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1.
Vox Sang ; 118(5): 384-391, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36912154

RESUMO

BACKGROUND AND OBJECTIVES: Corrected count increment (CCI) measurements monitor the effectiveness of platelet transfusions in haemato-oncology, but they usually fail in patients undergoing cardiac surgery. We investigated whether polymerase chain reaction (PCR) of mitochondrial single-nucleotide polymorphisms (SNPs) is able to monitor the survival of transfused platelets in these patients. MATERIALS AND METHODS: Leukocyte-free, platelet-rich plasma was prepared from patients' blood to measure platelet counts based on patient-/donor-specific SNPs by digital PCR after DNA extraction. Platelet counts in samples from patients with severe thrombocytopenia were analysed by both PCR and flow cytometry. Ten patients undergoing cardiac surgery with the use of heart lung machine and without overt bleeding received a single apheresis platelet concentrate because of either dual platelet inhibition during a non-elective intervention or a complex procedure. Blood samples were collected at nine defined intervals (0-120 h) post transfusion. RESULTS: The digital PCR of the seven SNPs reliably quantified levels ≥0.6 G/L platelets, in good agreement with flow cytometry and without interference by other SNPs or by platelet activation. A mean 24-h CCI of 11.8 (range: 5.6-19.8) and a mean 120-h area under the curve (AUC) of 1386 (915-1821) hxG/L were observed for the transfused platelets. The mean AUC of 14,103 (3415-27,305) hxG/L for the patients' endogenous platelets indicates that transfused platelets represented only 11% (5-25) of the total platelet counts during 120 h post transfusion. CONCLUSION: PCR of mitochondrial SNPs offers a tool to assess the survival of platelets from apheresis concentrates in cardiac surgery patients to facilitate the implementation of improved transfusion strategies.


Assuntos
Procedimentos Cirúrgicos Cardíacos , Trombocitopenia , Humanos , Transfusão de Plaquetas/métodos , Plaquetas/fisiologia , Reação em Cadeia da Polimerase
2.
Cytotherapy ; 22(6): 337-342, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32223996

RESUMO

BACKGROUND AIMS: At the frontier of transfusion medicine and transplantation, the field of cellular therapy is emerging. Most novel cellular therapy products are produced under investigational protocols with no clear standardization across cell processing centers. Thus, the purpose of this study was to uncover any variations in manufacturing practices for similar cellular therapy products across different cell processing laboratories worldwide. METHODS: An exploratory survey that was designed to identify variations in manufacturing practices in novel cellular therapy products was sent to cell processing laboratory directors worldwide. The questionnaire focused on the manufacturing life cycle of different cell therapies (i.e., collection, purification, in vitro expansion, freezing and storage, and thawing and washing), as well as the level of regulations followed to process each product type. RESULTS: The majority of the centers processed hematopoietic progenitor cells (HPCs) from peripheral blood (n = 18), bone marrow (n = 16) or cord blood (n = 19), making HPCs the most commonly processed cells. The next most commonly produced cellular therapies were lymphocytes (n = 19) followed by mesenchymal stromal cells (n = 14), dendritic cells (n = 9) and natural killer (NK) cells (n = 9). A minority of centers (<5) processed pancreatic islet cells (n = 4), neural cells (n = 3) and induced-pluripotent stem cells (n = 3). Thirty-two laboratories processed products under an investigational status, for either phase I/II (n = 27) or phase III (n = 17) clinical trials. If purification methods were used, these varied for the type of product processed and by institution. Environmental monitoring methods also varied by product type and institution. CONCLUSION: This exploratory survey shows a wide variation in cellular therapy manufacturing practices across different cell processing laboratories. A better understanding of the effect of these variations on the quality of these cell-based therapies will be important to assess for further process evaluation and development.


