Cerebellar astrocyte transduction as gene therapy for megalencephalic leukoencephalopathy

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare genetic disorder belonging to the group of vacuolating leukodystrophies. It is characterized by megalencephaly, loss of motor functions, epilepsy, and mild mental decline. In brain biopsies of MLC patients, vacuoles were observed in myelin and in astrocytes surrounding blood vessels. There is no therapy for MLC patients, only supportive treatment. We show here a preclinical gene therapy approach for MLC using the Mlc1 knock-out mouse. An adeno-associated virus coding for human MLC1 under the control of the glial fibrillary acidic protein promoter was injected in the cerebellar subarachnoid space of Mlc1 knock-out and wild-type animals at 2 months of age, before the onset of the disease, as a preventive approach. We also tested a therapeutic strategy by injecting the animals at 5 months, once the histopathological abnormalities are starting, or at 15 months, when they have progressed to a more severe pathology. MLC1 expression in the cerebellum restored the adhesion molecule GlialCAM and the chloride channel ClC-2 localization in Bergmann glia, which both are mislocalized in Mlc1 knock-out model. More importantly, myelin vacuolation was extremely reduced in treated mice at all ages and correlated with the amount of expressed MLC1 in Bergmann glia, indicating not only the preventive potential of this strategy but also its therapeutic capacity. In summary, here we provide the first therapeutic approach for patients affected with MLC. This work may have also implications to treat other diseases affecting motor function such as ataxias

Gene therapy for overexpressing Neuregulin 1 type I in skeletal muscles promotes functional improvement in the SOD1G93A ALS mice

Altres ajuts: Fundació La Marato-TV3: TV3201428-10 ; AFM-Telethon: 20289Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motoneurons (MNs), with no effective treatment currently available. The molecular mechanisms that are involved in MN death are complex and not fully understood, with partial contributions of surrounding glial cells and skeletal muscle to the disease. Neuregulin 1 (NRG1) is a trophic factor highly expressed in MNs and neuromuscular junctions. Recent studies have suggested a crucial role of the isoform I (NRG1-I) in the collateral reinnervation process in skeletal muscle, and NRG1-III in the preservation of MNs in the spinal cord, opening a window for developing novel therapies for neuromuscular diseases like ALS. In this study, we overexpressed NRG1-I widely in the skeletal muscles of the SOD1G93A transgenic mouse. The results show that NRG1 gene therapy activated the survival pathways in muscle and spinal cord, increasing the number of surviving MNs and neuromuscular junctions and reducing the astroglial reactivity in the spinal cord of the treated SOD1G93A mice. Furthermore, NRG1-I overexpression preserved motor function and delayed the onset of clinical disease. In summary, our data indicates that NRG1 plays an important role on MN survival and muscle innervation in ALS, and that viral-mediated overexpression of NRG1 isoforms may be considered as a promising approach for ALS treatment

Extrauterine Growth Restriction in Very Low Birth Weight Infants: Concordance Between Fenton 2013 and INTERGROWTH-21st Growth Charts

Postnatal growth restriction has high prevalence in very low birth weight (VLBW) preterm neonates, and this could affect their long-term prognosis. Nowadays, there is no consensus on how to monitor growth in these neonates.Objective: This study aimed to compare prevalence of intra- and extrauterine growth restriction (IUGR and EUGR) in a sample of VLBW infants according to the Fenton 2013 charts and INTERGROWTH-21st (IW-21) standards and to analyze concordance between both in the different EUGR definitions criteria (cross-sectional, dynamic, and true).Patients and Methods: An observational retrospective study of 635 VLBW preterm was performed. The study was carried out in Central University Hospital of Asturias. Body measurements (weight, length, and head circumference) were collected at birth and at hospital discharge and expressed in z-scores for the two references (Fenton 2010 and IW-21). Kappa concordance was calculated.Results: Kappa concordance between Fenton and IW-21 was 0.887 for IUGR and 0.580 for static EUGR. Prevalence was higher according to Fenton in IUGR (36.5 vs. 35.1%), in static EUGR (73.8 vs. 59.3%), and in dynamic EUGR (44.3 vs. 29.3%). Despite observing low prevalence of EUGR when IW-21 was used to define EUGR, a statistical association between neonatal morbidity and diagnosis of EUGR was observed.Conclusion: The Fenton and IW-21 concordance for IUGR is good. IW-21 is more restrictive than Fenton in EUGR. Patients diagnosed by IW-21 as EUGR are more likely to have neonatal morbidity, especially if we use EUGR dynamic definition. In our study, we cannot conclude that one graph is better than the other

