Pharmacokinetics and Biodistribution of Rhopalurus junceus Scorpion Venom in Tumor-Bearing Mice after Intravenous and Oral Administration

Background: Rhopalurus junceus scorpion venom has shown potential for anticancer treatment. However, there are no scientific evidence about venom pharmacokinetic (PK) and biodistribution (BD) in tumor-bearing mice. Methods: 131I-labeled venom was administrated by intravenous (IV) and oral (PO) routes at the single dose of 12.5 mg/kg. Mice were sacrificed and blood samples, major organs, and tumor were taken at 10, 20, 40, 90, 180, 300, 480, and 1440 min. Results: For IV route, maximum peak concentration (Cmax), elimination half-lives, total body clearance (CL), distribution volume (Vd), mean residence time (MRT), and area under curve (AUC) were 21.77 ± 2.45 %Dosis•h/mL, 12.65 ± 2.1 h, 4.59 ± 0.23 mL/h, 83.80 ± 12 mL, 12.49 ± 2.71 h, and 21.77 ± 2.45 %Dosis•h/mL, respectively. For PO route, they were 0.60 ± 0.07 %Dosis•h/mL, 9.33 ± 1.35 h, 36.94 ± 4.01 mL/h, 497.33 ± 30 mL, 12.40 ± 1.87 h, and 6.89 ± 1.18 %Dosis•h/mL, respectively. PK parameters (Cmax, CL, Vd, and AUC) showed significant differences between IV and PO routes. Bioavailability was 31.6 ± 4% for PO dose. Kidney, stomach, liver, and lung for IV and stomach, kidney, spleen, and lung for PO routes showed the major uptakes for 131I-labeled venom. In tumor tissue, after the maximum uptake for both routes, there was a consistent behavior of radioactivity respect to the major organs during the first 480 min. Conclusion: The PK and BD of R. junceus venom in mice depend on the administration route. These data represent a starting point for future experiments with this scorpion venom in experimental models of cancer.

The cytotoxic effects of propolis on breast cancer cells involve PI3K/Akt and ERK1/2 pathways, mitochondrial membrane potential, and reactive oxygen species generation.

Propolis has been extensively used to improve health and prevent inflammatory diseases. Different types of Cuban propolis (red, brown and yellow) have been documented. The purpose of this research was to investigate the cytotoxic effects of Cuban red propolis (CP) on MDA MB-231 cell line, since breast cancer is considered one of the most common causes of mortality among women. Antiproliferative and cytotoxic activity of CP against MDA MB-231 cells were determined by the 3-[4,5-dimethylth-iazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) and lactate dehydrogenase (LDH) assays. Apoptosis/necrosis, involvement of PI3K/Akt and ERK1/2 pathways, mitochondrial membrane potential and expression of genes were investigated. CP extract exhibited antiproliferative and cytotoxic effects on MDA MB-231 cells, what may be probably related to PI3K/Akt and ERK1/2 pathways. A decreased expression of apoptosis-related genes (TP53, CASP3, BAX and P21) was seen, whereas the expressions of BCL-2, BCL-XL, NOXA and PUMA were unaffected. CP extract induced mitochondrial dysfunction and LDH release, what indicated cell necrosis associated with reactive oxygen species production and decreased cell migration. Our findings provide a basis for future investigation of chemopreventive and/or therapeutic studies against apoptosis-resistant breast cancer, in animals and humans.

Diferencias en la fagocitosis de Mycobacterium habana TMC 5135 en cultivos celulares de macrófagos murinos / Differences in phagocytosis of Mycobacterium ´habana´ TMC 5135 by murine macrophages cellular cultures

La tuberculosis es uno de los mayores problemas que enfrenta la salud mundial en la actualidad. La única vacuna disponible contra esta enfermedad es la BCG, esta protege solo contra la tuberculosis grave de la infancia, lo cual plantea un reto en la búsqueda de nuevos candidatos vacunales. Teniendo en cuenta el antecedente protector de Mycobacterium ´habana´ contra la tuberculosis experimental, nos propusimos aportar elementos que avalen el empleo de M. ´habana´ TMC 5135 como candidato vacunal contra la tuberculosis, mediante estudios de infección en cultivos celulares de macrófagos murinos. Se caracterizó el proceso de fagocitosis de esta micobacteria por cultivos primarios de macrófagos peritoneales murinos y por la línea celular RAW 264.7, para lo cual se determinó el porcentaje de fagocitosis y el número fagocítico. El presente trabajo demostró que el proceso de fagocitosis de M. ´habana´ TMC 5135 está influenciado por la fuente celular empleada como célula hospedadora, así como por la carga bacteriana infectante y el tiempo de exposición a la misma. La presente investigación contribuye a la caracterización de la infección por esta micobacteria en sus principales células blanco de la inmunidad innata y traza el camino de futuras investigaciones para evaluar la activación de mecanismos efectores de la inmunidad innata frente a este candidato(AU)

Tuberculosis remains as a major problem in the global health. BCG is the available vaccine against tuberculosis but only protects against severe form of disease during childhood, so the search for new vaccine candidates is a challenge. Taking into account the protective capacity of Mycobacterium ´habana´ against experimental tuberculosis, we proposed in vitro experiments using murine macrophages (peritoneal macrophages and cell line Raw 264.7) to characterize phagocytic process of this candidate. Phagocitic index and phagocytic number were calculated. The present work demonstrated that the phagocytosis process of M. ‘habana’ TMC 5135 is influenced by the cellular source used as host cell, as well as by the infecting bacterial load and the time of exposure. The present investigation contributes to the characterization of the infection by this mycobacteria in its main target cells of innate immunity and it suggest future investigations to evaluate the activation of effector mechanisms of the innate immunity against this candidate(AU)

Mechanisms underlying the cytotoxic effect of propolis on human laryngeal epidermoid carcinoma cells.