Assuntos
Biotecnologia/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Biotecnologia/normas , Medula Óssea , Sangue Fetal , Células-Tronco Hematopoéticas , Humanos , Células Matadoras Naturais , Laboratórios/normas , Células-Tronco Mesenquimais
3.
Transfusion ; 58(2): 461-469, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29210068

RESUMO

BACKGROUND: Cell therapy products are often stored and transported between sites. The aim of this study was to determine the effect of storage temperature, solution, and cell concentration on nonmobilized, peripheral blood-derived mononuclear cells (MNCs). STUDY DESIGN AND METHODS: This was a multicenter prospective study involving healthy volunteers who underwent nonmobilized MNC collection by apheresis. Products were processed at local laboratories and concentrated to either 100 × 106 or 300 × 106 nucleated cells/mL in 5% human serum albumin (HSA) or HypoThermosol FRS (HT; BioLife Solutions). Products were stored at room temperature (RT; 20-25°C) or refrigerated temperatures (2-8°C) with assessment at 0, 24, 48, and 72 hours. NC and MNC concentration, viability, and flow cytometric analysis for CD3, CD4, CD8, CD14, CD19, CD25, and CD56 were measured. RESULTS: Viability decreased over time for all conditions tested. Refrigerated storage preserved viability greater than RT storage, especially for products with a higher cell concentration. RT maintenance with a high cell concentration was associated with a relative loss of CD14- and CD4-positive cells, whereas the concentration of cells positive for other markers tested did not vary. Finally, there was delayed decrease in pH when using HT compared with HSA; however, there was no difference in viability between the two solutions. CONCLUSION: Low cell concentrations (approx. 100 × 106 cells/mL), refrigerated temperatures, and HT storage solution appear to be the optimal conditions for storing nonmobilized, peripheral blood-derived MNC products.


Assuntos
Antígenos CD/metabolismo , Remoção de Componentes Sanguíneos , Transfusão de Componentes Sanguíneos , Preservação de Sangue/métodos , Leucócitos/citologia , Leucócitos/metabolismo , Adulto , Feminino , Humanos , Masculino , Estudos Prospectivos
4.
Transfusion ; 58(9): 2184-2191, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30204955

RESUMO

BACKGROUND: Viability testing is a common practice in laboratories. The goal of this study was to ascertain current laboratory practices internationally for performing viability testing for cryopreserved cord blood (CB) products and glean information about how to standardize the method to improve interlaboratory reproducibility. STUDY DESIGN AND METHODS: A survey to evaluate current laboratory practices for viability testing was designed and distributed internationally. The question topics included sampling and testing methods, responses to unexpected results, and the rating of the reliability of the CB quality tests, together with expectations for standardization. RESULTS: There were 32 respondents to the survey, of whom 28 responded to the more detailed questionnaire about viability methods. Overall, responses indicated that various stains were used among the laboratories, and when multiple sites used the same viability stain the methods differed. The majority of the respondents were in favor of standardizing the viability testing methods. A wide variety of preferences were communicated about how to standardize the method, but a majority did advocate the use of 7-aminoactinomycin D (7-AAD) with flow cytometry. CONCLUSIONS: The survey results revealed a variety of tests and inconsistent interlaboratory practices for performing the viability assay. Flow cytometry with a 7-AAD dye was suggested as a first step toward standardization.


Assuntos
Preservação de Sangue/métodos , Segurança do Sangue , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Criopreservação/métodos , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Núcleo Celular/ultraestrutura , Separação Celular/métodos , Sobrevivência Celular , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Transplante de Células-Tronco de Sangue do Cordão Umbilical/normas , Dactinomicina/análogos & derivados , Citometria de Fluxo/métodos , Corantes Fluorescentes , Pesquisas sobre Atenção à Saúde , Células-Tronco Hematopoéticas/ultraestrutura , Humanos , Recém-Nascido , Cooperação Internacional , Internet , Laboratórios/normas , Utilização de Procedimentos e Técnicas , Reprodutibilidade dos Testes
5.
Transfusion ; 58(1): 151-157, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29030954

RESUMO

BACKGROUND: Typical practice is to transfuse group-specific plasma units; however, there are situations where group AB plasma (universal donor) is issued to group A, B, or O recipients. If demand for group AB plasma exceeds collections, there is potential for shortage. This project explored the patterns of group AB plasma utilization at hospitals around the world. STUDY DESIGN AND METHODS: The study had two phases: a survey that inquired about hospital group AB plasma inventory, policies, and transfusion practices and a retrospective review of 2014 calendar year data where participants submitted information on plasma disposition including ABO group of unit and recipient, transfusion location, and select indications. Recruitment occurred through snowball sampling. Descriptive analyses were performed. RESULTS: Survey data were received from 25 centers across 10 countries; of those, 15 participants contributed to the data collection component. These 15 centers transfused a total of 43,369 AB plasma units during the study period. Only 1496 of 5541 (27%) group AB plasma units were transfused to group AB recipients. Transfusion policies, practices, and patterns were variable across sites. CONCLUSION: Group AB plasma units are frequently transfused to non-AB recipients. Whether transfusing 73% of group AB plasma units to non-AB recipients is the ideal inventory management strategy remains to be determined.


