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.

Role of coagulation tests in the management of acute appendicitis in children.

BACKGROUND: Acute appendicitis (AA) is one of the most common acute surgical conditions in children. Coagulation tests (CoTs) are usually utilized in preoperative assessment to rule out hemorrhagic risks. Our study aimed to evaluate the role of CoTs as predictors for the severity of AA. METHODS: In a retrospective study, we compared the blood tests of two cohorts of pediatric patients with AA (group A and B) evaluated in the Emergency Department of a Pediatric Tertiary care hospital between January 2017 and January 2020. Children in Group A underwent appendectomies while those in Group B were treated with conservative management per hospital protocol. Group A was then subdivided into non-complicated (NCA) and complicated appendicitis (CA), and the CoTs were compared between the two subgroups. RESULTS: Group A consisted of 198 patients and Group B of 150 patients. Blood tests, including CoTs and inflammatory markers, were compared between the 2 groups. We found a statistically significant difference in PT ratio mean value between Group A and B, suggesting that those who underwent appendicectomies had higher PT ratio values. From a pathophysiological point of view, we speculated that the variation of PT ratio in AA might be secondary to a vitamin K absorption deficit due to enteric inflammation. CONCLUSIONS: Our study underlined that a longer PT ratio could be helpful to distinguish CA from NCA. Further investigations may lead to the role of the PT ratio in the choice between conservative and surgical management.

Fetal Lung Interstitial Tumor (FLIT): Review of The Literature.

Fetal lung interstitial tumor (FLIT) is an extremely rare pediatric lung tumor that shares radiological features with congenital pulmonary malformations (cPAM) and other lung neoplasms. A review of the literature, together with the first European case, are herein reported. A systematic and manual search of the literature using the keyword "fetal lung interstitial tumor" was conducted on PUBMED, Scopus, and SCIE (Web of Science). Following the PRISMA guidelines, 12 articles were retrieved which describe a total of 21 cases of FLIT, and a new European case is presented. A prenatal diagnosis was reported in only 3 out of 22 (13%) cases. The mean age at surgery was 31 days of life (1-150); a lobectomy was performed in most of the cases. No complications or recurrence of disease were reported at a mean follow-up of 49 months. FLIT is rarely diagnosed during pregnancy, may present at birth with different levels of respiratory distress, and requires prompt surgical resection. Histology and immunohistochemistry allow for the differentiation of FLIT from cPAM and other lung tumors with poor prognosis, such as pleuropulmonary blastoma, congenital peri-bronchial myofibroblastic tumor, inflammatory myofibroblastic tumor, and congenital or infantile fibrosarcoma.

SARS-CoV-2 infection and replication in human gastric organoids.

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.

Complex open elbow fracture-dislocation with severe proximal ulna bone loss: a case report of massive osteochondral allograft surgical treatment.

We report a case of a 69-year-old right-dominant man who had an open Monteggia-like lesion of the right elbow (Gustilo-Andersen IIIA) with severe proximal ulna bone loss associated with an ipsilateral ulnar shaft fracture due to a motorcycle accident. The patient underwent two-stage surgery. Wound debridement and bridging external fixation were performed at first. Three months later, a frozen massive osteochondral ulnar allograft was implanted and fixed with a locking compression plate. A superficial wound infection appeared 5 weeks after the second surgery. Superficial wound debridement, negative pressure therapy, and antibiotics were administered for 3 months, achieving infection healing. At 3 years post-surgery, the elbow range of motion was satisfactory with a Disabilities of the Arm, Shoulder and Hand (DASH) score of 16.7. Radiographs and computed tomography scans showed good allograft-bone integration without allograft reabsorption or hardware loosening. Although not complication-free, massive ulna osteochondral allograft implantation can be considered a valid option in cases of open Monteggia-like lesions associated with ulnar shaft fracture and severe bone loss in active patients, whenever osteosynthesis or joint replacement is not a proper solution. This type of bone stock restoration allows for future surgery, if needed.

Preclinical stem cell therapy in fetuses with myelomeningocele: A systematic review and meta-analysis.

