Heteroctenus junceus Scorpion Venom Modulates the Concentration of Pro-Inflammatory Cytokines in F3II Tumor Cells.

The ability of Heteroctenus junceus scorpion venom to modulate the concentration of cytokines related to its antitumoral effect is unknown. F3II cells were treated with » IC50, ½ IC50 and the IC50 of H. junceus scorpion venom. Tumor growth kinetics in F3II-bearing mice were evaluated after 24 days of oral administration of venom doses. The effect of tumor lysates on F3II cell viability was evaluated by MTT assay, while cytokines present in each sample were determined by ELISA. In supernatant, H. junceus scorpion venom decreased the concentration of IL-6 (p < 0.001), IFN-γ (p < 0.001), IL-1ß (p < 0.01); meanwhile IL-12 (p < 0.001) and TNF-α (p < 0.001) levels increased significantly, according to the concentration and the time of incubation. Heteroctenus junceus scorpion venom effectively inhibits in vivo tumor progression. In the sera, a significant decrease was observed in TNF-α levels (p < 0.05). In tumor lysates, IL-6 decreased significantly in the groups treated with 12.5 mg/kg (p < 0.001) and 25 mg/kg (p < 0.05). Heteroctenus junceus scorpion venom is capable of modulating other proinflammatory and protumoral cytokines involved in the inflammation associated with cancer.

Molecular evolution of coxsackievirus A24v in Cuba over 23-years, 1986-2009.

Coxsackievirus A24 variant (CVA24v) is a major causative agent of acute hemorrhagic conjunctivitis outbreaks worldwide, yet the evolutionary and transmission dynamics of the virus remain unclear. To address this, we analyzed and compared the 3C and partial VP1 gene regions of CVA24v isolates obtained from five outbreaks in Cuba between 1986 and 2009 and strains isolated worldwide. Here we show that Cuban strains were homologous to those isolated in Africa, the Americas and Asia during the same time period. Two genotypes of CVA24v (GIII and GIV) were repeatedly introduced into Cuba and they arose about two years before the epidemic was detected. The two genotypes co-evolved with a population size that is stable over time. However, nucleotide substitution rates peaked during pandemics with 4.39 × 10-3 and 5.80 × 10-3 substitutions per site per year for the 3C and VP1 region, respectively. The phylogeographic analysis identified 25 and 19 viral transmission routes based on 3C and VP1 regions, respectively. Pandemic viruses usually originated in Asia, and both China and Brazil were the major hub for the global dispersal of the virus. Together, these data provide novel insight into the epidemiological dynamics of this virus and possibly other pandemic viruses.

Mycobacterium "habana" TMC 5135 as a vaccine candidate against tuberculosis: In vitro studies.

Background: Tuberculosis (TB) is one of the biggest problems of global health, at present. Bacillus-Calmette-Guérin is the only vaccine available against this disease. It protects only against the severe forms of TB in the childhood, which is a challenge in the search of new vaccine candidates. Taking into account the protective history of Mycobacterium "habana" against experimental TB, we proposed to provide the elements that support the use of M. "habana" TMC 5135 as a vaccine candidate against TB by infection studies in murine macrophages cell cultures. Methods: The production of microbicidal compounds dependent on oxygen metabolism as nitric oxide and hydrogen peroxide by murine peritoneal macrophages was detected. The invasive and toxigenic capacity of M. "habana" to infect this cell type was also evaluated through the quantification of intracellular alive bacillus and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay, respectively. Results: The results suggest that M. "habana" TMC 5135 is able to persist into peritoneal macrophages, to resist the effectors mechanisms of respiratory burst, and to keep the viability of the target cell. The demonstration of these effector mechanisms and the survival capacity of M. "habana" in this niche are relevant aspects of this research assuring the continuity of this candidate to next phases of preclinical development. Conclusion: 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.

Sticholysin II-mediated cytotoxicity involves the activation of regulated intracellular responses that anticipates cell death.