Assuntos
Sistema ABO de Grupos Sanguíneos , Transfusão de Componentes Sanguíneos/estatística & dados numéricos , Inventários Hospitalares/estatística & dados numéricos , Plasma , Adulto , América , Bancos de Sangue/estatística & dados numéricos , Incompatibilidade de Grupos Sanguíneos , Criança , Coleta de Dados , Grupos Diagnósticos Relacionados , Europa (Continente) , Pesquisas sobre Atenção à Saúde , Necessidades e Demandas de Serviços de Saúde , Humanos , Recém-Nascido , Internacionalidade , Japão , Nova Zelândia , Estudos de Amostragem
6.
Transfusion ; 57(10): 2329-2337, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28840943

RESUMO

BACKGROUND: Transfusion of group O blood to non-O recipients, or transfusion of D- blood to D+ recipients, can result in shortages of group O or D- blood, respectively. This study investigated RBC utilization patterns at hospitals around the world and explored the context and policies that guide ABO blood group and D type selection practices. STUDY DESIGN AND METHODS: This was a retrospective study on transfusion data from the 2013 calendar year. This study included a survey component that asked about hospital RBC selection and transfusion practices and a data collection component where participants submitted information on RBC unit disposition including blood group and D type of unit and recipient. Units administered to recipients of unknown ABO or D group were excluded. RESULTS: Thirty-eight hospitals in 11 countries responded to the survey, 30 of which provided specific RBC unit disposition data. Overall, 11.1% (21,235/191,397) of group O units were transfused to non-O recipients; 22.6% (8777/38,911) of group O D- RBC units were transfused to O D+ recipients, and 43.2% (16,800/38,911) of group O D- RBC units were transfused to recipients that were not group O D-. Disposition of units and hospital transfusion policy varied within and across hospitals of different sizes, with transfusion of group O D- units to non-group O D- patients ranging from 0% to 33%. CONCLUSION: A significant proportion of group O and D- RBC units were transfused to compatible, nonidentical recipients, although the frequency of this practice varied across sites.


Assuntos
Transfusão de Eritrócitos/estatística & dados numéricos , Eritrócitos/imunologia , Sistema ABO de Grupos Sanguíneos/imunologia , Incompatibilidade de Grupos Sanguíneos , Hospitais , Humanos , Estudos Retrospectivos , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Inquéritos e Questionários
7.
BMC Biotechnol ; 15: 10, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25888378

RESUMO

BACKGROUND: Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. RESULTS: The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10(10) independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. CONCLUSION: The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.


Assuntos
Biblioteca de Peptídeos , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Sequência de Aminoácidos , Autoantígenos/química , Autoantígenos/imunologia , Bacteriófagos/genética , Bacteriófagos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/imunologia , Anticorpos de Cadeia Única/química
8.
Mol Cell Proteomics ; 12(5): 1099-114, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23315794

RESUMO

The recent Natural Killer (NK) cell maturation model postulates that CD34(+) hematopoietic stem cells (HSC) first develop into CD56(bright) NK cells, then into CD56(dim)CD57(-) and finally into terminally maturated CD56(dim)CD57(+). The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56(bright) NK cells versus CD56(dim) NK cells and CD56(dim)CD57(-) NK cells versus CD56(dim)CD57(+) NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions. Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa.


Assuntos
Células Matadoras Naturais/fisiologia , Proteoma/metabolismo , Sequência de Aminoácidos , Antígeno CD56/metabolismo , Antígenos CD57/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Separação Celular , Células Cultivadas , Humanos , Sinapses Imunológicas/metabolismo , Ativação Linfocitária , Anotação de Sequência Molecular , Dados de Sequência Molecular , Miosina não Muscular Tipo IIA/metabolismo , Transporte Proteico , Proteoma/química , Proteína A6 Ligante de Cálcio S100 , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/química , Proteínas S100/metabolismo , Transdução de Sinais
9.
Cytotherapy ; 15(3): 255-62, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23579058

