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Objetivos: O fator de crescimento de hepatócitos (HGF) é um mitógeno secretado por células estromais mesenquimais (MSCs) que regula o crescimento, a motilidade e a morfogênese celular. No microambiente tumoral da medula óssea (MO) (nicho), o HGF interage com o receptor MET nas células leucêmicas ativando vias de sinalização que contribuem para a resistência quimioterápica e, consequentemente, o desenvolvimento de novos compostos que atuam na interação HGF/MET, torna-se crucial para o tratamento da leucemia mieloide aguda (LMA). Assim, investigamos os efeitos de três novos inibidores de MET, B2000, B6000 e C1000 (Brigham University, WO 2011/127192 A2) in vitro e in vivo. Métodos: Células mononucleares da medula óssea humana (BMNCs) de pacientes com LMA e doadores saudáveis (HD) e linhagens leucêmicas (OCI-AML3, MOLM-13) foram tratadas com inibidores de MET e as taxas de apoptose foram avaliadas. O valor de IC50 foi calculado através de resultados de ensaios de viabilidade. A expressão proteica de HGF foi avaliada por imunofluorescência. In vivo, camundongos portadores de LMA agressiva foram tratados com os inibidores de MET (10 mg/kg; 1x/dia; 5 dias) e medula óssea, sangue periférico e baço foram coletados. Um sistema 3D que mimetiza o nicho da MO foi desenvolvido usando uma esponja hemostática para cultura de células estromais mesenquimais (MSCs), células endoteliais da veia umbilical humana (HUVECs) e células leucêmicas. Análises de sinergia foram realizadas usando o pacote Synergy Finder em R. Resultados: Os inibidores promoveram apoptose de BMNCs de pacientes com LMA (p < .01) sem modular células saudáveis. Nas células Molm-13 e OCI-AML3, o tratamento com os compostos resultou em valores baixos de IC50 (< 10 μM). Para validar esses achados em um modelo fisiopatológico, camundongos leucêmicos foram tratados com os inibidores de MET. O composto C1000 demonstrou o efeito mais citotóxico, inibindo a proliferação de células leucêmicas na medula óssea, baço e sangue periférico (p 10). Conclusão: Nossos achados pré-clínicos sugerem que inibidores de MET representam uma classe de agentes terapêuticos com potencial para melhorar significativamente o tratamento de pacientes com LMA. A combinação com Venetoclax pode oferecer uma opção terapêutica mais eficaz e com menor toxicidade, aumentando as chances de cura e melhorando a qualidade de vida dos pacientes. Apoio: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.

Aims: Acute lymphoblastic leukemia (ALL), the most prevalent childhood cancer, disrupts the hematopoietic bone marrow (BM) niche, a specialized microenvironment that supports normal hematopoiesis. During ALL development, this niche undergoes remodeling and reprogramming to support tumor growth, hindering blood cell formation. Mesenchymal stromal cells (MSCs), particularly those located in the perivascular niche, play a pivotal role by shielding leukemic lymphoblasts from external stressors, impacting drug responses and potentially contributing to relapse. Understanding this complex interplay between ALL cells and the BM niche is vital for developing effective drug screening models. Therefore, our study aimed to develop an in vitro coculture system that mimics the ALL BM tumor microenvironment. Methods: T-ALL (Jurkat), B-ALL (RS4-11), and MSCs (HS5) cell lines were maintained in RPMI-1640 supplemented with 10% FBS. Tridimensional cocultures were established using hemostatic sponges (Gelfoam, Pfizer) and spheroids (Hanging Drop or Liquid Overlay Technique containing 0, 5, 10 and 20% Matrigel). These cocultures combined normal MSCs (HS5) and ALL cells to mimic different cellular environments within the tumor microenvironment. Different cell ratios (MSC to T-ALL proportion: 1:1, 1:2, 1:5, 2:5) were tested to simulate different cellular environments. To evaluate the system's applicability, the cocultures were treated for 72h with a BCL2 inhibitor, Venetoclax, or an MCL1 inhibitor, AZD5991, as single agents or in combination. Both agents were used at their 10% maximal response concentration (IC10 - 20 nM). Following the treatment, the 3D systems were dissociated using collagenase (for Gelfoam) or trypsin (for spheroids), and cell populations were analyzed by flow cytometry. Results: In comparison to monocultures, T-ALL cell viability was enhanced in cocultures with MSCs in Gelfoam 3D system, with ̃80% of viable ALL cells in all cell ratios tested. Conversely, proliferation of MSCs was diminished compared to monocultures. Although spheroid formation was obtained using hanging drop technique, increasing Matrigel concentrations impaired spheroid formation, except at the highest cell seeding ratio (2:5), which still resulted in highly irregular spheroids with a near-equal distribution of ALL cells and MSCs (1:1). Thus, considering the bone marrow microenvironment, Gelfoam (1:5 cell ratio) was the 3D system that most closely resembled the cellular composition observed in ALL. Cocultures in Gelfoam were treated with Venetoclax and/or AZD5991. The combination therapy effectively inhibited the proliferation of ALL cells in monoculture. However, the combined treatment was not enough to overcome the protective niche action. Importantly, the treatments did not affect MSCs proliferation. Conclusions: Our findings demonstrate an innovative 3D coculture system that successfully replicates the BM stromal niche. This novel platform holds promise for the development of new therapeutic strategies for ALL. Apoio: FAPESP 2017/21801-2, 2019/25247-5, 2021/05320-0; 2023/13850-4; CNPq 303405/2018-0.

