RESUMO
Recientemente se ha descubierto que diversos tejidos dentales son fuente importante de Células Madre Mesenquimales (CMM). En la cavidad oral podemos encontrar CMM en la pulpa, en el folículo dental, papila y en la encía entre otros lugares. Varios estudios avalan el extenso potencial terapéutico de las CMM en terapias de regeneración. El objetivo de este estudio es aislar, cultivar células madres mesenquimales de pulpa y folículo dental humano, caracterizar su inmunofenotipo y su potencial de diferenciación a linaje osteogénico, condrogénico y osteogénico. Se cultivaron células de pulpa y folículo dental de terceros molares de dientes permanentes jóvenes humanos. Los cultivos de CMM fueron monitoreados por microscopia óptica, las células se inmunotipificaron por citometría de flujo. Posteriormente se evaluó su capacidad de diferenciaron a los tres linajes mencionados. En estas condiciones experimentales se comprobó que las células aisladas y cultivadas de pulpa y folículo dental correspondían a células madre mesenquimales humanas, siendo éstas últimas más fáciles de obtener y proliferar. Las CMM de folículo dental poseen mayor potencial de crecimiento y capacidad de diferenciación en comparación a las CMM de pulpa dental, probablemente debido a su estado evolutivo más inmaduro.
It was recently discovered that dental tissues are important sources of mesenchymal stem cells (MSCs). In the oral cavity MSCs can be found in the pulp, dental follicle, apical papilla and gingival tissue, among others. Many studies support the therapeutic potential of MSCs in regenerative therapies. The objective of this study was to isolate and culture mesenchymal stem cells from human dental pulp and follicle, and to characterize their immunophenotype and differentiation potential to adipogenic, chondrogenic and osteogenic lineages. Oral cavity stem cells were cultured from pulp and dental follicle of wisdom teeth from young permanent teeth. Immunotypification of MSCs was performed by flow cytometry and cultures were evaluated for their ability to differentiate into the three lineages mentioned. Our results corroborate that cultured oral MSC cells isolated from pulp and dental follicle were mesenchymal in origin, being the latter more easy to obtain. Dental follicle MSCs have greater growth potential and differentiation capacity compared to dental pulp MSCs, probably due to their more immature developmental state.
Assuntos
Humanos , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Proliferação de Células , Polpa Dentária/citologia , Saco Dentário/citologia , Técnicas de Cultura de Células , ImunofenotipagemRESUMO
Las células madres mesenquimales (CMM) pueden ser afectadas en su capacidad de proliferar in vitro bajo estimulación física o bioquímica, siendo esta una capacidad esencial para un adecuado cultivo celular. Un método de estimulación física que ha demostrado ser eficiente en este sentido es el Ultrasonido pulsátil de baja intensidad (USBI), aplicado en intensidades iguales o inferiores a 100 mW/cm2, habitualmente entre 30 y 50 mW/cm2. El objetivo de esta investigación fue determinar el nivel de intensidad de ultrasonido pulsátil de baja intensidad óptimo entre 30 y 50 mW/cm2 para estimular la proliferación de CMM de médula ósea de ratas Sprague Dawley, in vitro. CMM (1x106cls/kg) de medula ósea de rata Sprague-Dawley fueron cultivadas (alfa-MEM, 20 por ciento FBS y 1 por ciento antibiotico) y estimuladas con USBI (0,02 milisegundos), por 20 minutos dos veces al día por 10 días con intensidades de 0, 30 y 50 mW/cm2 (grupos A [control], B, C respectivamente). Se contabilizó el número de células en el cultivo y se evaluó morfología celular en microscopio óptico. Se utilizaron tests de ANOVA on Ranks y Bonferroni. Los cultivos estimulados con USBI presentaron mayores recuentos celulares, y se observaron diferencias estadísticamente significativas entre los grupos A y C (p<0,05). Se observaron diferencias morfológicas entre células de grupos estimulados con USBI y el control. La estimulación de las CMM en cultivos bidimensionales con USBI influencia cambios en la morfología celular y se concluye que 50mW/cm2 es la intensidad óptima dentro de las evaluadas para producir aumento en la proliferación celular (p<0.05).