Propolis has been used as a traditional remedy for centuries because of its beneficial effects, including anticancer properties. The aim of this study was to compare the cytotoxic mechanism of Cuban red propolis (CP) and Brazilian green propolis (BP) on human laryngeal carcinoma (HEp-2) cells. Cell viability, leakage of lactate dehydrogenase, fluorescence staining, mitochondrial membrane potential (ΔΨm) and the expression of pro/anti-apoptotic genes were assessed. Cell viability and cytotoxic assays suggested a dose-dependent effect of CP and BP extracts with a possible association of intracellular reactive oxygen species production and decreased ΔΨm. Both samples induced apoptosis via activation of TP53, CASP3, BAX, P21 signalling, and downregulation of BCL2 and BCL-XL. CP exerted a higher cytotoxic effect than BP extract. Our findings suggest further investigation of the main components of each propolis sample, what may lead to the development of strategies for the treatment of laryngeal cancer.

Diferencias en la fagocitosis de Mycobacterium habana TMC 5135 en cultivos celulares de macrófagos murinos / Differences in phagocytosis of Mycobacterium ´habana´ TMC 5135 by murine macrophages cellular cultures

La tuberculosis es uno de los mayores problemas que enfrenta la salud mundial en la actualidad. La única vacuna disponible contra esta enfermedad es la BCG, esta protege solo contra la tuberculosis grave de la infancia, lo cual plantea un reto en la búsqueda de nuevos candidatos vacunales. Teniendo en cuenta el antecedente protector de Mycobacterium habana contra la tuberculosis experimental, nos propusimos aportar elementos que avalen el empleo de M. habana TMC 5135 como candidato vacunal contra la tuberculosis, mediante estudios de infección en cultivos celulares de macrófagos murinos. Se caracterizó el proceso de fagocitosis de esta micobacteria por cultivos primarios de macrófagos peritoneales murinos y por la línea celular RAW 264.7, para lo cual se determinó el porcentaje de fagocitosis y el número fagocítico. El presente trabajo demostró que el proceso de fagocitosis de M. habana TMC 5135 está influenciado por la fuente celular empleada como célula hospedadora, así como por la carga bacteriana infectante y el tiempo de exposición a la misma. La presente investigación contribuye a la caracterización de la infección por esta micobacteria en sus principales células blanco de la inmunidad innata y traza el camino de futuras investigaciones para evaluar la activación de mecanismos efectores de la inmunidad innata frente a este candidato(AU)

Tuberculosis remains as a major problem in the global health. BCG is the available vaccine against tuberculosis but only protects against severe form of disease during childhood, so the search for new vaccine candidates is a challenge. Taking into account the protective capacity of Mycobacterium ´habana´ against experimental tuberculosis, we proposed in vitro experiments using murine macrophages (peritoneal macrophages and cell line Raw 264.7) to characterize phagocytic process of this candidate. Phagocitic index and phagocytic number were calculated. The present work demonstrated that the phagocytosis process of M. habana TMC 5135 is influenced by the cellular source used as host cell, as well as by the infecting bacterial load and the time of exposure. The present investigation contributes to the characterization of the infection by this mycobacteria in its main target cells of innate immunity and it suggest future investigations to evaluate the activation of effector mechanisms of the innate immunity against this candidate(AU)

Rhopalurus junceus scorpion venom induces apoptosis in the triple negative human breast cancer cell line MDA-MB-231.

Rhopalurus junceus scorpion venom has demonstrated high cytotoxic activity in epithelial cancer cells. In the present study, the effect of scorpion venom on cell viability and apoptosis was evaluated in the MDA-MB-231 human breast carcinoma cell line. Cell viability was analyzed using MTT assay. The cell death event was examined trough end-point RT-PCR to identify the expression of apoptosis-related genes, fluorescent microscopy and mitochondrial membrane potential (ΔΨm) alteration. The results demonstrated that scorpion venom induced apoptosis in MDA-MB-231 cells in a time-dependent manner. Besides, scorpion venom treatment also resulted in p53, bax, noxa, puma, caspase 3 and p21 over-expression, while the expression of bcl-2 and bcl-xl was down-regulated. Apoptosis was associated with depolarization of ΔΨm. The overall effect indicates that the selective cytotoxic effect of the scorpion venom is associated with its apoptosis-inducing effect through the mitochondrial pathway. Therefore, R. junceus scorpion venom may be an interesting natural extract for further investigation in breast cancer treatment strategies.

Evaluación de la citotoxicidad de extractos marinos sobre un panel de líneas celulares / Evaluation of cytotoxicity of different marine extracts in a cell line panel

Introducción: los extractos naturales provenientes de fuentes marinas representan una importante fuente en el descubrimiento de nuevos compuestos con potencialidades como anticarcinogénicos. Objetivos: determinar el efecto de cinco extractos provenientes de diferentes organismos marinos sobre la viabilidad de un panel de cinco líneas celulares (A549, HEp-2, MDA-MB-231, SiHa y MRC-5). Metodos: el efecto de los extractos (Physalia physali, Cassiopea xamachana, Tripneustes ventricosus, Echinometra lucunter) se determinó mediante el ensayo colorimétrico con el empleo de bromuro de 3(4,5 dimetil-2-tiazoil)-2,5-difeniltetrazolio. Mediante de RT-PCR se determinó adicionalmente el efecto del extracto de Echinometra lucunter sobre la expresión de los genes apoptóticos p53, survivin, bcl-xL y noxa en SiHa. Resultados: todos los extractos afectaron la viabilidad celular de la línea normal MRC-5 de pulmón humano. Sin embargo, no disminuyeron la viabilidad de las líneas celulares de origen tumoral con excepción del extracto de Echinometra lucunter. Este extracto solo afectó la viabilidad de la línea celular tumoral SiHa. Los valores de las concentraciones inhibitorias medias (CI50) mostraron que solo para el extracto de Echinometra lucunter, la línea celular tumoral SiHa evidenció una CI50 de 52,07±11 μg/mL que es significativamente inferior a MRC-5 con una CI50 de 98,6±14 μg/mL, por lo que se muestra selectividad frente a las células tumorales. Adicionalmente, el extracto disminuyó significativamente la expresión de los genes proapoptóticos lo que sugiere la muerte celular por necrosis en las células SiHa. Conclusiones: el extracto proveniente de Echinometra lucunter resultó selectivo frente a las células tumorales SiHa. Experimentos que incluyan otras líneas celulares de cáncer cérvicouterino podrían confirmar el potencial de este extracto frente a esta variedad histológica de cáncer(AU)