Objetivos: Embora a terapia da leucemia mieloide aguda (LMA) tenha registado progressos na última década, a recaída continua a ser o maior desafio terapêutico. As Pim quinases regulam a proliferação e sobrevivência celular e inibem a apoptose ativando vias anti-apoptóticas, como a BCL2, conferindo um pior prognóstico em pacientes com LMA e contribuindo para a recidiva da doença. Portanto, direcionar especificamente as quinases PIM é uma estratégia para o tratamento da LMA. Dessa forma, investigamos os efeitos de um novo inibidor da Pan-PIM quinase, PIM447 (Novartis), em células leucêmicas e exploramos a combinação de PIM447 e Venetoclax, um quimioterápico inibidor de BCL2. Métodos: Células mononucleares (CMs) da medula óssea (MO) de pacientes com LMA e doadores saudáveis (HD) e linhagens de leucemia (OCI-AML3, MOLM-13) foram tratadas com PIM447 e as taxas de apoptose foram avaliadas. A eficácia do PIM447 foi testada em um ensaio organotípico de microscopia, usando CMs de MO de AML cocultivadas com células estromais mesenquimais (MSCs) em meio AIMV-10% FBS e 1,5 mg/mL de colágeno I. Em um modelo xenográfico, camundongos NSG leucêmicos (Molm-13-GFP) foram tratados com PIM447 (2 mg/kg) por 5 dias e medula óssea, sangue periférico e baço foram coletados. A expressão das proteínas BCL2, MCL1 e BCLXL foi avaliada por citometria de. Cocultura de células estromais mesenquimais (MSCs), células endoteliais da veia umbilical humana (HUVECs) e células (Molm-13) foi realizada em um sistema 3D. O pacote Synergy Finder em R foi usado para análises de sinergia. Resultados: O tratamento aumentou em 57% as taxas de apoptose nas CMs de MO leucêmicas (p -10). Em um modelo de nicho da MO, o tratamento com PIM447 inibiu em 70% a proliferação de células Molm-13 (p >,0001) e em 17% blastos de pacientes com LMA (p >,05), sendo mais potente quando combinado com Venetoclax (Molm13: redução de 90%, p < 0001; blastos: redução de 60%). Conclusão: Nossos resultados indicam que o PIM447 possui potente atividade citotóxica contra células leucêmicas, tornando-o um candidato promissor para terapia direcionada em LMA. A combinação sinérgica com um inibidor de BCL2 permite superar a resistência conferida pelo microambiente da medula óssea, abrindo novas perspectivas para o tratamento dessa doença. Apoio: FAPESP 2017/21801-2, 2019/25247-5, 2021/05320-0; CNPq 303405/2018-0.

Epigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, has shown a range of beneficial effects, including the inhibition of M2 macrophage polarization. Macrophages are indispensable tissue components and play a significant role in the pathophysiology of hematopoietic malignancies. In myelodysplastic syndrome (MDS), macrophages exhibit more M2-related characteristics (alternatively activated), which generally have pro-tumor effects. Thus, the aim of this study was to assess the immunomodulatory potential of EGCG on macrophages in MDS. Female NHD13 transgenic mice and their wild-type littermates, at 10 weeks old, were administered EGCG at a dosage of 50 mg/kg per day, five times a week, intraperitoneally for four weeks. Prior to receiving treatment, NHD13+ mice displayed a decrease in leukocyte and platelet counts as well as hemoglobin levels, consistent with characteristics of the disease and documented findings. EGCG promoted the polarization of macrophages within the bone marrow (BM) milieu. This effect was in part through heightened expression of pro-inflammatory cytokines/M1 macrophage markers, specifically interleukin 6 (IL6) (p = 0.05) and tumor necrosis factor-alpha (TNF-α) (p = 0.05). Concurrently, there was a noteworthy attenuation in the levels of interleukin 4 (IL4) (p = 0.03), an established anti-inflammatory marker associated with the M2 macrophage phenotype. Additionally, the percentage of M2 macrophages (CD45+CD11b+F4/80+CD206+) underwent a substantial reduction (p = 0.002). In summary, EGCG effectively steers macrophage polarization towards the M1 phenotype within the bone marrow microenvironment. These findings highlight EGCG's potential therapeutic role as an immunomodulator in MDS, providing new perspectives for its use in the treatment of hematopoietic malignancies by influencing the immune response in the context of the disease.

