Efeito da melanina e do oxigenio singlete na morte celular e fluxo de cálcio em células Melan-A e B16-F10

Orientadora : Prof. Glaucia Regina MartinezDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba, 12/02/2010Bibliografia: 65-74Área de concentração: BioquímicaResumoResumo: O melanoma e um tipo de cancer bastante relevante ja que as opcoes de tratamento eficazes sao limitadas. A presenca da melanina protege os individuos de pele escura contra os efeitos da radiacao solar, a principal causa de formacao do melanoma pela geracao de especies reativas de oxigenio (ROS). Porem, a melanina tambem pode ter um papel duplo, que e a de gerar especies reativas durante sua sintese que podem prejudicar a celula. Portanto, o objetivo deste trabalho foi a avaliacao das caracteristicas de morte celular causadas por uma ROS, o oxigenio singlete (1O2), nas celulas de melanoma murino B16-F10 com e sem estimulo para producao de melanina e nas celulas de melanocito murino Melan-a. O estimulo para a sintese de melanina foi obtido incubando-se as celulas por 48 horas com meio RPMI 1640 enriquecido com 400 ƒÊmol/L de L-tirosina e 10 mmol/L de cloreto de amonio. A concentracao de melanina aumentou em mais de nove vezes nas celulas B16-F10 e as celulas Melan-a tiveram aumento de menos de duas vezes. Foi utilizado o endoperoxido DHPNO2 10 mmol/L por 2 horas para a geracao de 1O2. Essa condicao causou queda na viabilidade avaliada pelo metodo do MTT para 78,0% nas celulas B16-F10, 70,2% nas B16-F10 estimuladas (B16-F10 Y) e 79,3% nas Melan-a. O ensaio foi feito apos 24 horas do inicio do tratamento com DHPNO2 e a viabilidade caiu para 49,7% nas B16-F10, 53,3% nas B16- F10 Y e 72,5% nas Melan-a. A avaliacao da morte celular utilizando laranja de acridina e brometo de etidio mostrou que apos o tratamento por 2 horas, somente as celulas B16- F10 tiveram aumento significativo na quantidade de celulas em apoptose, e as B16-F10 Y tiveram leve queda na quantidade de celulas viaveis, com tendencia ao aumento de celulas em apoptose. As celulas Melan-a nao tiveram diferenca entre os tratamentos. A liberacao do citocromo c foi determinada por HPLC e mostrou-se que apos 2 horas, as celulas B16-F10 tratadas com 1O2 tiveram mais citocromo c liberado para o citoplasma comparado com o controle. Nos demais grupos, nao houve alteracao com o tratamento. Porem, as celulas controle com mais melanina tiveram maior liberacao de citocromo comparado com o controle das celulas nao estimuladas, mostrando que as celulas estavam sofrendo algum dano inerente da sintese de melanina. A analise da fragmentacao do DNA apos 2 e 24 horas mostrou que nao houve aparecimento de quebras caracteristicas de apoptose pelo tratamento com 1O2 em nenhum dos grupos testados. As celulas B16-F10 Y controle apresentaram DNA fragmentado inespecificamente, representado como um arraste no gel de agarose, que nao foi alterado pelo tratamento. A razao entre ADP e ATP foi quantificada para avaliar o estado energetico da celula, que pode refletir algumas caracteristicas de morte. Nenhuma das celulas teve diferenca estatistica apos o tratamento de 2 horas com 1O2, mas foi observado que as razoes ADP/ATP das celulas controle B16-F10 com e sem estimulo apresentaram valores acima do valor considerado para celulas viaveis/proliferativas. Os resultados da celula Melan-a foram bem proximos dos valores ditos normais. O fluxo de calcio tambem foi avaliado e o 1O2 foi capaz de liberar calcio das reservas intracelulares para o citoplasma nas celulas B16-F10, sendo que nas celulas estimuladas, o aumento do calcio citoplasmatico foi menor, indicando a possivel recaptacao do calcio pela melanina. As celulas Melan-a nao sofreram grandes alteracoes na quantidade de calcio liberado para o citoplasma. Nao houve diferenca na liberacao de AIF em nenhuma das celulas. Em conjunto, os resultados mostram que a sintese da melanina estimulada pela suplementacao do meio foi deleteria as celulas, pois causou fragmentacao no DNA, liberacao de citocromo c e aumentou a razao ADP/ATP para valores considerados de celula em apoptose. Por outro lado, a presenca da melanina parece ter protegido as celulas da acao do 1O2, pois alguns resultados indicam uma tendencia de