Pesquisa de terapias para o melanoma em modelos de metástases pulmonares em camundongos

Orientador : Prof. Dr. Edvaldo da Silva TrindadeOrientadora : Profª. Drª. Dorly de Freitas BuchiTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 28/08/2014Inclui referênciasResumo: Melanoma é um câncer de pele cujas metástases para órgãos distantes levam à óbito milhares de pessoas todos os anos. Até o presente momento, não existe uma forma de terapia que possua alta frequencia de respostas duradouras. Sendo assim, muitos pacientes com câncer utilizam homeopatia como uma terapia integrativa e complementar, e relatam terem vivenciado os benefícios do tratamento. Entretanto, existem poucas evidências científicas que comprovem sua real eficácia. Sendo assim, no presente estudo foi investigado o potencial anti-melanoma de um medicamento homeopático chamado M1, com base em sua ação imunoestimulante relatada anteriormente. Para tanto camundongos C57BL/6 foram injetados intravenosamente com células de melanoma B16F10 ou LWT1 para formação de metástases pulmonares e tratados com M1 ou seu veículo (água). Após 14 días de tratamento, os camundongos foram eutanasiados e os pulmões foram removidos para análises microscópicas e por citometria de fluxo. As análises histológicas revelaram que M1 reduziu a formação de metástases em ambos os modelos de melanoma. Adicionalmente, o tamanho dos nódulos, bem como a proliferação e angiogênese tumorais estavam todas reduzidas em metástases de melanoma B16F10 de camundongos tratados com M1. Interessantemente, a ação do M1 foi independente da imunidade inata e adaptativa mediada por linfócitos. Por outro lado, pulmões de camundongos com B16F10 e tratados com M1 posuíram menores frequencias de células mielóides imunossupressoras caracterizadas pela marcação CD11b+ Gr-1baixo AT1R+. A presença de AT1R nessas células sugere um papel na angiogênese tumoral, inibida pelo M1. Adicionalmente, as metástases de melanoma apresentaram um fenótipo de marcações para imunofluorescência associado à menor malignidade tumoral, como maior expressão de Beta-Catenina e menor expressão de Ácido Hialurônico. Esses dados revelaram que o tratamento com M1 resultou em menor carga tumoral de metástases de melanoma em camundongos e sugerem um possível mecanismo de ação celular: o controle da angiogênese pela inibição de células CD11b+ Gr-1baixo AT1R+ com consequente menor desenvolvimento tumoral e aumento de apoptose. Esses dados comprovam a eficiência do tratamento com M1 para o câncer de pele tipo melanoma.Abstract: Melanoma is a skin cancer with increasing incidence and incurable metastases, which annually kills thousands of people worldwide. Until now, there is not a therapy with high efficacy rate and durable response. Therefore many cancer patients use integrative therapies such as Homeopathy and claim the benefits from these treatments. However, there are not enough scientific evidences to support efficacy of these homeopathic medicines. Therefore, in this study was investigated the antimelanoma potential of a homeopathic medicine termed M1, based on M1's immunestimulatory action that has been previously reported. C57BL/6 mice were injected intravenously with B16F10 or LWT1 mouse melanoma cells and treated with M1 or vehicle (water). Following 14 days of treatment, mice were euthanized, lungs removed and analysed by microscopy or flow cytometry. Microscopic analysis revealed the quantity of metastases was reduced by treatment with M1 for both melanoma cell lines. Furthermore, metastases' size, proliferation and angiogenesis were all reduced by M1 treatment for the B16F10 model. Surprisingly, these effects by M1 treatment were not associated but independent of innate and adoptive immune responses for B16F10 and LWT1 melanoma metastases. On the other hand, mice's lungs with B16F10 and treated with M1 had fewer frequency of CD11b+ Gr-1low AT1R+ myeloid-derived suppressor cells. The expression of AT1R on these cells suggests a possible role for tumour angiogenesis, inhibited by M1. Additionally, melanoma metastases had a phenotype determined by microscopy typical for decreased malignancy, such as increased Beta-Catenin and decreased Hyaluronic Acid expression. In summary, these data reveal M1 treatment reduces tumor burden of melanoma metastases in mice and suggest a possible mechanism of action: control of angiogenesis by inhibition of CD11b+ Gr-1low AT1R+ cells with consequent reduction of tumor development and increased tumour apoptosis. These data show the efficacy of treatment with M1 and provide insights in the importance of treatment with homeopathic medicines. Key words: Melanoma. Homeopathy. M1. Angiogenesis. CD11b+ Gr-1low AT1R+ cells

Avaliação da ação do medicamento M1 sobre o melanoma murino "in vivo"