Objetivos: O sistema endocanabinoide consiste em uma complexa rede de sinalização celular primariamente responsável por manutenção da homeostase e regulação de processos fisiológicos e, por isso, tem sido alvo de interesse pelo potencial terapêutico em diversas condições patológicas. Um de seus receptores, CB2, é preferencialmente expresso nos tecidos periféricos, tendo sido demonstrado em todas as populações de leucócitos humanos. Portanto, o objetivo deste trabalho foi avaliar o efeito do tratamento com Cannabis medicinal sobre as subpopulações de leucócitos em paciente portadora de Doença Falciforme (DF). Material e métodos: Os dados clínicos e sociodemográficos foram compilados através de análise retrospectiva de prontuário médico, e a avaliação de subtipos de leucócitos foi realizada por citometria de fluxo. Para isso, 4 mL de sangue periférico foram coletados em tubo EDTA antes do início do tratamento e após 15 e 21 dias de tratamento. A imunofenotipagem foi realizada nos 3 períodos utilizando-se anticorpos monoclonais conjugados à fluorescência: CD3, CD4, CD8, CD14, CD16, CD19, CD25, CD45, CD56, CD66b, CD68, CD80, CD163, FoxP3, HLA-DR, IFN-y, IL-4, IL-10, Granzima e Perforina. As amostras foram adquiridas no citômetro de fluxo CytoFLEX - Beckman Coulter e analisadas no FlowJo™. Resultados: Paciente do sexo feminino, 42 anos, em seguimento por Sβ-talassemia zero, tendo como importante complicação da doença múltiplos microinfartos ósseos em coluna lombar levando a quadro de dor crônica incapacitante. Em uso de Hidroxiureia (20 mg/kg/dia), Gabapentina 1200 mg/dia, com necessidade de doses diárias de Dipirona e Tramal; faz seguimento com fisioterapia tendo sido avaliada por ortopedista sem indicação cirúrgica. Diante do quadro de dor crônica refratária, iniciado tratamento complementar com Cannabis Medicinal: 25 mg de canabidiol, divididos em duas doses diárias (óleo sublingual 50 mg/mL, 0,25 mL 2x ao dia), mais 5 mg de THC (óleo sublingual 50 mg/mL, 0,05 mL 2x ao dia). Após 21 dias de tratamento, além da importante melhora no quadro de dor, a avaliação das subpopulações leucocitárias por citometria de fluxo evidenciou: i) aumento da porcentagem de linfócitos T CD4+ e diminuição de T CD8+, ii) aumento da porcentagem de Tregs FoxP3+, iii) diminuição no conteúdo de granzima em linfócitos T CD8+, iv) diminuição progressiva na porcentagem de neutrófilos e v) polarização macrofágica com tendência ao fenótipo M2. Discussão: O tratamento com cannabis medicinal neste caso levou a importantes alterações quantitativas nos elementos do sistema imune, sugerindo uma importante mudança para um perfil anti-inflamatório (supressão de neutrófilos e aumento no número de Tregs FoxP3+) e de reparo tecidual (com menor produção de granzima, aumento da relação CD4/CD8 e polarização macrofágica M2). O perfil observado antes do uso da medicação era característico das DF e foi parcialmente revertido com apenas poucos dias de tratamento. Conclusão: Estes resultados sugerem a modulação de algumas subpopulações de leucócitos com o uso de Cannabis Medicinal em paciente com Doença Falciforme, revertendo o ambiente inflamatório característico desse grupo de doenças e evidenciando um potencial terapêutico que deve ser analisado em maior profundidade em ensaios clínicos.

A hipercoagulabilidade é uma característica importante da doença falciforme (DF), e sua fisiopatologia está relacionada à ativação concomitante da hemostasia e da imunidade inata, em um processo conhecido como imunotrombose. Recentemente, estudos destacam a via podoplanina/CLEC-2 como importante no processo de ativação plaquetária e trombose. A podoplanina (PDPN) é uma proteína que já foi descrita em monócitos, e tem como único ligante conhecido a CLEC-2, expressa principalmente em plaquetas. Apesar do papel da via PDPN/CLEC-2 não seja totalmente esclarecida, evidências sugerem que ela pode se encaixar como uma nova via dentro do conceito de imunotrombose. O objetivo do estudo foi avaliar a expressão de PDPN e CLEC-2 em pacientes com DF e explorar sua relação com a gravidade da doença. Foram incluídos 31 pacientes com DF (genótipo SS = 23; genótipo SC = 8), além de 10 controles saudáveis, com aprovação ética (CAAE: 53121421.0.0000.5404). A avaliação da expressão de PDPN e CLEC-2 foram feitas por citometria de fluxo do sangue periférico de pacientes DF na fase estável da doença, além dos indivíduos saudáveis. Foram utilizados anticorpos anti-CD45, Anti-CD41, Anti-P-Selectina, Anti-PDPN e Anti-CLEC-2. O FSC (tamanho) e SSC (granulosidade) foi utilizado para separar as populações celulares. Para tal, foram analisados “gates” de células monocitárias e granulocíticas, e os resultados foram descritos como porcentagem. Os dados clínicos e laboratoriais foram obtidos dos prontuários eletrônicos. A expressão de PDPN em monócitos mostrou tendência a aumento nos pacientes DF-SS quando equiparados aos indivíduos saudáveis (teste Kruskal-Wallis [X2 (2) = 3,69; p = 0,156]; p = 0,06, Bonferroni, post-hoc). Nas células granulocíticas não houve diferença na expressão de PDPN entre os grupos, teste Kruskal-Wallis [x2 (2) = 1,06; p = 0,58]. Já a expressão de CLEC-2 em monócitos foi diferente entre os grupos, teste Kruskal-Wallis [x2 (2) = 19,82; p < 0,0001], com os pacientes DF-SS apresentando aumento da porcentagem de expressão de CLEC-2 quando equiparados aos pacientes DF-SC (p = 0,03) e indivíduos saudáveis (p < 0,0001). Não observamos diferença entre os pacientes DF-SC e saudáveis (p = 0,57). Também observamos diferenças entre os grupos na expressão de CLEC-2 em granulócitos, teste Kruskal-Wallis [x2 (2) = 10,11; p = 0,006], com maior expressão em pacientes DF-SS quando comparados aos pacientes DF-SC (p = 0,02) e indivíduos saudáveis (p = 0,006). Houve correlação de monócitos PDPN+ com marcadores laboratoriais, tais como: monócitos CLEC-2+ (R = 0,646), monócitos P-Selectina+ (R = 0,540), monócitos CD41+ (R = 0,525), granulócitos PDPN+ (R = 0,664) e reticulócitos (R = 0,434). Também houve correlação de monócitos CLEC-2+ com marcadores laboratoriais, como: monócitos P-Selectina+ (R = 0,823), monócitos CD41+ (R = 0,720), granulócitos CLEC-2+ (R = 0,884), hemoglobina (R = -0,533), plaquetas (R = 0,415) e reticulócitos (R = 0,563). Estes resultados sugerem que a via PDPN/CLEC-2 possa fazer parte da fisiopatologia da DF, de forma análoga a outras vias imunotrombóticas já descritas como a expressão de fator tecidual em vesículas extracelulares e a geração de NETs. Estudos para explorar o papel da mediação das interações plaqueta:leucócitos nestes resultados estão em andamento. Embora preliminares, nossos dados representam a primeira avaliação da participação desta via da DF, que pode abrir caminho para novas estratégias prognósticas e terapêuticas para esta condição.