OBJECTIVE: We performed a systematic review to summarize the efficacy and safety of in utero stem cells application in preclinical models with myelomeningocele (MMC). METHODS: The study was registered with PROSPERO (CRD42019160399). We searched MEDLINE, Embase, Web of Science, Scopus and CENTRAL for publications articles on stem cell therapy in animal fetuses with MMC until May 2020. Publication quality was assessed by the SYRCLE's tool. Meta-analyses were pooled if studies were done in the same animal model providing similar type of stem cell used and outcome measurements. Narrative synthesis was performed for studies that could not be pooled. RESULTS: Nineteen and seven studies were included in narrative and quantitative syntheses, respectively. Most used mesenchymal stem cells (MSCs) and primarily involved ovine and rodent models. Both intra-amniotic injection of allogeneic amniotic fluid (AF)-MSCs in rat MMC model and the application of human placental (P)-MSCs to the spinal cord during fetal surgery in MMC ovine model did not compromise fetal survival rates at term (rat model, relative risk [RR] 1.03, 95% CI 0.92-1.16; ovine model, RR 0.94, 95% CI 0.78-1.13). A single intra-amniotic injection of allogeneic AF-MSCs into rat MMC model was associated with a higher rate of complete defect coverage compared to saline injection (RR 16.35, 95% CI 3.27-81.79). The incorporation of human P-MSCs as a therapeutic adjunct to fetal surgery in the ovine MMC model significantly improved sheep locomotor rating scale after birth (mean difference 5.18, 95% CI 3.36-6.99). CONCLUSIONS: Stem cell application during prenatal period in preclinical animal models is safe and effective.

Decellularized skeletal muscles display neurotrophic effects in three-dimensional organotypic cultures.

Skeletal muscle decellularization allows the generation of natural scaffolds that retain the extracellular matrix (ECM) mechanical integrity, biological activity, and three-dimensional (3D) architecture of the native tissue. Recent reports showed that in vivo implantation of decellularized muscles supports muscle regeneration in volumetric muscle loss models, including nervous system and neuromuscular junctional homing. Since the nervous system plays pivotal roles during skeletal muscle regeneration and in tissue homeostasis, support of reinnervation is a crucial aspect to be considered. However, the effect of decellularized muscles on reinnervation and on neuronal axon growth has been poorly investigated. Here, we characterized residual protein composition of decellularized muscles by mass spectrometry and we show that scaffolds preserve structural proteins of the ECM of both skeletal muscle and peripheral nervous system. To investigate whether decellularized scaffolds could per se attract neural axons, organotypic sections of spinal cord were cultured three dimensionally in vitro, in presence or in absence of decellularized muscles. We found that neural axons extended from the spinal cord are attracted by the decellularized muscles and penetrate inside the scaffolds upon 3D coculture. These results demonstrate that decellularized scaffolds possess intrinsic neurotrophic properties, supporting their potential use for the treatment of clinical cases where extensive functional regeneration of the muscle is required.

Neuronal Development and Onset of Electrical Activity in the Human Enteric Nervous System.

BACKGROUND & AIMS: The enteric nervous system (ENS) is the largest branch of the peripheral nervous system, comprising complex networks of neurons and glia, which are present throughout the gastrointestinal tract. Although development of a fully functional ENS is required for gastrointestinal motility, little is known about the ontogeny of ENS function in humans. We studied the development of neuronal subtypes and the emergence of evoked electrical activity in the developing human ENS. METHODS: Human fetal gut samples (obtained via the MRC-Wellcome Trust Human Developmental Biology Resource-UK) were characterized by immunohistochemistry, calcium imaging, RNA sequencing, and quantitative real-time polymerase chain reaction analyses. RESULTS: Human fetal colon samples have dense neuronal networks at the level of the myenteric plexus by embryonic week (EW) 12, with expression of excitatory neurotransmitter and synaptic markers. By contrast, markers of inhibitory neurotransmitters were not observed until EW14. Electrical train stimulation of internodal strands did not evoke activity in the ENS of EW12 or EW14 tissues. However, compound calcium activation was observed at EW16, which was blocked by the addition of 1 µmol/L tetrodotoxin. Expression analyses showed that this activity was coincident with increases in expression of genes encoding proteins involved in neurotransmission and action potential generation. CONCLUSIONS: In analyses of human fetal intestinal samples, we followed development of neuronal diversity, electrical excitability, and network formation in the ENS. These processes are required to establish the functional enteric circuitry. Further studies could increase our understanding of the pathogenesis of a range of congenital enteric neuropathies.

