Modeling Lysosomal Storage Disorders in an Innovative Way: Establishment and Characterization of Stem Cell Lines from Human Exfoliated Deciduous Teeth of Mucopolysaccharidosis Type II Patients.

Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.

Help Comes from Unexpected Places: How a Tiny Fairy and a Tropical Fish may help us Model Mucopolysaccharidoses.

INTRODUCTION: When it comes to disease modeling, countless models are available for Lysosomal Storage Diseases (LSD). Historically, two major approaches are well-established: in vitro assessments are performed in patient fibroblasts, while in vivo pre-clinical studies are performed in mouse models. Still, both platforms have a series of drawbacks. Thus, we implemented two alternative and innovative protocols to mimic a particular sub-group of LSDs, the Mucopolysaccharidoses both in vitro and in vivo. METHODS: The first one relies on a non-invasive approach using dental pulp stem cells from deciduous teeth (SHEDs). SHEDs are multipotent neuronal precursors that can easily be collected. The second uses a state-of-the-art gene editing technology (CRISPR/Cas9) to generate zebrafish disease models. RESULTS: Even though this is an ongoing project, we have already established and characterized two MPS II and one MPS VI SHED cell models. These cells self-maintain through several passages and can give rise to a variety of cells including neurons. Furthermore, all MPS-associated sub-cellular phenotypes we have assessed so far are easily observable in these cells. Regarding our zebrafish models, we have successfully knocked down both naglu and hgsnat and the first results we got from the behavioral analysis are promising ones, as we can observe altered activity and sleep patterns in the genetically modified fish. For this particular approach we chose MPS III forms as our target disorders, since their neurological features (hyperactivity, seizures and motor impairment) and lifespan decrease would be easily recognizable in zebrafish. CONCLUSION: Now that these methods are well-established in our lab, their potential is immense. On one hand, the newly developed models will be of ultimate value to understand the mechanisms underlying MPS sub-cellular pathology, which have to be further elucidated. On the other hand, they will constitute an optimal platform for drug testing in house. Also noteworthy, our models will be published as lab resources and made available for the whole LSD community.

Challenges in the Definitive Diagnosis of Niemann-Pick Type C-Leaky Variants and Alternative Transcripts.

Niemann-Pick type C (NPC, ORPHA: 646) is a neuro-visceral, psychiatric disease caused predominantly by pathogenic variants in the NPC1 gene or seldom in NPC2. The rarity of the disease, and its wide range of clinical phenotypes and ages of onset, turn the diagnosis into a significant challenge. Other than the detailed clinical history, the typical diagnostic work-up for NPC includes the quantification of pathognomonic metabolites. However, the molecular basis diagnosis is still of utmost importance to fully characterize the disorder. Here, the authors provide an overview of splicing variants in the NPC1 and NPC2 genes and propose a new workflow for NPC diagnosis. Splicing variants cover a significant part of the disease-causing variants in NPC. The authors used cDNA analysis to study the impact of such variants, including the collection of data to classify them as leaky or non-leaky pathogenic variants. However, the presence of naturally occurring spliced transcripts can misdiagnose or mask a pathogenic variant and make the analysis even more difficult. Analysis of the NPC1 cDNA in NPC patients in parallel with controls is vital to assess and detect alternatively spliced forms. Moreover, nonsense-mediated mRNA decay (NMD) analysis plays an essential role in evaluating the naturally occurring transcripts during cDNA analysis and distinguishing them from other pathogenic variants' associated transcripts.

MicroRNA Profile, Putative Diagnostic Biomarkers and RNA-Based Therapies in the Inherited Lipid Storage Disease Niemann-Pick Type C.

Lipids are essential for cellular function and are tightly controlled at the transcriptional and post-transcriptional levels. Dysregulation of these pathways is associated with vascular diseases, diabetes, cancer, and several inherited metabolic disorders. MicroRNAs (miRNAs), in particular, are a family of post-transcriptional gene repressors associated with the regulation of many genes that encode proteins involved in multiple lipid metabolism pathways, thereby influencing their homeostasis. Thus, this class of non-coding RNAs (ncRNAs) has emerged as a promising therapeutic target for the treatment of lipid-related metabolic alterations. Most of these miRNAs act at an intracellular level, but in the past few years, a role for miRNAs as intercellular signaling molecules has also been uncovered since they can be transported in bodily fluids and used as potential biomarkers of lipid metabolic alterations. In this review, we point out the current knowledge on the miRNA signature in a lysosomal storage disorder associated with lipid dysfunction, Niemann-Pick type C, and discuss the potential use of miRNAs as biomarkers and therapeutic targets for RNA-based therapies.

Self-efficacy, organizational commitment, workload as predictors of life satisfaction in elementary school teachers: the mediating role of job satisfaction.