Introduction: natural extracts from marine sources represent an important source for the discovery of new compounds with anti-carcinogenic potentialities. Objectives: to determine the effect of five extracts from several marine organisms pm the viability of a panel of five cell lines (A549, HEp-2, MDA-MB-231, SiHa y MRC-5). Methods: the effects of the extracts (Physalia physali, Cassiopea xamachana, Tripneustes ventricosus, Echinometra lucunter) were then determined by using the colorimetric assay with 3 (4,5 dimethyl-2-tiazoil)/2,5-difeniltetrazolium bromide. Additionally, the effect of extract of Echinometra lucunter was determined on the expression of apoptotic genes p53, survivin, bcl-xL and noxa in SiHa. Results: all the extracts affected the cell viability of the normal cell line MRC-5 of the human lung. However, viability of tumoral cell lines did not decrease except for the extract from Echinometra lucunter. This extract just affected the viability of tumor cell line SiHa. The mean inhibitory concentrations (IC50) showed that only for Echinometra lucunter extract, the tumor cell line SiHa revealed a IC50 of 52,07±11 μg/mL that is significantly lower than that of MRC-5 with IC50 of 98,6±14 μg/mL, therefore the selectivity against the tumor cells was shown. Moreover, the extract markedly decreased the expression of propapoptosis genes, thus indicating the cell death from necrosis in SIHa cells.Conclusions: extract from Echinometra lucunter was selective against tumor cells SiHa. Other experiments that will include other cervix uterine cancer cell lines can confirm the potential of this extract to have an effect on this histological cancer type(AU)

Evaluación de la citotoxicidad de extractos marinos sobre un panel de líneas celulares / Evaluation of cytotoxicity of different marine extracts in a cell line panel

Introducción: los extractos naturales provenientes de fuentes marinas representan una importante fuente en el descubrimiento de nuevos compuestos con potencialidades como anticarcinogénicos. Objetivos: determinar el efecto de cinco extractos provenientes de diferentes organismos marinos sobre la viabilidad de un panel de cinco líneas celulares (A549, HEp-2, MDA-MB-231, SiHa y MRC-5). Metodos: el efecto de los extractos (Physalia physali, Cassiopea xamachana, Tripneustes ventricosus, Echinometra lucunter) se determinó mediante el ensayo colorimétrico con el empleo de bromuro de 3(4,5 dimetil-2-tiazoil)-2,5-difeniltetrazolio. Mediante de RT-PCR se determinó adicionalmente el efecto del extracto de Echinometra lucunter sobre la expresión de los genes apoptóticos p53, survivin, bcl-xL y noxa en SiHa. Resultados: todos los extractos afectaron la viabilidad celular de la línea normal MRC-5 de pulmón humano. Sin embargo, no disminuyeron la viabilidad de las líneas celulares de origen tumoral con excepción del extracto de Echinometra lucunter. Este extracto solo afectó la viabilidad de la línea celular tumoral SiHa. Los valores de las concentraciones inhibitorias medias (CI50) mostraron que solo para el extracto de Echinometra lucunter, la línea celular tumoral SiHa evidenció una CI50 de 52,07±11 µg/mL que es significativamente inferior a MRC-5 con una CI50 de 98,6±14 µg/mL, por lo que se muestra selectividad frente a las células tumorales. Adicionalmente, el extracto disminuyó significativamente la expresión de los genes proapoptóticos lo que sugiere la muerte celular por necrosis en las células SiHa. Conclusiones: el extracto proveniente de Echinometra lucunter resultó selectivo frente a las células tumorales SiHa. Experimentos que incluyan otras líneas celulares de cáncer cérvicouterino podrían confirmar el potencial de este extracto frente a esta variedad histológica de cáncer(AU)

Introduction: natural extracts from marine sources represent an important source for the discovery of new compounds with anti-carcinogenic potentialities. Objectives: to determine the effect of five extracts from several marine organisms pm the viability of a panel of five cell lines (A549, HEp-2, MDA-MB-231, SiHa y MRC-5). Methods: the effects of the extracts (Physalia physali, Cassiopea xamachana, Tripneustes ventricosus, Echinometra lucunter) were then determined by using the colorimetric assay with 3 (4,5 dimethyl-2-tiazoil)/2,5-difeniltetrazolium bromide. Additionally, the effect of extract of Echinometra lucunter was determined on the expression of apoptotic genes p53, survivin, bcl-xL and noxa in SiHa. Results: all the extracts affected the cell viability of the normal cell line MRC-5 of the human lung. However, viability of tumoral cell lines did not decrease except for the extract from Echinometra lucunter. This extract just affected the viability of tumor cell line SiHa. The mean inhibitory concentrations (IC50) showed that only for Echinometra lucunter extract, the tumor cell line SiHa revealed a IC50 of 52,07±11 µg/mL that is significantly lower than that of MRC-5 with IC50 of 98,6±14 µg/mL, therefore the selectivity against the tumor cells was shown. Moreover, the extract markedly decreased the expression of propapoptosis genes, thus indicating the cell death from necrosis in SIHa cells. Conclusions: extract from Echinometra lucunter was selective against tumor cells SiHa. Other experiments that will include other cervix uterine cancer cell lines can confirm the potential of this extract to have an effect on this histological cancer type(AU)

Enzymatic analysis of venom from Cuban scorpion Rhopalurus junceus.