Mesenchymal stem cells (MSCs) can be affected in their capabilities to proliferate in vitro under physical and/or biochemical stimulation. The aim of this study was to select an optimal intensity level for low intensity pulsed ultrasound (LIPUS) stimulation of Sprague-Dawley bone marrow MSCs proliferation in vitro. Bone marrow MSCs of Sprague-Dawley rats where cultured (a-MEM, 20 percent FBS and 1 percent antibiotic) and stimulated with LIPUS (0,02 milisec), for 20 minutes twice daily for 10 days, at intensities of 0 (control), 30 and 50mW/cm2 (groups A, B, C). Cellular count and morphological evaluation were performed. ANOVA and Bonferroni tests were performed. LIPUS-stimulated cultures displayed greater cellular counts, and significant differences were observed between groups A and C (p<0,05). Morphological differences were observed between cells from LIPUS-stimulated and control groups. An intensity of 50mW/cm2 elicits increased cellular proliferation (p<0.05). Stimulation of MSCs cultures with LIPUS influences cellular morphology.
Assuntos
Animais , Ratos , Células-Tronco Mesenquimais/citologia , Proliferação de Células , Ultrassom , Técnicas de Cultura de Células , Células Cultivadas , Células da Medula Óssea/citologia , Ratos Sprague-DawleyRESUMO
Introducción: El Herpes Labial Recurrente supone una condición inmunológica alterada, tal como una hiperactividad de células T-reguladoras CD4+CD25+ (Treg). Éstas ejercen control sobre la tolerancia periférica y reducen el riesgo inmunopatológico, suprimiendo otras líneas celulares. Por ende, la supresión ejercida sobre la reacción inmune antiviral podría afectar negativamente el curso de la infección. Este contexto ha impulsado la búsqueda de nuevas alternativas inmunomoduladoras como la Equinácea purpúrea. Dada su propiedad inmunosupresora, se propone en el tratamiento del Herpes Labial Recurrente. Metodología: Estudio clínico prospectivo que analiza las subpoblaciones linfocitarias en 12 pacientes con Herpes Labial Recurrente, antes y después de recibir Equinácea purpúrea (30 gotas tres veces al día durante siete días).Resultados: En comparación con individuos sanos, los pacientes presentan una respuesta aumentada de células Treg. Esta condición se reduce significativamente tras recibir Equinácea purpúrea (515 + 145 y 432 + 113 cel/mm3 antes y después del tratamiento, respectivamente, p < 0,005). Conclusión: La hiperactividad de células Treg podría explicar el estado de inmunosupresión de estos pacientes y favorecería la persistencia viral. Se propone esta fitomedicina como una alternativa inmunoterapéutica beneficiosa.
Background: Recurrent Herpes Labialis patients may suffer from immunological alterations, such as CD4+CD25+Regulatory-T Cell (Treg) hyperactivity. These cells control peripheral tolerance and reduce immunopathology risk by suppressing other immunological cells. Hence, the Treg cell suppression on the antiviral immune reaction may perturb adversely the herpes infection outcome. This scenario has forced physicians to explore new immunomodulatory alternatives in Phytomedicine, such as Echinacea purpurea. Regarding the immunosuppressive property, it has been challenged to be employed in the Recurrent Herpes Labialis management. Methods: Clinical prospective study that analyzed lymphocytic subpopulation profile in twelve patients with Recurrent Herpes Labialis, before and after receiving E. purpurea (30 drops three times a day during seven days). Results: Comparing to healthy subjects, patients presented an enlarged Treg cell response. This condition became significantly reduced after receiving E. purpurea. (515 + 145 and 432 + 113 cel, before and after treatment respectively, p < 0.005). Conclusion: The intensified Treg cell activity may elucidate the immune suppression these patients undergo, aiding the viral persistence and survival. This proposes E. purpurea asa beneficial immunotherapeutic alternative.
