Venom of the Phoneutria nigriventer spider alters the cell cycle, viability, and migration of cancer cells

The mechanisms of cancer involve changes in multiple biological pathways. Multitarget molecules, which are components of animal venoms, are therefore a potential strategy for treating tumors. The objective of this study was to screen the effects of Phoneutria nigriventer spider venom (PnV) on tumor cell lines. Cultured human glioma (NG97), glioblastoma (U-251) and cervix adenocarcinoma (HeLa) cells, and nontumor mouse fibroblasts (L929) were treated with low (14?µg/ml) and high (280?µg/ml) concentrations of PnV, and analyzed through assays for cell viability (thiazolyl blue tetrazolium blue), proliferation (carboxyfluorescein succinimidyl ester), death (annexin V/propidium iodide [Pi]), the cell cycle (Pi), and migration (wound healing and transwell assay). The venom decreased the viability of U-251 cells, primarily by inducing cell death, and reduced the viability of NG97 cells, primarily by inhibiting the cell cycle. The migration of all the tumor cell lines was delayed when treated with venom. The venom significantly affected all the tumor cell lines studied, with no cytotoxic effect on normal cells (L929), although the nonglial tumor cell (HeLa) was less sensitive to PnV. The results of the current study suggest that PnV may be composed of peptides that are highly specific for the multiple targets involved in the hallmarks of cancer. Experiments are underway to identify these molecules.

Venom of the Phoneutria nigriventer spider alters the cell cycle, viability, and migration of cancer cells

The mechanisms of cancer involve changes in multiple biological pathways. Multitarget molecules, which are components of animal venoms, are therefore a potential strategy for treating tumors. The objective of this study was to screen the effects of Phoneutria nigriventer spider venom (PnV) on tumor cell lines. Cultured human glioma (NG97), glioblastoma (U-251) and cervix adenocarcinoma (HeLa) cells, and nontumor mouse fibroblasts (L929) were treated with low (14?µg/ml) and high (280?µg/ml) concentrations of PnV, and analyzed through assays for cell viability (thiazolyl blue tetrazolium blue), proliferation (carboxyfluorescein succinimidyl ester), death (annexin V/propidium iodide [Pi]), the cell cycle (Pi), and migration (wound healing and transwell assay). The venom decreased the viability of U-251 cells, primarily by inducing cell death, and reduced the viability of NG97 cells, primarily by inhibiting the cell cycle. The migration of all the tumor cell lines was delayed when treated with venom. The venom significantly affected all the tumor cell lines studied, with no cytotoxic effect on normal cells (L929), although the nonglial tumor cell (HeLa) was less sensitive to PnV. The results of the current study suggest that PnV may be composed of peptides that are highly specific for the multiple targets involved in the hallmarks of cancer. Experiments are underway to identify these molecules.

Thymic atrophy in infectious diseases

The thymus is a primary lymphoid organ that plays an important role in the development of the immune system and in the differentiation and maturation of the vast majority of the T cell repertoire. During the normal life span, this organ undergoes involution in situations such as pregnancy, aging and in the presence of a wide variety of infectious diseases. This atrophy is characterized by intense structural and morphological alterations associated with an increased level of apoptosis, for which many direct and indirect mechanisms have been proposed. The fact that the thymus is a target for infectious diseases could make the host enviroment propitious for the maintenance or enhancement of infection.

Thymic atrophy and fungal virulence during experimental paracoccidioidomycosis

A imunossupressão observada em micoses sistêmicas pode estar relacionada a danos nos órgãos linfóides primários. Assim, nosso laboratório tem estudado uma possível ação do Paracoccidioides brasiliensis sobre o timo de camundongos. Neste estudo, analisamos o timo de camundongos susceptíveis e resistentes ao fungo utilizando cepas de alta e baixa virulência. Todos os grupos apresentaram atrofia tímica, perda de delimitação corticomedular e aumento da taxa de apoptose. Entretanto, as alterações foram mais precoces e pronunciadas em camundongos inoculados com a cepa virulenta do fungo, sugerindo que a virulência fúngica pode estar diretamente ligada à atrofia tímica e indução de imunossupressão.