Isolation and characterisation of mouse intestinal mesoangioblasts.

AIMS AND OBJECTIVES: Children suffering from intestinal failure (IF) endure considerable morbidity and overall have poor survival rates, complicated by the shortage of organs available for transplantation. Therefore, new therapeutic approaches are pivotal if outcomes are to be improved. Over the past years, tissue engineering (TE) has emerged as a possible alternative treatment for many congenital and acquired conditions. TE aims at creating bioengineered organs by means of combining scaffolds with appropriate cell types, which in the intestine are organised within a multilayer structure. In order to generate functional intestine, this cellular diversity and organisation will need to be recreated. While the cells for the epithelial, neural and vascular compartments have been well defined, so far, less attention has been put on the muscular compartment. More recently, mesoangioblasts (MABs) have been identified as a novel source for tissue regeneration since they are able to give rise to vascular and other mesodermal derivatives. To date MABs have not been successfully isolated from intestinal tissue. Therefore, our aim was to demonstrate the possibility of isolating MABs from adult mouse small intestine. MATERIALS AND METHODS: All experiments were carried out using small intestinal tissues from C57BL/6J mice. We applied an established protocol for MAB isolation from the isolated neuromuscular layer of the small intestine. Cultured cells were stained for Ki67 to assess proliferation rates as well as for a panel of pericyte markers to determine their phenotype. RESULTS: Cells were successfully isolated from gut biopsies. Cultured cells showed good proliferative capacity and positivity for at least three pericytes markers found in vessels of the gut neuromuscular wall: neuron-glial antigen 2, alkaline phosphatase and platelet-derived growth factor ß. CONCLUSION: This proof-of-principle study lays the foundation for further characterization of MABs as a possible cell source for intestinal smooth muscle regeneration and TE.

Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration.

Pathological conditions affecting skeletal muscle function may lead to irreversible volumetric muscle loss (VML). Therapeutic approaches involving acellular matrices represent an emerging and promising strategy to promote regeneration of skeletal muscle following injury. Here we investigated the ability of three different decellularised skeletal muscle scaffolds to support muscle regeneration in a xenogeneic immune-competent model of VML, in which the EDL muscle was surgically resected. All implanted acellular matrices, used to replace the resected muscles, were able to generate functional artificial muscles by promoting host myogenic cell migration and differentiation, as well as nervous fibres, vascular networks, and satellite cell (SC) homing. However, acellular tissue mainly composed of extracellular matrix (ECM) allowed better myofibre three-dimensional (3D) organization and the restoration of SC pool, when compared to scaffolds which also preserved muscular cytoskeletal structures. Finally, we showed that fibroblasts are indispensable to promote efficient migration and myogenesis by muscle stem cells across the scaffolds in vitro. This data strongly support the use of xenogeneic acellular muscles as device to treat VML conditions in absence of donor cell implementation, as well as in vitro model for studying cell interplay during myogenesis.

Update on Foregut Molecular Embryology and Role of Regenerative Medicine Therapies.

Esophageal atresia (OA) represents one of the commonest and most severe developmental disorders of the foregut, the most proximal segment of the gastrointestinal (GI) tract (esophagus and stomach) in embryological terms. Of intrigue is the common origin from this foregut of two very diverse functional entities, the digestive and respiratory systems. OA appears to result from incomplete separation of the ventral and dorsal parts of the foregut during development, resulting in disruption of esophageal anatomy and frequent association with tracheo-oesophageal fistula. Not surprisingly, and likely inherent to OA, are associated abnormalities in components of the enteric neuromusculature and ultimately loss of esophageal functional integrity. An appreciation of such developmental processes and associated defects has not only enhanced our understanding of the etiopathogenesis underlying such devastating defects but also highlighted the potential of novel corrective therapies. There has been considerable progress in the identification and propagation of neural crest stem cells from the GI tract itself or derived from pluripotent cells. Such cells have been successfully transplanted into models of enteric neuropathy confirming their ability to functionally integrate and replenish missing or defective enteric nerves. Combinatorial approaches in tissue engineering hold significant promise for the generation of organ-specific scaffolds such as the esophagus with current initiatives directed toward their cellularization to facilitate optimal function. This chapter outlines the most current understanding of the molecular embryology underlying foregut development and OA, and also explores the promise of regenerative medicine.

