Introdução: A mielofibrose (MF), a trombocitemia essencial (TE) e a policitemia vera (PV) são neoplasias mieloproliferativas (NMP) associadas a uma elevada carga de sintomas constitucionais, redução da qualidade de vida e da sobrevida global. Em geral, estes pacientes têm um risco muito aumentado de eventos trombóticos e a progressão para MF (no caso de pacientes com TE e PV) ou leucemia mieloide aguda permanece como uma grande preocupação. Até o momento, existem poucos dados na literatura sobre a epidemiologia das NMP no Brasil. Objetivo: Analisar e descrever dados epidemiológicos, clínicos, laboratoriais e complicações de pacientes diagnosticados com NMP em 5 centros brasileiros. Métodos: Estudo retrospectivo e prospectivo, em 5 centros no Brasil, com dados coletados do prontuário e incluídos em ficha clínica eletrônica na plataforma REDCap. A análise foi realizada com o pacote estatístico R. Resultados: Foram incluídos 201 pacientes com NMP, dos quais, 56.7% eram do sexo feminino e 43.3% do sexo masculino, com mediana de idade ao diagnóstico igual a 63 anos (IQR: 51 a 72). A NMP mais comum foi a mielofibrose (40,2%), que incluiu MF primária, secundária e pré-fibrótica, seguida da TE (34.2%) e PV (25.6%). A mutação mais frequente foi JAK2 que estava presente em 96%, 58.8% e 50% dos pacientes com PV, MF e TE, respectivamente (p < 0.001). CALR e MPL estavam presentes em 30.9% e 4.4% dos pacientes com TE, e em 29.5% e 2.6% daqueles com MF, respectivamente. Em 84.8% dos pacientes com mielofibrose o cariótipo não foi realizado. Os sintomas mais frequentes ao diagnóstico foram fadiga (32%), emagrecimento (20.8%) e prurido (18%). Esplenomegalia estava presente em 33.5% dos pacientes ao diagnóstico. A complicação mais frequente ao diagnóstico foi trombose (12%) e evolução para leucemia aguda (LA) ocorreu em 2.8%. Conclusão: Entre os resultados preliminares encontrados, destacamos que: (a) os resultados de idade, gênero e a frequência de PV e TE foram semelhantes aos descrito na literatura; (b) a fadiga é também na população brasileira o sintoma mais frequente e severo; (c) a maior prevalência de MF pode estar correlacionada ao maior diagnóstico de pacientes com MF pré fibrótica; e (d) a mutação em MPL teve uma frequência menor comparada a outros estudos.
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.
Aims: The establishment of a multi-user laboratory for metabolomics, proteomics, and lipidomics at the university aims to improve the quality of life for patients with sickle cell disease by enhancing transfusion practices. The primary objective is to utilize these advanced technologies to identify biomarkers that can guide the personalization of transfusions, reducing complications and optimizing treatments. Materials and methods: The new laboratory is equipped with state-of-the-art technologies, including mass spectrometry and high-performance liquid chromatography. It features a structured biobank with stored samples from a large cohort of sickle cell disease participants, collected before and after transfusions for analysis. This setup allows for various analyses, particularly in the search for biomarkers for sickle cell disease. Metabolomics and proteomics are applied to detect changes in metabolic and protein profiles, while lipidomics identifies alterations in plasma lipids. The methodology includes comparing the obtained data with clinical parameters of the patients, aiming to correlate biochemical changes with clinical outcomes of transfusions. Results: Preliminary results from pilot projects conducted in the newly implemented laboratory indicate that metabolomics can reveal significant alterations in the metabolic profiles of patients with sickle cell disease, identifying biomarkers that can predict adverse reactions to transfusions and complications of the disease. Proteomic analysis highlights specific proteins that change in response to transfusions, providing insights into treatment efficacy and potential new therapeutic targets. Lipidomics contributes to understanding changes in lipids that may impact the cardiovascular health of patients. Discussion: The results obtained so far suggest that integrating metabolomics, proteomics, and lipidomics in the context of transfusions can offer a more personalized and effective approach to treating patients with sickle cell disease. The identification of specific biomarkers allows for adjusting transfusions according to the individual needs of patients, potentially reducing complications and improving clinical outcomes. Additionally, the laboratory serves as a platform for future research that can explore new aspects of sickle cell disease biology and transfusions. Conclusion: The structuring of this new laboratory represents a significant advancement for research in the quest to improve the quality of life for patients with sickle cell disease. Metabolomic, proteomic, and lipidomic technologies not only enable a deeper understanding of transfusion responses but also pave the way for treatment personalization. These advances highlight the importance of technological infrastructure in clinical practice and research, evidencing the positive impact on the health of patients with sickle cell disease. Furthermore, this infrastructure will support researchers at the university and other institutions.
