Bothrops moojeni L-amino acid oxidase induces apoptosis and epigenetic modulation on Bcr-Abl+ cells

Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl+ leukemic cells and improve the disease treatment. Methods: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl+ cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro. Results: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl+ cells. Conclusion: BmooLAAO-I exerts selective antitumor action mediated by H2O2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy.(AU)

BthTX-I from Bothrops jararacussu induces apoptosis in human breast cancer cell lines and decreases cancer stem cell subpopulation

Breast cancer is the neoplasm with both the highest incidence and mortality rate among women worldwide. Given the known snake venom cytotoxicity towards several tumor types, we evaluated the effects of BthTX-I from Bothrops jararacussu on MCF7, SKBR3, and MDAMB231 breast cancer cell lines. Methods: BthTX-I cytotoxicity was determined via MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay. Cell death was measured by a hypotonic fluorescent solution method, annexin-V-FITC/propidium iodide staining and by apoptotic/autophagic protein expression. Cancer stem cells (CSCs) were quantified by flow cytometry using anti-CD24-FITC and anti-CD44-APC antibodies and propidium iodide. Results: BthTX-I at 102 µg/mL induced cell death in all cell lines. The toxin induced apoptosis in MCF7, SKBR3, and MDAMB231 in a dose-dependent manner, as confirmed by the increasing number of hypodiploid nuclei. Expression of pro-caspase 3, pro-caspase 8 and Beclin-1 proteins were increased, while the level of the antiapoptotic protein Bcl-2 was diminished in MCF7 cells. BthTX-I changed the staining pattern of CSCs in MDAMB231 cells by increasing expression of CD24 receptors, which mediated cell death. Conclusions: BthTX-I induces apoptosis and autophagy in all breast cancer cell lines tested and also reduces CSCs subpopulation, which makes it a promising therapeutic alternative for breast cancer.(AU)

Philadelphia-negative myeloproliferative neoplasms as disorders marked by cytokine modulation

ABSTRACT Background: Cytokines are key immune mediators in physiological and disease processes, whose increased levels have been associated with the physiopathology of hematopoietic malignancies, such as myeloproliferative neoplasms. Methods: This study examined the plasma cytokine profiles of patients with essential thrombocythemia, primary myelofibrosis, polycythemia vera and of healthy subjects, and analyzed correlations with JAK2 V617F status and clinical-hematological parameters. Results: The proinflammatory cytokine levels were increased in myeloproliferative neoplasm patients, and the presence of the JAK2 V617F mutation was associated with high IP-10 levels in primary myelofibrosis patients. Conclusions: Essential thrombocythemia, primary myelofibrosis, and polycythemia vera patients exhibited different patterns of cytokine production, as revealed by cytokine network correlations. Together, these findings suggest that augmented cytokine levels are associated with the physiopathology of myeloproliferative neoplasms.

Cytotoxic and pro-apoptotic action of MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, towards leukemic cells

A leucemia mieloide crônica (LMC) é uma neoplasia mieloproliferativa BCR-ABL1 + marcada por aumento da mieloproliferação e presença de células leucêmicas resistentes à apoptose. A terapia de primeira linha atual para a LMC é a administração de inibidores da tirosina quinase, mesilato de imatinibe, dasatinibe ou nilotinibe. Embora eficaz no tratamento da LMC, alguns pacientes se tornaram resistentes a essa terapia, levando à progressão da doença e à morte. Assim, a descoberta de novos compostos para melhorar a terapia da LMC ainda é um desafio. Aqui, os destinatários se MjTX-I, uma fosfolipase A 2 isolado a partir de Bothrops moojeni de veneno de cobra, afecta a viabilidade de Bcr-Abl de mesilato de imatinib-resistente + linhas celulares. Métodos: Examinamos o efeito citotóxico e pró-apoptótico de MjTX-I em células K562-S e K562-R Bcr-Abl + e na linha de células HEK-293 não tumorais e células mononucleares de sangue periférico, usando o 3- (4, Brometo de 5-dimetiltiazol-2-il) -2,5-difeniltetrazólio e os métodos de solução fluorescente hipotônica, associados à detecção de ativação de caspases 3, 8 e 9 e clivagem de poli (ADP-ribose) polimerase (PARP). Também analisamos o potencial MjTX-I para modular a expressão de genes relacionados à apoptose em células K562-S e K562-R. Resultados: O MjTX-I diminuiu a viabilidade das células K562-S e K562-R em 60 a 65%, sem afetar a viabilidade das células não tumorais, ou seja, exerceu citotoxicidade seletiva para as linhagens celulares Bcr-Abl + . Em linhas de células leucêmicas, a toxina induziu apoptose, caspases 3, 8 e 9 ativadas, PARP clivada, expressão negativa do gene anti-apoptótico BCL-2 e expressão aumentada do gene pró-apoptótico BAD. Conclusão: O efeito antitumoral de MjTX-I está associado ao seu potencial para induzir apoptose e citotoxicidade em linhagens celulares positivas para Bcr-Abl sensíveis e resistentes ao mesilato de imatinibe, indicando que MjTX-I é um candidato promissor a fármaco para atualizar a terapia de LMC.(AU)