Objetivos: PIM-quinases, associadas à progressão de cânceres induzidos pelo fator de crescimento de hepatócitos (HGF) por meio de seu receptor, MET, interagem com a serina protease tipo kunitz-2 (SPINT2), no microambiente tumoral (TME) e sua interação pode representar uma importante estratégia terapêutica multialvo na leucemia mieloide aguda (LMA). Analisamos a interação entre SPINT2, HGF e pan-PIM quinases avaliando a expressão gênica e os efeitos de um inibidor de pan-PIM quinase, PIM447 (Novartis), em células leucêmicas e no TME. Métodos: Expressão de mRNA foi obtida do estudo OHSU (2018) e TCGA (cBio Portal). A análise de correlação foi obtida pelo coeficiente de Spearman (r). Células mesenquimais estromais (MSC) da medula óssea (BM) de 5 doadores saudáveis (HD) e 5 pacientes com LMA foram tratadas com 5-Azacitidina (Aza). OCIAML3, MOLM13, U937, RS4;11, Jurkat foram tratadas com PIM447 e as taxas de apoptose foram determinadas. Sangue periférico e células mononucleares (MC) da BM (PBMC/BMMC) de HD foram testados quanto à citotoxicidade. Células leucêmicas foram examinadas para espécies reativas de oxigênio (ROS), produção de óxido nítrico (NO) e potencial de membrana mitocondrial (ΔΨm). Foram realizadas coculturas de células de leucemia com monócitos/macrófagos derivados de monócitos (MDMs) ou MSC com SPINT2 superexpresso. Estatística foi realizada por Kruskal Wallis, ANOVA ou Mann-Whitney (p < 0,05). Resultados: A expressão gênica de 188 pacientes com LMA mostrou que os níveis de HGF correlacionaram-se positivamente com PIM1/PIM2/PIM3 ((r) =,8727;.9094;.9093), p

Objetivos: A leucemia mieloide aguda (LMA) é uma doença sistêmica que continua sendo um grave problema médico em todo o mundo. O acúmulo de células leucêmicas na medula óssea, leva a supressão da hematopoiese normal e promove processos inflamatórios que despertam células-tronco hematopoiéticas quiescentes que proliferam, se diferenciam e geram uma resposta imune desregulada que pode contribuir para a resistência à terapia. Nesse cenário, regular a inflamação pode ser uma estratégia para melhorar as respostas antitumorais. Assim, buscamos compreender o papel dos macrófagos derivados de monócitos no microambiente tumoral da medula óssea (TME) e sua contribuição para a progressão da leucemia, avaliando os efeitos de um composto de tocotrienol derivado da semente de urucum (New Max), uma isoforma da vitamina E. Métodos: Monócitos de células mononucleares de sangue periférico (PBMC) de doadores saudáveis foram isolados de buffy coats. Para ensaios de polarização de macrófagos derivados de monócitos (MDMs), os monócitos foram estimulados com GM-CSF/LPS/INFy ou M-CSF/IL-4 para M1/M2. Células leucêmicas foram adicionadas a uma monocamada de monócitos/macrófagos. Camundongos BALB/c receberam uma única injeção intraperitoneal de LPS para estimular um choque inflamatório e foram tratados com tocotrienol. Análises foram analisadas por citometria de fluxo. Resultados: A análise in vitro mostrou aumento de monócitos clássicos após o tratamento com tocotrienol. Os monócitos tratados com tocotrienol levaram a um aumento da apoptose nas células leucêmicas, sem alterar o microambiente normal. A análise revelou uma população menor de M2-MDMs (HLA-DR-CD206+) e aumento da população de M1-MDMs (HLA-DR+CD80+) (p < 0,0007) em MDMs tratados com tocotrienol. Apoptose de células leucêmicas foi aumentada em coculturas com MDMs tratados com tocotrienol (p < 0,0001). Observou-se aumento da frequência de macrófagos M1 (CD11b+F4/80+CD80+) e diminuição da população M2 (CD11b+F4/80+CD206+) em camundongos tratados com tocotrienol. Conclusão: O tocotrienol foi capaz de ativar o fenótipo de morte de monócitos contra células leucêmicas, sem alterar a sobrevida de monócitos normais, além de ter aumentado a imunidade antitumoral in vitro e in vivo, promovendo a polarização do macrófago M1 e reduzindo o estresse inflamatório com uma ação significativa no TME. Nossos resultados destacam um papel importante do tocotrienol derivado da semente de urucum contra neoplasias hematológicas por meio de uma ação imunológica, sugerindo novos caminhos para estudos futuros. Suporte: FAPESP 2017/21801-2, 2019/25247-5, 2021/05320-0; CNPq303405/2018-0.

Objetivos: Na leucemia mieloide aguda (LMA), MET, um receptor do fator de crescimento de hepatócitos (HGF), contribui para polarização de macrófagos para um fenótipo M2, os quais têm uma ação imunossupressora e interagem com células leucêmicas, promovendo sua sobrevivência, evasão imune e resistência à quimioterapia. Portanto, analisamos a expressão do gene MET em células de pacientes com LMA e avaliar a contribuição de um inibidor específico de MET, B2000 (Brigham University), para a progressão da leucemia com foco no microambiente (TME) inflamatório. Métodos: Expressão de mRNA de MET foram obtidos do estudo TCGA AML, realizado em células isoladas de 290 pacientes com LMA (idade média de 61 anos). Células mononucleares (MC) de sangue periférico (PB) humano e medula óssea (BM) (n = 10) foram obtidas de doadores saudáveis (HD) e usadas para o isolamento de monócitos. OCIAML3, MOLM13 e BMNC de HD e pacientes com LMA (n = 10) foram tratadas com B2000 e as taxas de apoptose foram avaliadas. Para obter macrófagos derivados de monócitos (MDMs), monócitos foram estimulados com GM-CSF/LPS/IFN-y ou M-CSF/IL-4, para polarização M1 ou M2, e usados em coculturas. BALB/c receberam uma injeção intraperitoneal de lipopolissacarídeo (LPS) e tratados com B2000. As análises foram realizadas por citometria de fluxo. Resultados: Observou-se alta expressão de MET em pacientes FAB M1/M3/M7 (p