Objetivo: Descrição de caso de síndrome MonoMAC em paciente com mutação GATA2 e Síndrome de Falência Medular com mutação TP53. Material e métodos: Relato de caso descritivo, retrospectivo e observacional realizado a partir de dados obtidos por meio da análise de prontuários eletrônicos de paciente internado em instituição privada de referência em Hematologia no estado de São Paulo. Resultados: Paciente sexo feminino, 24 anos, iniciou quadro de febre de origem indeterminada há 60 dias da admissão, com quadro de linfonodomegalia supraclaviular esquerda, lesões cutâneas nodulares em membro superior esquerdo e perda de peso nos últimos três meses. Referia acompanhamento irregular em outro serviço devido a histórico de plaquetopenia aos 19 anos e antecedente familiar de 1 irmão com diagnóstico de mielodisplasia e 1 irmão que foi a óbito aos 19 anos por LMA. Foi investigada com biópsia de medula óssea evidenciando mielodisplasia com celularidade de 40%, sequenciamento de exoma com GATA2 e CHEK2 mutados e DEB test sem alterações. Na admissão no nosso serviço, paciente apresentava hemograma com anemia normocromica e normocítica, linfopenia e monocitopenia, DHL aumentado e sorologias para HIV, HBV, HCV, CVM e EBV negativas. Também realizadas sorologias para doenças fúngicas (Histoplasmose, Paracococcidiomicose e Criptococose) com resultado negativo assim como sorologia para Bartonelose. Novo aspirado de medula óssea evidenciou celularidade normal com dismegacariopoese e série granulocítica hipogranular. Não foram encontradas populações anômalas na imunofenotipagem de medula óssea e o cariótipo mostrou 47, XX, +8. Realizado FISH para SMD/LMA com trissomia do 8 e NGS com mutação missense em região hotspot de TP53 (VAF 45.4%). Realizada linfadenectomia supraclaviular à esquerda além de biópsia excisional de nódulo de MSE e enviados materiais para análise com crescimento de Mycobacteroides abscessus em cultura. Foi iniciado tratamento para micobacteriose com rifampicina, etambutol, levofloxacino, amicacina e claritromicina. Paciente evoluiu com rash cutâneo difuso e biópsia de pele compatível com Síndrome de DRESS, tratada adequadamente com corticoterapia e retomado tratamento droga-a-droga para avaliar reações adversas. Em última avaliação ambulatorial, paciente sem citopenias e sem indicação de tratamento específico para SMD no momento, em uso antibioticoterapia para tratamento de micobacteriose e aguardando resolução de quadro infeccioso para programar transplante alogênico de medula óssea devido a Síndrome de Falência medular com mutação TP53 que confere alto risco para doença. Discussão/Conclusão: A síndrome de monocitopenia e infecção micobacteriana (MonoMAC) é causada por mutações heterozigóticas em GATA2, e resulta na perda da função de um gene que regula a hematopoiese e é associada a um risco aumentado de infecções oportunistas e malignidades hematológicas (SMD/LMA familiar). Essa imunodeficiência genética tem múltiplas manifestações, incluindo anemia aplástica, doença micobacteriana, infecções fúngicas, doenças pulmonares, câncer de células escamosas associado ao papilomavírus humano. A mortalidade da síndrome MonoMAC pode chegar a 28%. O transplante alogênico de células-tronco hematopoiéticas tem se mostrado uma estratégia eficaz para reconstituir os compartimentos hematopoiéticos esgotados e reverter o fenótipo clínico observado nos pacientes afetados.