Sticholysin II (StII) is a pore-forming toxin of biomedical interest that belongs to the actinoporin protein family. Sticholysins are currently under examination as an active immunomodulating component of a vaccinal platform against tumoral cells and as a key element of a nucleic acids delivery system to cell cytosol. These proteins form pores in the plasma membrane leading to ion imbalance and cell lysis. However, the intracellular mechanisms triggered by actinoporins upon binding to membranes and its consequences for cell death are barely understood. Here, we have examined the cytotoxicity and intracellular responses induced by StII upon binding to human B-cell lymphoma Raji in vitro. StII cytotoxicity involves a functional actin cytoskeleton, induces cellular swelling, lysis and the concomitant release of cytosol content. In addition, StII induces calcium release mainly from the Endoplasmic Reticulum, activates Mitogen-Activated Protein Kinase ERK and impairs mitochondrial membrane potential. Furthermore, StII stimulates the expression of receptor interacting protein kinase 1 (RIP1), normally related to different forms of regulated cell death such as apoptosis and necroptosis. In correspondence, necrostatin-1, an inhibitor of this kinase, reduces StII cytotoxicity. However, the mechanism of cell death activated by StII does not involve caspases activation, typical molecular features of apoptosis and pyroptosis. Our results suggest that, beyond pore-formation and cell lysis, StII-induced cytotoxicity could involve other regulated intracellular mechanisms connected to RIP1-MEK1/2 -ERK1/2- pathways. This opens new perspectives and challenges the general point of view that these toxins induce a completely unregulated mechanism of necrotic cell death. This study contributes to a better understanding of the molecular mechanisms involved in toxin-cell interaction and the implications for cell functioning, with connotation for the exploitations of these toxins in clinical settings.

In vitro antiplasmodial activity, cytotoxicity and chemical profiles of sponge species of Cuban coasts.

Aqueous and organic fractions from the crude extracts of 17 sponge species collected at Boca de Calderas, Havana, Cuba were analysed. The organic fractions of Mycale laxissima, Clathria echinata and Agelas cerebrum exhibited values of concentrations causing 50% inhibition of in vitro growth of Plasmodium berghei (IC50) of 42.3 ± 5.1, 52 ± 9.7 and 60.3 ± 10.6 µg/mL, respectively, while their selectivity indexes for fibroblast cell lines were 9.45, 4.24 and 8.7, correspondingly. These fractions reduced parasitemia of infected Balb/c mice as well. Selective cytotoxicity indexes against tumour HeLa cells focused an interest on the aqueous fraction of M. laxissima (>7.12) and organic fractions of Polymastia nigra (5.95), A. cerebrum (5.48) and Niphates erecta (>4.2). Triterpenoids/steroids and alkaloids detected in the organic fractions of M. laxissima, C. echinata and A. cerebrum should be isolated for future investigation.

Normalización de un sistema de reacción en cadena de la polimerasa en tiempo real para la cuantificación de papilomavirus humano de alto riesgo oncogénico / Standardization of a real-time based polymerase chain reaction system for the quantification of human papilomavirus of high oncogenic risk

El objetivo de este trabajo fue normalizar e implementar un sistema de reacción en cadena de la polimerasa en tiempo real, para determinar la carga viral de 7 genotipos de papilomavirus humano (PVH) de alto riesgo oncogénico. Se evaluó la especificidad del sistema y se construyeron las curvas estándar para PVH 16 y 18, que se emplearon para la cuantificación de ADN viral en diferentes muestras de pacientes identificados como positivos a PVH, mediante reacción en cadena de la polimerasa (RCP) cualitativa y secuenciación nucleotídica. Se obtuvieron dos curvas estándar para PVH 16 y 18, a partir del ADN genómico de las líneas celulares SiHa y HeLa, las que mostraron una buena correlación lineal ( r = -0,99) y valores bajos de error. El límite inferior de detección a partir del ADN de las líneas celulares fue de hasta 10 copias para ambos genotipos. No se obtuvo reacción cruzada entre los diferentes tipos de PVH ni con otros virus ADN. La reacción en cadena de la polimerasa en tiempo real (RCP-TR) normalizada probó ser un sistema simple, rápido, específico y altamente sensible. Además, permitirá desarrollar investigaciones sobre la prevalencia de infección por PVH en Cuba, con vistas a la aplicación de las vacunas que se encuentran disponibles en el mercado internacional, así como la evaluación de otros candidatos vacunales diseñados en el futuro(AU)

The objective of the present study is to standardize a real-time based polymerase chain reaction system in order to detect and quantify 7 high risk human papillomavirus in different clinical samples from patients suspected of this type of infection. The validation of a 5´ exonuclease fluorescent probe real-time PCR assay (TaqMan format) for the detection and quantification of the 7 most frequent HR-HPV types (16, 18, 31, 33, 45, and 58) which account for over 87% of cervical carcinomas world-wide was carried out. Simultaneous PCR reactions are required to detect the designated HPV types. Specificity tests for each HPV type and other DNA viruses were performed. Standard external curve constructions were achieved, which allow determining the number of target DNA copies in the previously HPV tested samples. HPV 16 and 18 standard curves were obtained from purified genomic DNA of SiHa and HeLa cell lines, respectively. The pattern curves were constructed on the basis of each of the resulting standard DNA, which showed good linear correlation (r = -0, 99) and low error values. The lower detection limit was 10 copies for both HPV 16 and 18. No cross reactions between HPV types and other DNA viruses were observed. Real-Time Polymerase Chain Reaction system, standardized for 7 HPV types, proved to be a rapid, specific and highly sensitive system for better diagnosis and follow-up of patients with high grade intraepithelial lesions. In addition, this assay will allow the development of coming researches in relation with the prevalence and pathogenesis of human papillomavirus infections in different samples from Cuban patients(AU)

Isolation of Coxsackievirus A24 variant from patients with hemorrhagic conjunctivitis in Cuba, 2008-2009.