Background: Life satisfaction as well as job satisfaction of teachers has a significant impact on educational outcomes. Objective: To evaluate a model of factors predicting life satisfaction through the mediating role of job satisfaction. Methods: This was a cross-sectional study, with a sample of 300 primary school teachers of both sexes (68% female, 32% male) and with a mean age of 42.52 years (SD = 10.04). They were administered the General Self-Efficacy Scale, the Satisfaction with Life Scale (SWLS), the Workload Scale (ECT), the Generic Job Satisfaction Scale, and the Organizational Commitment Questionnaire (OCQ). Structural equation modeling (SEM) was used for data analysis. Results: The SEM analysis found significant goodness-of-fit indices: (χ2 = 13.739; df = 5; p = <0.001; CFI = 0.99, TLI = 0.98, RMSEA = 0.05, SRMR = 0.04). Specifically, self-efficacy and organizational commitment were positive predictors of job satisfaction, while workload was a negative predictor of job satisfaction. The mediating effect of job satisfaction between self-efficacy, life satisfaction, workload, and overall life satisfaction was confirmed. Conclusion: The results confirm the importance of self-efficacy, organizational commitment, and workload in job satisfaction and overall life satisfaction of elementary education teachers. Job satisfaction acts as a mediator in this relationship. It is important to reduce workload and promote self-efficacy and organizational commitment to improve the well-being and satisfaction of teachers.

Neurological Disease Modeling Using Pluripotent and Multipotent Stem Cells: A Key Step towards Understanding and Treating Mucopolysaccharidoses.

Despite extensive research, the links between the accumulation of glycosaminoglycans (GAGs) and the clinical features seen in patients suffering from various forms of mucopolysaccharidoses (MPSs) have yet to be further elucidated. This is particularly true for the neuropathology of these disorders; the neurological symptoms are currently incurable, even in the cases where a disease-specific therapeutic approach does exist. One of the best ways to get insights on the molecular mechanisms driving that pathogenesis is the analysis of patient-derived cells. Yet, not every patient-derived cell recapitulates relevant disease features. For the neuronopathic forms of MPSs, for example, this is particularly evident because of the obvious inability to access live neurons. This scenario changed significantly with the advent of induced pluripotent stem cell (iPSC) technologies. From then on, a series of differentiation protocols to generate neurons from iPSC was developed and extensively used for disease modeling. Currently, human iPSC and iPSC-derived cell models have been generated for several MPSs and numerous lessons were learnt from their analysis. Here we review most of those studies, not only listing the currently available MPS iPSC lines and their derived models, but also summarizing how they were generated and the major information different groups have gathered from their analyses. Finally, and taking into account that iPSC generation is a laborious/expensive protocol that holds significant limitations, we also hypothesize on a tempting alternative to establish MPS patient-derived neuronal cells in a much more expedite way, by taking advantage of the existence of a population of multipotent stem cells in human dental pulp to establish mixed neuronal and glial cultures.

Blood collection under anesthesia, peripheral blood cells, plasma biochemistry, and plasma protein electrophoresis in a living fossil: the Spotted Ratfish (Hydrolagus colliei).

This report describes the safe and effective handling under anesthesia, blood collection and main clinical pathology values determination for three adult Spotted Ratfish (Hydrolagus colliei) (two males and one female) successfully maintained under human care for more than 20 years. The anesthetic MS-222 diluted in tamponed salt water at 50 ppm provided deep narcosis with mean induction and recovery times of 5 and 20 min, allowing safe handling and blood collection in the three animals sampled. Major leukocyte types were similar to other teleost and elasmobranch species, identifying lymphocytes as the predominant leukocyte (75.5%), followed by the fine eosinophilic granulocytes (10.25%), the coarse eosinophilic granulocytes (9.75%), and a small percentage of monocytes (5.50%). Plasma biochemistry values in this species were like those seen in elasmobranchs, with the highest levels of blood urea nitrogen described in any Chondrichthyes (mean 679.7 mmol/l). Plasma protein electrophoresis analysis in the Spotted Ratfish showed five consistent fractions, like those previously described in other fish species, with a negligible quantity of proteins migrating in the region equivalent to albumin, and with fraction 4 (mean 53.20%) as the predominant fraction. Despite the limitations imposed by the small sample size and the challenging access to the studied species under human care, this study will shed light on and enhance clinical knowledge regarding Ratfish handling, anesthesia, blood collection, and analysis. It aims to deliver a comprehensive clinical pathology description, presenting valuable data for professionals engaged in the care and management of Chimaerans.

Rituximab biosimilar RTXM83 versus reference rituximab in combination with CHOP as first-line treatment for diffuse large B-cell lymphoma: a randomized, double-blind study.