melhora dos parametros avaliados.Abstract: Melanoma is a relevant type of cancer since the options for efficient treatment are limited. The presence of melanin protects the dark-skinned people against the effects of solar radiation, the main cause for melanoma development by the generation of reactive species of oxygen (ROS). However, melanin may also have a role on the generation of reactive species during its synthesis, which may harm the cell. So, the objective of this work was the evaluation of the characteristics of cell death caused by a ROS, the singlet oxygen (1O2) on murine melanoma cells B16-F10 with or without stimulation for synthesis of melanin and on murine melanocytes cells Melan-a. The stimulus for the synthesis of melanin was obtained treating the cells for 48 hours with medium RPMI 1640 supplemented with 400 ìmol/L of L-Tyrosine and 10 mmol/L of ammonium chloride. The concentration of melanin increased more than nine times in the B16-F10 cells and the Melan-a cells increase was almost twice the amount of the control. It was used the endoperoxide DHPNO2 10 mmol/L for 2 hours for the generation of 1O2. This condition caused the decrease in the viability determined by the method of MTT to 78% in B16-F10, 70,2% in B16-F10 that were stimulated (B16-F10 Y) and 79,3% in Melana cells. The test was performed after 24 hours from the beginning of the treatment with DHPNO2 and the viability decreased to 49,7% in B16-F10, 53,3% in B16-F10 Y and 72,5 % in Melan-a. The evaluation of cell death using acridine orange and ethidium bromide showed that after the two-hour treatment, only the B16-F10 cells had a significant increase of the number of apoptotic cells, and the B16-F10 Y cells had a slight decrease of the amount of viable cells, with the tendency of the increase of apoptotic cells. Melan-a cells did not show difference among the treatments. The release of cytochrome c was determined by HPLC and it showed that after two hours, B16-F10 cells had more cytochrome c released to the cytoplasm compared to the control. There was not any alteration for other groups of cells with the treatment. However, the control cells that had more melanin showed increased cytochrome c release compared to the control of the not-stimulated cells, demonstrating that the previous cells were suffering some kind of damage from the melanin synthesis. The analysis of DNA fragmentation revealed the absence of typical apoptosis fragmentation in any of the groups. B16-F10 Y control cells displayed unspecific DNA damage observed as a smear in the agarose gel, which was not altered by 1O2 treatment. The same result was observed after the treatment for 2 and 24 hours. The ratio between ADP and ATP was quantified to evaluate the energetic state of the cell, which may reflect some characteristics of cell death. None of the cells showed results statistically significant after the two-hour treatment 1O2, but it was shown that the values of ADP/ATP ratio of the B16-F10 control cells with and without stimulation were above of the threshold accepted for viable/proliferative cells. The results of Melan-a cells were very close to the values considered normal. The calcium flux was also evaluated and it was evidenced that 1O2 was capable of releasing calcium from the intracellular stores to the cytoplasm in B16- F10 cells, and the release of calcium was lower in B16-F10 Y, indicating the possibility of the binding of the metal to melanin. The Melan-a cells did not showed much increase in the quantities of calcium released to the cytoplasm. There was no difference in the release of AIF in any group. Over all, the results support that the synthesis of melanin that was stimulated by the supplementation of the medium was deleterious to the cells, since it caused DNA fragmentation, release of cytochrome c and increase of the ratio ADP/ATP to values of cells in apoptosis. On the other hand, the presence of melanin seemed to protect the cells against the action of 1O2, because some results indicate a tendency of improvement in the parameters evaluated

Why not "do simple things in a simple way": Use of the Pap test as the first step in screening genetic stability for human cultured stem cell therapy?