Background and aims: Green tea catechins, particularly, epigallocatechin-3-gallate (EGCG), exhibit a range of beneficial effects, like apoptosis and differentiation-promoting effect which can help to counteract the uncontrolled proliferation and impaired differentiation trait of leukemia and myelodysplastic syndrome. This study aims to evaluate the effect of EGCG in vivo in mice model of myelodysplastic syndrome (NHD13 mice). Methods: To assess the effects of EGCG on MDS, 10-week-old female NUP98-HOXD13 mice and their littermate WT mice were treated with EGCG (50 mg/kg/day 5x/week i.p. for 4 weeks) or vehicle. Animals were sacrificed and bone marrow and spleen were obtained to evaluate immune system by flow cytometry and histology analyses. In addition, automated hematological counts and clinical biochemistry parameter analysis were done. Moreover, a 71-year-old man diagnosed with high-risk myelodysplastic syndrome (MSD) with 9% blast presence in the bone marrow, underwent a daily treatment of 800 mg EGCG intake, administered orally. Throughout this period, the patient underwent weekly regular clinical assessments and laboratory studies for monitor any potential toxicities. Additionally, peripheral blood samples were evaluated before and after the treatment to observe any alterations in immune cell composition. Results and discussion: NDH13+ exhibited, before the treatment, a reduction in the number of leukocytes, platelets and hemoglobin levels, which was in line with literature. EGCG treatment ameliorates the hematological parameters, visualized by the increase in the number of leukocytes (monocytes and neutrophils) and platelets; with no signals of toxicity - not morphological features nor enzyme levels changes in the liver. The expansion of Foxp3+ regulatory T cells leads the suppression of host antitumor responses and is a characteristic of progression to aggressive subtypes of the disease. Interestingly EGCG did not alter the percentage/frequency Foxp3+ regulatory T cells (CD4+CD25+ and/or CD4+FOXP3+), as well as the percentage/frequency of immature cells (CD45IntCD34+), in bone marrow and spleen. Our preliminary results showed an increase in extramedullary hematopoiesis in the spleen. One patient treated by EGCG for a month, exhibit reduction of regulatory T cells CD4+CD25+ and CD4+FOXP3+), and increased CD8+ T cells (CD3+CD8+) and natural killer cells (CD16+CD56+) accompanied with an increase of mature leukocytes (monocytes and neutrophils), with no changes in hemoglobin levels and platelets number. Conclusion: Our initial findings indicate that EGCG treatment may reduce ineffective hematopoiesis leading to an improvement of hematological parameters. These results align with the data obtained from one patient with high-risk myelodysplastic syndrome HR-SMD who received EGCG treatment. In summary, EGCG might be a promising adjuvant compound in the treatment of MDS. Further research and clinical studies are warranted to validate and explore its therapeutic benefits fully.

Objectives Studies have demonstrated high rates of mortality in people with proximal femoral fracture and SARS-CoV-2, but there is limited published data on the factors that influence mortality for clinicians to make informed treatment decisions. This study aims to report the 30-day mortality associated with perioperative infection of patients undergoing surgery for proximal femoral fractures and to examine the factors that influence mortality in a multivariate analysis.Setting Prospective, international, multicentre, observational cohort study.Participants Patients undergoing any operation for a proximal femoral fracture from 1 February to 30 April 2020 and with perioperative SARS-CoV-2 infection (either 7 days prior or 30-day postoperative).Primary outcome 30-day mortality. Multivariate modelling was performed to identify factors associated with 30-day mortality.Results This study reports included 1063 patients from 174 hospitals in 19 countries. Overall 30-day mortality was 29.4% (313/1063). In an adjusted model, 30-day mortality was associated with male gender (OR 2.29, 95% CI 1.68 to 3.13, p80 years (OR 1.60, 95% CI 1.1 to 2.31, p=0.013), preoperative diagnosis of dementia (OR 1.57, 95% CI 1.15 to 2.16, p=0.005), kidney disease (OR 1.73, 95% CI 1.18 to 2.55, p=0.005) and congestive heart failure (OR 1.62, 95% CI 1.06 to 2.48, p=0.025). Mortality at 30 days was lower in patients with a preoperative diagnosis of SARS-CoV-2 (OR 0.6, 95% CI 0.6 (0.42 to 0.85), p=0.004). There was no difference in mortality in patients with an increase to delay in surgery (p=0.220) or type of anaesthetic given (p=0.787).Conclusions Patients undergoing surgery for a proximal femoral fracture with a perioperative infection of SARS-CoV-2 have a high rate of mortality. This study would support the need for providing these patients with individualised medical and anaesthetic care, including medical optimisation before theatre. Careful preoperative counselling is needed for those with a proximal femoral fracture and SARS-CoV-2, especially those in the highest risk groups.Trial registration number NCT04323644

The repair of lesions of the central nervous system (CNS) varies widely throughout the animal kingdom. At the level of neuronal replacement lie the major differences in CNS regeneration. At one extreme are the amniote vertebrates (reptile, avian and mammalian groups), which have very limited capacity for neuronal replacement, and therefore for neural regeneration; at the other extreme, animals such as planarians (flatworms) and colonial tunicates can repair their entire CNS after major injuries. These differences can be attributed to the abundance of multipotent and/or pluripotent stem cells and/or undifferentiated precursors among the general cell population. In this review we discuss recent advancements in knowledge of regeneration of the CNS of invertebrates. We focus on ascidians, which are a sister group of vertebrates, but we also address other invertebrate groups. Because neurogenesis is central to the events that allow regeneration of the adult CNS, we address this issue focusing on crustaceans, which have provided a paradigm to study the mechanisms underlying this phenomenon. The attraction of hemocytes toward a neurogenic niche and respecification of these cells toward a neural fate has been strongly suggested. Based on recent and emerging research, we suggest that cells of the blood lineage are not only associated with the roles that are generally attributed to them, but are the cells that either signal other cell types to differentiate into neural cells, or even eventually themselves transdifferentiate into neural cells.

