Efecto del Medio Condicionado de la Línea Celular H9C2 Tratada con Dehidroepiandrosterona y Expuesta a Daño sobre la Motilidad de Células Troncales Mesenquimales / Effect of the Conditioned Medium from H9C2 Cell Line Treated with Dehydroepiandrosterone and Exposed to Damage on the Motility of Mesenchymal Stem Cells

RESUMEN: Las enfermedades cardiovasculares (ECV) son la principal causa de muerte a nivel mundial, donde la terapia con Células Troncales Mesenquimales (CTM) representa una alternativa para los pacientes que no logran recuperarse con los tratamientos actuales. El lograr que las CTM residentes se movilicen al órgano afectado representaría una ventaja para el manejo terapéutico de las ECV. La dehidroepiandrosterona (DHEA) es un precursor hormonal cuyos niveles disminuyen a lo largo de la vida, lo que se ha asociado al desarrollo de ECV. Diversos estudios han demostrado que el consumo de DHEA previene y mejora la condición cardiaca, aunque no se sabe si esto ocurre porque se ejerce un efecto en los cardiomiocitos y estos, a su vez, hacia las CTM. El objetivo del presente estudio fue determinar el efecto del medio condicionado procedente de la línea H9C2 pretratada con DHEA y sometida a daño, sobre la motilidad de CTM, llevando a cabo un ensayo de cierre de herida. El pretratamiento con DHEA y el daño en la línea H9C2, promueve la motilidad de CTM. El estímulo de la motilidad de CTM por un efecto indirecto de DHEA podría ser una estrategia terapéutica para el daño cardiaco.

ABSTRACT: Cardiovascular diseases (CVD) are the leading cause of death worldwide. Mesenchymal Stem Cell (MSC) therapy is an alternative for patients who cannot recover with current treatments. Ensure movilization of MSC to the affected organs would represent an advantage for therapeutic management of CVD. Dehydroepiandrosterone (DHEA) is a hormone precursor whose levels decrease throughout life, which has been associated with the onset of CVD. Several studies have shown that DHEA consumption, prevents and improves heart condition, although it is not known if this is because an effect on cardiomyocytes is exercised on these cells and this, in turn, to CTM. The aim of this study was to determine the effect of conditioned medium from H9C2 cell line pretreated with DHEA and subjected to damage, on the motility of CTM, performing a wound healing assay. Pretreatment with DHEA and damage to H9C2 cell line, promotes motility of CTM. Stimulation of CTM motility by an indirect effect of DHEA could be a therapeutic strategy for heart damage.

Comparación entre Células Troncales Mesenquimales obtenidas de Médula Ósea, Tejido Adiposo y Gelatina de Wharton en base a los Criterios de la ISCT / Comparison between Mesenchymal Stem Cells obtained from Bone Marrow, Adipose Tissue and Wharton Gelatin according to the ISCT Criteria

RESUMEN: Las células troncales mesenquimales (CTM) representan una población heterogénea con capacidad para auto-renovarse y diferenciarse a distintos tipos celulares. Estas fueron descritas en un inicio en médula ósea (MO) a mediados del siglo pasado, desde entonces este tejido se ha convertido en el estándar de oro para la obtención y caracterización de CTM. Actualmente se sabe que este tipo de células se encuentran alojadas en nichos distribuidos por todo el organismo, donde contribuyen a los procesos de regeneración del tejido donde se localizan. No obstante, encontrar una fuente alterna de CTM con las mismas características que las de MO, pero que su extracción no suponga riesgo para el donador es fundamental para su utilización con fines terapéuticos. En este trabajo se aislaron células troncales de médula ósea, y se compararon con tejido adiposo y gelatina de Wharton y caracterizaron de acuerdo a los criterios de la Sociedad Internacional para la Terapia Celular (ISCT). Los resultados mostraron que la morfología, diferenciación osteogénica y adipogénica, así como la expresión de los antígenos de superficie CD90, CD73 y CD105 cumplen con los estándares, señalando a las provenientes de gelatina de Wharton como mejor opción.