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways

Background Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked byBothrops snakes. The present study aimed to assess whether Bothrops jararacussuand Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways.Methods Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t ½values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model.Results At the highest concentration tested (120 μg/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 μg/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 μg/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum.Conclusion Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system byB. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.(AU)

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways

Background Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked byBothrops snakes. The present study aimed to assess whether Bothrops jararacussuand Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways.Methods Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t ½values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model.Results At the highest concentration tested (120 g/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 g/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 g/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum.Conclusion Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system byB. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.

Desregulação da apoptose em neoplasias mieloproliferativas crônicas / Apoptosis deregulation in myeloproliferative neoplasms

As neoplasias mieloproliferativas crônicas cromossomo Filadélfia negativas são doenças hematológicas clonais que se caracterizam pela independência ou pela hipersensibilidade dos progenitores hematopoiéticos às citocinas. Os mecanismos celulares e moleculares envolvidos na fisiopatologia das neoplasias mieloproliferativas crônicas ainda não estão totalmente esclarecidos. Achados fisiopatológicos relevantes para as neoplasias mieloproliferativas crônicas estão associados às alterações genéticas como, por exemplo, a mutação somática no gene que codifica o JAK2 (JAK2V617F). A desregulação do processo de morte celular programada, denominada apoptose, parece participar da patogênese dessas desordens. Sabe-se que a desregulação da expressão dos genes pró- e antiapoptóticos promove a resistência das células à apoptose, culminando com o acúmulo das células mieloides e estabelecendo a neoplasia. Esta revisão enfocou as alterações na regulação da apoptose em neoplasias mieloproliferativas crônicas e a importância da melhor compreensão desse mecanismo para o desenvolvimento de novas terapias para essas doenças.

Philadelphia-chromosome negative chronic myeloproliferative neoplasms are clonal hematologic diseases characterized by hematopoietic progenitor independence from or hypersensitivity to cytokines. The cellular and molecular mechanisms involved in the pathophysiology of myeloproliferative neoplasms have not yet been fully clarified. Pathophysiologic findings relevant for myeloproliferative neoplasms are associated with genetic alterations, such as, somatic mutation in the gene that codifies JAK-2 (JAK V617F). Deregulation of the process of programmed cellular death, called apoptosis, seems to participate in the pathogenesis of these disorders. It is known that expression deregulation of pro- and anti-apoptotic genes promotes cell resistance to apoptosis, culminating with the accumulation of myeloid cells and establishing neoplasms. This review will focus on the alterations in apoptosis regulation in myeloproliferative neoplasms, and the importance of a better understanding of this mechanism for the development of new therapies for these diseases.

Upregulation of hsa-miR-125b in HTLV-1 asymptomatic carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis patients

The retrovirus human T lymphotropic virus type 1 (HTLV-1) promotes spastic paraparesis, adult T cell leukaemia and other diseases. Recently, some human microRNAs (miRNAs) have been described as important factors in host-virus interactions. This study compared miRNA expression in control individuals, asymptomatic HTLV-1 carriers and HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis patients. The proviral load and Tax protein expression were measured in order to characterize the patients. hsa-miR-125b expression was significantly higher in patients than in controls (p = 0.0285) or in the HAM group (p = 0.0312). Therefore, our findings suggest that miR-125b expression can be used to elucidate the mechanisms of viral replication and pathogenic processes.