The repair of lesions of the central nervous system (CNS) varies widely throughout the animal kingdom. At the level of neuronal replacement lie the major differences in CNS regeneration. At one extreme are the amniote vertebrates (reptile, avian and mammalian groups), which have very limited capacity for neuronal replacement, and therefore for neural regeneration; at the other extreme, animals such as planarians (flatworms) and colonial tunicates can repair their entire CNS after major injuries. These differences can be attributed to the abundance of multipotent and/or pluripotent stem cells and/or undifferentiated precursors among the general cell population. In this review we discuss recent advancements in knowledge of regeneration of the CNS of invertebrates. We focus on ascidians, which are a sister group of vertebrates, but we also address other invertebrate groups. Because neurogenesis is central to the events that allow regeneration of the adult CNS, we address this issue focusing on crustaceans, which have provided a paradigm to study the mechanisms underlying this phenomenon. The attraction of hemocytes toward a neurogenic niche and respecification of these cells toward a neural fate has been strongly suggested. Based on recent and emerging research, we suggest that cells of the blood lineage are not only associated with the roles that are generally attributed to them, but are the cells that either signal other cell types to differentiate into neural cells, or even eventually themselves transdifferentiate into neural cells.

The authors review and discuss the medical treatment in the prehospital phase of acute myocardial infarction while working at the Referral Centre for Emergency Patients of the National Institute for Medical Emergencies (CODU-INEM). The diagnosis of myocardial infarction should be suspected immediately by the doctor on duty at CODU-INEM in all patients with acute thoracic pain. Then it is essential to send a medical team to the scene as soon as possible after the onset of symptoms in order to treat and to prevent acute fatal complications.

The Fgfr2cC342Y/+ Crouzon syndrome mouse model carries a cysteine to tyrosine substitution at amino acid position 342 (Cys342Tyr; C342Y) in the fibroblast growth factor receptor 2 (Fgfr2) gene equivalent to a FGFR2 mutation commonly associated with Crouzon and Pfeiffer syndromes in humans. The Fgfr2c C342Y mutation results in constitutive activation of the receptor and is associated with upregulation of osteogenic differentiation. Fgfr2cC342Y/+ Crouzon syndrome mice show premature closure of the coronal suture and other craniofacial anomalies including malocclusion of teeth, most likely due to abnormal craniofacial form. Malformation of the mandible can precipitate a plethora of complications including disrupting development of the upper jaw and palate, impediment of the airway, and alteration of occlusion necessary for proper mastication. The current paradigm of mandibular development assumes that Meckel’s cartilage (MC) serves as a support or model for mandibular bone formation and as a template for the later forming mandible. If valid, this implies a functional relationship between MC and the forming mandible, so mandibular dysmorphogenesis might be discerned in MC affecting the relationship between MC and mandibular bone. Here we investigate the relationship of MC to mandible development from the early mineralization of the mandible (E13.5) through the initiation of MC degradation at E17.7 using Fgfr2cC342Y/+ Crouzon syndrome embryos and their unaffected littermates (Fgfr2c+/+). Differences between genotypes in both MC and mandibular bone are subtle, however MC of Fgfr2cC342Y/+ embryos is generally longer relative to unaffected littermates at E15.5 with specific aspects remaining relatively large at E17.5. In contrast, mandibular bone is smaller overall in Fgfr2cC342Y/+ embryos relative to their unaffected littermates at E15.5 with the posterior aspect remaining relatively small at E17.5. At a cellular level, differences are identified between genotypes early (E13.5) followed by reduced proliferation in MC (E15.5) and in the forming mandible (E17.5) in Fgfr2cC342Y/+ embryos. Activation of the ERK pathways is reduced in the perichondrium of MC in Fgfr2cC342Y/+ embryos and increased in bone related cells at E15.5. These data reveal that the Fgfr2c C342Y mutation differentially affects cells by type, location, and developmental age indicating a complex set of changes in the cells that make up the lower jaw.

In this study, the Al–20at%Ca alloy was synthesized by mechanical alloy from the elemental powder mixture. Subsequently, the alloy particles were reacted at room temperature to determine the amount of hydrogen released. For these purposes, the powders reacted with different types of water, such as distilled water, tap water, and seawater, and also in the presence of NaCl and CaO additives. Both milled samples and reaction powders were analyzed by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), FT-IR, and Raman spectroscopy (RS). The XRD patterns of the powders prepared show a nanocrystalline alloy consisting of a solid-cubic solution of Al and the tetragonal intermetallic phase CaAl4. Studies of XRD and SEM, as well as direct measurements of H2, indicated that the best results of H2 generation were obtained when the alloy reacts with distilled water. Both NaCl and CaO additives improve hydrogen generation, reaching 100% efficiency in distilled water and seawater, and without induction time. Samples with a combination of NaCl and distilled water showed the best reaction times to generate the entire theoretical amount of hydrogen. The XRD and DSC–TGA standards also confirmed the presence of bayerite Al(OH)3 as a secondary reaction product.