ABSTRACT: Mesenchymal stem cells (MSC) represent a heterogeneous population with the capacity to self-renew and differentiate into different cell types. At the middle of the last century these cells initially were described in bone marrow (BM), thence this tissue has become the gold standard for obtaining and characterization of MSC. It is known that these cells are housed in specific areas called niches distributed throughout all body, where they contribute to tissue regeneration processes of self-tissue were they are located. However, finding an alternative source of CTM with the same characteristics that have showed in MO, but its obtention no represent a risk since the donor is essential to their use for therapeutic purposes. In this study we isolated mesenchymal stem cells from bone marrow, adipose tissue and Wharton's jelly and they were compared in their characteristics in according to the standards of the International Society for Cellular Therapy (ISCT). The results showed that the morphology as well as adipogenic and osteogenic differentiation and also the expression of surface antigens (CD90, CD73, and CD105) from all tissues accomplished the standards, although Wharton's jelly represented the best option.

Efecto de la dehidroepiandroesterona y el acetónido de triamcinolona sobre la línea celular 3T3-L1 / Effect of dehydroepiandroesterone and triamcinolone acetonide on 3T3-L1 cell line

RESUMEN: La piel es el órgano más extenso en el ser humano, su integridad representa protección contra diferentes agentes químicos, biológicos y mecánicos. Las lesiones ocasionadas en este tejido se resuelven mediante la formación de una cicatriz, sin embargo, diferentes alteraciones moleculares pueden sobre estimular este proceso, lo que conlleva a la formación de cicatrices aberrantes (hipertrófica o queloide). El tratamiento más recomendado para este tipo de lesiones es la aplicación intralesional del acetónido de triamcinolona (AT) y por otro lado, la dehidroepiandrosterona (DHEA) es una pro-hormona que posee una gran variedad de efectos biológicos como: regulación de la síntesis de fibras de colágeno, protección celular, propiedades antitumorales, antiinflamatorias y antioxidante. En este trabajo, se estudió la combinación de AT-DHEA sobre la proliferación y muerte celular en la línea celular de fibroblastos 3T3-L1. Los resultados mostraron que la AT a 100 M y la DHEA a 1000 M inhiben la proliferación en un 50 y 40% respectivamente. La combinación de AT-DHEA (10000-10 M) inhibe la proliferación celular e inducen muerte celular programada, entonces esta combinación pudiera utilizarse en cicatrices hipertróficas o queloides para su eliminación.

ABSTRACT: The skin in the human is the largest organ, his integrity represents protection against various chemical, biological and mechanical agents. The injuries in this tissue are solved by forming a scar, however, different molecular alterations may overstimulate this process, leading to the formation of aberrant scars (hypertrophic or keloid). The most recommended treatment for such injuries is the intralesional application of triamcinolone acetonide (TA) and on the other hand, dehydroepiandrosterone (DHEA) is a pro-hormone that has a wide variety of biological effects such as regulation of the synthesis of collagen fibers, cell protection, anti-tumor properties, anti-inflammatory and antioxidant. In this paper, the combination of AT-DHEA on proliferation and cell death in fibroblast cell line 3T3-L1 was studied. The results showed that the AT 100 and 1000 M DHEA to inhibit proliferation by 50 and 40% respectively. The combination of AT-DHEA (10000-10 M) inhibits cell proliferation and induce programmed cell death, so this combination could be used in hypertrophic or keloid scars for disposal.

[Lung eosinophilic syndrome: clinical presentation and cases report]. / Síndrome de eosinofília pulmonar presentación clínica y serie de casos.

INTRODUCTION: Pulmonary eosinophilia syndrome is characterized by a group of diseases that present clinical-radiological conditions, pulmonary eosinophilia or peripheral lung parenchyma in its evolution. We described the clinical and radiological presentation. METHODS: Retrospective descriptive analysis of medical records of 7 patients between 2007 and 2010. RESULTS: The highest numbers of cases were observed in women, with peripheral eosinophilia with values between 550 and 10,000 cells/mm3. The more frequent signs and symptoms were cough, dyspnea, fever and wheezing. The more prevalent radiological findings were alveolar interstitial and alveolar pattern. At CT scan, the most frequent pattern was ground glass. The main diagnoses made were acute and chronic eosinophilic pneumonia in equal proportions, both with response to steroids. CONCLUSIONS: The pulmonary eosinophilia syndrome shares common features with clinical and radiological entities most prevalent, particularly community-adquired pneumonia.

