Trk Inhibition Reduces Tumorsphere Formation and Changes Expression of Stemness Markers in SK-ES-1 Ewing Sarcoma Cells / Inibição de Trk Reduz a Formação de Tumoresferas e Modifica a Expressão de Marcadores Tronco Tumorais em Células de Sarcoma de Ewing SK-ES-1 / Inhibición de Trk Reduce la Formación de Esferoides Tumorales y Cambia la Expresión de Marcadores de Stemness en Células de Sarcoma de Ewing SK-ES-1

Introduction: Ewing sarcoma (ES) is a highly aggressive type of childhood cancer characterized by a chromosomal translocation resulting in fusions between the gene encoding EWS RNA Binding Protein 1 (EWSR1) and one gene of the ETS family, most frequently FLI-1, resulting in the EWS-FLI1 aberrant transcription factor. ES tumors can contain a subpopulation of cells showing cancer stem cell (CSC) features, which express stemness markers including CD133, OCT4 (Octamer-binding transcription factor 4), and NANOG, and display capacity to form tumorspheres likely enriched in CSCs. Neurotrophin (NT) receptors of the tropomyosin receptor kinase (Trk) family (TrkA, TrkB, and TrkC) may play a role in stimulating ES progression, but their possible role in CSCs remains unknown. Objective: To verify the effect of Trks inhibition on the formation of tumorspheres as well as the gene expression of stem markers. Method: The cells were dissociated and the formation of spheres was induced with supplemented culture medium and the K252a treatment was performed. After RNA extraction, mRNA expression levels of target genes Prom1 (CD133), OCT4 (POU5F1), SOX2, and Musashi-1 (MSI1) were analyzed by qPCR. Results: The pan-Trk inhibitor K252a (100 or 500 mM) hindered tumorsphere formation in human SK-ES-1 ES cell cultures. K252a also reduced mRNA expression of Prom1 (CD133-coding gene) while enhancing expression of OCT4. No changes in mRNA levels of SOX2 or Musashi-1 were observed. Conclusion: These findings provide the first evidence suggesting that Trk activity can influence stemness in ES cells

Introdução: O sarcoma de Ewing (SE) é um tipo altamente agressivo de câncer infantil caracterizado por uma translocação cromossômica que resulta em fusões entre o gene que codifica a proteína de ligação a RNA EWS 1 (EWSR1) e um gene da família ETS, mais frequentemente o FLI-1, resultando no fator de transcrição aberrante EWS-FLI1. Os tumores de SE podem conter uma subpopulação de células com características de células-tronco tumorais (CTT), que expressam marcadores de pluripotência como CD133, OCT4 e NANOG, e têm a capacidade de formar esferas tumorais provavelmente enriquecidas em CTT. Os receptores de neurotrofinas (NT) da família de receptor de quinase de tropomiosina (Trk) (TrkA, TrkB e TrkC) podem desempenhar um papel no estímulo à progressão do SE, mas seu possível papel nas CTT permanece desconhecido. Objetivo: Verificar o efeito da inibição dos Trk na formação de tumoresferas, bem como na expressão gênica de marcadores de pluripotência. Método: As células foram dissociadas, a formação de esferas com meio de cultura suplementado foi induzida e realizou-se o tratamento com K252a. Após a extração de RNA, os níveis de expressão de mRNA dos genes-alvo Prom1 (CD133), OCT4 (POU5F1), SOX2 e Musashi-1 (MSI1) foram analisados por qPCR. Resultados: O inibidor pan-Trk K252a (100 ou 500 mM) impediu a formação de esferas tumorais em culturas de células de SE humanas SK-ES-1. O K252a também reduziu a expressão de mRNA de Prom1 (o gene que codifica CD133), enquanto aumentou a expressão de OCT4. Não foram observadas mudanças nos níveis de mRNA de SOX2 ou Musashi-1. Conclusão: Essas descobertas fornecem as primeiras evidências, sugerindo que a atividade dos Trk possa influenciar a pluripotência nas células de SE