Orientador : Prof. Dr. Edvaldo da Silva TrindadeCo-orientadora : Profª Drª Dorly de Freitas BuchiDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 11/11/2011Inclui referênciasResumoResumo: O melanoma metastático é o câncer de pele com maior agressividade, sendo responsável por milhões de mortes a cada ano. Muitos pacientes com câncer buscam terapias complementares para serem utilizadas em conjunto com as terapias convencionais. A homeopatia está entre as terapias complementares mais utilizadas para o tratamento do câncer. O complexo altamente-diluído codificado como 'M1' é um produto manipulado de acordo com a técnica homeopática padrão. Estudos anteriores constataram que esse produto possui atividade anti-metastática contra o melanoma de camundongo em modelo de colonização pulmonar. No presente estudo, utilizando um modelo de melanoma subcutâneo em camundongos, foi avaliado se ele possui também atividade contra a formação de um tumor sólido. Para isso, camundongos foram inoculados subcutaneamente com células de melanoma murino (linhagem B16-F10), e após 24 horas da inoculação foram tratados por inalação durante 14 dias. Após esse período, os tumores foram removidos a analisados. Cortes histológicos dos tumores foram processados para histologia convencional e imunohistoquímica para PCNA, ácido hialurônico (AH), CD44 e AT1R. O tratamento com M1 foi capaz de reduzir o crescimento tumoral do melanoma (p<0,05). Este efeito pode ser decorrente da regulação negativa da angiogênese tumoral, avaliada pela imunohistoquímica para AH, uma vez que os tumores oriundos de animais que foram tratados apresentaram uma significante redução no número de novos vasos sanguineos (p<0,05). A angiotensina II por sí só já é um fator angiogênico e a cascata de sinalização intracelular iniciada pela sua ligação com o receptor celular AT1R desencadeia a formação de novos vasos. Talvez essa redução na neoangiogenese dos tumores do grupo M1 seja decorrente do menor número de células positivas para o AT1R na periferia tumoral (p < 0,05). Não foram encontradas diferenças com o uso dos demais marcadores. É fundamental a continuidade de estudos envolvendo esse produto, pois além de não apresentarem efeitos colaterais, até agora mostraram efeitos rápidos e impactantes na redução da evolução tumoral.Abstract: Metastatic melanoma is the most aggressive skin cancer and it is responsible for millions of deaths every year. Many patients with cancer look for some kind of complementary therapy in order to use it together with the conventional ones. Homeopathy is among the most used complementary therapies against cancer. The highly diluted complex called 'M1' is prepared according to standard homeopathy technique. Previous studies showed that this complex has anti-metastatic activity against mouse melanoma, when analyzed using experimental metastasis model. Here, using a model for mouse melanoma growth, it was verified M1 has activity on the development of a solid tumor. For this purpose, mice were subcutaneously injected with B16-F10 mouse melanoma cells. The treatment with M1 was started 24 hours after the injection by inhalation for 14 days. Subsequently, tumor were removed and analyzed. Sections from tumors were processed for conventional histology and immunohistochemistry techniques for PCNA, hyaluronic acid, CD44 and AT1R. M1 treatment was effective in reducing melanoma tumor growth (p<0.05). This can be a consequence of the modulation of tumor angiogenesis, analyzed by hyaluronic acid immunohistochemistry, since M1 tumors have fewer vessels (p<0.05). Angiotensin II receptor 1 (AT1R) plays a role in angiogenesis and its interaction with angiotensin II molecule initializes intracellular signaling that leads to new vessels formation. The angiogenesis reduction by M1 would be related with fewer cells AT1R+ on tumor peripheries (p<0.05). There were not found differences between M1 and no treated tumors related to the others immunohistochemistries. It is fundamental to continue the study of this product, since it does not have side effects, and has been showed its activity against tumor development

Erratum: Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017

Interpretation: By quantifying levels and trends in exposures to risk factors and the resulting disease burden, this assessment offers insight into where past policy and programme efforts might have been successful and highlights current priorities for public health action. Decreases in behavioural, environmental, and occupational risks have largely offset the effects of population growth and ageing, in relation to trends in absolute burden. Conversely, the combination of increasing metabolic risks and population ageing will probably continue to drive the increasing trends in non-communicable diseases at the global level, which presents both a public health challenge and opportunity. We see considerable spatiotemporal heterogeneity in levels of risk exposure and risk-attributable burden. Although levels of development underlie some of this heterogeneity, O/E ratios show risks for which countries are overperforming or underperforming relative to their level of development. As such, these ratios provide a benchmarking tool to help to focus local decision making. Our findings reinforce the importance of both risk exposure monitoring and epidemiological research to assess causal connections between risks and health outcomes, and they highlight the usefulness of the GBD study in synthesising data to draw comprehensive and robust conclusions that help to inform good policy and strategic health planning

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Ação do complexo homeopático em macrófagos peritoneais e no sangue periférico de camundongos