Humanized medium (h7H) allows long-term primary follicular thyroid cultures from human normal thyroid, benign neoplasm, and cancer

Mechanisms of thyroid physiology and cancer are principally studied in follicular cell lines. However, human thyroid cancer lines were found to be heavily contaminated by other sources, and only one supposedly normal-thyroid cell line, immortalized with SV40 antigen, is available. In primary culture, human follicular cultures lose their phenotype after passage. We hypothesized that the loss of the thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture, including hormones with marked differences in its affinity for the relevant rodent/human receptor.|The objective of the study was to define conditions that allow the proliferation of primary human follicular thyrocytes for many passages without losing phenotype.|Concentrations of hormones, transferrin, iodine, oligoelements, antioxidants, metabolites, and ethanol were adjusted within normal homeostatic human serum ranges. Single cultures were identified by short tandem repeats. Human-rodent interspecies contamination was assessed.|We defined an humanized 7 homeostatic additives medium enabling growth of human thyroid cultures for more than 20 passages maintaining thyrocyte phenotype. Thyrocytes proliferated and were grouped as follicle-like structures; expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin, and thyroperoxidase showed iodine-uptake and secreted thyroglobulin and free T3. Using these conditions, we generated a bank of thyroid tumors in culture from normal thyroids, Grave's hyperplasias, benign neoplasms (goiter, adenomas), and carcinomas.|Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The bank of thyroid tumors in culture generated under humanized humanized 7 homeostatic additives culture conditions will provide a much-needed tool to compare similarly growing cells from normal vs pathological origins and thus to elucidate the molecular basis of thyroid disease.Ministerio de Ciencia e InnovaciónInstituto de Salud Carlos IIIXunta de GaliciaFondo Social Europeo of the European Communit

Physiopathological and molecular characterization of a transgenic mouse overexpressing TNFalpha in schwann cells reveals a model for chronic peripheral neuropathy