Introducción: El sarcoma de Ewing (SE) es un tipo de cáncer infantil altamente agresivo caracterizado por una translocación cromosómica que resulta en fusiones entre el gen que codifica la proteína de unión a RNA EWS 1 (EWSR1) y un gen de la familia ETS, más frecuentemente FLI-1, lo que resulta en el factor de transcripción aberrante EWS-FLI1. Los tumores del SE pueden contener una subpoblación de células que presentan características de células madre cancerosas (CMC), las cuales expresan marcadores de pluripotencia como CD133, OCT4 y NANOG, y muestran la capacidad de formar esferas tumorales probablemente enriquecidas en CMC. Los receptores de neurotrofinas (NT) de la familia del receptor de quinasa de tropomiosina (Trk) (TrkA, TrkB y TrkC) podrían desempeñar un papel en el estímulo de la progresión del SE, pero su posible papel en las CMC aún es desconocido. Objetivo: Verificar el efecto de la inhibición de los Trk en la formación de esferoides tumorales, así como en la expresión génica de marcadores de pluripotencia. Método: Las células fueron disociadas e inducidas a formar esferas con un medio de cultivo suplementado y se realizó el tratamiento con K252a. Después de la extracción de ARN, los niveles de expresión de ARNm de los genes objetivo Prom1 (CD133), OCT4 (POU5F1), SOX2 y Musashi-1 (MSI1) se analizaron mediante qPCR. Resultados: El inhibidor pan-Trk K252a (100 o 500 mM) evitó la formación de esferas tumorales en cultivos de células de SE humanas SK-ES-1. El K252a también redujo la expresión de ARNm de Prom1 (el gen que codifica CD133), mientras que aumentaba la expresión de OCT4. No se observaron cambios en los niveles de ARNm de SOX2 o Musashi-1. Conclusión: Estos hallazgos proporcionan las primeras evidencias que sugieren que la actividad de Trk puede influir en la pluripotencia en las células del SE

Temporo-spatial distribution of stem cell markers CD146 and p75NTR during odontogenesis in mice

Abstract Mesenchymal and epithelial stem cells were identified in dental tissues; however, knowledge about the odontogenic stem cells is limited, and there are some questions regarding their temporo-spatial dynamics in tooth development. Objective Our study aimed to analyze the expression of the stem cell markers CD146 and p75NTR during the different stages of odontogenesis. Methodology The groups consisted of 13.5, 15.5, 17.5 days old embryos, and 14 days postnatal BALB/c mice. The expression of CD146 and p75NTR was evaluated by immunohistochemistry. Results Our results showed that positive cells for both markers were present in all stages of tooth development, and the number of positive cells increased with the progression of this process. Cells of epithelial and ectomesenchymal origin were positive for CD146, and the expression of p75NTR was mainly detected in the dental papilla and dental follicle. In the postnatal group, dental pulp cells were positive for CD146, and the reduced enamel epithelium and the oral mucosa epithelium showed immunostaining for p75NTR. Conclusions These results suggest that the staining pattern of CD146 and p75NTR underwent temporal and spatial changes during odontogenesis and both markers were expressed by epithelial and mesenchymal cell types, which is relevant due to the significance of the epithelial-ectomesenchymal interactions in tooth development.

Phenotype changes of oral epithelial stem cells after in vitro culture

Abstract The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.