Murine virus contaminant of Trypanosoma cruzi experimental infection

A possibilidade de que alguma contaminacao por virus poderia estar alterando a resposta do hospedeiro a infeccao experimental por Trypasoma cruzi foi investigada. Os dados obtidos mostraram que camundongos CBA/J infectados com estoques de parasitos mantidos em camundongos (Y IUEC) apresentavam maior parasitemia e menor periodo de sobrevivencia do que os infectados com um estoque(Y ITC) que tambem foi mantido em animal mas tinha sido previamente passado em culturas de celulas. Testes de producao de anticorpos em camundongos (Map tests) realizados com o plasma de animais infectados com Y IUEC indicaram a presenca do virus da hepatite do camundongo (MHV) enquanto nenhum virus foi detectado no plasma de animais infectados com Y ITC. O plasma filtrado obtido de camundongos infectados com Y IUEC continha um fator capaz de aumentar o nivel de parasitemia e reduzir o tempo medio de sobrevivencia dos camundongos desafiados com 10 elevado a quinta potencia Y ITC. Testes de neutralizacao mostraram que este fator que podia ser passado seriadamente para camundongos era um coronavirus.

Thymic atrophy and fungal virulence during experimental paracoccidioidomycosis

The immunosuppression observed in systemic mycosis can be related to primary lymphoid organs damage. Thus, our laboratory has studied the effects of the Paracoccidioides brasiliensis infection on the thymus of mice. Here, thymuses of susceptible and resistant mice were evaluated after inoculation with highly and slightly virulent isolates of the fungus. All groups presented thymic atrophy, loss of corticomedullary delimitation and increase of apoptotic index. However, mice inoculated with high virulent strain showed earlier and stronger alterations suggesting that thymic atrophy can be directly related to the fungal virulence and to the immunosuppression.

A imunossupressão observada em micoses sistêmicas pode estar relacionada a danos nos órgãos linfóides primários. Assim, nosso laboratório tem estudado uma possível ação do Paracoccidioides brasiliensis sobre o timo de camundongos. Neste estudo, analisamos o timo de camundongos susceptíveis e resistentes ao fungo utilizando cepas de alta e baixa virulência. Todos os grupos apresentaram atrofia tímica, perda de delimitação corticomedular e aumento da taxa de apoptose. Entretanto, as alterações foram mais precoces e pronunciadas em camundongos inoculados com a cepa virulenta do fungo, sugerindo que a virulência fúngica pode estar diretamente ligada à atrofia tímica e indução de imunossupressão.

Thymic atrophy and fungal virulence during experimental paracoccidioidomycosis

The immunosuppression observed in systemic mycosis can be related to primary lymphoid organs damage. Thus, our laboratory has studied the effects of the Paracoccidioides brasiliensis infection on the thymus of mice. Here, thymuses of susceptible and resistant mice were evaluated after inoculation with highly and slightly virulent isolates of the fungus. All groups presented thymic atrophy, loss of corticomedullary delimitation and increase of apoptotic index. However, mice inoculated with high virulent strain showed earlier and stronger alterations suggesting that thymic atrophy can be directly related to the fungal virulence and to the immunosuppression.

A imunossupressão observada em micoses sistêmicas pode estar relacionada a danos nos órgãos linfóides primários. Assim, nosso laboratório tem estudado uma possível ação do Paracoccidioides brasiliensis sobre o timo de camundongos. Neste estudo, analisamos o timo de camundongos susceptíveis e resistentes ao fungo utilizando cepas de alta e baixa virulência. Todos os grupos apresentaram atrofia tímica, perda de delimitação corticomedular e aumento da taxa de apoptose. Entretanto, as alterações foram mais precoces e pronunciadas em camundongos inoculados com a cepa virulenta do fungo, sugerindo que a virulência fúngica pode estar diretamente ligada à atrofia tímica e indução de imunossupressão.