RESUMO

BACKGROUND AIMS: Wide acceptance of the colony-forming unit (CFU) assay as a reliable potency test for stem cell products is hindered by poor inter-laboratory reproducibility. The goal of this study was to ascertain current laboratory practices for performing the CFU assay with an eye towards identifying practices that could be standardized to improve overall reproducibility. METHODS: A survey to evaluate current laboratory practices for performing CFU assays was designed and internationally distributed. RESULTS: There were 105 respondents to the survey, of whom 68% performed CFU assays. Most survey recipients specified that an automated rather than a manual cell count was performed on pre-diluted aliquots of stem cell products. Viability testing methods employed various stains, and when multiple sites used the same viability stain, the methods differed. Cell phenotype used to prepare working cell suspensions for inoculating the CFU assay differed among sites. Most respondents scored CFU assays at 14-16 days of incubation, but culture plates were read with various microscopes. Of 57 respondents, 42% had not performed a validation study or established assay linearity. Only 63% of laboratories had criteria for determining if a plate was overgrown with colonies. CONCLUSIONS: Survey results revealed inconsistent inter-laboratory practices for performing the CFU assay. The relatively low number of centers with validated CFU assays raises concerns about assay accuracy and emphasizes a need to establish central standards. The survey results shed light on numerous steps of the methodology that could be targeted for standardization across laboratories.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Ensaio de Unidades Formadoras de Colônias/normas , Células-Tronco Hematopoéticas , Células-Tronco/citologia , Antígenos CD34/metabolismo , Transplante de Células-Tronco Hematopoéticas , Humanos , Padrões de Referência , Reprodutibilidade dos Testes
10.
Front Immunol ; 14: 1288794, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38022629

RESUMO

Introduction: The evolution of novel SARS-CoV-2 variants significantly affects vaccine effectiveness. While these effects can only be studied retrospectively, neutralizing antibody titers are most used as correlates of protection. However, studies assessing neutralizing antibody titers often show heterogeneous data. Methods: To address this, we investigated assay variance and identified virus infection time and dose as factors affecting assay robustness. We next measured neutralization against Omicron sub-variants in cohorts with hybrid or vaccine induced immunity, identifying a gradient of immune escape potential. To evaluate the effect of individual mutations on this immune escape potential of Omicron variants, we systematically assessed the effect of each individual mutation specific to Omicron BA.1, BA.2, BA.2.12.1, and BA.4/5. Results: We cloned a library of pseudo-viruses expressing spikes with single point mutations, and subjected it to pooled sera from vaccinated hosts, thereby identifying multiple mutations that independently affect neutralization potency. Discussion: These data might help to predict antigenic features of novel viral variants carrying these mutations and support the development of broad monoclonal antibodies.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Estudos Retrospectivos , SARS-CoV-2/genética , COVID-19/prevenção & controle , Mutação , Vacinação , Anticorpos Neutralizantes
11.
Pediatr Transplant ; 15(8): 809-18, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21967552

RESUMO

The presence of microchimerism in peripheral blood of solid organ transplant recipients has been postulated to be beneficial for allograft acceptance. Kinetics of donor cell trafficking and accumulation in pediatric allograft recipients are largely unknown. In this study, we implemented SNPs of the HVRs I and II of mitochondrial DNA to serve as molecular genetic markers to detect donor-specific cell chimerism after pediatric renal transplantation. Serial dilution of artificial chimeric DNA samples showed a linear correlation coefficient of R > 0.98 and a detection sensitivity of 0.01% with high reproducibility. Longitudinal semiquantitative analysis of donor-specific SNPs was then performed in peripheral blood mononuclear cells samples up to two yr post-transplant. Quantity of donor-specific cell chimerism in peripheral blood was highest in the early post-transplant period reaching values of ~10% after liver-kidney and 2.8% after renal transplantation. From one wk after transplantation, renal transplant patients exhibited an amount of donor-specific mtDNA ranging from 0.01% to 0.1%. We developed a highly accurate, sensitive, and rapid real-time quantitative PCR method using sequence-specific primers and fluorescent hydrolysis probes for the detection of at least 0.01% donor-specific cells in the recipient's peripheral blood after renal transplantation.


