Determinação da sobrecarga de ferro na talassemia pela IRM hepática e ferritina

Accumulation of iron in thalassemia causes organ damage and reduces patient survival due to heart lesions in the second decade of life. Iron deposits are monitored by direct (biopsy) and indirect methods (ferritin) with sequential data being better than isolated measurements. This paper compares two indirect measurements of iron overload; a single hepatic iron concentration (HIC) by magnetic resonance and mean ferritin levels over four years. A retrospective study of 25 patients from the Centro Regional de Hemoterapia in Ribeirão Preto, Brazil was carried out. High HIC (above 7 mg per gram of dry weight) was found in 20 patients and high mean serum ferritin (above 2500 μg/L) in 10 patients. Stratification into three levels (low, moderate and high) of iron overload gave similar results in both tests. Many other factors influence de degree of iron overload in thalassemia. No correlation was found using a non-parametric statistical test between HIC and mean serum ferritin. Both methods provide better planning of chelation therapy.O acúmulo de ferro na talassemia causa lesões orgânicas e reduz a sobrevida do paciente por lesão cardíaca na segunda década da vida, e tem sido avaliado por medidas diretas (biópsia) e indiretas (ferritina). As medidas isoladas carecem de valor, sendo preferidas as sequenciais. Este trabalho pretende comparar medidas indiretas de sobrecarga de ferro, uma medida da concentração de ferro hepático por ressonância magnética, e a ferritina sérica média dos últimos quatro anos. Trata-se de estudo retrospectivo de 25 pacientes do Centro Regional de Hemoterapia, em Ribeirão Preto, Brasil. Encontrou-se em vinte pacientes ferro hepático acima de 7 mg/g peso seco e ferritina média elevada acima de 2.500 ug/l em dez. Estratificação em três níveis de sobrecarga (leve, moderada e grave) produziu resultados semelhantes em ambos os testes. Vários outros fatores influenciam o grau de sobrecarga de ferro na talassemia. Não houve correlação significativa com aplicação de testes não-paramétricos. Ambos os métodos usados concomitantemente levarão a um melhor planejamento da terapia quelante.FapespCNPqCapes and Fundação Hemocentr

Modification of T lymphocytes with lentiviral vectors for expression of anti-CD19 chimeric antigen receptor (CAR)

The use of immunotherapy with modified T lymphocytes with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemias and lymphomas resistant to chemotherapy. CAR possess an extracellular domain derived from variable regions of antibodies and costimulation intracellular domains of T lymphocytes. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors as well as normal B cells, but not in other types of cells. Recent clinical studies involving anti-CD19 CAR T-cells have shown excellent responses in a variety of B-cell tumors, even in patients with relapse after high-dose chemotherapy. This study aimed to produce CD4+ lymphocyte lineage Jurkat (ATCC® TIB-152 ™) modified with a second generation anti-CD19 CAR with 4-1BB as intracellular costimulation domain. Lentiviral vectors were produced in HEK293T (ATCC® CRL-3216 ™) transiently transfected with plasmids containing the coding sequence of the CAR, viral envelope VSV-G, and viral capsid. The viral titer was calculated by real time PCR after transduction of HEK293T cells, resulting in 1.65 x 105 IU/mL. The literature indicates an MOI (multiplicity of infection) from 5 to 10 IU/cell for transduction of lymphocytes. A new batch of virus was produced, and the supernatant was ultracentrifuged at 19200 rpm (Beckman Coulter, SW28 rotor) in order to concentrate the viral particles. The viral titer of the concentrated batch was 1.26 x 108 IU/mL. This new titer is compatible with the necessary to infect 107 cells, amount of pre-expansion cells necessary to obtain the number of cells suitable for infusion into patients (2.5 x 109 to 5 x 109 cells). Then, the infection of Jurkat was performed in a 6-well plate with RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 µg/mL Polybrene®, and centrifugation at 1000 rpm for 20 minutes at room temperature. After 16 hours of incubation (37°C, 5% CO2 and 85% humidity), the medium was exchanged for fresh RPMI 1640 10% FBS. After additional 48 hours of incubation under the same conditions, the cells were collected and was their DNA was extracted. We obtained by real-time PCR that the number of integrated viral copies per genome was 35.3 ± 4.5 (mean ± standard deviation) for transduction with MOI of 5 IU/cell. While for MOI of 10 IU/cell, it was obtained 42.6 ± 0.1 copies per genome. It was observed that there was not a significant increase in viral copies when the MOI increased from 5 to 10. This may occur because cell’s surface receptors have been saturated by the large number of viruses. The lentiviral vector used by us has been shown to transduce T lymphocyte satisfactorily. The next steps of the study are the transduction of T lymphocytes from healthy donors and verification of the CAR receptor effectiveness to bind to CD19 of cell B lymphocyte lineages. Grant #2016/08374-5, São Paulo Research Foundation (FAPESP)