Se ha relacionado la implicación de Tumor Necrosis Factor (TNF) alpha en la patogénesis de la neuropatía diabética periférica (DPN) así como en otras enfermedades inflamatorias cursadas con desmielinización y dolor neuropático. TNF-∝ es una citoquina proinflamatoria que puede actuar a diferentes niveles en el proceso de desmielinización. Tras una lesión en el nervio periférico, las células de Schwann producen esta citoquina en el sistema nervioso periférico (PNS) y se secreta localmente para atraer y activar a los macrófagos circulantes hacia la lesión, contribuyendo así a la degeneración Walleriana. En diferentes neuropatías periféricas conocidas, el incremento de los niveles de TNF-∝ en plasma se encuentran implicados en la progresión maligna de la enfermedad. Estudios en ratas inyectas con TNF-∝ directamente en el nervio ciático, demuestran una inflamación local seguida de desmielinización y degeneración axonal. Además, la administración de TNF-∝ ocasiona hiperalgesia mecánica aguda, una de las principales características del dolor neuropático, así como se ha postulado a TNF-∝ como biomarcador de dolor tras lesión nerviosa. Hoy en día no existen terapias efectivas para frenar y revertir la degeneración axonal y el dolor que cursan las neuropatías. Datos preclínicos obtenidos con los modelos animales actuales demuestran la carencia de un modelo óptimo y de los parámetros adecuados para establecer la patogénesis de la enfermedad. La obtención de un modelo animal apropiado es crítico para mimetizar las neuropatías periféricas humanas. Aunque el incremento mundial de la prevalencia de la diabetes ha potenciado el desarrollo de numerosos modelos, las principales discrepancias para la obtención de un correcto modelo in vivo para las neuropatías asociadas a la Diabetes Mellitus, residen en las importantes diferencias anatómicas y la esperanza de vida entre humanos y roedores. Con el objetivo de caracterizar los efectos de TNF-∝ en el desarrollo de neuropatía periférica crónica y dolor neuropático, se generó un ratón transgénico que sobreexpresa TNF-∝ en células de Schwann, bajo control del promotor de la proteína de la mielina P0. En el trabajo que se presenta a continuación se caracteriza la sobreexpresión de TNF-∝ en las células de Schwann pro-mielinizantes en diferentes estadíos de mielinización (a 5, 21 y 65 días de vida), reflejando que los altos niveles alcanzados en los nervios ciáticos transgénicos ocasionan una desregulación de las principales proteínas de la mielina (P0, MBP, PMP22, MAG), correlacionando con una desestructuración de la vaina de mielina y el incremento de células positivas para p75NTR, marcador específico de células de Schwann inmaduras y no-mielinizantes. También detectamos una inflamación local debido a una una alta infiltración de macrófagos en nervios ciáticos y médula espinal en estos animales. Tras la lesión del nervio ciático, la recuperación de la función motora y posterior remielinización se retrasa en los ratones transgénicos, como demuestra la evaluación mediante el Sciatic Functional Index, y los test electrofisológicos. Sin embargo, estudios de algesimetría muestran inalterada la nocicepción frente a estímulos mecánicos, aunque los ratones transgénicos presentan hipersensibilidad ante estímulos térmicos, incrementada tras la lesión y correlacionada con la activación microglial y astrocitaria en la médula espinal. Además, se detectaron altos niveles de BDNF and CCL2, junto con una sobreexpresión de los canales iónicos Nav1.7 y Nav1.8, relacionados con dolor. Estudios morfométricos no reflejan diferencias en el tamaño de los nervios, ni siquiera tras la lesión, aunque los animales transgénicos sí presentan una ligera reducción en el diámetro axonal y un menor grosor de la mielina. Por consiguiente, este modelo podría ayudar a elucidar el papel de TNF-∝ en el desarrollo de dolor, la regeneración y las neuropatías periféricas, así como en el desarrollo de nuevas terapias eficientes para paliar estas patologías.Tumor Necrosis Factor (TNF) alpha has been implicated in the pathogenesis of diabetic peripheral neuropathy (DPN), among other inflammatory demyelinating diseases and neuropathic pain. TNF-∝ is a pro-inflammatory cytokine that can act at several steps in the demyelination process. It is produced by Schwann cells in the peripheral nervous system (PNS) after nerve injury and released into the local environment to attract and activate macrophages at the site of injury, contributing to Wallerian degeneration and demyelination. An important observation in different peripheral neuropathies is the increased levels of TNF-∝ in plasma, being implicated in the onset and/or malignant progression of peripheral nerve diseases. In vivo studies demonstrated a local inflammation in the sciatic nerve of rats after injection of TNF-∝, followed by demyelination and axonal degeneration. Furthermore, the administration of TNF-∝ resulted in acute mechanical hyperalgesia, a main characteristic of neuropathic pain and therefore TNF-∝ is postulated as a biomarker for painful alterations after nerve injury. Nowadays, there is not an effective therapy to stop and reverse the axonal degeneration and pain that characterize peripheral neuropathies. Unfortunately, the preclinical data using animal models demonstrated the lack of optimum models and outcome measures to underlay the pathogenesis of the disease. An appropriate animal model is critical for replicating the essential features of peripheral neuropathies, understanding DPN pathophysiology and to develop effective strategies. Although the increasing worldwide prevalence of diabetes has fueled the development of several mouse models, the main discrepancies related to the proper generation of a mouse model for the study of peripheral neuropathies, and complications of DM, like diabetic peripheral neuropathy, are a consequence of the anatomical differences and the incomparable life expectancies between humans and rodents. With the aim to characterize TNF-∝ effects in the development of peripheral neuropathy and chronic neuropathic pain, a transgenic mouse model overexpressing TNF-∝ in Schwann cells, under the peripheral myelin protein P0 promoter, was generated. Here we characterized the overexpression of TNF-∝ in myelinated Schwann cells at different stpes of myelination (postnatal days 5, 21 and 65) showing that high levels of TNF-∝ in sciatic nerve leads to the downregulation of the major PNS myelin proteins (P0, MBP, PMP22, MAG) compared to wild type mice, correlating with the loss of structured myelin and an increase in p75NTR in the sciatic nerve, a marker for immature and non-myelinated Schwann cells. Local inflammation was also demonstrated by high levels of macrophage infiltration in both sciatic nerve and spinal cord, compared with wild type animals. Furthermore, stress conditions were induced by sciatic nerve crush after which recovery and subsequent remyelination were delayed in the transgenic mice, as evaluated by the Sciatic Functional Index and electrophysological tests. On the other hand, algesimetrical tests revealed unaltered mechanical nociception, with or without injury, although transgenic animals showed thermal hypersensivity, higher after peripheral injury, correlating with the microglial and astrocyte activation in the spinal cord. Moreover, high expression of BDNF and CCL2, as well as overexpression of Nav1.7 and Nav1.8 channels, all related to the maintenance of chronic inflammatory pain, were detected in DRGs of TNF-∝ transgenic mice. A morphometrical study of tibialis nerves showed no differences in the total nerve surface between genotypes and injury. However, transgenic mice exhibited a slight reduction in the axonal diameter and a significant thinner myelin sheath than wild type animals. This model could be helpful in the characterization of the role TNF-∝ in pain development, injury and DPN as well as in developing efficient therapeutic strategies to modulate such pathological conditions