Assuntos
DNA Mitocondrial/genética , Transplante de Rim/imunologia , Reação em Cadeia da Polimerase/métodos , Quimeras de Transplante/genética , Análise Mutacional de DNA , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Transplante de Fígado/imunologia , Transportador 1 de Ânion Orgânico Específico do Fígado , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Doadores de Tecidos
12.
Transfusion ; 50(4): 843-55, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20003054

RESUMO

BACKGROUND: Dendritic cells (DCs) are applied worldwide in several clinical studies of immune therapy of malignancies, autoimmune diseases, and transplantations. Most legislative bodies are demanding high standards for cultivation and transduction of cells. Closed-cell cultivating systems like cell culture bags would simplify and greatly improve the ability to reach these cultivation standards. We investigated if a new polyolefin cell culture bag enables maturation and adenoviral modification of human DCs in a closed system and compare the results with standard polystyrene flasks. STUDY DESIGN AND METHODS: Mononuclear cells were isolated from HLA-A*0201-positive blood donors by leukapheresis. A commercially available separation system (CliniMACS, Miltenyi Biotec) was used to isolate monocytes by positive selection using CD14-specific immunomagnetic beads. The essentially homogenous starting cell population was cultivated in the presence of granulocyte-macrophage-colony-stimulating factor and interleukin-4 in a closed-bag system in parallel to the standard flask cultivation system. Genetic modification was performed on Day 4. After induction of maturation on Day 5, mature DCs could be harvested and cryopreserved on Day 7. During the cultivation period comparative quality control was performed using flow cytometry, gene expression profiling, and functional assays. RESULTS: Both flasks and bags generated mature genetically modified DCs in similar yields. Surface membrane markers, expression profiles, and functional testing results were comparable. The use of a closed-bag system facilitated clinical applicability of genetically modified DCs. CONCLUSIONS: The polyolefin bag-based culture system yields DCs qualitatively and quantitatively comparable to the standard flask preparation. All steps including cryopreservation can be performed in a closed system facilitating standardized, safe, and reproducible preparation of therapeutic cells.


Assuntos
Técnicas de Cultura de Células/métodos , Células Dendríticas/fisiologia , Adulto , Antígenos CD/análise , Antígenos CD/genética , Antígenos de Superfície/análise , Contagem de Células , Técnicas de Cultura de Células/instrumentação , Divisão Celular/fisiologia , Senescência Celular/fisiologia , Criança , Células Dendríticas/citologia , Células Dendríticas/ultraestrutura , Feminino , Técnicas de Transferência de Genes , Humanos , Leucaférese/métodos , Leucócitos/citologia , Leucócitos/fisiologia , Receptores de Lipopolissacarídeos/análise , Receptores de Lipopolissacarídeos/genética , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos
13.
Transfusion ; 50(4): 831-42, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20003055

RESUMO

BACKGROUND: Dendritic cells (DCs) play a central role in the initiation and regulation of immune responses. DCs for clinical applications can be generated with high yield from leukapheresis products. Using adenoviral transduction we genetically modified human DCs to produce and present melanoma-associated antigens. Coexpression of green fluorescent protein and epitope tags were used to monitor genetic modification. Generation, genetic modification, and cryoconservation of gene modified human DCs on a clinical scale in a closed system is feasible. STUDY DESIGN AND METHODS: CD14-positive monomuclear cells were isolated from leukapheresis products of HLA-A* 0201 positive voluntary blood donors using immunomagnetic beads. Selected cells were cultivated for 7 days. Adenovirus transduction was optimal on Day 4. Maturation was induced on Day 5. Mature DC were aliquoted and cryoconserved on Day 7. Quality control was performed using flow cytometry, expression profiling, and functional assays (ELISPOT, CBA). RESULTS: We were able to generate sufficient genetically modified mature DCs in serum-free cultures that could be stored by cryopreservation. The use of a closed system facilitated development of methods for standardized production of clinically applicable genetically modified DCs. The adenoviral transduction system allowed simultaneous and flexible expression of tumor-associated antigens for prolonged presentation of multiple epitopes. CONCLUSION: The feasibility of a closed-bag system for the cultivation of genetically modified human DCs is shown. The immature DCs were genetically modified by recombinant replication-deficient adenoviruses to express multiple epitopes of tumor-associated proteins and then differentiated to mature antigen-presenting DCs.