Introducción: El síndrome de eosinofilia pulmonar se caracteriza por un grupo de patologías que presentan afección clínico ­ radiológica pulmonar con eosinofilia periférica o en parénquima pulmonar en su evolución. Materiales y métodos: Se describen las características de presentaciones clínico-radiológicas y evolutivas de pacientes atendidos entre 2007 y 2010 en Hospital Rawson. Resultados: Sobre 8 casos, se observó mayor número de casos en mujeres. Los signos y síntomas principales fueron tos, disnea, fiebre y sibilancias. Los hallazgos radiológicos más prevalentes fueron patrón alveolar y alveolointersticial. En la TAC el más frecuente fue el patrón en vidrio esmerilado. La eosinofilia periférica presentó valores entre 550 y 10.000 cel/mm3. Los pacientes fueron abdordados inicialmente como neumonía adquirida en la comunidad en el 62% de los casos. Los diagnósticos principales realizados fueron neumonía eosinofílica aguda y crónica, ambas con respuesta a esteroides. Conclusiones: El síndrome de eosinofilias pulmonares comparte características clínico-radiológicas comunes con entidades de mayor prevalencia, particularmente NAC.

High prevalence of asymptomatic Leishmania spp. infection among liver transplant recipients and donors from an endemic area of Brazil.

Visceral leishmaniasis is an uncommon disease in transplant recipients; however, if left untreated, the mortality can be high. If an organ donor or recipient is known to be an asymptomatic Leishmania spp. carrier,monitoring is advised. This study proposes to assess the prevalence of asymptomatic Leishmania spp.infection in liver transplant donors and recipients from an endemic area. A total of 50 liver recipients and 17 liver donors were evaluated by direct parasite search, indirect fluorescent antibody test (IFAT), anti-Leishmania rK39 rapid test and Leishmania spp.DNA detection by polymerase chain reaction (PCR).Leishmania spp. amastigotes were not observed in liver or spleen tissues. Of the 67 serum samples, IFAT was reactive in 1.5% and indeterminate for 17.9%, and the anti-Leishmania rK39 rapid test was negative for all samples. The PCR test was positive for 7.5%, 8.9%, and 5.9% of blood, liver and spleen samples, respectively(accounting for 23.5% of the donors and 8% of the recipients). Leishmania infantum-specific PCR confirmed all positive samples. In conclusion, a high prevalence of asymptomatic L. infantum was observed in donors and recipients from an endemic area, and PCR was the most sensitive method for screening these individuals.

Síndrome de eosinofília pulmonar presentación clínica y serie de casos / [Lung eosinophilic syndrome: clinical presentation and cases report].

INTRODUCTION: Pulmonary eosinophilia syndrome is characterized by a group of diseases that present clinical-radiological conditions, pulmonary eosinophilia or peripheral lung parenchyma in its evolution. We described the clinical and radiological presentation. METHODS: Retrospective descriptive analysis of medical records of 7 patients between 2007 and 2010. RESULTS: The highest numbers of cases were observed in women, with peripheral eosinophilia with values between 550 and 10,000 cells/mm3. The more frequent signs and symptoms were cough, dyspnea, fever and wheezing. The more prevalent radiological findings were alveolar interstitial and alveolar pattern. At CT scan, the most frequent pattern was ground glass. The main diagnoses made were acute and chronic eosinophilic pneumonia in equal proportions, both with response to steroids. CONCLUSIONS: The pulmonary eosinophilia syndrome shares common features with clinical and radiological entities most prevalent, particularly community-adquired pneumonia.

Síndrome de eosinofília pulmonar presentación clínica y serie de casos. / [Lung eosinophilic syndrome: clinical presentation and cases report].