Rhopalurus junceus scorpion venom has been identified as a natural extract with anticancer potential. Interestingly, this scorpion venom does not cause adverse symptoms in humans. However, there is scarce information about its composition and enzymatic activity. In this work, we determined the electrophoretic profile of the venom, the gelatinase and caseinolytic activity, and the phospholipase A2 (PLA2) and hemolytic activity. The effect of different venom doses (6.25, 12.5 and 25 mg/kg) on gastrocnemius muscle was also measured as CK and LDH activity in serum. The presence of hyaluronidase was determined by turbidimetric assay. The effect of different fractions obtained by gel filtration chromatography were evaluated at different concentrations (0.05, 0.1, 0.2, 0.4, 0.6mg/ml) against lung cancer cell A549 and lung normal cell MRC-5 using MTT assay. The electrophoretic profile demonstrated the presence of proteins bands around 67kDa, 43kDa, 18.4kDa and a majority band below 14.3kDa. The venom did not showed caseinolytic, gelatinase, PLA2 and hemolytic activity even at highest venom concentration used in the study. Scorpion venom only showed a significant toxic effect on gastrocnemius muscles identified by CK and LDH release after subcutaneous injection of 12.5 and 25mg/kg. Low molecular weight fractions (<4kDa) induced a significant cytotoxicity in A549 cells while high molecular weight proteins (45-60kDa) were responsible for hyaluronidase activity and toxic effect against MRC-5. Experiments indicate that Rhopalurus junceus scorpion venom has low enzymatic activity, which could contribute to the low toxic potential of this scorpion venom.

Brazilian green propolis induced apoptosis in human lung cancer A549 cells through mitochondrial-mediated pathway.

OBJECTIVES: Propolis effect on the growth and apoptosis of human lung adenocarcinoma (A549 cells) was investigated as well as its mechanisms. METHODS: Cells were incubated with propolis for 72 h, and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were employed to assess cell viability and the inhibitory concentration (IC). Apoptosis was detected by Acridine Orange/Ethidium Bromide and 4',6-diamidino-2-phenylindole staining after 24 and 48 h of incubation with » IC50 of propolis by testing the mitochondrial membrane potential (ΔΨm) and the expression of apoptosis-related genes (p53, Caspase-3, Bax, Bcl-2, Bcl-XL , Noxa, Puma and p21) by reverse transcription polymerase chain reaction. KEY FINDINGS: Propolis displayed antiproliferative and cytotoxic effects on A549 cells in a dose- and time-dependent manner, but it did not suppress the growth of normal Vero cells. An enhanced apoptosis was seen in A549 propolis-treated cells after 48 h compared with the control cells. Propolis decreased mitochondrial membrane potential by overexpression of pro-apoptotic genes (Bax and Noxa) and reduction of the antiapoptotic gene Bcl-XL . The expression level of other genes remained unchanged (p53, Caspse-3 and Bax), whereas p21 expression was increased. Propolis induced caspase-independent apoptosis through a p53-independent mitochondrial pathway, and cell cycle arrest by upregulation of p21. CONCLUSIONS: Although propolis induces apoptosis mainly by p53-independent manner, it may be induced by another pathway, and new insights may arise for preventing or treating lung cancer.

Normalización de un método inmunoquímico para detectar Papilomavirus humano tipo 16 en lesiones cérvico-uterinas / Standardization of immunochemical method for detection of type 16 human papilomavirus in cervix uterine lesions

Introducción: la infección por Papilomavirus Humano (PVH) es la condición necesaria para la aparición y desarrollo del cáncer cérvico-uterino. Los genotipos de alto riesgo oncogénico son los causantes de este tipo de neoplasia y dentro de ellos el más frecuente es el PVH 16, que se encuentra aproximadamente en el 60 por ciento de los casos. Los métodos de diagnóstico comerciales resultan costosos para países con escasos recursos económicos, lo que sugiere la búsqueda de alternativas empleando protocolos sencillos y baratos. Objetivos: normalizar un método inmunoquímico para la detección del antígeno L1 de PVH tipo 16 en muestras cérvico-uterinas de pacientes con lesiones intraepiteliales escamosas y determinar la coincidencia entre el método normalizado y la Reacción en Cadena de la Polimerasa en Tiempo Real (RCP-TR), como técnica de referencia, para estimar la utilidad de dicho método en el diagnóstico de la infección por este genotipo viral. Métodos: se compararon tres procedimientos de inmunotinción (Indirecto de inmunoperoxidasa en dos pasos, Estreptavidina-Biotina y Amplificación por polímero) respecto a sensibilidad analítica, tinción inespecífica de fondo y tiempo de terminación, para la detección de la proteína L1 de PVH 16 en líneas celulares derivadas de carcinomas cervicales humanos y en muestras cérvico-uterinas utilizadas como controles. El protocolo normalizado se aplicó a muestras cérvico-uterinas de mujeres entre 30 y 59 años, 82 con lesiones intraepiteliales cervicales y 10 sin antecedentes de alteraciones citológicas, a las que además se les determinó PVH 16 mediante RCP-TR. Resultados: el procedimiento de Estreptavidina-Biotina resultó el más sensible y específico...

Introduction: Human Papillomavirus (HPV) infection is the necessary condition for the occurernce and development of cervical cancer. The high oncogenic risk genotypes are the responsible for this type of neoplasia and the most frequent is HPV 16 that affects roughly 60 percent of cases. Commercial kits for HPV detection are expensive for resource-poor countries, which suggests the search for alternative throguh non-expensive simple protocoles. Objectives: to standardize an immunochemical method for the detection of HPV 16 L1 antigen in cervical samples of patients with squamous intraepithelial lesions and to determine the diagnostic coincidence between the immunochemical method and the real-time polymerase chain reaction to estimate the usefulness of this method for the detection of cervical infection with this viral genotype. Methods: three immunostaining methods (Two-Step Indirect Immunoperoxidase, Labelled Streptavidin-Biotin and Enhanced Polymer) were compared in terms of analytical sensitivity, nonspecific background staining and time of completion, for the detection of protein L1 of HPV-16 in a cell line derived from human cervical carcinoma and clinical samples from uterine cervix. The optimized protocol was applied to 82 cervical samples from women aged 30-59 years with squamous intraepithelial lesions and to 10 samples of sexually active women without previous signals of positive cytology. The presence of type 16 HPV was also detected with the aid of RT-PCR. Results: the Streptavidin-Biotin system was the most sensitive and specific...