O objetivo deste estudo foi o de relatar a extração dentária de um paciente com a anemia de Blackfan Diamond. Paciente VGSL, 26 anos, sexo feminino, portador da anemia de Blackfan-Diamond com aplasia de medula óssea, histórico de recorrentes internações para transfusões de concentrados de hemácias e plaquetas, alérgico a prednisona. Durante uma consulta ambulatorial de rotina, referiu dor de dente exacerbada nos dentes 26, 36 e 37 e a equipe de odontologia foi solicitada. No exame clínico e radiográfico apresentou lesão extensa de cárie, dor continua e localizada, espessamento do ligamento periodontal. Foi solicitada a internação para a extração dentária e realização da profilaxia antibiótica em ambiente hospitalar. Apresentava eritrócitos 2,94 M/μL, hemoglobina 8,2 g/dL, hematócrito a 24% e plaquetas a 22 k/μL. No dia da extração a paciente recebeu um concentrado de plaquetas filtradas e dois concentrados de hemácias, duas horas antes do procedimento e utilizou-se da manobra compressiva e Exogel® como medida hemostática. A paciente foi orientada a realizar higiene oral com clorexedina 0,12%, 12/12 horas, por duas semanas. A mesma permaneceu por uma semana em vigilância após o procedimento, recebeu alta hospitalar e segue em acompanhamento clínico e radiográfico por via ambulatorial. Devido ao quadro de anemia instável onde a paciente oscila com hemoglobina entre 4 e 9 g/dL, optamos pela transfusão previa ao procedimento cirúrgico e foi prescrito antibiótico profilaxia para diminuir risco de infecção do sitio cirúrgico no pós operatório. Como a paciente necessitava de extração de três molares, optamos também pela reposição previa de plaquetas já que a mesma se encontrava abaixo de 30 k/μL assim diminuindo o risco de sangramento trans e pós operatório. Com as medidas transfusionais previas ao procedimento cirúrgico, associado a medidas hemostáticas locais e um rígido controle pós operatório, a paciente seguiu com ausência e sangramento trans e pós-operatórios e sem quadro de infecção local do sitio cirúrgico.
Introduction: Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) patients are exposed to acute and chronic nephrotoxic events (drugs, hypotension, infections, and microangiopathy). The need for hemodialysis (HD) may be associated with high mortality rates. However, the risk factors and clinical impact of HD are poorly understood. Aim: To analyze survival and risk factors associated with HD in allo-HSCT Patients and methods: single-center cohort study 185 (34 HD cases versus 151 controls) consecutive adult allo-HSCT patients from 2007-2019. We performed univariate statistical analysis, then logistic regression and competing risk regression were used to multivariate analysis. Survival was analyzed by Kaplan-Meier and Cox proportional-hazards models. Results: The one-year HD cumulative incidence was 17.6%. Univariate analysis revealed that HD was significantly associated with male gender, age (p 0.056), haploidentical donor, grade II-IV acute GVHD, polymyxin B, amikacin, cidofovir, microangiopathy, septic shock (norepinephrine use) and steroid exposure. The median days of glycopeptides exposure (teicoplanin/vancomycin) was 16 (HD) versus 10 (no HD) (p 0.088). In multivariate analysis, we found: norepinephrine (hazard ratio, HR:3.3; 95% confidence interval, 95%CI:1.2-8.9; p 0.024), cidofovir drug (HR:11.0; 95%CI:4.6- 26.0; p < 0.001), haploidentical HSCT (HR:1.94; 95%CI:0.81-4.65; p 0.14) and Age (HR:1.01; 95%CI: 0.99-1.03; p 0.18) . The HD group had higher mortality rate (HR:6.68; 95% CI: 4.1-10.9; p < 0.001). Conclusion: HD was associated with decreased survival in allo-HSCT. Carefully use of nephrotoxic drugs and improving immune reconstitution could reduce severe infections (shock) and patients requiring cidofovir, which taken together may result in lower rates of HD, therefore improving survival.