Physiopathological and molecular characterization of a transgenic mouse overexpressing TNFalpha in schwann cells reveals a model for chronic peripheral neuropathy /

Se ha relacionado la implicación de Tumor Necrosis Factor (TNF) alpha en la patogénesis de la neuropatía diabética periférica (DPN) así como en otras enfermedades inflamatorias cursadas con desmielinización y dolor neuropático. TNF-∝ es una citoquina proinflamatoria que puede actuar a diferentes niveles en el proceso de desmielinización. Tras una lesión en el nervio periférico, las células de Schwann producen esta citoquina en el sistema nervioso periférico (PNS) y se secreta localmente para atraer y activar a los macrófagos circulantes hacia la lesión, contribuyendo así a la degeneración Walleriana. En diferentes neuropatías periféricas conocidas, el incremento de los niveles de TNF-∝ en plasma se encuentran implicados en la progresión maligna de la enfermedad. Estudios en ratas inyectas con TNF-∝ directamente en el nervio ciático, demuestran una inflamación local seguida de desmielinización y degeneración axonal. Además, la administración de TNF-∝ ocasiona hiperalgesia mecánica aguda, una de las principales características del dolor neuropático, así como se ha postulado a TNF-∝ como biomarcador de dolor tras lesión nerviosa. Hoy en día no existen terapias efectivas para frenar y revertir la degeneración axonal y el dolor que cursan las neuropatías. Datos preclínicos obtenidos con los modelos animales actuales demuestran la carencia de un modelo óptimo y de los parámetros adecuados para establecer la patogénesis de la enfermedad. La obtención de un modelo animal apropiado es crítico para mimetizar las neuropatías periféricas humanas. Aunque el incremento mundial de la prevalencia de la diabetes ha potenciado el desarrollo de numerosos modelos, las principales discrepancias para la obtención de un correcto modelo in vivo para las neuropatías asociadas a la Diabetes Mellitus, residen en las importantes diferencias anatómicas y la esperanza de vida entre humanos y roedores. Con el objetivo de caracterizar los efectos de TNF-∝ en el desarrollo de neuropatía periférica crónica y dolor neuropático, se generó un ratón transgénico que sobreexpresa TNF-∝ en células de Schwann, bajo control del promotor de la proteína de la mielina P0. En el trabajo que se presenta a continuación se caracteriza la sobreexpresión de TNF-∝ en las células de Schwann pro-mielinizantes en diferentes estadíos de mielinización (a 5, 21 y 65 días de vida), reflejando que los altos niveles alcanzados en los nervios ciáticos transgénicos ocasionan una desregulación de las principales proteínas de la mielina (P0, MBP, PMP22, MAG), correlacionando con una desestructuración de la vaina de mielina y el incremento de células positivas para p75NTR, marcador específico de células de Schwann inmaduras y no-mielinizantes. También detectamos una inflamación local debido a una una alta infiltración de macrófagos en nervios ciáticos y médula espinal en estos animales. Tras la lesión del nervio ciático, la recuperación de la función motora y posterior remielinización se retrasa en los ratones transgénicos, como demuestra la evaluación mediante el Sciatic Functional Index, y los test electrofisológicos. Sin embargo, estudios de algesimetría muestran inalterada la nocicepción frente a estímulos mecánicos, aunque los ratones transgénicos presentan hipersensibilidad ante estímulos térmicos, incrementada tras la lesión y correlacionada con la activación microglial y astrocitaria en la médula espinal. Además, se detectaron altos niveles de BDNF and CCL2, junto con una sobreexpresión de los canales iónicos Nav1.7 y Nav1.8, relacionados con dolor. Estudios morfométricos no reflejan diferencias en el tamaño de los nervios, ni siquiera tras la lesión, aunque los animales transgénicos sí presentan una ligera reducción en el diámetro axonal y un menor grosor de la mielina. Por consiguiente, este modelo podría ayudar a elucidar el papel de TNF-∝ en el desarrollo de dolor, la regeneración y las neuropatías periféricas, así