Assuntos
Células Dendríticas/citologia , Células Dendríticas/fisiologia , Antígenos HLA-A/imunologia , Proteínas de Neoplasias/análise , Organismos Geneticamente Modificados/fisiologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD19/imunologia , Sobrevivência Celular/imunologia , Células Dendríticas/imunologia , Antígenos HLA-A/genética , Antígeno HLA-A2 , Teste de Histocompatibilidade/métodos , Humanos , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Ativação Linfocitária/imunologia , Reação em Cadeia da Polimerase , Linfócitos T/imunologia , Vacinação/métodos
14.
Transfus Med Hemother ; 37(2): 57-64, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20737047

RESUMO

Cellular therapies that either use modifications of a patient's own cells or allogeneic cell lines are becoming in vogue. Besides the technical issues of optimal isolation, cultivation and modification, quality control of the generated cellular products are increasingly being considered to be more important. This is not only relevant for the cell's therapeutic application but also for cell science in general. Recent changes in editorial policies of respected journals, which now require proof of authenticity when cell lines are used, demonstrate that the subject of the present paper is not a virtual problem at all. In this article we provide 2 examples of contaminated cell lines followed by a review of the recent developments used to verify cell lines, stem cells and modifications of autologous cells. With relative simple techniques one can now prove the authenticity and the quality of the cellular material of interest and therefore improve the scientific basis for the development of cells for therapeutic applications. The future of advanced cellular therapies will require production and characterization of cells under GMP and GLP conditions, which include proof of identity, safety and functionality and absence of contamination.

15.
Transfusion ; 49(2): 252-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18980617

RESUMO

BACKGROUND: Genotyping of single-nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, where finally tools for end users have become available to design primers and analyze SNPs of their own interest. This study investigated the potential of this technique in platelet (PLT) genotyping and developed a validated method for genotyping of clinical relevant human PLT antigens (HPAs). STUDY DESIGN AND METHODS: A multiplex assay using MALDI-TOF MS to analyze six HPA loci (HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, and HPA-15) simultaneously in a single reaction was applied for the genotyping of 100 DNA samples from a cohort of plateletpheresis donors and a patient population (n = 20) enriched for rare alleles. The genotyping results using MALDI-TOF MS were validated by the comparison with the results from typing by polymerase chain reaction with sequence-specific primers and conventional DNA sequencing. RESULTS: Both homozygous and heterozygous genotypes of HPA-1 to -5 and -15 of the 120 individuals were easily identified by a six-plexed assay on MALDI-TOF MS. The three approaches achieved a 100 percent concordance for the genotyping results of the six HPA loci. CONCLUSION: Compared to conventional methods, the MALDI-TOF MS showed several advantages, such as a high velocity, the ability to perform multiplexed assays in a single reaction, and automated high-throughput analysis of samples. This enables cost-efficient large-scale PLT genotyping for clinical applications.


Assuntos
Antígenos de Plaquetas Humanas/genética , Microquímica/métodos , Nanotecnologia/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Alelos , Estudos de Coortes , DNA/química , DNA/genética , DNA/isolamento & purificação , Primers do DNA/química , Primers do DNA/genética , Frequência do Gene , Triagem de Portadores Genéticos , Genótipo , Homozigoto , Humanos , Plaquetoferese , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Análise de Sequência de DNA
16.
Transfus Med Hemother ; 36(5): 302-308, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-21076548

RESUMO

Adoptive transfer in animal models clearly indicate an essential role of CD4+ CD25+ FOXP3+ regulatory T (T(reg)) cells in prevention and treatment of autoimmune and graft-versus-host disease. Thus, T(reg) cell therapies and development of drugs that specifically enhance T(reg) cell function and development represent promising tools to establish dominant tolerance. So far, lack of specific markers to differentiate human T(reg) cells from activated CD4+ CD25+ effector T cells, which also express FOXP3 at different levels, hampered such an approach. Recent identification of the orphan receptor glycoprotein-A repetitions predominant (GARP or LRRC32) as T(reg) cell-specific key molecule that dominantly controls FOXP3 via a positive feedback loop opens up new perspectives for molecular and cellular therapies. This brief review focuses on the role of GARP as a safeguard of a complex regulatory network of human T(reg) cells and its implications for regulatory T cell therapies in autoimmunity and graft-versus-host disease.