INTRODUCTION: Pulmonary eosinophilia syndrome is characterized by a group of diseases that present clinical-radiological conditions, pulmonary eosinophilia or peripheral lung parenchyma in its evolution. We described the clinical and radiological presentation. METHODS: Retrospective descriptive analysis of medical records of 7 patients between 2007 and 2010. RESULTS: The highest numbers of cases were observed in women, with peripheral eosinophilia with values between 550 and 10,000 cells/mm3. The more frequent signs and symptoms were cough, dyspnea, fever and wheezing. The more prevalent radiological findings were alveolar interstitial and alveolar pattern. At CT scan, the most frequent pattern was ground glass. The main diagnoses made were acute and chronic eosinophilic pneumonia in equal proportions, both with response to steroids. CONCLUSIONS: The pulmonary eosinophilia syndrome shares common features with clinical and radiological entities most prevalent, particularly community-adquired pneumonia.

Impact of Model for End-Stage Liver Disease in the occurrence of infectious events and survival in a cohort of liver transplant recipients.

The Model for End-Stage Liver Disease (MELD), which predicts mortality on the waiting list before liver transplantation, has changed organ allocation criteria to prioritize severely ill patients. The aim of this study was to investigate the impact of the new criteria on the incidence of Healthcare Associated Infections (HAI) and patient survival after liver transplantation. This retrospective cohort included liver transplant recipients from 2005 to 2007. Infection notification followed the recommended criteria of the National Healthcare Safety Network (NHSN). Statistical analysis was performed using the Statistical Package for the Social Sciences. Of 142 patients, 67 (47.2%) underwent transplantation before June 2006. There were no differences between the 2 periods considering patient gender, diagnosis, age, length of hospitalization, and mean time to first infection occurrence. However, the length of intensive care unit (ICU) hospitalization (P = .006) and central venous catheter (CVC) use (P = .025) were higher in the first period of the study. Comparison of time until first systemic infection before and after changes in allocation criteria showed no significant difference (log-rank = 0.06; P = .81). There was a trend toward greater lethality during the second period of the study (P = .09). There was no difference in time to death between the 2 periods (log-rank = 0.9; P = .76). However, when comparing time to death of all patients with systemic infection versus those without this event, patients without infection showed a higher mortality rate (log-rank = 15.7; P < .001).

[Superior Cava Venous syndrome as presentation of neoplasic disease]. / Síndrome de vena cava superior: forma inicial de presentacion en enfermedades neoplásicas.

Superior Cava Venous Syndrome (SVCS) encompasses a constellation of signs and symptoms resulting from partial or complete obstruction of blood flow through the superior vena cava (SVC) to the right atrium. Thrombosis can be caused by intrinsic (primary) or extrinsic compression (with or without secondary thrombosis). The causes of SVCS can be grouped according to their etiology in non-neoplastic and neoplastic. The causes malignant correspond to 65-90% of them, thrombosis and nonmalignant conditions are another causes. The presentation may be acute, clinically characterized by dyspnea and a characteristic triad (facial, neck and arms edema, cyanosis and collateral circulation). Early detection improves prognosis and is based on clinical data and imaging studies. We describe 8 cases where the SVCS was the first manifestation of neoplastic diseases. Patients were predominantly of middle-aged, male, without association with consumption of tobacco and lymphoma was most frequent diagnosis.

Se denomina Síndrome de Vena Cava Superior (SVCS) al conjunto de signos y síntomas derivados de la obstrucción parcial o completa del flujo sanguíneo a través de la vena cava superior (VCS) hacia la aurícula derecha. Puede ser causada por trombosis intrínseca (primaria) o a compresión extrínseca (asociada o no a trombosis secundaria) Las causas de SVCS pueden ser agrupadas según su etiología en no neoplásicas y neoplásicas. Las causas malignas corresponden a un 65-90% del total de las causas, la trombosis y las condiciones no malignas son las causantes del resto de los casos. La forma de presentación puede ser aguda, caracterizándose clínicamente por disnea y la triada característica (edema en esclavina, cianosis facial y presencia de circulación colateral). La detección precoz mejora el pronóstico y se asienta sobre los datos clínicos y estudios por imágenes. Presentamos 8 casos registrados en nuestro hospital, donde el SVCS fue la manifestación inicial de enfermedades neoplásicas. En esta serie hubo predominancia de varones de edad media, sin asociación con consumo de tabaco y el diagnóstico más frecuente fue linfoma.