Thromboembolic diseases are usually inherited in the family. The tendency to repeat in an individual is a phenomenon that allows it to be studied. The inheritance and recurrence of thromboembolic diseases, of course, have individual risk factors for this occurrence. In the past, the ABO blood group was only needed for transfusion and organ transplant therapy. Over time, scientists think that blood type is a risk factor for certain diseases, including thromboembolism. Many studies divide between type O and non-O blood groups, both of which are distinguished by the presence of antigens on the cell surface and antibodies in the plasma of individuals. Type O does not have A, B antigens but has antibodies against A, B antigens, and vice versa for the non-O type. Many studies have shown that the non-O blood group has a risk factor for thromboembolic diseases, commonly due to higher levels of von Willebrand factor (VWF) and factor VIII (FVIII). These thromboembolic events can occur in arteries or venous. Thromboembolic manifestations are often associated with cardiovascular diseases for arterial thrombosis; and deep vein thrombosis (DVT) and pulmonary embolism (PE) for venous thromboembolism (VTE).
Abstract Background Both coronavirus disease‐2019 (COVID‐19) and myeloproliferative neoplasms (MPNs) are associated with systemic inflammation and risk of thrombosis. Risk of thrombosis in patients with COVID with and without MPNs has not been extensively studied. Methods Retrospective cohort study of 44 patients with MPNs and 1114 patients without MPNs positive for SARS‐COV‐2. Outcomes were arterial thrombosis (AT), venous thromboembolism (VTE), bleeding, and death. Time‐to‐event analysis was performed using competing risk regression model and Cox proportional hazards. Results AT occurred more frequently in patients with MPN (7% vs. 1%, p = 0.03). Rates of VTE (7% vs. 5%, p = 0.73), bleeding (7% vs. 2%, p = 0.06), and death (9% vs. 6%, p = 0.32) were similar. MPN patients were older and had more cardiovascular comorbidities. After time‐to‐event competing‐risk regression adjusting for age, MPN patients had higher risk of AT (subdivision hazards ratio 3.95, 95% CI 1.09–14.39) but not VTE, bleeding, or death. Conclusions Among patients with COVID‐19, MPN patients had higher risk of arterial thrombosis but not VTE, bleeding, and death compared with non‐MPN patients. Larger studies are needed to confirm our findings given the limited sample size.
Shilpi Yadav, Jonathan K. Williamson, Maria A. Aronova
et al.
Platelets are small, anucleate cell fragments that are central to hemostasis, thrombosis, and inflammation. They are derived from megakaryocytes from which they inherit their organelles. As platelets can synthesize proteins and contain many of the enzymes of the secretory pathway, one might expect all mature human platelets to contain a stacked Golgi apparatus, the central organelle of the secretory pathway. By thin section electron microscopy, stacked membranes resembling the stacked Golgi compartment in megakaryocytes and other nucleated cells can be detected in both proplatelets and platelets. However, the incidence of such structures is low and whether each and every platelet contains such a structure remains an open question. By single-label, immunofluorescence staining, Golgi glycosyltransferases are found within each platelet and map to scattered structures. Whether these structures are positive for marker proteins from multiple Golgi subcompartments remains unknown. Here, we have applied state-of-the-art techniques to probe the organization state of the Golgi apparatus in resting human platelets. By the whole cell volume technique of serial-block-face scanning electron microscopy (SBF-SEM), we failed to observe stacked, Golgi-like structures in any of the 65 platelets scored. When antibodies directed against Golgi proteins were tested against HeLa cells, labeling was restricted to an elongated juxtanuclear ribbon characteristic of a stacked Golgi apparatus. By multi-label immunofluorescence microscopy, we found that each and every resting human platelet was positive for cis, trans, and trans Golgi network (TGN) proteins. However, in each case, the proteins were found in small puncta scattered about the platelet. At the resolution of deconvolved, widefield fluorescence microscopy, these proteins had limited tendency to map adjacent to one another. When the results of 3D structured illumination microscopy (3D SIM), a super resolution technique, were scored quantitatively, the Golgi marker proteins failed to map together indicating at the protein level considerable degeneration of the platelet Golgi apparatus relative to the layered stack as seen in the megakaryocyte. In conclusion, we suggest that these results have important implications for organelle structure/function relationships in the mature platelet and the extent to which Golgi apparatus organization is maintained in platelets. Our results suggest that Golgi proteins in circulating platelets are present within a series of scattered, separated structures. As separate elements, selective sets of Golgi enzymes or sugar nucleotides could be secreted during platelet activation. The establishment of the functional importance, if any, of these scattered structures in sequential protein modification in circulating platelets will require further research.