Normalización de un método inmunoquímico para detectar Papilomavirus humano tipo 16 en lesiones cérvico-uterinas / Standardization of immunochemical method for detection of type 16 human papilomavirus in cervix uterine lesions

Introducción: la infección por Papilomavirus Humano (PVH) es la condición necesaria para la aparición y desarrollo del cáncer cérvico-uterino. Los genotipos de alto riesgo oncogénico son los causantes de este tipo de neoplasia y dentro de ellos el más frecuente es el PVH 16, que se encuentra aproximadamente en el 60 % de los casos. Los métodos de diagnóstico comerciales resultan costosos para países con escasos recursos económicos, lo que sugiere la búsqueda de alternativas empleando protocolos sencillos y baratos. Objetivos: normalizar un método inmunoquímico para la detección del antígeno L1 de PVH tipo 16 en muestras cérvico-uterinas de pacientes con lesiones intraepiteliales escamosas y determinar la coincidencia entre el método normalizado y la Reacción en Cadena de la Polimerasa en Tiempo Real (RCP-TR), como técnica de referencia, para estimar la utilidad de dicho método en el diagnóstico de la infección por este genotipo viral. Métodos: se compararon tres procedimientos de inmunotinción (Indirecto de inmunoperoxidasa en dos pasos, Estreptavidina-Biotina y Amplificación por polímero) respecto a sensibilidad analítica, tinción inespecífica de fondo y tiempo de terminación, para la detección de la proteína L1 de PVH 16 en líneas celulares derivadas de carcinomas cervicales humanos y en muestras cérvico-uterinas utilizadas como controles. El protocolo normalizado se aplicó a muestras cérvico-uterinas de mujeres entre 30 y 59 años, 82 con lesiones intraepiteliales cervicales y 10 sin antecedentes de alteraciones citológicas, a las que además se les determinó PVH 16 mediante RCP-TR. Resultados: el procedimiento de Estreptavidina-Biotina resultó el más sensible y específico. La coincidencia entre el método inmunoquímico y la RCP-TR fue de un 98,6 por ciento, la sensibilidad fue de un 98,57 por ciento y la especificidad de un 91,67 por ciento, con valores predictivos negativo y positivo por encima del 90 por ciento. Conclusiones: se demostró la validez del método inmunoquímico como prueba confirmatoria de la infección por PVH 16. Dicho método probó ser sensible, sencillo y no requiere de una compleja infraestructura para detectar PVH 16 en muestras cervicales. Además, esta técnica permite obtener información rápidamente y evita el uso de métodos invasivos(AU)

Introduction: Human Papillomavirus (HPV) infection is the necessary condition for the occurernce and development of cervical cancer. The high oncogenic risk genotypes are the responsible for this type of neoplasia and the most frequent is HPV 16 that affects roughly 60 percent of cases. Commercial kits for HPV detection are expensive for resource-poor countries, which suggests the search for alternative throguh non-expensive simple protocoles. Objectives: to standardize an immunochemical method for the detection of HPV 16 L1 antigen in cervical samples of patients with squamous intraepithelial lesions and to determine the diagnostic coincidence between the immunochemical method and the real-time polymerase chain reaction to estimate the usefulness of this method for the detection of cervical infection with this viral genotype. Methods: three immunostaining methods (Two-Step Indirect Immunoperoxidase, Labelled Streptavidin-Biotin and Enhanced Polymer) were compared in terms of analytical sensitivity, nonspecific background staining and time of completion, for the detection of protein L1 of HPV-16 in a cell line derived from human cervical carcinoma and clinical samples from uterine cervix. The optimized protocol was applied to 82 cervical samples from women aged 30-59 years with squamous intraepithelial lesions and to 10 samples of sexually active women without previous signals of positive cytology. The presence of type 16 HPV was also detected with the aid of RT-PCR. Results: the Streptavidin-Biotin system was the most sensitive and specific. The diagnostic agreement between the immunochemical method and the real-time polymerase chain reaction reached 98.6 percent, sensitivity was 98.57 percent and specificity was 91.67 %, with positive and negative predictive values above 90 percent. Conclusions: the validity of the immunochemical method as a confirmatory test for infection by HPV-16 has been demonstrated. The normalized immunochemical method proved to be a sensitive, simple, relatively fast method to detect HPV from clinical samples of cervical cells. Furthermore, this method provides information quickly, avoiding the use of invasive methods in patients(AU)

In vitro anticancer effect of venom from Cuban scorpion Rhopalurus junceus against a panel of human cancer cell lines.

In Cuba the endemic species of scorpion Rhopalurus junceus has been used in traditional medicine for cancer treatment. However, there is little scientific evidence about its potential in cancer therapy. The effect of a range of scorpion venom concentrations (0.1, 0.25, 0.5, 0.75 and 1mg/ml) against a panel of human tumor cell lines from epithelial (Hela, SiHa, Hep-2, NCI-H292, A549, MDA-MB-231, MDA-MB-468, HT-29), hematopoietic origins (U937, K562, Raji) and normal cells (MRC-5, MDCK, Vero) was determined by the MTT assay. Additionally, the effect of venom on tumor cell death was assayed by Fluorescence microscopy, RT-PCR and western blot. Only the epithelial cancer cells showed significant cell viability reduction, with medium cytotoxic concentration (IC50) ranging from 0.6-1mg/ml, in a concentration-dependent manner. There was no effect on either normal or hematopoietic tumor cells. Scorpion venom demonstrated to induce apoptosis in less sensitive tumor cells (Hela) as evidenced by chromatin condensation, over expression of p53 and bax mRNA, down expression of bcl-2 mRNA and increase of activated caspases 3, 8, 9. In most sensitive tumor cells (A549), scorpion venom induced necrosis evidenced by acridine orange/ethidium bromide fluorescent dyes and down-expression of apoptosis-related genes. We concluded the scorpion venom from R. junceus possessed a selective and differential toxicity against epithelial cancer cells. This is the first report related to biological effect of R. junceus venom against a panel of tumor cells lines. All these results make R. junceus venom as a promise natural product for cancer treatment.