The aim of this study was to analyze adipose tissue-derived mesenchymal stem cells (AT-MSCs) using the Pap test as a first screening step to evaluate genetic stability. Human adipose tissue from six healthy female donors was obtained from elective liposuction procedures. The cells were isolated, cultivated at P2/P3, characterized by flow cytometric analysis, and differentiation induced. The AT-MSCs were stained by Papanicolaou staining and analyzed according to the Bethesda classification, and viability-apoptosis relationships were evaluated. The results of the Pap test for Sample I indicated high-grade alterations consistent with genetic instability; for Samples II-V, atypical cells of undetermined significance; and for Sample VI, normal cells. These results demonstrate the potential of using the Pap test as an initial screening step to evaluate the genetic stability of cultured AT-MSCs and also suggest its use for other adherent cells such as embryonic stem cells or induced pluripotent stem cells

Anti-tetanus human monoclonal antibodies.

Anticorpos monoclonais (AcMos) para uso terapêutico correspondem a uma área importante na indústria de biofármacos, em especial os AcMos humanos, que apresentam menor probabilidade de elicitar imunogenicidade. O objetivo deste trabalho consistiu em obter AcMos humanos antitetânicos através da separação de linfócitos B produtores de anticorpos específicos utilizando o antígeno ou de plasmablastos. As células foram coletadas de doadores após vacinação e separadas por equipamento de cell sorter. As regiões variáveis dos anticorpos foram amplificadas e clonadas em vetores de expressão, que foram usados para transfectar transitoriamente células HEK293-F. O uso da toxina tetânica conjugada independentemente com dois marcadores, biotina e Alexa Fluor® 647, possibilitou a separação específica de linfócitos B produtores de AcMos antitetânicos, que foram avaliados por ELISA, western blotting e pela inibição da ligação da toxina ao gangliosídio GT1b. O ensaio in vivo mostrou proteção total dos animais contra a toxina tetânica quando três AcMos foram usados em conjunto.Monoclonal antibodies (mAbs) for therapeutic use correspond to a major area of the biopharmaceutical industry, especially human mAbs that are less prone to elicit immunogenicity. The objective of this work was to obtain anti-tetanus human mAbs through separation of memory B lymphocytes producing specific antibodies stained with the antigen or plasmablasts. Cells were collected from peripheral blood of donors after vaccination and separated through cell sorting. The variable regions of the antibodies were amplified and cloned in expression vectors for transient transfection of HEK293-F cells. The staining with the tetanus toxin labeled independently with two markers, biotin and Alexa Fluor® 647 allowed the separation of specific B lymphocytes producing anti-tetanus mAbs. The antibodies expressed were evaluated by ELISA, western blotting and the inhibition of the binding of the tetanus toxin to the ganglioside GT1b. The in vivo neutralization assay showed that a pool of three different mAbs were able to protect mice against the tetanus toxin

Recombinant measles vaccine expressing malaria antigens induces long-term memory and protection in mice

Malaria: Recombinant measles virus malaria vaccine Following the limited success of the RTS,S recombinant malaria vaccine there is a pressing need for second generation malaria vaccines. Frédéric Tangy and colleagues at the Pasteur Institute, Paris, generate novel vaccines based on recombinant measles virus (rMV) expressing the major circumsporozoite antigen CS from either Plasmodium berghei (rMV-CSPb) or P. falciparum (rMV-CSPf). rMV is a strong vector candidate because of its widespread use, safety profile and efficacy. Mice permissive to rMV infection show rapid and durable (at least 22 weeks) CS antibody responses as well as activation of cell-mediated immunity and type 1 helper responses following vaccination with rMV-CSPb or rMV-CSPf. rMV-CSPb vaccination protects mice from lethal challenge with Pb sporozoites, and in a subset of mice leads to sterile immunity. The rMV vector offers the potential of incorporating further antigens from other Plasmodium infection stages and thereby enhancement of vaccine efficacy

