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)

HTLV-1 affects the adipogenic differentiation of human mesenchymal stem cells

Financial support: CTC, INCTC, FAPESP, FUNDHERP and CNPq

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

Epidemiology of HIV/HCV coinfection in patients cared for at the Tropical Medicine Foundation of Amazonas

The association of HIV infection and hepatitis C virus (HCV) infection often occurs because both viruses share the same transmission routes, increasing the possibility of HIV/HCV coinfection. World prevalence greater than 30% of coinfected cases is estimated, and it can reach 90% depending on the transmission route. With the aim of determining the frequency and profile of HIV/HCV coinfected patients, a descriptive analysis was carried out with patients with HIV/AIDS whose serology was positive for hepatitis C virus (HCV), cared for at the Fundação de Medicina Tropical do Amazonas from 2000 to 2007. In the present study, of the 2,653 AIDS cases notified in SINAN, 1,582 patients underwent serology test for hepatitis C, and a frequency of 4.42% (n = 70) of HIV/HCV coinfected patients was identified in the period studied. The most frequent infection route was sexual transmission (84.3%), 68.6% among heterosexual individuals. Most patients were males (72.9%), aged between 25 and 40 years (60.1%), of low income (50% earning up to one minimum wage), and low educational level (80% had completed only middle school). A high percentage of deaths were observed during the study (34.3%). The results indicate a low seroprevalence of HIV/HCV coinfection in this population, in which sexual transmission, characterized by sexual promiscuity among heterosexual individuals, is the major transmission route of the virus rather than the use of injection drugs, as shown in world statistics

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

Quantitative correlation between transcriptional levels of ER chaperone, peroximal protein and FVIII productivity in human Hek-293 cell line

Hek-293 cell line presents good production platform for recombinant therapeutic proteins, however little is known about the components that contribute to the cellular control of recombinant protein production. In this study, we generated a Hek-293 producing recombinant factor VIII (FVIII) and we evaluated the immunoglobulin-binding protein (BiP) and phytanoil-CoA α-hydroxylase (PAHX) expression levels which are known for diminishing FVIII production. Our analyses showed that the recombinant cell population expresses 3.1 ± 1.4 fold of BIP mRNA (P = 0.0054) and 97.8 ± 0.5 fold of PAHX mRNA (P = 0.0016) compared to nontransduced cells. The amount of these proteins was inversely correlated to the secreted FVIII. In conclusion, BIP and PAHX expression are augmented in human cells producing FVIII and they antagonize the amount of therapeutic factor VIII in the cell culture.This study was supported by the Center for Cell-based Therapy (CTC) and Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP. The authors acknowledge Dr. Garry P. Nolan for contributing with the pBMN-I-GFP vector and David Marco Antonio for statistical support. We thank Fernanda Udinal and Alessandra Almeida for English language support. We also thank Sandra Navarro Bresciani for preparing the figures.This study was supported by the Center for Cellbased Therapy (CTC) and Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP. The authors acknowledge Dr. Garry P. Nolan for contributing with the pBMNIGFP vector and David Marco Antonio for statistical support. We thank Fernanda Udinal and Alessandra Almeida for English language support. We also thank Sandra Navarro Bresciani for preparing the figures

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

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