Efecto del anticuerpo monoclonal 4B3 en la infección experimental por Cryptococcus neoformans / Effect of 4B3 monoclonal antibody on the experimental Cryptococcus neoformans infection

Introducción: las frecuencias de recaídas y elevada letalidad de la criptococosis se mantienen altas, lo cual incentiva la búsqueda de nuevas estrategias terapéuticas. Objetivos: evaluar el efecto del anticuerpo monoclonal 4B3 sobre la infección criptocócica en ratones BALB/c. Métodos: se determinó la cinética de la concentración sérica del anticuerpo monoclonal para su administración intraperitoneal (500 µg), que fue medida por ELISA cuantitativo de doble anticuerpo. La capacidad protectora se observó mediante el registro de supervivencia de ratones BALB/c administrados con 500 µg de anticuerpo monoclonal 4B3 e inoculados con 2 x 10(2,2) células/mL de Cryptococcus neoformans, así como por evaluación de la diseminación de la levadura a los principales órganos diana. Se identificó el efecto del anticuerpo monoclonal 4B3 sobre la fagocitosis y lisis del microorganismo por células de la línea de macrófagos P338.D1. Resultados: la dosis empleada fue suficiente para mantener valores séricos elevados del 4B3 (38 µg/mL) durante al menos 46 d. Se determinó que el 4B3 no confiere protección, lo cual potencia la diseminación del microorganismo y disminuye el tiempo de vida de los animales. El ensayo de fagocitosis mostró que el anticuerpo evaluado incrementa la actividad fagocítica de los macrófagos sin lograr un efecto fungicida. Conclusiones: el anticuerpo monoclonal 4B3 estimula la fagocitosis de C. neoformans por macrófagos, pero sin efecto fungicida. Con ello favorece la diseminación de la levadura y disminuye el tiempo de vida de los ratones a la infección.

Introduction: frequent relapses and high lethality of criptococcosis has encouraged the search for new therapeutic strategies. Objectives: to evaluate the effect of the monoclonal antibody 4B3 on the cryptococcal infection in Balb/c mice. Methods: the kinetics in serum concentration of the studied monoclonal antibody was determined for intraperitoneal administration (500 µg) by quantitative sandwich ELISA. In order to assess its protective capability, were administered 500 µg of 4B3 and innoculated 2 x 10(2.2) cells/mL of Cryptococcus neoformans. The survival of mice was recorded and the yeast dissemination to the main target organs was evaluated. Macrophages P338.D1 cell lines were used to measure the effect of the monoclonal antibody 4B3 on the phagocytosis and lysis of the microorganism. Results: the used dose helped to keep high values (38 µg/m) of 4B3 in serum for at least 46 days. It was found that the monoclonal antibody does not give protection, which makes the microorganism dissemination possible, along with the reduction in the survival of mice. Finally, the phagocytosis test showed that 4B3 increased the phagocytic activity of macrophages without any fungicidal effect. Conclusions: the monoclonal antibody 4B3 stimulates C. neoformans phagocytosis by macrophages without fungicidal effect, thus favoring yeast dissemination and decreasing the survival of mice due to cryptococcal infection.

Efecto del anticuerpo monoclonal 4B3 en la infección experimental por Cryptococcus neoformans / Effect of 4B3 monoclonal antibody on the experimental Cryptococcus neoformans infection

Introducción: las frecuencias de recaídas y elevada letalidad de la criptococosis se mantienen altas, lo cual incentiva la búsqueda de nuevas estrategias terapéuticas. Objetivos: evaluar el efecto del anticuerpo monoclonal 4B3 sobre la infección criptocócica en ratones BALB/c. Métodos: se determinó la cinética de la concentración sérica del anticuerpo monoclonal para su administración intraperitoneal (500 Ág), que fue medida por ELISA cuantitativo de doble anticuerpo. La capacidad protectora se observó mediante el registro de supervivencia de ratones BALB/c administrados con 500 Ág de anticuerpo monoclonal 4B3 e inoculados con 2 x 10(2,2) células/mL de Cryptococcus neoformans, así como por evaluación de la diseminación de la levadura a los principales órganos diana. Se identificó el efecto del anticuerpo monoclonal 4B3 sobre la fagocitosis y lisis del microorganismo por células de la línea de macrófagos P338.D1. Resultados: la dosis empleada fue suficiente para mantener valores séricos elevados del 4B3 (38 Ág/mL) durante al menos 46 d. Se determinó que el 4B3 no confiere protección, lo cual potencia la diseminación del microorganismo y disminuye el tiempo de vida de los animales. El ensayo de fagocitosis mostró que el anticuerpo evaluado incrementa la actividad fagocítica de los macrófagos sin lograr un efecto fungicida. Conclusiones: el anticuerpo monoclonal 4B3 estimula la fagocitosis de C. neoformans por macrófagos, pero sin efecto fungicida. Con ello favorece la diseminación de la levadura y disminuye el tiempo de vida de los ratones a la infección(AU)

Introduction: frequent relapses and high lethality of criptococcosis has encouraged the search for new therapeutic strategies. Objectives: to evaluate the effect of the monoclonal antibody 4B3 on the cryptococcal infection in Balb/c mice. Methods: the kinetics in serum concentration of the studied monoclonal antibody was determined for intraperitoneal administration (500 Ág) by quantitative sandwich ELISA. In order to assess its protective capability, were administered 500 Ág of 4B3 and innoculated 2 x 10(2.2) cells/mL of Cryptococcus neoformans. The survival of mice was recorded and the yeast dissemination to the main target organs was evaluated. Macrophages P338.D1 cell lines were used to measure the effect of the monoclonal antibody 4B3 on the phagocytosis and lysis of the microorganism. Results: the used dose helped to keep high values (38 Ág/m) of 4B3 in serum for at least 46 days. It was found that the monoclonal antibody does not give protection, which makes the microorganism dissemination possible, along with the reduction in the survival of mice. Finally, the phagocytosis test showed that 4B3 increased the phagocytic activity of macrophages without any fungicidal effect. Conclusions: the monoclonal antibody 4B3 stimulates C. neoformans phagocytosis by macrophages without fungicidal effect, thus favoring yeast dissemination and decreasing the survival of mice due to cryptococcal infection(AU)

Citotoxicidad de extractos de plantas medicinales sobre la línea celular de carcinoma de pulmón humano A549 / Cytotoxicity of medicinal plant extracts on the human lung carcinoma cell line A549