como en el desarrollo de nuevas terapias eficientes para paliar estas patologías.Tumor Necrosis Factor (TNF) alpha has been implicated in the pathogenesis of diabetic peripheral neuropathy (DPN), among other inflammatory demyelinating diseases and neuropathic pain. TNF-∝ is a pro-inflammatory cytokine that can act at several steps in the demyelination process. It is produced by Schwann cells in the peripheral nervous system (PNS) after nerve injury and released into the local environment to attract and activate macrophages at the site of injury, contributing to Wallerian degeneration and demyelination. An important observation in different peripheral neuropathies is the increased levels of TNF-∝ in plasma, being implicated in the onset and/or malignant progression of peripheral nerve diseases. In vivo studies demonstrated a local inflammation in the sciatic nerve of rats after injection of TNF-∝, followed by demyelination and axonal degeneration. Furthermore, the administration of TNF-∝ resulted in acute mechanical hyperalgesia, a main characteristic of neuropathic pain and therefore TNF-∝ is postulated as a biomarker for painful alterations after nerve injury. Nowadays, there is not an effective therapy to stop and reverse the axonal degeneration and pain that characterize peripheral neuropathies. Unfortunately, the preclinical data using animal models demonstrated the lack of optimum models and outcome measures to underlay the pathogenesis of the disease. An appropriate animal model is critical for replicating the essential features of peripheral neuropathies, understanding DPN pathophysiology and to develop effective strategies. Although the increasing worldwide prevalence of diabetes has fueled the development of several mouse models, the main discrepancies related to the proper generation of a mouse model for the study of peripheral neuropathies, and complications of DM, like diabetic peripheral neuropathy, are a consequence of the anatomical differences and the incomparable life expectancies between humans and rodents. With the aim to characterize TNF-∝ effects in the development of peripheral neuropathy and chronic neuropathic pain, a transgenic mouse model overexpressing TNF-∝ in Schwann cells, under the peripheral myelin protein P0 promoter, was generated. Here we characterized the overexpression of TNF-∝ in myelinated Schwann cells at different stpes of myelination (postnatal days 5, 21 and 65) showing that high levels of TNF-∝ in sciatic nerve leads to the downregulation of the major PNS myelin proteins (P0, MBP, PMP22, MAG) compared to wild type mice, correlating with the loss of structured myelin and an increase in p75NTR in the sciatic nerve, a marker for immature and non-myelinated Schwann cells. Local inflammation was also demonstrated by high levels of macrophage infiltration in both sciatic nerve and spinal cord, compared with wild type animals. Furthermore, stress conditions were induced by sciatic nerve crush after which recovery and subsequent remyelination were delayed in the transgenic mice, as evaluated by the Sciatic Functional Index and electrophysological tests. On the other hand, algesimetrical tests revealed unaltered mechanical nociception, with or without injury, although transgenic animals showed thermal hypersensivity, higher after peripheral injury, correlating with the microglial and astrocyte activation in the spinal cord. Moreover, high expression of BDNF and CCL2, as well as overexpression of Nav1.7 and Nav1.8 channels, all related to the maintenance of chronic inflammatory pain, were detected in DRGs of TNF-∝ transgenic mice. A morphometrical study of tibialis nerves showed no differences in the total nerve surface between genotypes and injury. However, transgenic mice exhibited a slight reduction in the axonal diameter and a significant thinner myelin sheath than wild type animals. This model could be helpful in the characterization of the role TNF-∝ in pain development, injury and DPN as well as in developing efficient therapeutic strategies to modulate such pathological conditions