Early histopathological changes in bone marrow of Paracoccidioides brasiliensis-infected mice

The involvement of bone marrow in the pathology of experimental P. brasiliensis infection in BALB/c mice was investigated. The histopathological features of bone marrow induced by the fungus were correlated with hematological changes in peripheral blood from 1 to 28 days post-infection. Intense lymphopenia and moderate neutrophilia were detected. The early changes in bone marrow included (i) maturation arrest characterized by an increase immature blood cell precursors, mainly of granulocytic origin, (ii) intense vascular congestion when compared with the vessels of normal marrow, and (iii) an increased number of megakaryocytes. The normal histological pattern of bone marrow was restored by 28 days post-infection. No histologically recognizable lesion, such as granuloma formation or an abnormal cellular infiltrate, which could indicate the presence of the P. brasiliensis in bone marrow, was observed. In addition, special stains were unable to detect the fungus. The mechanisms responsible for the alterations described here are still unclear but are probably related to more general phenomena affecting the host rather than the direct damage of the precursors cells by P. brasiliensis.

Imunoterapia ativa especifica no tratamento do carcinoma renal estadio IV: reaçoes linfoproliferativa determinadas por um antigeno purificado da vacina / Specific active immunotherapy foe renal carcinoma stage IV: lymphooproliferitive reactions determined from a purified antigen

Proteinas associadas ao tecido tumoral (PATT) foram extraidas e utilizadas tanto para a preparaçao de uma vacina como para a comparaçao, quanto ao seu perfil imunoquimico com proteinas associadas ao tecido renal normal (PATN). A analise eletroforetica em gel de poliacrilamida na presença de SDS demonstrou a existencia de um componente na fraçao PATT praticamente ausente em PATN. A cromatografia de filtraçao em Sephadex G-100 da fraçao PATT evidenciou seis fraçoes (F1 a F6). A analise eletroforetica das fraçoes obtidas em gel de poliacrimida na presença de SDS, evidenciou que a fraçao F2 encontrada em PATT estava em PATN, sugerindo que a imunogenicidade da vacina em estudo poderia ser atribuida ao antigeno F2. A reaçao linfoproliferativa de celulas do paciente imunizado com a vacina bruta frente a F2 foi cinco vezes maior do que em celulas de individuos normais. A determinaçao de TNF (Fator de Necrose Tumoral) foi realizada no paciente, antes e depois do inicio da imunoterapia, e mostrou a queda desta citocina em torno de 47//apos seis meses de tratamento com a vacina.

Effect of Helium-Neon (HeNe) laser irradiacation on lesions in experimental paracoccidioidomycosis

Cutaneous lesions caused by the fungus Paracoccidioides brasiliensis lead to ulcerative wounds, which are difficult to be cured by conventional treatment with anti-fungal drugs due to their adverse effects and the multidrug resistance that some fungal isolates present. So, the laser therapy emerges as an alternative treatment since its anti-inflammatory effects and wound healing properties are already known. In this work, we showed that lesions caused by the inoculation of yeast cells into the back foot-pad of BALB/c mice and treated with HeNe laser present greater histologic organization, milder inflammatory infiltrate, higher organization of the granulation tissue around the lesion, and enhanced immunolabeling for iNOS. In addition, an increased SOD activity and NO concentration, higher percentage of macrophages, and lower neutrophil numbers were observed. The percentage of NK and NKT cells were also slightly higher after the first laser session. Altogether these results point towards a dual effect of the laser treatment, decreasing the inflammatory response and accelerating the wound healing of the lesions. In this sense, the HeNe laser can be considered as an effective adjunctive treatment to be combined with pharmacologic therapies for Effect of Helium-Neon (HeNe) laser irradiation on lesions in experimental paracoccidioidomycosis improving the treatment of painful non-healing paracoccidioidomycotic wounds.