The present research had as objective to investigate the perceptions of high school students regarding their knowledge as well as the use of pesticides. The investigation was carried out by means of a questionnaire to 83 high school students from the Juscelino Kubitschek de Oliveira State School in the district of Lucialva, in the municipality of Jauru-Mato Grosso, addressing issues related to pesticides as well as the frequency of use of these products, the use of personal protective equipment, knowledge about the harmful effects of agrotoxic use on the environment, human health and the evils of this product, and the need to work on the agrotoxic theme in the classroom. The analysis of the results showed that the interviewed students mostly have contact with agrotoxicos, little knowledge about the information and the handling of these products as well as the inappropriate use of Personal Protective Equipment being critical the risk of intoxication by the agrotoxicos. Therefore, it is necessary to work on this theme at school and in the community in order to raise awareness and raise awareness among families about the harmfulness and the risks of poisoning with pesticides.

Electrical storm (ES) is a potentially lethal syndrome defined as three or more sustained episodes of ventricular tachycardia or ventricular fibrillation within 24 h. There are multiple inciting factors for ES, one of which involves excess catecholamine (endogenous and exogenous) effects. Exogenous catecholamines used for hemodynamic support can paradoxically engender or exacerbate an underling arrhythmia leading to ES. We report on an 63-year-old man who presented for repair of an ascending aortic dissection. After cardiopulmonary bypass separation assisted with high-dose epinephrine, ES developed requiring over 40 defibrillatory shocks. The epinephrine infusion was held and within 5 min, the ES self-terminated. ES in the context of cardiovascular surgery with the use of epinephrine for hemodynamic support has not be previously reported. Clinicians need to be cognizant of the seemingly paradoxical effect of epinephrine to induce ES. Initial ES treatment involves acute stabilization (treating or removing exacerbating factors (i.e., excess catecholamines)).

Background: Acute respiratory infections are the leading cause of mortality in children worldwide, especially in developing countries. Pneumonia accounts for 16% of all deaths of children under 5 years of age and was the cause of death of 935 000 children in 2015. Despite its frequency and severity, information regarding its etiology is limited. The aim of this study was to identify respiratory viruses associated with community-acquired pneumonia (CAP) in children younger than 5 years old. Methods: One thousand four hundred and four children younger than 5 years of age with a clinical and/or radiological diagnosis of CAP in 11 hospitals in Mexico were included. Nasal washes were collected, placed in viral medium, and frozen at −70 °C until processing. The first 832 samples were processed using the multiplex Bio‐Plex/Luminex system and the remaining 572 samples using the Anyplex multiplex RT-PCR. Clinical data regarding diagnosis, clinical signs and symptoms, radiographic pattern, and risk factors were obtained and recorded. Results: Of the samples tested, 81.6% were positive for viruses. Respiratory syncytial virus (types A and B) was found in 23.7%, human enterovirus/rhinovirus in 16.6%, metapneumovirus in 5.7%, parainfluenza virus (types 1–4) in 5.5%, influenza virus (types A and B) in 3.6%, adenovirus in 2.2%, coronavirus (NL63, OC43, 229E, and HKU1) in 2.2%, and bocavirus in 0.4%. Co-infection with two or more viruses was present in 22.1%; 18.4% of the samples were negative. Using biomass for cooking, daycare attendance, absence of breastfeeding, and co-infections were found to be statistically significant risk factors for the presence of severe pneumonia. Conclusions: Respiratory syncytial virus (types A and B), human enterovirus/rhinovirus, and metapneumovirus were the respiratory viruses identified most frequently in children younger than 5 years old with CAP. Co-infection was present in an important proportion of the children.

La economía artificial es uno de los métodos o enfoques de investigación para el estudio de sistemas socioeconómicos complejos con mayor crecimiento durante los últimos años. Este artículo presenta una visión crítica sobre sus características, su potencial y los riesgos relativos al uso de esta metodología. Para ello, encontramos útil relacionar y comparar a la economía artificial con la economía teórica más tradicional. Desde nuestro análisis, la economía teórica y la economía artificial comparten los mismos objetivos, presentan menos diferencias metodológicas de las que a primera vista pudiera parecer, y sus aproximaciones son sin duda complementarias. ------------------------------------ Artificial Economics is one of the fastest growing approaches to analyse complex socio-economic systems. In this paper we present our views on the distinguishing features of Artificial Economics and on its relation with Theoretical Economics — the field that in our opinion lies closest to Artificial Economics. In this context, we discuss various reasons why conducting research on Artificial Economics may be worthwhile, and provide general guidelines on how to go about it. Our view is that Artificial Economics and Theoretical Economics share the same goals, do not differ conceptually as much as it is sometimes perceived, and their approaches are certainly complementary.

Recent studies suggest that some monoclonal antibodies (mAbs) specific for ebolavirus glycoprotein (GP) can protect experimental animals against infections. Most mAbs isolated from ebolavirus survivors appeared to target the glycan cap or the stalk region of the viral GP, which is the envelope protein and the only antigen inducing virus-neutralizing antibody response. Some of the mAbs were demonstrated to be protective in vivo. Here, a panel of mAbs from four individual survivors of ebolavirus infection that target the glycan cap or stem region were selected for investigation of the mechanisms of their antiviral effect. Comparative characterization of the inhibiting effects on multiple steps of viral replication was performed, including attachment, post-attachment, entry, binding at low pH, post-cleavage neutralization of virions, viral trafficking to endosomes, cell-to-cell transmission, viral egress, and inhibition when added early at various time points post-infection. In addition, Fc-domain related properties were characterized, including activation and degranulation of NK cells, antibody-dependent cellular phagocytosis and glycan content. The two groups of mAbs (glycan cap versus stem) demonstrated very different profiles of activities suggesting usage of mAbs with different epitope specificity could coordinate inhibition of multiple steps of filovirus infection through Fab- and Fc-mediated mechanisms, and provide a reliable therapeutic approach.