Protective Malaria Vaccine in Mice Based on the Plasmodium vivax Circumsporozoite Protein Fused with the Mumps Nucleocapsid Protein

International audiencePlasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP—All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and P. vivax-like) were fused with the mumps virus nucleocapsid protein in the absence (NLP-CSPR) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSPCT). To elicit stronger humoral and cellular responses, Pichia pastoris yeast was used to assemble them as nucleocapsid-like particles (NLPs). Mice were immunized with each recombinant protein adjuvanted with Poly (I:C) and presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, these immunized mice with NLP-CSPCT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria

The Anopheles leucine-rich repeat protein APL1C is a pathogen binding factor recognizing Plasmodium ookinetes and sporozoites

We thank the Institut Pasteur core facility, the Center for the Production and Infection of Anopheles (CEPIA) for rearing and infection of mosquitoes; the UtechS Photonic BioImaging facility (Imagopole C2RT), Institut Pasteur for confocal microscopy instruments and assistance, and the Image Analysis Hub, Institut Pasteur for assistance with image analysis.International audienceLeucine-rich repeat (LRR) proteins are commonly involved in innate immunity of animals and plants, including for pattern recognition of pathogen-derived elicitors. The Anopheles secreted LRR proteins APL1C and LRIM1 are required for malaria ookinete killing in conjunction with the complement-like TEP1 protein. However, the mechanism of parasite immune recognition by the mosquito remains unclear, although it is known that TEP1 lacks inherent binding specificity. Here, we find that APL1C and LRIM1 bind specifically to Plasmodium berghei ookinetes, even after depletion of TEP1 transcript and protein, consistent with a role for the LRR proteins in pathogen recognition. Moreover, APL1C does not bind to ookinetes of the human malaria parasite Plasmodium falciparum , and is not required for killing of this parasite, which correlates LRR binding specificity and immune protection. Most of the live P . berghei ookinetes that migrated into the extracellular space exposed to mosquito hemolymph, and almost all dead ookinetes, are bound by APL1C, thus associating LRR protein binding with parasite killing. We also find that APL1C binds to the surface of P . berghei sporozoites released from oocysts into the mosquito hemocoel and forms a potent barrier limiting salivary gland invasion and mosquito infectivity. Pathogen binding by APL1C provides the first functional explanation for the long-known requirement of APL1C for P . berghei ookinete killing in the mosquito midgut. We propose that secreted mosquito LRR proteins are required for pathogen discrimination and orientation of immune effector activity, potentially as functional counterparts of the immunoglobulin-based receptors used by vertebrates for antigen recognition

A Multistage Formulation Based on Full-Length CSP and AMA-1 Ectodomain of Plasmodium vivax Induces High Antibody Titers and T-cells and Partially Protects Mice Challenged with a Transgenic Plasmodium berghei Parasite

International audienceInfections with Plasmodium vivax are predominant in the Americas, representing 75% of malaria cases. Previously perceived as benign, malaria vivax is, in fact, a highly debilitating and economically important disease. Considering the high complexity of the malaria parasite life cycle, it has been hypothesized that an effective vaccine formulation against Plasmodium should contain multiple antigens expressed in different parasite stages. Based on that, we analyzed a recombinant P. vivax vaccine formulation mixing the apical membrane antigen 1 ectodomain (PvAMA-1) and a full-length circumsporozoite protein (PvCSP-AllFL) previously studied by our group, which elicits a potent antibody response in mice. Genetically distinct strains of mice (C57BL/6 and BALB/c) were immunized with the proteins, alone or in combination, in the presence of poly(I:C) adjuvant, a TLR3 agonist. In C57BL/6, high-antibody titers were induced against PvAMA-1 and the three PvCSP variants (VK210, VK247, and P. vivax-like). Meanwhile, mixing PvAMA-1 with PvCSP-AllFL had no impact on total IgG antibody titers, which were long-lasting. Moreover, antibodies from immunized mice recognized VK210 sporozoites and blood-stage parasites by immunofluorescence assay. However, in the BALB/c model, the antibody response against PvCSP-AllFL was relatively low. PvAMA-1-specific CD3+CD4+ and CD3+CD8+ T-cell responses were observed in C57BL/6 mice, and the cellular response was impaired by PvCSP-AllFL combination. More relevant, the multistage vaccine formulation provided partial protection in mice challenged with a transgenic Plasmodium berghei sporozoite expressing the homologous PvCSP protein