Effects of HeNe laser irradiation on experimental paracoccidioidomycotic lesions

Paracoccidioidomycosis (PCM) is the most prevalent human mycosis in Latin America. The infection is thought to take place firstly in the lungs and then may disseminate to other organs and tissues. Treatment by currently available antifungals is lengthy, the drugs may have undesirable side effects, and some are costly. Occasional resistant strains of Paracoccidioides brasiliensis, the causative agent of PCM, have been reported. So, the search for more efficient treatments or adjuvant therapies has to be continued. In this work, we evaluated the effects of HeNe laser irradiation on cutaneous inflammatory lesions caused by the inoculation of 5 x 10(6)/0.1ml yeasts cells into the back footpad of Balb/c mice. HeNe irradiation (lambda=632.8nm, 3mW, incident energy of 3J/cm(2)) was applied at days 7, 8 and 9 post-infection and histological and immunohistochemical analysis were done. Unirradiated animals were used as controls. The results showed that laser-treated mice presented reduction of footpad edema, faster cutaneous wound healing, confluent granuloma, diffuse- and more loosely distributed immunolabeling for TNF-alpha, enhanced labeling of IFN-gamma and any P. brasiliensis form detected, whereas multiple viable fungi were seen in diffuse widespread granulomas obtained from non-treated mice foot-pad. Fungi that were harvested from laser-treated animals presented no capability of growth in vitro as compared to those obtained from non-treated mice. We conclude that HeNe laser irradiation was able to inhibit the progress of inflammatory local reaction produced by P. brasiliensis infection and influence local cytokines production. We suggest that this treatment modality can be a useful coadjuvant tool to be combined with antifungal agents in the treatment of PCM ulcerations. The mechanisms involved in laser therapy of PCM lesions need further investigation.

Thymic atrophy in infectious diseases

The thymus is a primary lymphoid organ that plays an important role in the development of the immune system and in the differentiation and maturation of the vast majority of the T cell repertoire. During the normal life span, this organ undergoes involution in situations such as pregnancy, aging and in the presence of a wide variety of infectious diseases. This atrophy is characterized by intense structural and morphological alterations associated with an increased level of apoptosis, for which many direct and indirect mechanisms have been proposed. The fact that the thymus is a target for infectious diseases could make the host environment propitious for the maintenance or enhancement of infection.

Programmed cell death in thymus during experimental paracoccidioidomycosis

Many works have shown that immunosuppressive effects induced by systemic mycosis can be related to primary lymphoid organ damage. Previous studies in our laboratory showed that Paracoccidioides brasiliensis was able to invade the thymus, inducing a severe atrophy with significant reduction of cortical area along with a loss of cortico-medullary boundary. The objective of the present study was to investigate whether thymic atrophy is caused by programmed cell death (PCD) and to examine the ultrastructural characteristics of the thymus in experimentally infected BALB/c mice. The results revealed an eightfold increase in the apoptotic index occurring by day 5 post infection, i.e., during early stages of the infection, shown by immunohistochemistry. In addition, typical cell alterations of autophagic PCD were observed by transmission electron microscopy. Taken together, these results reinforce the idea that thymic alterations may be involved in the immunosuppressive phenomenon frequently associated with paracoccidioidomycotic infection.

Thymus invasion and atrophy induced by Paracoccidioides brasiliensis in BALB/c mice

Literature has shown that immunosuppression observed in systemic mycosis can be related to damage in primary lymphoid organs. We have studied the immunopathological alterations induced experimentally by Paracoccidioides brasiliensis in these organs. In this work, thymic alterations induced in BALB/c mice during acute and chronic stages of infection are described. It was observed that P. brasiliensis is able to invade the thymic microenvironment, inducing severe atrophy characterized by degeneration of the cortical area, organ weight decrease, loss of corticomedullary delimitation and increase in histiocyte number. Occurrence of polymorphonuclear infiltration in the subcapsular area was also observed. Our results demonstrate that P. brasiliensis induces profound thymic atrophy and raises the question of whether this could be a fungal strategy to achieve successful establishment in the host over the long term.