17.
Transfus Med Hemother ; 36(5): 325-328, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-21076551

RESUMO

SUMMARY: OBJECTIVE: In a significant proportion of patients with hematologic malignancies (5-30%) poor mobilization of hematopoietic stem cells (HSC) is observed. This compromises the application of effective and potentially curative high-dose chemotherapy (HDC) treatment. CASE REPORT: Here we report the case of a 38-year-old female patient who was treated for recurrent follicular B-cell non-Hodgkin's lymphoma grade III. In this patient, we failed twice to mobilize stem cells using chemotherapy followed by granulocyte-colony stimulating factor (G-CSF). Recently a new chemokine receptor CXCR4 antagonist, AMD3100 (plerixafor), was introduced which can be combined with G-CSF mobilization and has been reported to increase the number of harvested stem cells significantly. Using this protocol, we were able to harvest a HSC product. This product was transplanted 3 weeks after the harvest (after HDC), and the patient had an uncomplicated recovery of granulopoiesis (day 11 after transplantation of autologous HSC). CONCLUSION: Plerixafor has the potency to become an important tool in mobilizing HSC, especially in those patients in whom HSC cannot be mobilized by the combination of G-CSF and chemotherapy alone.

18.
Transfus Med Hemother ; 36(3): 181-187, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-21113259

RESUMO

Serology, defined as antibody-based diagnostics, has been regarded as the diagnostic gold standard in transfusion medicine. Nowadays however the impact of molecular diagnostics in transfusion medicine is rapidly growing. Molecular diagnostics can improve tissue typing (HLA typing), increase safety of blood products (NAT testing of infectious diseases), and enable blood group typing in difficult situations (after transfusion of blood products or prenatal non-invasive RhD typing). Most of the molecular testing involves the determination of the presence of single nucleotide polymorphisms (SNPs). Antigens (e.g. blood group antigens) mostly result from single nucleotide differences in critical positions. However, most blood group systems cannot be determined by looking at a single SNP. To identify members of a blood group system a number of critical SNPs have to be taken into account. The platforms which are currently used to perform molecular diagnostics are mostly gel-based, requiring time-consuming multiple manual steps. To implement molecular methods in transfusion medicine in the future the development of higher-throughput SNP genotyping non-gel-based platforms which allow a rapid, cost-effective screening are essential. Because of its potential for automation, high throughput and cost effectiveness the special focus of this paper is a relative new technique: SNP genotyping by MALDI-TOF MS analysis.

19.
Transfus Med Hemother ; 40(6): 384-5, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24474887
20.
J Psychopharmacol ; 20(4): 547-52, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16401665

RESUMO

Panic disorder is a common psychiatric disorder characterized by recurrent anxiety attacks and anticipatory anxiety. Due to the severity of the symptoms of the panic attacks and the frequent additional occurrence of agoraphobia, panic disorder is an often debilitating disease. Elevation of central serotonin levels by drugs such as clomipramine represents one of the most effective treatment options for panic disorder. This points to an important role of dysregulation of the serotonergic system in the genetic etiology of panic disorder. The novel brain-specific 5-HT synthesizing enzyme, tryptophan hydroxylase-2 (TPH2), which represents the rate-limiting enzyme of 5-HT production in the brain, may therefore be of particular importance in panic disorder. We focused on the putative transcriptional control region of TPH2 and identified two novel common single nucleotide polymorphisms (SNPs) of TPH2 in and close to this region. Moreover, a recently described loss-of-function mutation of TPH2 which results in an 80% reduction of serotonin production, was assessed. In an analysis of the putative transcriptional control region SNPs in a sample of panic disorder patients and controls no association of the disorder with the TPH2 SNPs or haplotypes was found. Moreover, the loss-of-function R441H mutation of TPH2 was not present in the panic disorder patients. The results of this first study of TPH2 in panic disorder argue against an importance of allelic variation of TPH2 in the pathogenesis of panic disorder with or without agoraphobia.


Assuntos
Química Encefálica/genética , Transtorno de Pânico/genética , Triptofano Hidroxilase/genética , Adulto , Agorafobia/genética , Agorafobia/psicologia , Alelos , Substituição de Aminoácidos , Éxons/genética , Feminino , Regulação da Expressão Gênica/genética , Frequência do Gene , Genótipo , Humanos , Masculino , Transtorno de Pânico/psicologia , Polimorfismo Genético/genética , Escalas de Graduação Psiquiátrica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica
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