Síndrome de vena cava superior: forma inicial de presentacion en enfermedades neoplásicas / [Superior Cava Venous syndrome as presentation of neoplasic disease].

Superior Cava Venous Syndrome (SVCS) encompasses a constellation of signs and symptoms resulting from partial or complete obstruction of blood flow through the superior vena cava (SVC) to the right atrium. Thrombosis can be caused by intrinsic (primary) or extrinsic compression (with or without secondary thrombosis). The causes of SVCS can be grouped according to their etiology in non-neoplastic and neoplastic. The causes malignant correspond to 65-90

of them, thrombosis and nonmalignant conditions are another causes. The presentation may be acute, clinically characterized by dyspnea and a characteristic triad (facial, neck and arms edema, cyanosis and collateral circulation). Early detection improves prognosis and is based on clinical data and imaging studies. We describe 8 cases where the SVCS was the first manifestation of neoplastic diseases. Patients were predominantly of middle-aged, male, without association with consumption of tobacco and lymphoma was most frequent diagnosis.

Síndrome de vena cava superior: forma inicial de presentacion en enfermedades neoplásicas. / [Superior Cava Venous syndrome as presentation of neoplasic disease].

Superior Cava Venous Syndrome (SVCS) encompasses a constellation of signs and symptoms resulting from partial or complete obstruction of blood flow through the superior vena cava (SVC) to the right atrium. Thrombosis can be caused by intrinsic (primary) or extrinsic compression (with or without secondary thrombosis). The causes of SVCS can be grouped according to their etiology in non-neoplastic and neoplastic. The causes malignant correspond to 65-90

of them, thrombosis and nonmalignant conditions are another causes. The presentation may be acute, clinically characterized by dyspnea and a characteristic triad (facial, neck and arms edema, cyanosis and collateral circulation). Early detection improves prognosis and is based on clinical data and imaging studies. We describe 8 cases where the SVCS was the first manifestation of neoplastic diseases. Patients were predominantly of middle-aged, male, without association with consumption of tobacco and lymphoma was most frequent diagnosis.

[Diastolic dysfunction in patients with human inmunodeficiency virus. Tissue Doppler study]. / Disfuncion diastolica en pacientes infectados con el virus de inmunodeficiencia humana. Estudio con Doppler tisular.

UNLABELLED: The heart has rarely been studied in patients infected with HIV. Diastolic dysfunction is the most frequently observed alteration , which could be due to direct viral action on the myocardium or due to autoimmune mechanism or concomitant infections by cardiotropic virus. The Doppler tissue is a more effective tool than the trans-mitral one to evaluate the diastolic function since is not influenced by preload, afterload or heart rate. OBJECTIVES: To study the prevalence of the left ventricular diastolic dysfunction (DDVI) in the HIV (+) group of patients, without symptoms or diagnosis of heart-disease and to analyze their relationship with the CD4 levels and the viral charge. MATERIAL AND METHODS: We prospectively studied 50 HIV(+) patients with no symptoms of cardiac involvement who underwent an echocardiogram using Doppler tissue compared with 50 healthy persons matched sex and age as control group. RESULTS: The infection of HIV was associated with DDVI and with CD4 Lymphocytes levels (p<0,05). The association between DDVI and viral load was not found. CONCLUSIONS: There is an association between the HIV infection and the presence of DDVI in asymptomatic patients. The long term monitoring studies should be considered in HIV (+) patients with silent diastolic dysfunction to evaluate their possible progression to the systolic dysfunction.

[Insomnia: prevalence in Cordoba city hospital]. / Insomnio: prevalencia en un hospital de la ciudad de Cordoba.