This multicenter, double-blind, randomized study compared the efficacy, pharmacokinetics (PKs)/pharmacodynamics (PDs), safety and immunogenicity profile of RTXM83 vs. reference rituximab (R-rituximab), both with CHOP, as first-line treatment of diffuse large B-cell lymphoma (DLBCL). A total of 272 patients <65 years of age, with good prognosis (136 per arm) were randomized (1:1) to receive six cycles of either RTXM83 or R-rituximab. The primary efficacy endpoint was achieved (overall response rate of 83.6% for RTXM83 and 82.9% for R-rituximab) with a difference 0.7% between arms (95%CI: [-8.77% to 10.17%]) fulfilling the predefined non-inferiority margin (-13%). Similar number of patients reported at least one adverse event (AE) (131 per arm) or one serious AE (47 with RTXM83 and 45 with R-rituximab). Anti-drug antibody development was comparable between the arms. PK/PD secondary endpoint results support similarity between the compounds. RTXM83 exhibits non-inferior efficacy and similar safety/immunogenicity to R-rituximab, being an accessible alternative for the treatment of patients with previously untreated DLBCL.

Decline in admissions for childhood asthma, a 26-year period population-based study.

BACKGROUND: The prevalence of childhood asthma has increased, although the rate of hospitalization for asthma seems to decrease. In Norway, the rate of hospital admission for childhood asthma from 1984 to 2000 increased. The aim of this study was to assess further trends in hospital admissions for childhood asthma up to 2010. METHODS: A population-based study including children 1-13 yrs of age hospitalized for asthma during six periods from 1984/1985 to 2009/2010 in Rogaland, Norway, was performed. Medical records from 1536 admissions (1050 children) were studied; and gender, age, number of admissions, length of hospital stay, medications and symptoms were recorded. RESULTS: For all age groups, the rate of admissions per 10.000 increased from 20.1 in 1984/85 to 33.7 in 1989/90, but declined to 14.4 in 2009/2010. Rates were highest in boys (OR 1.87; 95% CI: 1.69, 2.09), younger age groups (OR 2.51; 2.38, 2.64) and decreased from 1984 to 2010 (OR 0.92; 0.88, 0.94). The rates of readmissions were higher than for primary admissions (OR 1.33; 1.19, 1.47). From 1984 to 2010, there was an increased use of inhaled corticosteroids prior to admission (6 to 51%) and started at discharge (7 to 37%), and systemic steroids given during admission (19 to 83%). CONCLUSION: There has been a substantial decline in the rate of hospital admissions for childhood asthma after 1989/1990, with major differences between age groups and genders. The decline could be due to improved care of children with asthma or a real reduction in asthma exacerbations.

[Spinal muscular atrophy in the ENT area]. / Atrofia muscular espinal en el área otorrinolaringológica.

To review the clinical and pathological characteristics of spinal muscular atrophy (SMA) in general and in particular regarding the ENT. SMA is relatively rare in the head and neck. Dysphagia is the main symptom. Retrospective review of 9 patients from the same family comprising the original patient, brothers, sister, and cousins with a diagnosis of SMA. Dysphagia and muscular hypotonia in upper members were the main presenting symptoms. The ENT exploration, oesophageal transit, electromyography, and genetic tests were necessary for the diagnosis. In our experience, the treatment of dysphagia in neurological diseases is poor as we have confirmed through a literature review. Thanks to rehabilitation units for swallowing and voice disorders, ENT specialists are involved in the treatment and training of these patients for a better quality of life.

Atrofia muscular espinal en el área otorrinolaringológica / Spinal muscular atrophy in the ENT area

Se ha querido revisar las características de la atrofia muscular espinal (AME) en general y en especial en lo relacionado con la otorrinolaringología. La AME es una entidad poco frecuente en el área de cabeza y cuello. La disfagia es el síntoma principal. Presentamos el estudio retrospectivo de una familia compuesta por 9 personas entre el afecto, los hermanos y los primos, diagnosticados de AME. Los principales síntomas fueron la disfagia y la debilidad muscular en los brazos. Para el diagnóstico se realizó exploración completa de cabeza y cuello, tránsito esofágico, electromiografía y estudio genético. Nuestra experiencia en el manejo de la disfagia en los trastornos neurológicos es poca, como se constata al realizar una revisión bibliográfica de esta patología. Gracias a las unidades de rehabilitación de los trastornos de la deglución y de la voz, los otorrinolaringólogos nos vemos implicados en cómo tratar y entrenar a estos enfermos para que tengan una mejor calidad de vida

To review the clinical and pathological characteristics of spinal muscular atrophy (SMA) in general and in particular regarding the ENT. SMA is relatively rare in the head and neck. Dysphagia is the main symptom. Retrospective review of 9 patients from the same family comprising the original patient, brothers, sister, and cousins with a diagnosis of SMA. Dysphagia and muscular hypotonia in upper members were the main presenting symptoms. The ENT exploration, oesophageal transit, electromyography, and genetic tests were necessary for the diagnosics. In our experience, the treatment of dysphagia in neurological diseases is poor as we have confirmed through a literature review. Thanks to rehabilitation units for swallowing and voice disorders, ENT specialists are involved in the treatment and training of these patients for a better quality of life

[Hospital admissions for acute asthma in children]. / Innleggelser for akutt astma hos barn.