Therapeutic role of full-length Neuregulin 1 Type III in SOD1-linked Amyotrophic Lateral Sclerosis.

International audienc

Therapeutic Role of Neuregulin 1 Type III in SOD1-Linked Amyotrophic Lateral Sclerosis

International audienc

Gene Therapy Overexpressing Neuregulin 1 Type I in Combination With Neuregulin 1 Type III Promotes Functional Improvement in the SOD1^G93A ALS Mice

Motoneuron; Neuregulin 1; Spinal cordMotoneurona; Neuregulina 1; Médula espinalMotoneurona; Neuregulina 1; Medul·la espinalAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting the neuromuscular system for which currently there is no effective therapy. Motoneuron (MN) degeneration involves several complex mechanisms, including surrounding glial cells and skeletal muscle contributions. Neuregulin 1 (NRG1) is a trophic factor present particularly in MNs and neuromuscular junctions. Our previous studies revealed that gene therapy overexpressing the isoform I (NRG1-I) in skeletal muscles as well as overexpressing the isoform III (NRG1-III) directly in the central nervous system are both effective in preserving MNs in the spinal cord of ALS mice, opening novel therapeutic approaches. In this study, we combined administration of both viral vectors overexpressing NRG1-I in skeletal muscles and NRG1-III in spinal cord of the SOD1G93A mice in order to obtain a synergistic effect. The results showed that the combinatorial gene therapy increased preservation of MNs and of innervated neuromuscular junctions and reduced glial reactivity in the spinal cord of the treated SOD1G93A mice. Moreover, NRG1 isoforms overexpression improved motor function of hindlimb muscles and delayed the onset of clinical disease. However, this combinatory gene therapy did not produce a synergic effect compared with single therapies, suggesting an overlap between NRG1-I and NRG1-III activated pathways and their beneficial effects.This work was funded by grant TV3201428-10 of Fundació La Marato-TV3, grant #20289 of AFM-Telethon, cooperative project 2015-01 from CIBERNED, PID2020-116735RB-I00 from MICINN and TERCEL (RD16/0011/0014) funds from the Instituto de Salud Carlos III of Spain

Postnatal Growth Faltering: Growth and Height Improvement at Two Years in Children with Very Low Birth Weight between 2002–2017

The prevalence of postnatal growth faltering (PGF) in preterm infants with very low birth weight (VLBW) (n = 112; and 2013–2017, n = 92) were compared. Results. In the second five-year period, a decrease in prevalence of PGF was observed (36.6% vs. 22.8%, p = 0.033), an increase in growth rate in the first 28 days (5.22 (4.35–6.09) g/kg/day vs. 11.38 (10.61–12.15) g/kg/day, p p = 0.023). Probability of short stature at 2 years was directly related to daily weight gain in the first 28 days. Conclusions: when comparing two five-year periods in the last two decades, growth in VLBW preterm infants has improved, both during neonatal period and at two years of age