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1.
Leukemia ; 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578451

RESUMO

AML SCT-BFM 2007 was the first hematopoietic stem cell transplantation (HCT) trial in Germany to comply with the European Clinical Trials Directive, and aimed to standardize pediatric HCT for acute myeloid leukemia (AML) across centers in Germany, Austria, and the Czech Republic. Children with high-risk features and a good early response achieving a complete first remission (CR-1) and those in CR-2 after a first relapse were stratified to receive HCT from a matched donor after myeloablative conditioning consisting of busulfan, cyclophosphamide, and melphalan. Four-year EFS and OS were 61 and 70%. Cumulative incidence of relapse (CIR) was 22%. TRM was 15% and correlated with age reaching 9% (SE 3%) in children younger than 12 years and 31% (SE 9%) in older children and adolescents. Children with poorly responding primary disease or relapse were allocated to receive early HCT after a cytoreductive regimen with fludarabine, amsacrine, and cytarabine, followed by reduced intensity conditioning and prophylactic donor lymphocyte infusions. Four-year EFS and OS were 49 and 53%. CIR was 38% and TRM 11%. For patients with primary poor response disease, early use of RIC HCT followed by prophylactic DLI can induce long-term remissions in more than 50% (EFS 46% (SE 9%)).

2.
Front Immunol ; 10: 1484, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31316520

RESUMO

Interleukin 10 is a central regulator of the antigen-presenting function of myeloid cells. It exerts immunomodulatory effects in vivo and induces a regulatory phenotype in monocyte-derived cells in vitro. We analyzed phenotype and function of monocytic cells in vitro in relation to the cytokine milieu and the timing of TLR-based activation. In GM-CSF/IL-4 cultured human monocytic cells, we identified two, mutually exclusive cell populations arising from undifferentiated cells: CD83+ fully activated dendritic cells and CD14+ macrophage like cells. Re-expression of CD14 occurs primarily after a sequential trigger with a TLR signal following IL-10 preincubation. This cell population with re-expressed CD14 greatly differs in phenotype and function from the CD83+ cells. Detailed analysis of individual subpopulations reveals that exogenous IL-10 is critical for inducing the shift toward the CD14+ population, but does not affect individual changes in marker expression or cell function in most cases. Thus, plasticity of CD14 expression, defining a subset of immunoregulatory cells, is highly relevant for the composition of cellular products (such as DC vaccines) as it affects the function of the total product.

3.
Biol Blood Marrow Transplant ; 25(9): 1786-1791, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31082473

RESUMO

Gonadal impairment is an important late effect with a significant impact on quality of life of transplanted patients. The aim of this study was to compare gonadal function after busulfan (Bu) or treosulfan (Treo) conditioning regimens in pre- and postpubertal children. This retrospective, multicenter study included children transplanted in pediatric European Society for Blood and Marrow Transplantation (EBMT) centers between 1992 and 2012 who did not receive gonadotoxic chemoradiotherapy before the transplant. We evaluated 137 patients transplanted in 25 pediatric EBMT centers. Median age at transplant was 11.04 years (range, 5 to 18); 89 patients were boys and 48 girls. Eighty-nine patients were prepubertal at transplant and 48 postpubertal. One hundred eighteen children received Bu and 19 Treo. A higher proportion of girls treated with Treo in the prepubertal stage reached spontaneous puberty compared with those treated with Bu (P = .02). Spontaneous menarche was more frequent after Treo than after Bu (P < .001). Postpubertal boys and girls treated with Treo had significantly lower luteinizing hormone levels (P = .03 and P = .04, respectively) compared with the Bu group. Frequency of gonadal damage associated with Treo was significantly lower than that observed after Bu. These results need to be confirmed in a larger population.

4.
J Pediatr Hematol Oncol ; 41(2): e101-e107, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30557171

RESUMO

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) is a life-saving procedure for children with a variety of (non) malignant conditions. GvHD is a severe complication with high morbidity and mortality. The pathogenesis remains unclear. We studied dendritic cell (DC) reconstitution to detect potential differences, which may improve our knowledge in the development of chronic GvHD (cGvHD). PROCEDURE: We examined immune reconstitution (T, B, and NK cells and dendritic cells) at defined time points in a pediatric cohort who underwent 61 allogeneic HSCTs. RESULTS: Regarding DC reconstitution we found a fast reconstitution of the DC compartment negatively correlated with age. After HSCT, both myeloid DC (mDC) and plasmacytoid DC (pDC) counts recover to pre-HSCT levels within 2 months. Higher CCR7 positive cell counts were found in patients receiving TBI during engraftment and during the whole posttransplant period we found a correlation with an improved outcome. In cGVHD patients decreased total DC counts and increased pDCs were found after day+100. No relevant correlation was achieved regarding to HLA-matching, stem cell manipulation of the graft as well as HSCT-indication compared with different DC counts. DISCUSSION: Pathogenesis of cGvHD remains complex. Our data suggest an influence of dendritic cells, which may contribute to the clinical picture and should be further investigated in future studies.