BACKGROUND: Monitoring hospital admissions for acute asthma may give information regarding prevalence, severity and treatment of childhood asthma. In a retrospective population-based study, the medical records were studied for children aged one to 14 years admitted for asthma during four periods, each of two years, from 1984 to 2000. RESULTS: Children aged one and two had the highest admission rates; both primary admission and re-admissions increased substantially throughout the period. For children aged three and four, total admission rates went up during the 1980s, but were down again during the 1990s. For older children there was a tendency towards declining admission rates over the study period. The use of systemic steroids at admission and pre-hospital inhaled corticosteroids increased substantially over the period. INTERPRETATION: For one and two-year-old children there was a continuous increase in both primary and readmission rates. Given better pre-hospital treatment there is a potential for lowering admission rates.

Valor relativo do exame direto, após concentraçäo e por cultivo de escarro no diagnóstico bacteriológico da tuberculose pulmonar no Amazonas / Relative values of the direct simear exam, concentrated simear and culture of sputum in the diagnosis of pulmonares tuberculosis in the Amazonas - Brazil

Realizou-se o estudo do valor da baciloscopia direta no diagnóstico da tuberculose pulmonar, comparativamente à baciloscopia após concentraçäo e cultivo, de 610 amostras de escarro de pacientes que apresentavam sinais ou sintomas indicativos da doença. Detectou-se que a baciloscopia após concentraçäo e o cultivo säo quatro e dez vezes, respectivamente, mais eficazes que a baciloscopia direta. Observaram-se também resultados baciloscópicos falsos positivos para tuberculose devido à presença de micobactérias outras que näo o bacilo da tuberculose (MOTT), sugerindo doença micobacteriana (micobacteriose pulmonar) ou colonizaçäo da orofaringe

Prevalência e distribuiçäo de micobactérias nas águas de algumas regiöes do Estado de Säo Paulo - Brasil / Prevalence and distribution of mycobacteria in water of some regions of Säo Paulo State - Brazil

A ocorrência de micobactérias em águas de diferentes regiöes do Estado de Säo Paulo foi pesquisada em 302 amostras de origem diversa, como águas naturais (lago, rio, mina, poço artesiano e raso), águas tratadas (potável de torneira e piscina) e águas destinadas à criaçäo de peixes (aquários e tanque de psicicultura). As amostras de águas provenientes de aquários apresentaram a maior frequência de positividade (88,6%), seguidas pelas amostras de águas de poços rasos (33,3%), de lagos e rios (29,4%), de piscinas (28,2%) de tanque de psicicultura (16,6%), de minas (12,5% e de águas de torneira (4,2%). Näo foram encontradas micobactérias em águas de poços artesianos. Das cepas isoladas, 63,3% foram caracterizadas como micobactérias de crescimento rápido e as demais como de crescimento lento. M. fortuitum esteve presente nos diversos tipos de águas, constituindo 36,7% das cepas isoladas. Outras micobactérias potencialmente patogências foram encontradas em menor frequência. Verificou-se que a maioria das águas examinadas estavam contaminadas por micobactérias potencialmente patogênicas ou saprofíticas, sendo os principais reservatórios as águas que entram em contato com peixes e humanos como as de aquário e piscina

Presence of Mycobacterium marinum and other opportunist Mycobacteria in swimming pool waters in Araraquara, SP

O isolamento de Mycobacterium marinum é relatado pela primeira vez no Brasil. Analisaram-se amostras de águas de piscinas quanto à presença de micobactérias, para avaliar a participaçäo dessas águas como possível fonte de micobactérias patogênicas. Isolaram-se 24 cepas de micobactérias a partir de 72 amostras analisadas. A freqüência de isolamento foi de 37,5% para M. gordonae, de 20,8% para M. chelonae, de 4,2% para M. marinum, de 4,2% para M. scrofulaceum, de 4,2% para M. avium-intracellulare e de 29,2% para cepas näo pertencentes a qualquer espécie de micobactérias conhecida. As cepas näo identificadas constituem um novo grupo de micobactérias com características morfológicas e bioquímicas semelhantes às de M. gordonae mas com um perfil de ácidos micólicos distinto. Salienta-se, em conseqüência, a importância do estudo dos ácidos micólicos no rastreio de identificaçäo de micobactérias do ambiente. Concluiu-se que as águas das piscinas de Araraquara contêm micobactérias oportunistas potencialmente patogênicas para o homem