Expression of CD90 and P75NTR stem cell markers in ameloblastomas: a possible role in their biological behavior

Abstract Multicystic and unicystic ameloblastomas are benign odontogenic tumors that present distinct biological behavior. The investigation of stem cells has become an important branch of tumor biology, with several studies addressing the possible role of these cells in tumor growth, angiogenesis, progression, infiltration and invasiveness. This study evaluated the immunohistochemical expression of CD90(Thy-1) and P75NTR stem cell markers in multicystic and unicystic ameloblastomas. Seventeen (17) samples of ameloblastomas (multicystic, n = 10; unicystic, n = 7) were submitted to immunohistochemical reactions and graded semi-quantitatively. The Kolmogorov-Smirnov test was used to verify possible differences in CD90 and P75NTR expressions between multicystic and unicystic ameloblastomas (p < 0.05). CD90 immunostaining was observed in all multicystic ameloblastoma specimens (n = 10), in the cytoplasm of the fibroblasts and vascular endothelial cells of the tumor stroma, near the neoplastic odontogenic epithelia. The staining of stromal CD90 was significantly higher in multicystic than in unicystic ameloblastomas (p = 0.003). Nuclear P75NTR immunostaining was observed in all ameloblastoma specimens. A significant difference was seen in the epithelial staining of P75NTR between multicystic and unicystic types (p = 0.007). The increased expression of CD90 and P75NTR found in multicystic ameloblastomas suggests a behavioral biological difference between multicystic and unicystic ameloblastomas, as well as a difference in ameloblastoma development.

Effectiveness of hip muscle strengthening in patellofemoral pain syndrome patients: a systematic review

Introduction: Patellofemoral pain syndrome (PFPS) is characterized by anterior knee pain, which may limit the performance of functional activities. The influence of hip joint motion on the development of this syndrome has already been documented in the literature. In this regard, studies have investigated the effectiveness of hip muscle strengthening in patients with PFPS. Objectives: The aims of this systematic review were (1) to summarize the literature related to the effects of hip muscle strengthening on pain intensity, muscle strength, and function in individuals with PFPS and (2) to evaluate the methodological quality of the selected studies. Method: A search for randomized controlled clinical trials was conducted using the following databases: Google Scholar, MEDLINE, PEDro, LILACS, and SciELO. The selected studies had to distinguish the effects of hip muscle strengthening in a group of patients with PFPS, as compared to non-intervention or other kinds of intervention, and had to investigate the following outcomes: pain, muscle strength, and function. The methodological quality of the selected studies was analyzed by means of the PEDro scale. Results: Seven studies were selected. These studies demonstrated that hip muscle strengthening was effective in reducing pain. However, the studies disagreed regarding the treatments' ability to improve muscle strength. Improvement in functional capabilities after hip muscle strengthening was found in five studies. Conclusion: Hip muscle strengthening is effective in reducing the intensity of pain and improving functional capabilities in patients with PFPS, despite the lack of evidence for its ability to increase muscle strength. .

Neurotrophic factors in tension-type headache / Fatores neurotróficos na cefaleia do tipo tensional

Neurotrophic factors (NF) are involved in pain regulation and a few studies have suggested that they may play a pathophysiological role in primary headaches. The aim of this study was to investigate NF levels in patients with tension type headache (TTH). We carried out a cross sectional study including 48 TTH patients and 48 age and gender matched controls. Beck Depression and Anxiety Inventories, and Headache Impact Test were recorded. Serum levels of NF were determined by ELISA. There were not significant differences between NF levels between TTH patients and controls. Patients with chronic and episodic TTH had not significant differences in NF levels. The presence of headache at the time of evaluation did not significantly alter the levels of NF. Depression and anxiety scores as well as headache impact did not correlate with NF levels. Our study suggest that the serum levels of NF are not altered in TTH.

Os fatores neurotróficos (FN) participam da regulação da dor e podem ter um papel na fisiopatologia das cefaleias peimárias. O objetivo do presente estudo foi avaliar os níveis séricos de FN em pacientes com cefaleia do tipo tensional (CTT). Foi realizado corte transversal com 48 pacientes com CTT e 48 controles pareados por gênero e idade. Os inventários de Beck para depressão e ansiedade, bem como o inventário de impacto da cefaleia foram aplicados. Os níveis séricos de FN foram determinados por ELISA. Não houve diferenças significativas entre níveis de FN entre pacientes com TTH e controles, bem como entre pacientes com TTH episódica e crônica. Presença de cefaleia no momento da avaliação não alterou os níveis séricos de FN. Os escores de depressão, ansiedade e impacto da cefaleia não se correlacionaram com os níveis de FN. Nosso estudo sugere que não há alteração dos níveis de FN na TTH.