Midgut permeabilization enables antibody access to <i>P</i>. <i>berghei</i> parasites in all midgut locations.

IFA of midguts collected 24 h post-IBM. Midguts were permeabilized and immunostained with anti-GFP conjugated antibody. GFP-expressing ookinetes (green) were also associated with anti-GFP antibody (red) which confirmed antibody accessibility to all parasites in mosquito midguts. Nuclei and actin were stained with DAPI and Phalloidin (blue). The scale bar is 10 μm. (PDF)</p

APL1C binds to extracellular <i>Plasmodium berghei</i> ookinetes <i>in vivo</i>.

A. Immunostaining of non-permeabilized P. berghei-infected mosquito midguts 24 h post-infection detects APL1C protein binding to extracellular ookinetes. Extracellular location of parasites is indicated by staining with antibody against Pys25 ookinete surface protein (yellow), APL1C binding is indicated by anti-APL1C antibody staining (red), and live parasites are indicated by expression of GFP (green). Numbered ookinetes indicate representative combinations of attributes: ookinete 1, extracellular (yellow) APL1C-positive (red) and live (green); 2, extracellular, APL1C-postive, and dead (not green); 3, the same as 2; 4, extracellular, APL1C-negative (not red), and live. Nuclei and actin were stained with DAPI and phalloidin, respectively (blue). Scale bar, 10 μm. B. The majority of extracellular ookinetes and almost all dead extracellular ookinetes are labelled by APL1C protein. All ookinetes from P. berghei-infected midguts treated as in panel A were counted and scored for attributes in three biological replicates (N, with 5–14 midguts per replicate), n indicates number of ookinetes scored. Top pie chart depicts the outcome for all extracellular (Pys25-positive) parasites (red pie slice, APL1C-positive extracellular ookinetes, green pie slice, APL1C-negative extracellular parasites), shown as the mean percentage obtained from three replicates. Bottom pie chart depicts the outcome of all dead extracellular (Pys25-positive and GFP negative) parasites (red pie slice, APL1C-positive dead parasites, yellow pie slice, APL1C-negative dead parasites). Parasite images in boxes are unmerged projections of the same numbered parasites shown in panel A. Scale bars of enlarged projection, 2 μm.</p

APL1C gene silencing depletes transcript and protein for at least 10 days.

A. APL1C expression is reduced at the time of salivary gland dissections done at 10 d post dsAPL1C injection. APL1C silencing was verified by qPCR measurement between dsAPL1C and dsGFP (dotted line) treatments 10 d post-treatment. The ratio of normalized APL1C transcript in dsAPL1C versus dsGFP treatments was calculated using triplicates from the same cDNA dilution. Graph represents mean with ±SEM of the expression fold change between dsAPL1C and dsGFP control from four biological replicates (N = 4). Data for qPCR analysis was analyzed by unpaired t-test (significance level of t-test **** p-value B. Western blot analysis of APL1C protein level in mosquito 10 d after APL1C gene silencing. The efficiency of APL1C depletion was monitored at day 10 post dsAPL1C injection versus dsGFP control in whole mosquitoes using anti-APL1C antibody and anti-GADPH antibody as a loading control. C. APL1C protein level is still decreased 10 d after dsAPL1C injection. The loading control normalized APL1C protein band densities were compared between dsAPL1C and dsGFP (dotted line) injected mosquitoes. (PDF)</p