Assuntos
Humanos , Antivirais/uso terapêutico , Echinacea/uso terapêutico , Echinacea/química , Herpes Labial/tratamento farmacológico , Preparações de Plantas/uso terapêutico , Antivirais/farmacologia , Echinacea/farmacologia , Herpes Labial/imunologia , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1 , Imunomodulação , Estudos Multicêntricos como Assunto , Estudos Prospectivos , Preparações de Plantas/farmacologia , Recidiva/prevenção & controle , Linfócitos T ReguladoresRESUMO
BACKGROUND: Rheumatic heart disease (RHD) is a delayed consequence of a pharyngeal infection with Group A streptococcus (GAS), usually ascribed to a cross-reactive immune response to the host cardiac tissues. Acute rheumatic fever (ARF) and its ensuing valvular sequelae are thus considered the prototype of a post-infectious autoimmune disease, with no direct evidence of residual streptococcal antigen in diseased valvular tissues. However, recent studies concerning the antigenic specificity and clonality of intralesional lymphocytes have revealed oligoclonal expansions characteristic of an antigen specific response, that might be related to GAS. AIM: To search for bacterial DNA in valvular tissue from RHD patients and controls. MATERIAL AND METHODS: We extracted DNA from surgically excised valve specimens from 15 RHD patients and 6 non RHD controls and tested for the presence of bacterial DNA by Polymerase Chain Reaction (PCR) with primers for 16S rRNA. RESULTS: Eighty percent (12/15) of valve specimens from RHD patients were positive for bacterial DNA, as opposed to none of the valves (n =6) from non RHD controls. CONCLUSIONS: These results suggest that GAS might persist in valvular tissue in patients with ARF and contribute to the inflammatory scarring lesion that leads to cardiovascular sequelae.
Assuntos
DNA Bacteriano/isolamento & purificação , Valvas Cardíacas/microbiologia , Faringite/microbiologia , Cardiopatia Reumática/microbiologia , Infecções Estreptocócicas/complicações , Streptococcus/isolamento & purificação , Adulto , Idoso , Antígenos de Bactérias/análise , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Faringe/microbiologiaRESUMO
Background: Rheumatic heart disease (RHD) is a delayed consequence of a pharyngeal infection with Group A streptococcus (GAS), usually ascribed to a cross-reactive immune response to the host cardiac tissues. Acute rheumatic fever (ARF) and its ensuing valvular sequelae are thus considered the prototype of a post-infectious autoimmune disease, with no direct evidence of residual streptococcal antigen in diseased valvular tissues. However, recent studies concerning the antigenic specificity and clonality of intralesional lymphocytes have revealed oligoclonal expansions characteristic of an antigen specific response, that might be related to GAS. Aim: To search for bacterial DNA in valvular tissue from RHD patients and controls. Material and methods: We extracted DNA from surgically excised valve specimens from 15 RHD patients and 6 non RHD controls and tested for the presence of bacterial DNA by Polymerase Chain Reaction (PCR) with primers for 16S rRNA. Results: Eighty percent (12/15) of valve specimens from RHD patients were positive for bacterial DNA, as opposed to none of the valves (n =6) from non RHD controls. Conclusions: These results suggest that GAS might persist in valvular tissue in patients with ARF and contribute to the inflammatory scarring lesion that leads to cardiovascular sequelae.