Cutaneous lesions sensory impairment recovery and nerve regeneration in leprosy patients

It is important to understand the mechanisms that enable peripheral neurons to regenerate after nerve injury in order to identify methods of improving this regeneration. Therefore, we studied nerve regeneration and sensory impairment recovery in the cutaneous lesions of leprosy patients (LPs) before and after treatment with multidrug therapy (MDT). The skin lesion sensory test results were compared to the histopathological and immunohistochemical protein gene product (PGP) 9.5 and the p75 nerve growth factor receptors (NGFr) findings. The cutaneous neural occupation ratio (CNOR) was evaluated for both neural markers. Thermal and pain sensations were the most frequently affected functions at the first visit and the most frequently recovered functions after MDT. The presence of a high cutaneous nerve damage index did not prevent the recovery of any type of sensory function. The CNOR was calculated for each biopsy, according to the presence of PGP and NGFr-immunostained fibres and it was not significantly different before or after the MDT. We observed a variable influence of MDT in the recovery from sensory impairment in the cutaneous lesions of LPs. Nociception and cold thermosensation were the most recovered sensations. The recovery of sensation in the skin lesions appeared to be associated with subsiding inflammation rather than with the regenerative activity of nerve fibres.

Supraorganized collagen enhances Schwann cell reactivity and organization in vitro

We investigated the reactivity and expression of basal lamina collagen by Schwann cells (SCs) cultivated on a supraorganized bovine-derived collagen substrate. SC cultures were obtained from sciatic nerves of neonatal Sprague-Dawley rats and seeded on 24-well culture plates containing collagen substrate. The homogeneity of the cultures was evaluated with an SC marker antibody (anti-S-100). After 1 week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, S-100 and p75NTR (pan neurotrophin receptor) and for scanning electron microscopy (SEM). Positive labeling with antibodies to the cited molecules was observed, indicating that the collagen substrate stimulates SC alignment and adhesion (collagen IV labeling - organized collagen substrate: 706.33 ± 370.86, non-organized collagen substrate: 744.00 ± 262.09; S-100 labeling - organized collagen: 3809.00 ± 120.28, non-organized collagen: 3026.00 ± 144.63, P < 0.05) and reactivity (p75NTR labeling - organized collagen: 2156.33 ± 561.78, non-organized collagen: 1424.00 ± 405.90, P < 0.05; means ± standard error of the mean in absorbance units). Cell alignment and adhesion to the substrate were confirmed by SEM analysis. The present results indicate that the collagen substrate with an aligned suprastructure, as seen by polarized light microscopy, provides an adequate scaffold for SCs, which in turn may increase the efficiency of the nerve regenerative process after in vivo repair.

Importancia de las neurotrofinas en medicina humana veterinaria / Importance of neurotrophins in human medicine veterinary