Deregulated expression of A1, Bcl-2, Bcl-xL, and Mcl-1 antiapoptotic proteins and Bid, Bad, and Bax proapoptotic genes in polycythemia vera patients

Apoptosis deregulation might have a role in the pathophysiology of polycythemia vera (PV). This study evaluated Bcl-2 molecule expression in CD34+ cells and leukocytes in 12 PV patients. Gene expression was investigated by real time PCR using SybrGreen Quantitect kit and protein expression was evaluated by western-blotting. JAK2 V617F mutation was detected according to Baxter et al (2005). CD34+ cells from PV patients presented higher levels of A1 and Mcl-1 expression (median: 22.6 and 5.2, respectively) in comparison with controls (0.9 and 0.5, p=0.004 and p=0.020); while Bcl-2 and Bcl-xL expression decreased in PV patients (0.18 and 1.19) compared with controls (1.39 and 2.01, p=0.006 and p=0.020). CD34+ cells in PV patients showed an elevated Bid expression (14.4) in comparison with healthy subjects (1.0; p=0.002). Patients' leukocytes showed an A1 augmentation (7.41, p=0.001) and a reduced expression of Bax (0.19; p=0.040) and Bad (0.2; p=0.030). There was no correlation between JAK2 V617F allele burden and molecular expression. PV patients showed alterations in Bcl-2 members' expression, which may interfere with control of apoptotic machinery and contribute to disease pathogenesis.

A desregulação da apoptose parece participar da fisiopatologia da policitemia vera (PV). Este estudo avaliou a expressão das moléculas da família Bcl-2 em células hematopoéticas CD34 + e leucócitos de 12 pacientes com PV. Foram realizados: a quantificação da expressão gênica por PCR em tempo real utilizando kit Sybrgreen Quantitect, avaliação da expressão de proteínas por western-blot e detecção da mutação JAK2 V617F segundo Baxter et al. (2005). Células CD34 + dos pacientes com PV apresentaram maior expressão de A1 e Mcl-1 (mediana: 22,6 e 5,2, respectivamente) em comparação com controles (0,9 e 0,5, p = 0,004 e p = 0,020) e expressão de Bcl-2 e Bcl-xL diminuída nestes pacientes (0,18 e 1,19) em relação aos controles (1,39 e 2,01, p = 0,006 e p = 0,020). Células CD34 + dos pacientes com PV mostraram expressão elevada de bid (14,4) em comparação aos controles (1,0; p = 0,002). Leucócitos dos pacientes mostraram aumento de A1 (7,41, p = 0,001) e expressão reduzida do Bax (0,19; p = 0,04) e Bad (0,2; p = 0,030). Não houve correlação entre percentagem de alelos JAK2 V617F mutados e expressão molecular. Pacientes com PV apresentaram alterações na expressão de moléculas Bcl-2 que podem interferir no controle da apoptose e contribuir para a patogênese da doença.

Ribotyping of Salmonella Enteritidis strains reveals the spread of a single genotype in the Brazilian city of Ribeirão Preto

INTRODUÇAO: Na identificação de cepas de Salmonella, os métodos de sorotipagem e ribotipagem na detecção de marcadores epidemiológicos são os mais utilizados nos laboratórios de referência mundiais. Esses métodos moleculares são imprescindíveis na vigilância epidemiológica e permitem a detecção da fonte da infecção. OBJETIVOS: O presente trabalho objetivou caracterizar as cepas de Salmonella Enteritidis pela ribotipagem. MATERIAL E MÉTODOS: Trinta e oito cepas de S. Enteritidis foram isoladas de pacientes atendidos no Hospital das Clínicas da Universidade de São Paulo, Ribeirão Preto, SP, entre os anos de 1996 e 1998. As cepas foram isoladas de fezes (31 amostras), sangue (quatro amostras) e outros fluidos (três amostras). As cepas de Salmonella foram isoladas utilizando-se métodos bacteriológicos de rotina, sorotipadas e ribotipadas. RESULTADOS: As 38 cepas de S. Enteritidis apresentaram na ribotipagem a separação das cepas em dois ribotipos: A (94,7 por cento das amostras) e B (5,3 por cento das amostras). DISCUSSAO E CONCLUSAO: Esses dados sugerem que grande parte dos pacientes (94,7 por cento) foi infectada pela mesma cepa. Essa cepa pode ser endêmica na comunidade de Ribeirão Preto ou os pacientes foram expostos a uma fonte comum de infecção.