[Evaluation of the knowledge of health professionals at a pediatric hospital regarding the use of metered-dose inhalers]. / Avaliação do conhecimento sobre o uso de inaladores dosimetrados entre profissionais de saúde de um hospital pediátrico.

OBJECTIVE: To evaluate health professionals working at a tertiary pediatric hospital in terms of their knowledge regarding the practical use of metered-dose inhalers. METHODS: Practical and written tests on the use of metered-dose inhalers were applied to physicians, physical therapists, nurses and nursing assistants. A score from 0 to 10 was assigned to each evaluation, and median scores were calculated for each professional category. Questions with higher and lower correct values were identified, and a descriptive comparison was made regarding the performance of the various professional categories. Statistical analysis was performed using the Kruskal-Wallis method for comparison of medians. A sequential logistic multiple regression analysis was also performed. RESULTS: A total of 30 resident physicians or interns in the pediatrics department, 23 attending physicians, 29 physical therapists, 33 nurses and 31 nursing assistants were evaluated. Resident physicians, physical therapists and attending physicians performed significantly better than did nurses and nursing assistants. Only resident physicians had a median score greater than 6. CONCLUSIONS: The health professionals evaluated, particularly the nurses and nursing assistants, who are directly involved in the practical use of metered-dose inhalers, possess insufficient knowledge regarding the use of such inhalers.

Avaliação do conhecimento sobre o uso de inaladores dosimetrados entre profissionais de saúde de um hospital pediátrico / Evaluation of the knowledge of health professionals at a pediatric hospital regarding the use of metered-dose inhalers

OBJETIVO: Avaliar os conhecimentos sobre o uso e manejo de inaladores pressurizados dosimetrados entre profissionais de um hospital pediátrico terciário. MÉTODOS: Foram realizadas avaliações sobre o conhecimento do uso de inaladores pressurizados dosimetrados através de questionários teóricos e práticos, com médicos, fisioterapeutas, enfermeiras e auxiliares de enfermagem, atribuindo-se uma nota de 0 a 10 para cada avaliação. Calcularam-se as medianas das notas obtidas pelos profissionais de cada categoria, as questões com maiores e menores índices de erros, e foi feita a comparação descritiva do desempenho das diversas categorias profissionais. A análise estatística foi feita através do método de Kruskal-Wallis de comparação de medianas. Também foi realizada a análise através de regressão logística múltipla seqüencial. RESULTADOS: Foram avaliados 30 médicos residentes ou estagiários de pediatria, 23 médicos assistentes, 29 fisioterapeutas, 33 enfermeiras e 31 auxiliares de enfermagem. Os médicos residentes, fisioterapeutas e médicos assistentes obtiveram desempenho significativamente superior aos dos enfermeiros e auxiliares de enfermagem. Apenas os médicos residentes obtiveram mediana superior a 6. CONCLUSÕES: O conhecimento a respeito do uso de inaladores dosimetrados entre os profissionais de saúde da instituição é insatisfatório, principalmente entre enfermeiros e auxiliares de enfermagem, diretamente envolvidos na aplicação prática dos inaladores dosimetrados.

OBJECTIVE: To evaluate health professionals working at a tertiary pediatric hospital in terms of their knowledge regarding the practical use of metered-dose inhalers. METHODS: Practical and written tests on the use of metered-dose inhalers were applied to physicians, physical therapists, nurses and nursing assistants. A score from 0 to 10 was assigned to each evaluation, and median scores were calculated for each professional category. Questions with higher and lower correct values were identified, and a descriptive comparison was made regarding the performance of the various professional categories. Statistical analysis was performed using the Kruskal-Wallis method for comparison of medians. A sequential logistic multiple regression analysis was also performed. RESULTS: A total of 30 resident physicians or interns in the pediatrics department, 23 attending physicians, 29 physical therapists, 33 nurses and 31 nursing assistants were evaluated. Resident physicians, physical therapists and attending physicians performed significantly better than did nurses and nursing assistants. Only resident physicians had a median score greater than 6. CONCLUSIONS: The health professionals evaluated, particularly the nurses and nursing assistants, who are directly involved in the practical use of metered-dose inhalers, possess insufficient knowledge regarding the use of such inhalers.