Experimental and economic evaluation of different culture systems for mesenchymal stromal/stem cell expansion for clinical applications

The translation of cell therapies into clinical practice requires a scalable, efficient and cost-effective manufacturing process. This study presents an integrated experimental and cost analysis of different cell culture technologies for commercial manufacture of a novel umbilical cord-derived cell therapy, currently in early phase clinical trials for the treatment of acute graft-versus-host disease (aGvHD). The experimental analysis assessed the expansion and harvest potential of mesenchymal stromal cells (MSCs), derived from umbilical cord matrix (UCM-MSCs), in different scalable cell culture technologies: a multi-layer vessel (ML), a stirred tank bioreactor with microcarrriers (STR), a hollow fiber bioreactor (HF) and a packed-bed bioreactor (PB). The presentation will highlight differences in cell proliferation rate, expansion fold and harvesting efficiency across the technologies. The cells retained their functional properties post culture in all the technologies evaluated. The experimental results were incorporated into a bioprocess economics tool comprising a stochastic cost of goods (COG) and sizing model to evaluate the commercial economic feasibility and robustness of the technologies. The financial and risk rank orders predicted by the tool will be presented, as well as their sensitivity to the reimbursement scenario selected. The model determined industrially relevant scenarios for which no technology will yield a satisfactory gross margin, indicating that many studies are still needed to establish an optimized manufacturing process

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

Abstract\ud \ud \ud \ud Background\ud \ud Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C.\ud \ud \ud \ud Results\ud \ud We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection.\ud \ud \ud \ud Conclusion\ud \ud Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.The authors would like to acknowledge FAPESP (2008/51505-7) and FINEP (01.07.0652.00) for financial support

Development of a molecular platform for HTLV confirmatory diagnosis: importance of the internal amplification control

Finnancial Support: CTC/INCTC, FAPESP, FUNDHERP, FINEP

Epidemiological factors related to slow progression of the acquired immune deficiency syndrome (AIDS)