Las neurotrofinas (NTs) son factores de crecimiento que ejercen la totalidad o parte de sus funciones sobreel sistema nervioso central y periférico. Las neurotrofinas son una familia de péptidos con propiedades neurotróficas y neurotrópicas que incluyen los siguientes compuestos: factor de crecimiento nerviosos, (nerve growth factor, NGF), factor de crecimiento derivado del cerebro (Brain-derived neurotrophic factor, BDNF), y las neurotrofinas 3 (NT-3), 4/5 (NT/4/5), (NT-6). Se trata de un conjunto de péptidos relacionados estructural yfuncionalmente, que favorecen la supervivencia y diferenciación fenotípica de su-poblaciones neuronales del sistemanervioso periférico durante el desarrollo embrionario y su mantenimiento durante la vida adulta. Además actúansobre algunos tipos de neuronas del sistema nervioso central. Por otro lado, estudios llevados a cabo en los últimosaños sugieren que las neurotrofinas podrían controlar algunas funciones en tejidos no nerviosos, especialmente enlos órganos linfoides y en las células inmunocompetentes. En medicina Humana se hadeterminado que en el aparato de la visión la falta de BDNF lleva: 1) a la pérdida de las células ganglionares de laretina y también es responsable del glaucoma en el hombre; 2) En el Sistema nervios causa depresión, epilepsia, enla enfermedad de Huntington; .3) y en la parte metabólica causa la diabetes tipo. Está comprobado que las dosprimeras (NTs) están presentes en el carcinoma de colon, páncreas y glándulas prostáticas, para estas dos patologíasya están en venta la anti-neurotrofinas que neutralizan a los NGF, BDNF, NT-3, NT 4/5NT e incluyen anticuerposgenerados así como sus fragmentos.En Medicina veterinaria se han detectados a los receptores de alta afinidad(Proteínas Trks) en el timo de todos los vertebrados inferiores y superiores. También en los órganos linfáticossecundarios de Sus scrofa domesticus y Lama glama; en el aparato digestivo y reproductor de Rattus norvergicus.

Neurotrophins (NTs ) are growth factors that exert all or part of its functions onthe central and peripheral nervous system. Neurotrophins are a family of peptides with neurotrophic and neurotropic properties include the following compounds: nerve growth factor ( nerve growth factor , NGF) , growth factor , brain derived ( Brain- derived neurotrophic factor , BDNF) , and neurotrophin 3 (NT -3 ) , 4 /5 ( NT/4/5 ) , ( NT - 6) . This is a set of structural and related peptides functionally , favoring survival and phenotypic differentiation of neuronal populations system su-peripheral nervous during embryonic development and maintenance during adulthood . Besides actingon some types of neurons of the central nervous system . Moreover, studies conducted in recentyears suggest that neurotrophins may control some functions in non-nervous tissues, especially inand lymphoid organs in immunocompetent cells . In Human medicine has determined that in the apparatus of the absence of BDNF vision leads : 1) a loss of ganglion cellsretina and is also responsible for glaucoma in man; 2) In the nerve system causes depression, epilepsy,Huntington 's disease ; 3 ) and in metabolic diabetes causes type part . It is proved that the twofirst (NTs ) are present in the carcinoma of the colon, pancreas and prostate glands , for these two pathologies Information already in the anti - neurotrophins that neutralize NGF , BDNF , NT -3 , NT and include antibodies 4/5NTgenerated and their fragmentos.En Veterinary Medicine detected to have high affinity receptors (Proteins Trks ) in the thymus of all lower and higher vertebrates. Also in the lymphatic organsHis side scrofa domesticus and Lama glama ; in the digestive tract and Rattus norvegicus.

Neurotrofinas na epilepsia do lobo temporal / Neurotrophins in temporal lobe epilepsy