INTRODUCTION: Insomnia is a sleep disorder characterized by a deficient or poor quality sleep, with adverse daytime consequences. Prevalence is 30-50% in adults and can be associated with depression or lead to the development of this condition. Despite the high prevalence rates, is a unrecognized, misdiagnosis and undertreated. There is not much publications about its prevalence in patients with chronic diseases. OBJECTIVES: to determine prevalence and clinical characteristics of insomnia in outpatients with chronic diseases. MATERIALS AND METHODS: Prospective observational cross-sectional descriptive study. Insomnia was defined based on ICSD-2criteria. The data collection was performed by a questionnaire. RESULTS: We surveyed 100 patients who attended the consultation of various clinical specialties, mean age 50 years old, 57% were women. Sixty nine per cent of them met criteria for insomnia. The most prevalent diseases were hypertension: 57%, asthma 20%, diabetes: 18% and hypothyroidism: 17%. Among patients with insomnia, 62% were women, 35% had insomnia without another illness, and the remaining 65% had secondary conditions associated with insomnia (60% depression). 25% of patients consulting for insomnia ever. The prevalence of criteria for depression in outpatients with chronic diseases was 52%, amounting to 63% in patients suffering from insomnia. DISCUSSION: The prevalence of insomnia in patients with chronic diseases is high (in outpatients with chronic disease: 69%), higher than the average rate described in the general population. It is an undertreated condition. CONCLUSIONS: The prevalence of insomnia in patients with chronic diseases is high, it is underdiagnosed and undertreated. It is associated with depression in high rate.

Introducción: Insomnio es el trastorno caracterizado por sueño deficiente o de mala calidad con consecuencias diurnas adversas. La prevalencia es 30-50% en adultos y puede llevar al desarrollo de depresión. A pesar de las altas tasas de prevalencia es una entidad poco reconocida, subdiagnosticada y subtratada. Poco se ha publicado acerca de la prevalencia en pacientes con enfermedades crónicas. Objetivos: conocer prevalencia y características clínicas del insomnio en pacientes ambulatorios con enfermedades crónicas. Materiales y métodos: Estudio prospectivo descriptivo observacional de corte transversal. Se definió insomnio en base a los criterios del ICSD-2. La obtención de los datos se realizó por un cuestionario autoadministrado. Resultados: Encuestamos a 100 pacientes que acudieron a la consulta de diferentes especialidades clínicas, edad promedio 50 años. El 57% mujeres. El 69% cumplía criterios de insomnio. Las enfermedades más prevalente fueron HTA: 57%; asma: 20%, diabetes: 18% e hipotiroidismo: 17%. Entre los pacientes con insomnio, el 62% fueron mujeres, el 35 % lo presentaban de manera aislada y el restante 65% presentaba condiciones asociadas a insomnio secundario (el 60% presentaban depresión). La cuarta parte de los pacientes consultó por insomnio alguna vez. La prevalencia de criterios de depresión en los enfermos crónicos analizados fue de 52%, y asciende a 63% en los pacientes que padecen insomnio. Discusión: La prevalencia de insomnio en pacientes con enfermedades crónicas es alta, siendo en pacientes ambulatorios con enfermedades crónicas del 69%, muy superior a la media descripta en la población general. Es una entidad subtratada. Conclusiones: La prevalencia de insomnio en pacientes con enfermedades crónicas es alta, está subdiagnosticada y subtratada. Presenta una asociación significativa con depresión.

[Nutrition and chronic alcohol abuse]. / Nutrición y alcoholismo crónico.

Many patients with chronic alcohol abuse present a clinical picture of malnourishment either because of reduced usual intake of essential nutrients or because alcohol precludes an appropriate digestion and absorption of the different essential elements, vitamins, and minerals. A usual example is vitamin A deficiency in these patients. Besides, ethanol metabolic pathways themselves (through the ADH and the MEOS system) generate toxic intermediate products (acetaldehyde, free radicals) interfering with normal metabolism of essential elements, mainly lipids, leading to cellular damage through lipid peroxidation mechanisms and impairment of the membrane fluidity, fat deposits (hepatocellular steatosis), inflammation secondary to oxidative stress and proinflammatory cytokines, activation of stellate cells, fibrogenesis, etc. Nutritional supports may be effective to improve alcoholic liver disease. A balanced diet, vitamin supplements, and pharmacological therapy with antioxidants in order to recover depleted glutathione deposits are recommended. It is paramount that these patients have a multidisciplinary clinical approach to resolve the problem of alcohol dependency.

[Extensive condyloma acuminata of the penis successfully treated with 5% imiquimod cream]. / Condiloma acuminado extenso en pene tratado exitosamente con imiquimod crema al 5%.