Efeito da terapia de reposição hormonal sobre o estado férrico / Effect of hormone replacement therapy in iron status

Introdução: Na literatura, numerosas publicações relatam a determinação do estado férrico em crianças, adolescentes e mulheres em fase reprodutiva, no entanto são raras as pesquisas quanto às alterações do ferro em estoque e eritrograma pós-terapia de reposição hormonal (TRH) em pré-menopausadas e menopausadas. O aumento dos estoques de ferro em mulheres pré-menopausadas e menopausadas pode conduzir à elevação do estresse oxidativo e, conseqüentemente, ao risco de desenvolvimento de câncer e doenças cardiovasculares. Objetivo: Avaliar o efeito da TRH sobre o eritrograma e o estado férrico em mulheres na pré-menopausa e na menopausa. Métodos: Foram determinados os eritrogramas e as dosagens de ferro, capacidade total de ligação do ferro a transferrina (CTLF) e ferritina séricas em 30 mulheres no climatério antes e após seis meses de TRH com medroxiprogesterona e estradiol. Os eritrogramas, as dosagens de ferro e CTLF foram determinados por meio da utilização de métodos clássicos, e a ferritina, por quimiluminescência. Resultados: Após o uso da TRH, constataram-se significante redução do número de eritrócitos, elevação dos índices hematimétricos e tendência à diminuição nos níveis de ferro sérico e CTLF. Nenhuma alteração significante nos níveis de ferritina e no índice de saturação de transferrina foi detectada após a TRH. Discussão e conclusão: No presente estudo não foram encontradas alterações nos paramêtros hematimétricos e no estado férrico que impeçam a utilização da TRH no climatério e na menopausa. Os resultados sugerem que a TRH exerceu efeito benéfico sobre o estado férrico nas mulheres no climatério deste estudo, mantendo os estoques de ferro normais e promovendo a elevação dos índices hematimétricos.

Prevalence and antimicrobial susceptibility of Salmonella serotypes in patients from Ribeiräo Preto, Säo Paulo, Brazil, between 1985 and 1999

Salmonella strains isolated from 1,138 samples representing 28,199 biological materials (stool, urine, blood and other fluids), collected between January 1985 and January 1999 at a reference University Hospital in Ribeiräo Preto, Säo Paulo, Brazil, were studied. The most frequently detected serotypes were Salmonella enterica subspecies enterica serotype4,5,12:i:- (S. I 4,5,12:i:) (21.2 percent), S. agona (15.8 percent) and S. enteritidis (11.3 percent). A changing pattern of Salmonella serotypes was observed between 1985-1999. S. agona, which represented 27 percent of Salmonella serotypes isolated from 1985-1989, declined to 4 percent during the period from 1995 to 1999. S. enteritidis isolation remained below 1 percent until 1989; rose to 5.9 percent between 1990 and 1994, and increased to 32.3 percent between 1995-1999. S. I 4,5,12:i:-; S. Enteritidis; S. Typhimurium; S. dublin and S. infantis, showed low to moderate resistance profiles to most antimicrobial drugs. Nalidixic acid and tetracycline were the most and the least effective drugs, respectively, in the disk diffusion tests. We encountered changes in salmonellosis epidemiology in this geographical region

Frequência de Cryptosporidium sp associada à pacientes com diarréia em Ribeirão Preto - SP / Frequenct of Cryptosporidium sp among patients with diarrhea in Ribeirão Preto - SP

A criptosporidiose é causada por um protozoário que pode produzir enterite em humanos. A doença é diagnosticada principalmente em indivíduos imuno-comprometidos, mas sabe-se que este protozoário acomete pessoas hígidas. Os autores analisaram no período de junho de 1990 a junho de 1997, na Unidade de Emergência do Hospital das Clínicas de Ribeirão Preto – SP. 3.365 amostras de fezes destinadas a pesquisa de Cryptosporidium sp em pacientes com quadro diarréico a esclarecer. A positividade para Cryptosporidium foi de 14,00% em crianças e 24,30% em adultos. Das 409 amostras positivas em crianças, verificou-se associação com outros enteropatógenos 21,50% (85 amostras) dos casos. Essa mesma associação foi observada nas 110 amostras positivas em adultos, cuja porcentagem foi de 14,50% (16 amostras). Esses dados indicam que este protozoário é um dos mais frequentes agentes etiológicos da diarréia em crianças e adultos, com ou sem associação de outros enteropatógenos.