Summary: Some monoclonal antibodies (mAbs) recovered from survivors of filovirus infections can protect against infection. It is currently unknown whether natural infection also induces some antibodies with the capacity for antibody-dependent enhancement (ADE). A panel of mAbs obtained from human survivors of filovirus infection caused by Ebola, Bundibugyo, or Marburg viruses was evaluated for their ability to facilitate ADE. ADE was observed readily with all mAbs examined at sub-neutralizing concentrations, and this effect was not restricted to mAbs with a particular epitope specificity, neutralizing capacity, or subclass. Blocking of specific Fcγ receptors reduced but did not abolish ADE that was associated with high-affinity binding antibodies, suggesting that lower-affinity interactions still cause ADE. Mutations of Fc fragments of an mAb that altered its interaction with Fc receptors rendered the antibody partially protective in vivo at a low dose, suggesting that ADE counteracts antibody-mediated protection and facilitates dissemination of filovirus infections. : In this paper, Kuzmina et al. demonstrate that filovirus antibodies from human survivors present at low concentrations are capable of enhancement of infection, suggesting that low levels of antibodies in humans may facilitate virus spread. The enhancement can be caused by antibodies of various epitope specificities, neutralizing capacities, and subclasses. Keywords: Ebola virus, filovirus, antibody, enhancement of infection, FC receptor, epitope

Introduction – The estimate of relative renal function (RRF) through scintigraphy with dimercaptosuccinic acid labelled with Technetium-99 metastable (99mTc-DMSA) may be influenced by kidney depth (KD), due to attenuation by soft tissue surrounding the kidneys. Considering that rarely this KD is known, several methods for attenuation correction (AC) have been developed, namely those using empirical formulae, such as Raynaud, Taylor or Tonnesen methods, or by direct calculation of the geometric mean (GM). Objectives – To identify the influence of different AC methods on RRF estimateby scintigraphy with 99mTc-DMSA and to evaluate the respective KD variability. Methods: Thirty-one patients were referred for 99mTc-DMSA scintigraphy and underwent the same acquisition protocol. Processing was performed by 2 independent operators, three times per exam, changing for the same processing the methods for the FRR determination: Raynaud’s method, Taylor’s method, Tonnesen´s method, GM and without AC (WAC). Friedman’s test was used to identify the influence of the different AC methods on RRF estimate and Pearson’s correlation test was used to evaluate the association and significance between KD and the variables age, weight and height. Results – Friedman’s test indicated that there were significant differences between methods (p=0.000), except for WAC/Raynaud, Tonnesen/GM and Taylor/GM (p=1.000) comparisons, for both kidneys. Pearson’s test showed a strong positive correlation between weight and the three methods of KD estimation. Conclusions – Taylor’s method, regarding the three methods of KD calculation, is the closest to GM. The choice of the attenuation correction method influences significantly the quantitative parameters of FRR.

The current challenges in developing a cost-effective bioethanol industry include the production of not only high-volume, low cost biofuels but also high-value products with minimal downstream waste. The up-grading of side-stream lignins from bioethanol production plants to novel high-value products will improve the profitability of the bioethanol industry; to do that, a precise understanding of lignin is required. In the present study, lignin-enriched fermentation residues from bioethanol production (steam explosion pretreatment, saccharification, and fermentation) of fast-growing poplar and forage sorghum were characterized. In addition to the purity and composition, lignin structure (syringyl/guaiacyl (S/G) ratio, inter-unit linkages) was also analyzed with spectroscopy techniques such as Fourier transform infrared and two-dimensional nuclear magnetic resonance. Bioethanol processing and feedstock origins seemed to be the main factors determining the purity, composition, and structure of lignins. Residual lignins from poplar and forage sorghum contained significant amounts of sugar and protein impurities. Poplar lignin showed a very high S/G ratio associated with p-hydroxybenzoate. A lower S/G ratio together with H lignin units and p-hydroxycinnamates (p-coumarate and ferulate) was observed for forage sorghum lignin. The main inter-unit linkages present in both lignins were β-O-4´ aryl ether followed by resinols and phenylcoumarans.

Fatal outcomes of Ebola virus (EBOV) infections are typically preceded by a 'sepsis-like' syndrome and lymphopenia despite T cells being resistant to Ebola infection. The mechanisms that lead to T lymphocytes death remain largely unknown; however, the degree of lymphopenia is highly correlative with fatalities. Here we investigated whether the addition of EBOV or its envelope glycoprotein (GP) to isolated primary human CD4+ T cells induced cell death. We observed a significant decrease in cell viability in a GP-dependent manner, which is suggestive of a direct role of GP in T cell death. Using immunoprecipitation assays and flow cytometry, we demonstrate that EBOV directly binds to CD4+ T cells through interaction of GP with TLR4. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the significant upregulation of pathways associated with interferon signaling, pattern recognition receptors and intracellular activation of NFκB signaling pathway. Both transcriptome analysis and specific inhibitors allowed identification of apoptosis and necrosis as mechanisms associated with the observed T cell death following exposure to EBOV. The addition of the TLR4 inhibitor CLI-095 significantly reduced CD4+ T cell death induced by GP. EBOV stimulation of primary CD4+ T cells resulted in a significant increase in secreted TNFα; inhibition of TNFα-mediated signaling events significantly reduced T cell death while inhibitors of both necrosis and apoptosis similarly reduced EBOV-induced T cell death. Lastly, we show that stimulation with EBOV or GP augments monocyte maturation as determined by an overall increase in expression levels of markers of differentiation. Subsequently, the increased rates of cellular differentiation resulted in higher rates of infection further contributing to T cell death. These results demonstrate that GP directly subverts the host's immune response by increasing the susceptibility of monocytes to EBOV infection and triggering lymphopenia through direct and indirect mechanisms.