Infection by HPV is considered one of the most frequent causes of sexually transmitted infections. Multiple forms of treatment have been used that vary in their effectiveness. We describe 2 patients with Extensive Condyloma Acuminata in the penis successfully treated with treated with 5% Imiquimod cream.

Cytotoxic activity of Justicia spicigera is inhibited by bcl-2 proto-oncogene and induces apoptosis in a cell cycle dependent fashion.

Identification of organic compounds from plants is of clinical significance because of the effect that they might have in patients with haematopoietic disorders. We studied the effect of the plant extract Justicia spicigera (Acanthaceae) in different haematopoietic cells: human leukaemic cell lines, umbilical cord blood cells, and mouse bone marrow cells. By examining colony formation and performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay it was shown that the plant extract of Justicia spicigera contains cytotoxic factors for leukaemic cells and has no proliferative activity on normal haematopoietic progenitor cells. Our results show that this plant extract induces apoptosis in the human leukaemia cell line TF-1, but not in the bcl-2 transfectant cell line TB-1. Similar results were obtained using a haemopoietic cell line 32D and 32DBcl2. The cultures of umbilical cord blood cells and mouse bone marrow that contain granulocyte-macrophage colony-stimulating factor (GM-CSF) do not proliferate or become terminally differentiated in the presence of the infusion of Justicia spicigera. GM-CSF that acts by abrogating programmed cell death is not sufficient to inhibit the apoptotic stimulus in TF-1 and 32D cells. Moreover mouse fibroblasts (3T3) and two cervical carcinoma cell lines CALO and INBL, undergo apoptosis in the presence of different concentrations of an infusion from the plant. Our data show that there is a strong correlation between the cytotoxic effect and cell proliferation. Together, these results indicate that the plant infusion of Justicia spicigera does not contain any haematopoietic activity, induces apoptosis inhibited by bcl-2 and is linked to cell proliferation.

Implication of tyrosine kinase receptor and steel factor in cell density-dependent growth in cervical cancers and leukemias.

Cell-cell interaction is important in the expansion of leukemic cells and of solid tumors. Steel factor (SF) or Kit ligand is produced as a membrane-bound form (mSF) and a soluble form. Because both primary gynecological tumors and primary leukemic cells from patients with acute myeloblastic leukemia (AML) have been shown to coexpress c-Kit and SF, we addressed the question of whether mSF could contribute to cell interaction in these cancers. Investigations on primary cervical carcinomas have been hindered by the fact that the cells do not grow in culture. We report herein the establishment of two cervical carcinoma cell lines, CALO and INBL, that reproduce the pattern of SF/c-Kit expression observed in primary tumor samples. In addition, these cells exhibit marked density-dependent growth much in the same way as AML blasts. Using an antisense strategy with phosphorothioate-modified oligonucleotides that specifically target SF without affecting other surface markers, we provide direct evidence for a role of mSF and c-Kit in cell interaction and cell survival in these gynecological tumor cell lines as well as in primary AML blasts. Finally, our study defines the importance of juxtacrine stimulation, which may be as important, if not more, than autocrine stimulation in cancers.

Steel factor sustains SCL expression and the survival of purified CD34+ bone marrow cells in the absence of detectable cell differentiation.

CD34+ cells express the basic helix-loop-helix transcription factor SCL, which is essential for blood cell formation in vivo. In addition, their survival is critically dependent on hemopoietic growth factors. We therefore compared the effects of Steel factor (SF) and GM-CSF on the survival, proliferation, and differentiation of primary human CD34+ cells, as well as the role of SCL during these processes. GM-CSF suppresses apoptosis in CD34+ cells, which proliferate and differentiate into mature granulocytic and monocytic cells (CD34-CD13+) and loose SCL expression. In contrast, SF suppresses apoptosis without a significant increase in cell numbers, and the cells remain CD34+ and SCL+ with a blast-like morphology. Examination of apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) reaction and of the cell cycle status indicated that SF is both a survival factor and a mitogenic factor for CD34+ cells. There was, however, constant cell death in a fraction of the population, which could be rescued by GM-CSF. Co-addition of SF and GM-CSF prevents the downregulation of SCL observed in the presence of GM-CSF by itself, allows for prolonged survival and expansion of CD34+ cells in culture, inhibits monocytic differentiation and impairs granulocytic differentiation. Finally, exposure to an antisense SCL but not a control oligonucleotide decreases SCL protein levels and prevents the suppression of apoptosis by SF without affecting GM-CSF-dependent cell survival. These observations suggest that the hemopoietic transcription factor SCL regulates the survival of CD34+ cells in response to SF.