INTRODUÇÃO: A neurotrofinas NGF, BDNF, NT-3 e NT-4 são os principais representantes da família das neurotrofinas no sistema nervoso central de mamíferos. Estão presentes em estágios específicos do crescimento e sobrevivência neuronal como a divisão celular, diferenciação e axogênese e também nos processos naturais de morte celular neuronal. A atividade biológica das neurotrofinas é mediada pelos receptores de tropomiosina quinase Trk. NGF ativa principalmente os receptores TrkA, BDNF e NT-4 interagem com os receptores TrkB e NT-3 com TrkC. Todas as NTs também podem se ligar, com menor afinidade, ao receptor p75NTR. Nesta breve revisão serão levantadas as principais evidências sobre o papel e expressão das principais neurotrofinas no hipocampo, com ênfase nas alterações que ocorrem em modelos animais de epilepsia. RESULTADOS: As neurotrofinas parecem ter um papel chave na plasticidade sináptica relacionada à epilepsia, onde elas poderiam agir tanto como fatores promotores da epileptogênese quanto como substâncias anti-epiléptogênicas endógenas. Além disso a expressão dos genes que codificam os fatores neurotróficos e seus receptores pode ser alterada pela atividade de crises em diversos modelos de epilepsia. CONCLUSÃO: Vários estudos têm demonstrado a relação entre a expressão das neurotrofinas e as alterações na plasticidade dos circuitos neuronais que ocorrem após danos cerebrais, tais como a epilepsia. O conhecimento das alterações na expressão das neurotrofinas na plasticidade neuronal pode nos auxiliar a entender como estas moléculas participam dos mecanismos epileptogênicos e dessa forma, dar início ao estudo de novas terapias e ao desenvolvimento de novas drogas que auxiliem no tratamento da epilepsia.

INTRODUCTION: NGF, BDNF, NT-3 and NT-4 are the major neurotrophins in the mammal central nervous system. These proteins play key roles in development of the nervous system, but they are also responsible for important functions in the adult brain, such as trophic support of adult neurons, cell plasticity and death. The neurotrophins activate three different members of the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases. These three receptors exhibit distinct affinities for different neurotrophins, with NGF activating TrkA, BDNF and NT-4 activating TrkB, and NT-3 predominantly activating TrkC. All NTs can also interact with the receptor p75NTR, a member of the tumor necrosis factor receptor superfamily. RESULTS: NTs have a key role also in the neuronal plasticity related to epilepsy, and they are able to act as epileptogenic factors and anti-epileptogenic endogenous factors. Besides that, several studies have shown that status epilepticus and chronic seizures may alter gene and protein expression of these factors. CONCLUSION: Here, we briefly give a short review of current knowledge of the roles and expression of the major neurotrophins in the hippocampus, with emphasis to the changes that occur in animal models of epilepsy. The knowledge on how the mechanisms underlying the multiplicity of biological functions in which the neurotrophins take part may provide us key insights into the cellular mechanisms of neuronal function in health and disease.

An immunohistochemical, clinical and electroneuromyographic correlative study of the neural markers in the neuritic form of leprosy

The nerve biopsies of 11 patients with pure neuritic leprosy were submitted to routine diagnostic procedures and immunoperoxidase staining with antibodies against axonal (neurofilament, nerve growth factor receptor (NGFr), and protein gene product (PGP) 9.5) and Schwann cell (myelin basic protein, S-100 protein, and NGFr) markers. Two pairs of non-adjacent histological cross-sections of the peripheral nerve were removed for quantification. All the fascicles of the nerve were examined with a 10X-ocular and 40X-objective lens. The immunohistochemistry results were compared to the results of semithin section analysis and clinical and electroneuromyographic data. Neurofilament staining was reduced in 100 percent of the neuritic biopsies. NGFr positivity was also reduced in 81.8 percent, PGP staining in 100 percent of the affected nerves, S100 positivity in 90.9 percent, and myelin basic protein immunoreactivity in 90.9 percent. Hypoesthesia was associated with decreased NGFr (81.8 percent) and PGP staining (90.9 percent). Reduced potential amplitudes (electroneuromyographic data) were found to be associated with reduced PGP 9.5 (63.6 percent) and nerve fiber neurofilament staining (45.4 percent) by immunohistochemistry and with loss of myelinated fibers (100 percent) by semithin section analysis. On the other hand, the small fibers (immunoreactive dots) seen amid inflammatory cells continued to be present even after 40 percent of the larger myelinated fibers had disappeared. The present study shows an in-depth view of the destructive effects of leprosy upon the expression of neural markers and the integrity of nerve fiber. The association of these structural changes with the clinical and electroneuromyographic manifestations of leprosy peripheral neuropathy was also discussed.