Es objetivo del trabajo evaluar el efecto de 10 extractos de plantas medicinales sobre el crecimiento de la línea celular humana de carcinoma de pulmón A549. El efecto de los extractos sobre la células tumorales se midió a través de un ensayo colorimétrico mediante el empleo del bromuro de 3-(4,5-dimetil-tiazol-2-yl)-2,5-difenil tetrazolio a concentraciones entre 3,9-250 µg/mL durante 72 h y se calculó la concentración citotóxica media para cada uno. Del total de los extractos evaluados solo cuatro (Parthenium hysterophorus, Bixa orellana, Momordica charantia y Cucurbita maxima) evidenciaron concentraciones citotóxicas medias inferiores a 100 µg/mL. Excepto Parthenium hysterophorus, las restantes se emplean en la medicina tradicional para el tratamiento del cáncer. Los extractos de Cecropia peltata, Melia azedarach, Annona glabra, Artemisia absintium, Lepidium virginicum y Bidens pilosa no mostraron efectos citotóxicos significativos. Los extractos de plantas que se emplean en la medicina tradicional para el tratamiento del cáncer, mostraron citotoxicidad sobre las células tumorales. El conocimiento etnobotánico representa una herramienta importante en la selección de plantas medicinales, en la búsqueda de nuevos compuestos para el tratamiento del cáncer

To evaluate the effect of 10 Cuban medicinal plant extracts on the human lung tumor cell line A549. The effect of the plant extracts on tumor cells was determined by a colorimetric assay using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at concentrations ranging from 3,9-250 µg/mL for 72 hours and the mean cytotoxic concentration was calculated for each of them. The ethanolic extracts of Parthenium hysterophorus, Bixa orellana, Momordica charantia and Cucurbita maxima showed mean cytotoxic concentrations under 100 µg/mL. Except for P hysterophorus, the others are used in traditional medicine to fight cancer. The remaining extracts from Cecropia peltata, Melia azedarach, Annona glabra, Artemisia absintium, Lepidium virginicum and Bidens pilosa did not show significant cytotoxic effects. the plant extracts for cancer treatment in traditional medicine showed cytotoxic effect on the tumor cell lines. Ethnobotanical data represent an important tool for medicinal plants screening in the quest for new compounds to treat cancer

Citotoxicidad de extractos de plantas medicinales sobre la línea celular de carcinoma de pulmón humano A549 / Cytotoxicity of medicinal plant extracts on the human lung carcinoma cell line A549

Es objetivo del trabajo evaluar el efecto de 10 extractos de plantas medicinales sobre el crecimiento de la línea celular humana de carcinoma de pulmón A549. El efecto de los extractos sobre la células tumorales se midió a través de un ensayo colorimétrico mediante el empleo del bromuro de 3-(4,5-dimetil-tiazol-2-yl)-2,5-difenil tetrazolio a concentraciones entre 3,9-250 µg/mL durante 72 h y se calculó la concentración citotóxica media para cada uno. Del total de los extractos evaluados solo cuatro (Parthenium hysterophorus, Bixa orellana, Momordica charantia y Cucurbita maxima) evidenciaron concentraciones citotóxicas medias inferiores a 100 µg/mL. Excepto Parthenium hysterophorus, las restantes se emplean en la medicina tradicional para el tratamiento del cáncer. Los extractos de Cecropia peltata, Melia azedarach, Annona glabra, Artemisia absintium, Lepidium virginicum y Bidens pilosa no mostraron efectos citotóxicos significativos. Los extractos de plantas que se emplean en la medicina tradicional para el tratamiento del cáncer, mostraron citotoxicidad sobre las células tumorales. El conocimiento etnobotánico representa una herramienta importante en la selección de plantas medicinales, en la búsqueda de nuevos compuestos para el tratamiento del cáncer(AU)

To evaluate the effect of 10 Cuban medicinal plant extracts on the human lung tumor cell line A549. The effect of the plant extracts on tumor cells was determined by a colorimetric assay using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at concentrations ranging from 3,9-250 µg/mL for 72 hours and the mean cytotoxic concentration was calculated for each of them. The ethanolic extracts of Parthenium hysterophorus, Bixa orellana, Momordica charantia and Cucurbita maxima showed mean cytotoxic concentrations under 100 µg/mL. Except for P hysterophorus, the others are used in traditional medicine to fight cancer. The remaining extracts from Cecropia peltata, Melia azedarach, Annona glabra, Artemisia absintium, Lepidium virginicum and Bidens pilosa did not show significant cytotoxic effects. the plant extracts for cancer treatment in traditional medicine showed cytotoxic effect on the tumor cell lines. Ethnobotanical data represent an important tool for medicinal plants screening in the quest for new compounds to treat cancer(AU)

[Effect of 4B3 monoclonal antibody on the experimental Cryptococcus neoformans infection]. / Efecto del anticuerpo monoclonal 4B3 en la infección experimental por Cryptococcus neoformans.

INTRODUCTION: frequent relapses and high lethality of criptococcosis has encouraged the search for new therapeutic strategies. OBJECTIVES: to evaluate the effect of the monoclonal antibody 4B3 on the cryptococcal infection in Balb/c mice. METHODS: the kinetics in serum concentration of the studied monoclonal antibody was determined for intraperitoneal administration (500 microg) by quantitative sandwich ELISA. In order to assess its protective capability, were administered 500 microg of 4B3 and innoculated 2 x 10(22) cells/mL of Cryptococcus neoformans. The survival of mice was recorded and the yeast dissemination to the main target organs was evaluated. Macrophages P338.D1 cell lines were used to measure the effect of the monoclonal antibody 4B3 on the phagocytosis and lysis of the microorganism. RESULTS: the used dose helped to keep high values (38 microg/m) of 4B3 in serum for at least 46 days. It was found that the monoclonal antibody does not give protection, which makes the microorganism dissemination possible, along with the reduction in the survival of mice. Finally, the phagocytosis test showed that 4B3 increased the phagocytic activity of macrophages without any fungicidal effect. CONCLUSIONS: the monoclonal antibody 4B3 stimulates C. neoformans phagocytosis by macrophages without fungicidal effect, thus favoring yeast dissemination and decreasing the survival of mice due to cryptococcal infection

Aislamiento y propagación del virus de la hepatitis E en diferentes líneas celulares / Isolation and propagation of hepatitis E virus in several cell lines