Assuntos
Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , DNA Bacteriano/isolamento & purificação , Valvas Cardíacas/microbiologia , Faringite/microbiologia , Cardiopatia Reumática/microbiologia , Infecções Estreptocócicas/complicações , Streptococcus/isolamento & purificação , Antígenos de Bactérias/análise , Doença Crônica , Faringe/microbiologiaRESUMO
BACKGROUND: Adenovirus serotypes 7, 2 and 1 are the second most common cause of viral acute lower respiratory tract infection (ALRI) requiring hospitalization in Chile. Nosocomial outbreaks have high secondary attack and lethality rates, and call for rapid and specific diagnosis. OBJECTIVE: We compared the results obtained on ALRI specimens by immunofluorescence (IFA) and virus isolation, plus restriction enzyme digestion (RFLP) typing, with universal, species-specific and 7h-specific PCR typing of adenovirus. A second objective was to determine the type of adenovirus implicated in nosocomial infection and nosocomial cross-infection rates. METHODS: Infants hospitalized for ALRI in the Roberto del Río Children's Hospital (Santiago, Chile) in 1995-1996 had nasopharyngeal aspirates obtained at admission and tested by IFA and virus isolation. Adenovirus isolates were identified by RFLP. When an index case was identified, samples were collected from contacts for 2 consecutive days and twice weekly thereafter for 2 weeks. Further typing of adenovirus isolates was undertaken with universal, species-specific and 7h-specific PCR performed in 2003 on the stored frozen samples. RESULTS: Fifteen index cases of adenovirus and their 65 contacts were identified. The nosocomial secondary attack rate using PCR was estimated as 46%. PCR had a higher sensitivity (98.7%) compared to virus isolation (90%) and IFA (50%) and facilitated identification of adenovirus strains more easily and accurately than RFLP (91.6% versus 55.8%). Fifty-three percent of the contacts had severe outcomes. The case fatality rate was 16.6% and was associated with adenovirus 7h. CONCLUSIONS: Prompt, rapid and sensitive methods to identify adenovirus infection are necessary, especially for hospital-acquired adenovirus infections, because of their ease of spread and high fatality rate.
Assuntos
Infecções por Adenovirus Humanos/epidemiologia , Infecção Hospitalar/epidemiologia , Reação em Cadeia da Polimerase/métodos , Infecções Respiratórias/epidemiologia , Infecções por Adenovirus Humanos/diagnóstico , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/classificação , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Chile/epidemiologia , Busca de Comunicante , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/virologia , DNA Viral/análise , DNA Viral/isolamento & purificação , Imunofluorescência , Humanos , Lactente , Polimorfismo de Fragmento de Restrição , Valor Preditivo dos Testes , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Sensibilidade e Especificidade , Especificidade da Espécie , Cultura de VírusRESUMO
La capacidad de defensa de las células fagocíticas depende del llamado estallido respiratorio, el cual se caracteriza por una serie de reacciones generadas por la NADPH oxidasa, que lleva a la liberación de especies reactivas de oxígeno, letales para el microorganismo. El propósito de este estudio fue desarrollar un método simple para la evaluación cuantitativa del estallido respiratorio mediante Citometría de flujo para poder utilizarlo como estrategia diagnóstica en individuos con alteración inmunológicas u otras patologías clínicas. La estandarización se realizó con estudios de dosis-respuesta y tiempo-respuesta, tanto del activador forbol 12-miristato-13acetato (PMA) así como del fluorocromo 123-dihidrorodamina (DHR). Se analizaron 30 muestras de individuos normales provenientes de un banco de sangre para obtener los valores de referencia, los cuales fueron definidos como los valores de estallido respiratorio neto expresado en porcentaje de fluorescencia y que fluctuaron entre 85,3 y 95,0 con una desviación estándar de 2,4; y los expresados en intensidad media de fluorescencia (MFI), que fluctuaron entre 13,3 y 50,4 con una desviación estándar de 9,3. De esta forma, los valores se caracterizaron como aquellos iguales al promedio ± 2 desviaciones estándar, cubriéndose el 96,6 por ciento de la muestra. En conclusión, esta nueva forma de determinar el estallido respiratorio, ofrece la gran ventaja de visualizar directamente las células, dando la oportunidad de medir en forma simultánea tanto porcentaje como la intensidad media de fluorescencia celular, permitiendo obtener así resultados más exactos y precisos.