BACKGROUND: An outbreak of acute hemorrhagic conjunctivitis occurred in Cuba in 2008 and 2009. OBJECTIVE: To determinate the etiological agent associated with the Cuban outbreaks of acute hemorrhagic conjunctivitis during 2008 and 2009. STUDY DESIGN: Conjunctival swabs and/or faecal samples from 382 patients with clinical diagnosis suggestive of acute hemorrhagic conjunctivitis were subject to viral culture in HEp-2 human laryngeal epidermoid carcinoma cells. Positive samples were identified by a specific Coxsackievirus A24 variant PCR and the 3C protease region of 16 isolates was sequenced for phylogenetic analysis. RESULTS: Enterovirus cytopathic effect was observed in 138 cases (36%). A higher percent of CA24v was recovered from faecal samples, 19 out of 45 cases (42.2%), than from conjunctival swabs, 127 out of 355 samples (35.8%). All isolates were identified as Coxsackievirus A24 variant. Phylogenetic analysis revealed that 2008 and 2009 Cuban outbreaks were caused by the same virus strains and that isolates were closely related to those from Taiwan (2006-2007), China (2007-2008) and Singapore (2005) with a bootstrap value of 71%. CONCLUSIONS: Outbreaks of acute hemorrhagic conjunctivitis occurred in Cuba in 2008 and 2009 were caused by Coxsackievirus A24 variant. The faecal-oral route is another mode of transmission of CA24v in the acute hemorrhagic conjunctivitis outbreaks. Phylogenetic analysis of Cuban CA24v strains involved in an acute hemorrhagic conjunctivitis outbreak in 2008 and 2009 confirms a new introduction of the CA24 variant into the Americas from South-east Asia.

A myocarditis outbreak with fatal cases associated with adenovirus subgenera C among children from Havana City in 2005.

BACKGROUND: Among multiple causes of acute myocarditis, viral infection, especially that due to enteroviruses and adenoviruses, is the leading cause. In the summer 2005 an outbreak of a febrile syndrome accompanied by acute cardiac decompensation occurred in infants and young children in Havana City. Eleven patients had a rapid evolution of disease and there were 8 fatalities from cardiac failure secondary to myocarditis. OBJECTIVE: The aim of the present study was to determine the etiological agent responsible for this outbreak. STUDY DESIGN: Children admitted to the pediatric hospitals of Havana City from July 3 to August 2 with this clinical presentation were studied. Forty samples of necropsy tissue, cerebrospinal fluid, stools and serum were tested by molecular methods for 14 respiratory viruses, 6 herpesviruses and generic enteroviruses and flavirus and alfaviruses. Viral isolation was performed in A-549 cells. Isolated viruses were typed by sequence analysis. RESULTS: Adenovirus genome was detected in 6 of the 8 fatal cases-the lungs in 5 (63%) and the myocardium in 3 (37%). In two fatal cases, viral genome was detected in both lung and myocardium. Adenovirus was isolated in five fatal cases. In all three non-fatal cases, adenovirus genome was detected and adenovirus was isolated into two. Sequence analysis showed that adenovirus type 5 was the only isolate from fatal cases and adenovirus 1 the only isolate in non-fatal cases. No other viruses were found by PCR or isolation techniques. CONCLUSION: Adenovirus was the etiologic agent implicated in this myocarditis outbreak and adenovirus type 5 was associated with fatal outcome.

Amplificación dependiente de anticuerpos del virus dengue en cepas cubanas utilizando anticuerpos monoclonales / Dengue virus antibodies dependent amplification in Cuban strains by using monoclonal antibodies

Se describieron los diferentes comportamientos de anticuerpos monoclonales anti-dengue serotipo 2 (H3/6, 4G3) y anti-proteína recombinante de la envoltura del virus dengue cuando fueron enfrentados a diferentes cepas de los serotipos 2 y 4 de este virus (D2 Nueva Guinea C, A15 cepa cubana y A15 propagada 53 veces en cultivo de riñón de perro Beagly y D4 H-2412 cepa prototipo) en un ensayo de inmunoamplificación dependiente de anticuerpos. Los anticuerpos monoclonales han demostrado ser una herramienta eficaz para explicar que la neutralización y el incremento de la multiplicidad viral pueden realizarse como funciones biológicas separadas. Solamente el AcM H3/6 fue capaz de producir el fenómeno amplificación dependiente de anticuerpos frente a la cepa A15 con un incremento significativo en la multiplicación viral. Los AcMs 4G3 y 4B6 no fueron capaces de inmunoamplificar la multiplicación viral de las cepas estudiadas(AU)