Orientador: Dorly de Freitas BuchiMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Ciências BiológicasResumo : O sistema imunológico constitui-se de um conjunto de células e moléculas capacitadas para o reconhecimento e destruição de microorganismos invasores, células tumorais e células próprias infectadas por patógenos intracelulares. Alterações na efetividade do sistema imunológico de camundongos já foram previamente descritas em testes experimentais de complexos homeopáticos. Aqui, foi avaliada a atividade biológica de um complexo homeopático derivado de Calcarea carbonica com associações (M8). Três grupos experimentais foram analisados: sem tratamento, tratado com solução diluente e tratado com M8. No experimento in vivo o tratamento foi feito através da água de beber de camundongos Mus musculus por sete dias. Após esse período, os leucócitos do sangue periférico foram analisados por extensão sanguínea e imunofenotipagem por citometria de fluxo. Para o experimento in vitro, macrófagos peritoneais de Mus musculus não tratados foram cultivados em DMEM com 10% de soro fetal bovino e mantidos a 37ºC em atmosfera úmida com 5% de CO2. O tratamento consistiu na adição de 20% nas da solução na cultura, com dose reforço de 1% após 24h. Após 48h do início do cultivo, foi analisada a marcação de vesículas ácidas com vermelho neutro. Como ferramenta estatística foi utilizado ANOVA one way (P<0,05) com pós teste de Tukey (P<0,05) através do software GraphPad® Prism 5. No experimento in vivo, não foram detectadas alterações através da extensão sanguínea e imunofenotipagem. Também, nos experimentos in vitro, não foram detectadas alterações na marcação de vesículas ácidas. Resultados prévios em nosso laboratório mostraram um aumento na resposta do sistema imune, Th1 e Th2, após tratamento com M8. Nossos resultados aqui apresentados não mostram novas informação sobre os mecanismos de ação do tratamento com M8, mas nos permite concluir que em camundongos saudáveis não existem efeitos colaterais no sangue periférico e nas vesículas ácidas de macrófagos. Assim, mais estudos sobre Calcarea carbônica e associações são necessários

DNAM-1 control of natural killer cells functions through nectin and nectin-like proteins

Natural killer (NK) cells represent key innate immune cells that restrain viral infection and malignant transformation and help mount an adaptive immune response. To perform such complicated tasks, NK cells express a wide set of inhibitory and activating receptors that alert them against cellular stress without damaging healthy cells. A new family of receptors that recognize nectin and nectin-like molecules has recently emerged as a critical regulator of NK cell functions. The most famous member of this family, DNAX accessory molecule (DNAM-1, CD226), is an adhesion molecule that control NK cell cytotoxicity and interferon-γ production against a wide range of cancer and infected cells. Its ligands CD112 and CD155 have been described in different pathological conditions, and recent evidence indicates that their expression is regulated by cellular stress. Additional receptors have been shown to bind DNAM-1 ligands and modulate NK cell functions bringing another level of complexity. These include CD96 (TACTILE) and TIGIT (WUCAM, VSTM3). Here, we review the role of DNAM-1, TIGIT and CD96 in NK cell biology summarizing the recent advances made on the role of these receptors in various pathologies, such as cancer, viral infections and autoimmunity

Natural Killer cell control of BRAFV600E mutant melanoma during targeted therapy

Pharmacologic inhibition of the mutant BRAF protein in advanced BRAFmelanoma results in a high proportion of patients that respond, but few with durable responses. We have recently revealed that Natural Killer (NK) cells play an essential role in the BRAF inhibitor control of melanoma metastases in mice that may be therapeutically exploited to help overcome drug resistance

The Latest Breakthroughs in Immunotherapy for Acute Myeloid Leukemia, with a Special Focus on NKG2D Ligands

Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion of stem and myeloid progenitor cells. Immunotherapy has revolutionized the care for other cancers such as solid tumors and lymphomas, and has the potential to effectively treat AML. There has been substantial progress in the developments of immunotherapeutic approaches for AML over the last several years, including the development of antibodies that further increase the innate immunogenicity of leukemia cells by the inhibition of NKG2D ligand&mdash;particularly MICA and MICB&mdash;shedding, chimeric proteins such as IL-15 superagonist that expand natural killer (NK) cells, blockers of immunologic checkpoints such as NKG2A, and chemicals that indirectly increase expression of immune stimulatory proteins in leukemia stem cells. Furthermore, cellular therapies have been designed to enable alloreactive immunity by allogeneic NK cells or target leukemia antigens such as mutated NPM1. These immunotherapeutic approaches have demonstrated remarkable efficacies in preclinical studies and have successfully transitioned to early phase clinical trials, to establish safety and initial signal of clinical activity. Here, we briefly discuss some of the most recent and impactful developments in the AML immunotherapy field and provide our perspectives for the future directions of this exciting and new therapeutic opportunity

Pixel-level annotated dataset of computed tomography angiography images of acute pulmonary embolism

Abstract Pulmonary embolism has a high incidence and mortality, especially if undiagnosed. The examination of choice for diagnosing the disease is computed tomography pulmonary angiography. As many factors can lead to misinterpretations and diagnostic errors, different groups are utilizing deep learning methods to help improve this process. The diagnostic accuracy of these methods tends to increase by augmenting the training dataset. Deep learning methods can potentially benefit from the use of images acquired with devices from different vendors. To the best of our knowledge, we have developed the first public dataset annotated at the pixel and image levels and the first pixel-level annotated dataset to contain examinations performed with equipment from Toshiba and GE. This dataset includes 40 examinations, half performed with each piece of equipment, representing samples from two medical services. We also included measurements related to the cardiac and circulatory consequences of pulmonary embolism. We encourage the use of this dataset to develop, evaluate and compare the performance of new AI algorithms designed to diagnose PE