[Transfection of TF-1 cell line with human bcl-2 proto-oncogene provides short-term survival in absence of GM-CSF without changing the phenotype]. / La transfección de la línea celular TF-1 con el proto-oncogen bcl-2 humano proporciona supervivencia a corto plazo en ausencia de GM-CSF sin cambiar el fenotipo.

UNLABELLED: Apoptosis is a process genetically controlled. The produce of the bcl-2 gene, bcl-2, is an anti apoptotic protein that is linked to the external membrane of the mitochondria. OBJECTIVE: To explore the possibility that bcl-2 transfection could change phenotype, response to mitogenic factor, and cell morphology on the TF-1 parental cell line and the bcl-2 transfectant TB-1 or TF-1neo. METHODS: We look at the expression of CD13, CD34 and c-Kit surface markers by flow cytometry. We have measured cell proliferation in response to GM-CSF and cell survival after GM-CSF withdrawal by the MTT assay on the same cell lines. Apoptosis was evaluated by the apoptotic membrane blebbing set up at different times after serum and survival factor removal or tolerance to cytotoxic compounds from Justicia spicigera. RESULTS: According with our results, ectopic expression of the bcl-2 gene prevented apoptosis without changes in morphology or phenotype in the absence of GM-CSF and serum or the presence of the extract from Justicia spicigera. Consisting with the Bcl-2 function, we found that Bcl-2 did not change response to GM-CSF. Serum deprivation or GM-CSF withdrawal induces cell death at 36 hours in TF-1 and TF-1neo cells, whereas TB-1 cells undergo apoptotic membrane blebbing after 96 hours under the same conditions. CONCLUSIONS: Taken together, our data indicate that Bcl-2 is a short term anti apoptotic protein in TB-1 cell line, that does not affect response to GM-CSF neither CD13, CD34 nor c-Kit antigen expression.

[Stem cell factor (SCF) supports granulocyte progenitor survival in mouse bone marrow cultures]. / El factor de células totipotenciales (stem cell factor; SCF) sostiene la supervivencia de progenitores de granulocitos en cultivos de médula ósea de ratón.

The regulation of cell differentiation and cell death in crucial to the generation of hematopoietic cells both in vitro and in vivo. The biologic role of stem cell factor (SCF) in hematopoietic cell development is not well known. We monitored the survival, proliferation and differentiation of mouse hematopoietic cells in culture in the presence of SCF. Examination of colony formation, MTT and thymidine killing of mouse bone marrow indicated that SCF is mainly a survival factor. Our results show that SCF maintains cells in a "undifferentiated" state. Committed granulocytic and monocytic progenitors (CFU-GM) survive for seven days in the presence of SCF alone, under conditions where no maturing granulocytic monocytic cells could be recovered. On transfer to GM-CSF containing cultures, these cells proliferate and differentiate terminally. Together, our data indicate that SCF induces survival in hematopoietic progenitors. Furthermore, SCF favors the survival of granulocytic progenitors over that of monocytic progenitors. In the absence of later acting factors such as GM-CSF, cells that progress beyond the CFU-GM stage lose c-kit expression and die by default. Hence, lack of cell expansion in the presence of SCF by itself is due to constant cell proliferation and survival, which is counterbalanced by cell death. In contrast, the presence of both SCF and GM-CSF allows for the continuous survival and expansion of hematopoietic progenitor cells in culture, as well as favoring their terminal differentiation along granulocytic and monocytic pathways. Furthermore, GM-CSF induces colonies of macrophages that produce G-CSF and IL-6, two molecules involved in granulopoiesis, and these in turn stimulate granulocyte colony formation. Finally, our data suggest that survival signals by SCF are crucial during the differentiative process of granulocytes, giving strength to deterministic model.