Antecedentes: el virus de la hepatitis E es el agente causal de la hepatitis E. Las propiedades biológicas y moleculares de las cepas asiáticas del VHE ya han sido exploradas en cultivos celulares. Objetivos: aislar y propagar una cepa cubana del virus de la hepatitis E en diferentes líneas celulares. Métodos: la monocapa de las células A549 fue inoculada con una suspensión de heces obtenida de un paciente con diagnóstico serológico y molecular de hepatitis E. Estas células fueron observadas hasta el décimo pase. Mientras que, las líneas celulares MRC5, LLCMK2, HEP-2, FRhK4 y HeLa fueron utilizadas para propagar el virus de la hepatitis E, a partir del sobrenadante obtenido del tercer pase en A549. Estas células fueron seguidas hasta el tercer pase. El ARN y los antígenos de la cepa ECV/2349-03 fueron identificados por TR-RCP e inmunofluorescencia indirecta. Resultados: en las células A549 el ECP apareció desde el primer pase, a los 3 d de posinoculación. El genoma y los antígenos del virus fueron identificados en todos los pases seriados. En el resto de las líneas celulares estudiadas no se observó el ECP. En estas células el material genético del virus de la hepatitis E se detectó desde el primer pase y los antígenos a partir del segundo pase, excepto en las HeLa. Conclusiones: estos resultados confirman que las células A549 pueden ser utilizadas para aislar y propagar el virus de la hepatitis E, mientras que las células MRC5, LLCMK2, HEP-2 y FRhK4 son capaces de mantener el crecimiento viral.

Background: hepatitis E virus (HEV) is the causative agent of hepatitis E. Biological and molecular properties of Asian HEV strains have been explored in cells cultures. Objetives: the aim of this investigation was to isolate and propagate a Cuban HEV isolate in different cell lines. Methods: A549 cells monolayer was infected with faeces suspension from patient with sporadic hepatitis E and followed up until tenth passage. Lately, the supernatant harvested from third passage in A549 cells was inoculated in MRC5, HEP-2, LLCMK2, HeLa y FRhK4 for propagation study. These cells were observed up to third passage. RNA and viral antigen of ECV/2349-03 HEV strain were identified by RT-PCR and indirect immunofluorescence. Results: CPE appeared since first passage, at third day of post-inoculation in A549 cells. HEV antigens and genome were detected in all serial passages. CPE was not observed in the rest of cellular cultures. In the cells used for propagation the viral genome was observed from first passage, while the antigens were detected since second passage, except HeLa. Conclusions: these results confirm that A549 can be used to isolate and propagate HEV. Meanwhile, the MRC5, HEP-2, LLCMK2 and FRhK4 were able to support viral growing.

Aislamiento y caracterización de cepas de Adenovirus que colonizan el tracto gastrointestinal de pacientes cubanos seropositivos al VIH / Isolation and characterization of Adenovirus strains that invade the gastrointestinal tract of Cuban HIV-positive patients

Introducción: el tracto gastrointestinal es uno de los sistemas que se afecta con frecuencia en el paciente seropositivo al virus de la inmunodeficiencia humana (VIH), de manera que las alteraciones o enfermedades en este sitio representan una causa importante de morbilidad y mortalidad. El hallazgo de cepas de adenovirus, raramente aisladas en pacientes inmunocompetentes, ha sido asociado en ocasiones a la presencia de diarrea, lo cual refleja la importancia del estudio y la tipificación de estas cepas. Objetivos: conocer la prevalencia de los Adenovirus en muestras de heces fecales de pacientes infectados con el VIH, con diarrea aguda o crónica, así como la caracterización de los agentes aislados, mediante la implementación de métodos rápidos y confiables. Métodos: fueron analizadas 167 muestras de heces fecales de individuos seropositivos al VIH y 127 seronegativos como grupo control. La prevalencia fue investigada mediante el aislamiento en cultivo celular, se realizó además la tipificación de las cepas aisladas, mediante técnicas de biología molecular como la reacción en cadena de la polimerasa y la secuenciación nucleotídica. El análisis estadístico se efectuó mediante la prueba exacta de Fisher contenido en el paquete estadístico Epi Info (Versión 6.04). Resultados: la prevalencia en los pacientes VIH+ fue de 15 por ciento, mientras que en los VIH- fue de 4 por ciento; la diferencia resultó estadísticamente significativa; 96 por ciento de las cepas aisladas en los pacientes VIH+ correspondieron al subgénero D, asociadas en 62,5 por ciento a cuadros diarreicos y estadios avanzados de la infección por VIH. Conclusiones: los resultados sugieren la importancia de incluir a los adenovirus y su tipificación en el diagnóstico de rutina de los pacientes VIH+, lo cual sería de interés en el manejo clínico de la infección y aportaría datos claves en la vigilancia y el control de la transmisión de la infección hospitalaria y los análisis epidemiológicos.

Bacckground: the gastrointestinal tract is one of the most frequently affected systems in HIV-positive patients, therefore, changes or diseases occurring in this site represent an important cause of morbidity and mortality. The finding of adenovirus strains, rarely isolated in immunocompetent patients, has been occasionally associated to diarrheas, which reflects the importance of study and typing of these strains. Objectives: to find out the prevalence of Adenovirus in feces samples from HIV patients having chronic or acute diarrheas, as well as the characterization of isolated agents through the application of reliable and quick methods. Methods: one hundred and sixty seven fecal samples from HIV-positive patients and 127 from seronegative subjects as the control group were analyzed. Isolation in cell cultures determined the prevalence and molecular biology techniques as the polymerase chain reaction and nucleotide sequencing allowed typing of isolated strains. The statistical analysis was based on the Fisher´s exact test included in Epi Info (6.04) statistical software. RESULTS: the prevalence in HIV positive patients was 15 percent whereas in the HIV-negative was 4 percent. The difference was statistically significant; 96 percent of isolated strains in HIV positive patients belonged to subgenus D, which are linked in 62.5 percent of cases to acute diarrheal condition and advanced stages of HIV infection. Conclusions: the results indicated the importance of including adenovirus and their typing in the routine diagnosis of patients positive to HIV, all of which would be useful in the clinical management of infection and would contribute key data on surveillance and control of hospital infection transmission and the epidemiological analysis.