Amplificación dependiente de anticuerpos del virus dengue en cepas cubanas utilizando anticuerpos monoclonales / Dengue virus antibodies dependent amplification in Cuban strains by using monoclonal antibodies

Se describieron los diferentes comportamientos de anticuerpos monoclonales anti-dengue serotipo 2 (H3/6, 4G3) y anti-proteína recombinante de la envoltura del virus dengue cuando fueron enfrentados a diferentes cepas de los serotipos 2 y 4 de este virus (D2 Nueva Guinea C, A15 cepa cubana y A15 propagada 53 veces en cultivo de riñón de perro Beagly y D4 H-2412 cepa prototipo) en un ensayo de inmunoamplificación dependiente de anticuerpos. Los anticuerpos monoclonales han demostrado ser una herramienta eficaz para explicar que la neutralización y el incremento de la multiplicidad viral pueden realizarse como funciones biológicas separadas. Solamente el AcM H3/6 fue capaz de producir el fenómeno amplificación dependiente de anticuerpos frente a la cepa A15 con un incremento significativo en la multiplicación viral. Los AcMs 4G3 y 4B6 no fueron capaces de inmunoamplificar la multiplicación viral de las cepas estudiadas.

The different behaviors of antidengue monoclonal antibodies serotype 2 (H3/6, 4G3) and antiprotein recombinant from the dengue virus sheath were described when they faced diverse strains from the serotypes 2 and 4 of this virus (D2 New Guinea C, A15 Cuban strain and A15 propagated 53 times in culture of Beagley dog kidney and D4 H-22412 prototype strain) in an immunoamplification assay dependent of antibodies. Themonoclonal antibodies have prove to be an efficient tool to explain that the neutralizatrion and increase of viral multiplicity may be carried out as separate biological functions. Only the H3/6 monoclonal antibdoy was capable of producing the amplification assay dependent phenomenon against the A15 strain with a significant rise in the viral multiplication. The 4G3 and 4B6 monoclonal antibodies were not capable of immunoamplifying the viral multiplication of the studied strains.

Evaluación de un conjugado anti-IgG de ratón-fluoresceína mediante técnicas de inmunofluorescencia indirecta y citometría de flujo / Evaluation of an anti-IgG conjugate of mouse-fluorescin by the indirect immunofluorescent techniques and flow citomerty

Se purificó una inmunoglobulina G de ratón a partir de suero por cromatografía de afinidad en proteína A. Con esta preparación se inmunizaron los conejos cuyos sueros fueron capaces de reconocer al antígeno inyectado mediante inmunodifusión doble. Los anticuerpos fueron precipitados del suero de conejo y purificados mediante cromatografía de intercambio iónico. Esta preparación fue conjugada a isotiocianato de fluorescencia según la tecnología convencional. El conjugado obtenido fue evaluado con las cepas de referencia de virus Parainfluenza 1, 2, 3; Adenovirus; virus sincitial respiratorio y virus influenza A y B por una técnica de inmunofluorescencia indirecta y muestras positivas de VIH mediante citometría de flujo. En ambos casos se utilizaron anticuerpos monoclonales específicos. Se evaluaron muestras clínicas de pacientes con infección respiratoria aguda

Evaluación de un conjugado anti-IgG de ratón-fluoresceína mediante técnicas de inmunofluorescencia indirecta y citometría de flujo / Evaluation of an anti-IgG conjugate of mouse-fluorescin by the indirect immunofluorescent techniques and flow citomerty

Se purificó una inmunoglobulina G de ratón a partir de suero por cromatografía de afinidad en proteína A. Con esta preparación se inmunizaron los conejos cuyos sueros fueron capaces de reconocer al antígeno inyectado mediante inmunodifusión doble. Los anticuerpos fueron precipitados del suero de conejo y purificados mediante cromatografía de intercambio iónico. Esta preparación fue conjugada a isotiocianato de fluorescencia según la tecnología convencional. El conjugado obtenido fue evaluado con las cepas de referencia de virus Parainfluenza 1, 2, 3; Adenovirus; virus sincitial respiratorio y virus influenza A y B por una técnica de inmunofluorescencia indirecta y muestras positivas de VIH mediante citometría de flujo. En ambos casos se utilizaron anticuerpos monoclonales específicos. Se evaluaron muestras clínicas de pacientes con infección respiratoria aguda(AU)