Com o objetivo de identificação de fatores envolvidos na progressão lenta para aids, realizou-se estudo transversal para avaliação de dados epidemiológicos de indivíduos infectados pelo Vírus da Imunodeficiência Humana tipo 1 (HIV-1), atendidos no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto-USP. Foram selecionados pacientes, conforme critérios definidos, constituindo duas populações: população 1, composta por lentos progressores (P1), que possuía anticorpos anti-HIV há mais de oito anos e com ocorrência de menos de duas doenças oportunistas no último ano, e a população 2 (P2), pacientes rápidos progressores, com diagnóstico de infecção pelo HIV e doença manifesta a menos de dois anos e com mais de duas doenças oportunistas, diagnosticadas no último ano. Todos os indivíduos foram submetidos a questionário, contendo dados demográficos, profissão, ocorrência de outras doenças sexualmente transmissíveis, forma de contágio, data de diagnóstico e hábitos. O período do estudo foi de março de 1998 a outubro de 1999. Obtivemos na P1: doze homens e quatro mulheres, idade média 30,7 anos, forma de contágio predominantemente sangüínea, tempo de progressão da doença 10,5 anos; P2: 12 homens e 4 mulheres; idade média 34,8 anos, forma de contágio predominantemente sexual, tempo de progressão da doença de 1,5 anos. Tabagismo foi o principal vício em ambas as populações. Quando interrogados sobre a causa do bom estado de saúde, os indivíduos da P1 responderam estar ela relacionada à fé e ao uso adequado das medicações. Os pacientes da P2 não foram interrogados sobre a causa de seu estado de saúde. Quanto à prática sexual, nove pacientes da P1 mantinham relações, sendo cinco sem uso regular do preservativo. Na P2, apenas um paciente utilizava preservativo. Dois pacientes da P1 e um da P2 revelaram ter apresentado DST, Sífilis e Papiloma Vírus Humano. Em vista do reduzido número de pacientes, não foi possível estabelecer associação entre as variáveis estudadas e os padrões de progressão da doença. Os dados sobre hábitos não parecem contribuir para o padrão de desenvolvimento da doença. O estudo oferece um perfil epidemiológico dessas populações de pacientes.To determine the factors involved in slow progression to aids, a transverse study was conducted for the evaluation of the epidemiological data of individuals infected with type 1 human immunodeficiency virus seen at the University Hospital of the Faculty of Medicine of Ribeirão Preto-USP. Patients were choosed in conformity some judgment, establish two populations: population 1, consisting of slow progressors (P1), had been carrying HIV antibodies for more than 8 years, with the occurrence of less than 2 opportunistic diseases in the last year, and population 2 (P2), consisting of rapid progressors, had a diagnosis of HIV infection and overt disease detected less than 2 years before and more than 2 opportunistic diseases diagnosed during the last year. All patients responded to a questionnaire concerning demographic data, profession, occurrence of other sexually transmissible diseases, form of contagion, date of diagnosis, and habits. The study was conducted from March 1998 to October 1999. We obtain in the P1: 12 men and 4 women, mean age 30.7 years, predominant form of contagion: blood route, time of disease progression 10.5 years; P2 12 men and 4 women; mean age 34,8 years, predominant form of contagion: sexual, time of disease progression 1.5 years. Tabagisme was the principal vice in about populations. When asked about motivation yours good health, the subjects of P1 answered to be relationed the faith and medications use. The patients of the P2 did not answer about yours health state. Whatever the sexual custom, 9 patients of the P1 had sexual relations, five without regular use of condom. In the P2 only um patient used condom. Two patients of the P1 and one of P2 declared to have STD, syphilis and Human Papiloma Virus (HPV). Because there are reduces number of patients it’s impossible to make asociation between the variables studies and mesures of the disease progression. The dates about habits don’t contribute for the disease development. The study offers an epidemiological profile of these patient populations

Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis

<p>Abstract</p> <p>Background</p> <p>Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and <it>PRV1 </it>gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34⁺hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, <it>PRV1 </it>overexpression, and clinical and laboratory parameters.</p> <p>Results</p> <p>By real time PCR assay, we observed that <it>A1, MCL1, BIK and BID</it>, as well as <it>A1, BCLW </it>and <it>BAK </it>gene expression were increased in ET and PMF CD34⁺cells respectively, while pro-apoptotic <it>BAX </it>and anti-apoptotic <it>BCL2 </it>mRNA levels were found to be lower in ET and PMF CD34⁺cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes <it>A1, BCL2, BCL-X_L</it>and <it>BCLW</it>. In contrast, pro-apoptotic <it>BID </it>and <it>BIM_EL</it>expression were downregulated in ET leukocytes. Increased BCL-X_Lprotein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and <it>BAX, BIK and BAD </it>gene expression and between <it>A1, BAX </it>and <it>BIK </it>and <it>PRV1 </it>gene expression. A negative correlation between <it>PRV1 </it>gene expression and platelet count was observed, as well as a positive correlation between <it>PRV1 </it>gene expression and splenomegaly.</p> <p>Conclusions</p> <p>Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, <it>PRV1 </it>and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.</p

Influence of INF-gamma gene polymorphism in HTLV-1 proviral load

Financial Support FAPESP, CNPq, CTC/FUNDHERP and INCTC