key genes for ovarian development in mouse and human were also upregulated during ovarian differentiation in the tammar. In particular, there was early sexually dimorphic expression of <it>FOXL2 </it>and <it>WNT4</it>, suggesting that these genes are key regulators of ovarian development in all therian mammals. We next examined the effect of exogenous oestrogen on the development of the mammalian XY gonad. Despite the presence of <it>SRY</it>, exogenous oestrogen blocked the key male transcription factor SOX9 from entering the nuclei of male somatic cells, preventing activation of the testicular pathway and permitting upregulation of key female genes, resulting in ovarian development of the XY gonad.</p> <p>Conclusions</p> <p>We have uncovered a mechanism by which oestrogen can regulate gonadal development through the nucleocytoplasmic shuttling of SOX9. This may represent an underlying ancestral mechanism by which oestrogen promotes ovarian development in the gonads of nonmammalian vertebrates. Furthermore, oestrogen may retain this function in adult female mammals to maintain granulosa cell fate in the differentiated ovary by suppressing nuclear translocation of the SOX9 protein.</p> <p>See commentary: http://www.biomedcentral.com/1741-7007/8/110</p

Aldo Keto Reductase 1B7 and Prostaglandin F2α Are Regulators of Adrenal Endocrine Functions

Prostaglandin F2α (PGF2α), represses ovarian steroidogenesis and initiates parturition in mammals but its impact on adrenal gland is unknown. Prostaglandins biosynthesis depends on the sequential action of upstream cyclooxygenases (COX) and terminal synthases but no PGF2α synthases (PGFS) were functionally identified in mammalian cells. In vitro, the most efficient mammalian PGFS belong to aldo-keto reductase 1B (AKR1B) family. The adrenal gland is a major site of AKR1B expression in both human (AKR1B1) and mouse (AKR1B3, AKR1B7). Thus, we examined the PGF2α biosynthetic pathway and its functional impact on both cortical and medullary zones. Both compartments produced PGF2α but expressed different biosynthetic isozymes. In chromaffin cells, PGF2α secretion appeared constitutive and correlated to continuous expression of COX1 and AKR1B3. In steroidogenic cells, PGF2α secretion was stimulated by adrenocorticotropic hormone (ACTH) and correlated to ACTH-responsiveness of both COX2 and AKR1B7/B1. The pivotal role of AKR1B7 in ACTH-induced PGF2α release and functional coupling with COX2 was demonstrated using over- and down-expression in cell lines. PGF2α receptor was only detected in chromaffin cells, making medulla the primary target of PGF2α action. By comparing PGF2α-responsiveness of isolated cells and whole adrenal cultures, we demonstrated that PGF2α repressed glucocorticoid secretion by an indirect mechanism involving a decrease in catecholamine release which in turn decreased adrenal steroidogenesis. PGF2α may be regarded as a negative autocrine/paracrine regulator within a novel intra-adrenal feedback loop. The coordinated cell-specific regulation of COX2 and AKR1B7 ensures the generation of this stress-induced corticostatic signal