Assuntos
Humanos , Citometria de Fluxo , Explosão Respiratória , Acetato de Tetradecanoilforbol/farmacocinética , Estudos de Casos e Controles , Corantes Fluorescentes/farmacocinética , Fagocitose , Valores de Referência , Rodaminas/farmacocinética , Fatores de TempoRESUMO
BACKGROUND: Hyper-IgM syndronie (HIGM) is a rare primary immunodeficiency used to describe a heterogeneous group of disorders characterized by recurrey bacterial infrctions, normal or elevated serum IgM levels and low or absent serum IgG, IgA and IgE. AIM: To make definitive diagnosis, detect mutations in carriers and perform genetic counseling in patients with HIGM. PATIENTS AND METHODS: We studied the expression of CD40L, CD40 and made a mutation analysis of the CD40L gene in 3 males of 2 unrelated Chilean families diagnosed as a possible syndrome of hyper-IgM and 3 relatives. RESULTS: We identified a deletion frameshift in the exon 2 (delA225) of the extracellular domain of GD40L gene in one patient and verified the carrier stains of his mother and sister. The other patients showed a low expression of GD40L in activated T cells (65.3% ammd 65.5%) and a normal expressiomi of CD40. No alterations were found in the single strand conformation polymorphism analysis of the CD40L. CONCLUSIONS: These result allowed us to make a definite diagnosis of HIGM1 of a patient, detect female carriers and suggest a HIGM of recessive inheritance with normal CD40 expression in the patients of the second family.
Assuntos
Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Ligante de CD40 , Hipergamaglobulinemia/genética , Imunoglobulina M/genética , Mutação da Fase de Leitura/genética , Ligante de CD40 , Aconselhamento Genético , Chile , Hipergamaglobulinemia/diagnóstico , Imunoglobulina M/sangue , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral , SíndromeRESUMO
Background: DiGeorge syndrome is characterized by developmental defects of the heart, parathyroid glands and thymus. Aim: To describe the clinical variability of DiGeorge syndrome and its relation with immunodeficiency. Patients and methods: A three years retrospective chart review from three hospitals of Santiago, Chile was conducted. We included patients with neonatal diagnosis of DiGeorge syndrome. Clinical and immuno-logic data were collected from their initial evaluation. Results: We found 9 patients with DiGeorge syndrome. All had dysmorphic facies, hypocalcemia and congenital heart disease. Three patients had hypoparathyroidism, 4 had interrupted aortic arch type B, 4 had tetralogy of Fallot and 1 had coarctation of aorta. Six patients had other malformations and associated diseases. FISH studies, performed in 8 patients, found the 22q11.2 microdeletion in all. Most patients had low CD3, CD4 and CD8 T cell counts, that ranged for CD3 T cells, between 256/mm3 and 3,664/mm3, for CD4 T cells, between 224/mm3 and 2,649/mm3, for CD8 T cells, between 26/mm3 and 942/mm3. Three patients had CD4 T cells counts <400/mm3 and one had a phytohemagglutinin stimulation index <10. Airway malformations and primary hypoparathyroidism were present in 3 out of 4 patients that died before 18 months compared with the surviving patients (p=0.048). Conclusions: We found variable clinical manifestations as well as CD3, CD4 and CD8 T cell counts in patients with DiGeorge syndrome. Airway malformations were associated with a higher mortality (Rev Méd Chile 2004; 132: 26-32).
Assuntos
Humanos , Síndrome de DiGeorge/imunologia , Chile , Transtornos CromossômicosRESUMO
We report a 11 years old male diagnosed as a X-linked hyper-IgM syndrome that presented with recurrent infections and sclerosing cholangitis and later developed a gallbladder cancer. Immunological evaluation showed decreased levels of serum IgG and IgA with elevated levels of IgM. Study of CD40 ligand expression on mitogen activated peripheral blood mononuclear cells revealed total absence of this marker on T lymphocytes. Molecular analysis detected, in the patient and his mother, a nonsense mutation in exon 1 of the transmembrane segment of the CD40 ligand. He also presented elevation of alkaline phosphatases and mild elevation of liver enzymes. Liver biopsy demonstrated the presence of idiopathic sclerosing cholangitis. The patient was started on monthly IVIG therapy at 400 mg/kg, as well as ursodeoxycholic acid and vitamin E, with normalization of his IgG and IgM levels a decrease in the incidence of infections and normalization of liver function. Three years after diagnosis, we detected the presence of polyps inside the gallbladder that were reported at biopsy as adenocarcinoma. He underwent hepatic bisegmentectomy (VI B-V) and local lymphadenectomy