Assuntos
Células Dendríticas/imunologia , Transplante de Células-Tronco Hematopoéticas , Recuperação de Função Fisiológica/imunologia , Adolescente , Aloenxertos , Criança , Pré-Escolar , Doença Crônica , Células Dendríticas/patologia , Feminino , Humanos , Linfócitos/imunologia , Linfócitos/patologia , Masculino
5.
Artigo em Inglês | MEDLINE | ID: mdl-30418422

RESUMO

Hematopoietic stem cell transplantation (HSCT) is a life-saving procedure for children with a variety of malignant and nonmalignant conditions. However, even if immune reconstitution after HSCT has been studied extensively, until now, data on the comparison of immune reconstitution after autologous versus allogeneic HSCT are scarce, but might provide important clinical implications. We examined immune reconstitution (T cells, B cells, and NK cells) at defined timepoints in 147 children who received 182 HSCTs. Differences in the time course of immune reconstitution were analyzed in autologous versus allogeneic HSCT. We identified a quicker immune reconstitution in the T-cell compartment, especially in the CD4 and naive subset after autologous HSCT, whereas recipients of allogeneic transplants showed a higher TCRgd proportion. B-cell reconstitution showed a delayed immune reconstitution after allogeneic HSCT in the first 2 years after HSCT. However, a reconstitution of all lymphocyte subsets after HSCT could be achieved in all patients. Children undergoing an HSCT show a different pattern of immune reconstitution in the allogeneic and autologous setting. This might influence the outcome and should affect the clinical handling of infectious prophylaxis and revaccinations.

6.
Br J Haematol ; 183(1): 104-109, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30028016

RESUMO

Osteonecrosis (ON) was prospectively assessed in 557 children and adolescents in the Berlin-Frankfurt-Münster Stem Cell Transplantation in children with acute lymphoblastic leukaemia 2003 trial. Median age at haematopoietic stem cell transplantation (HSCT) was 10·3 years (range 0·5-26). Cumulative incidence of symptomatic ON (sON) was 9% at 5 years (standard deviation 1%), median time from HSCT to diagnosis of sON was 12·4 months (range 1-126). Multivariate analysis identified age at HSCT [10-15 years vs. <10 years: hazard ratio (HR) 3·73, P = 0·009; >15 years vs. <10 years: HR 5·46, P = 0·001], diagnosis of sON prior to HSCT and chronic graft-versus-host disease (yes versus no: HR 2·696, P = 0·015) as significant independent risk factors for the development of sON.

7.
Cancer Immunol Immunother ; 67(10): 1545-1558, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30054667

RESUMO

High-grade gliomas (HGG) exert systemic immunosuppression, which is of particular importance as immunotherapeutic strategies such as therapeutic vaccines are increasingly used to treat HGGs. In a first cohort of 61 HGG patients we evaluated a panel of 30 hematological and 34 plasma biomarkers. Then, we investigated in a second cohort of 11 relapsed HGG patients receiving immunomodulation with metronomic cyclophosphamide upfront to a DC-based vaccine whether immune abnormalities persisted and whether they hampered induction of IFNγ+ T-cell responses. HGG patients from the first cohort showed increased numbers of leukocytes, neutrophils and MDSCs and in parallel reduced numbers of CD4+/CD8+ T-cells, plasmacytoid and conventional DC2s. MDSCs and T-cell alterations were more profound in WHO IV° glioma patients. Moreover, levels of MDSCs and epidermal growth factor were negatively associated with survival. Serum levels of IL-2, IL-4, IL-5 and IL-10 were altered in HGG patients, however, without any impact on clinical outcome. In the immunotherapy cohort, 6-month overall survival was 100%. Metronomic cyclophosphamide led to > 40% reduction of regulatory T cells (Treg). In parallel to Treg-depletion, MDSCs and DC subsets became indistinguishable from healthy controls, whereas T-lymphopenia persisted. Despite low T-cells, IFNγ-responses could be induced in 9/10 analyzed cases. Importantly, frequency of CD8+VLA-4+ T-cells with CNS-homing properties, but not of CD4+ VLA-4+ T-cells, increased during vaccination. Our study identifies several features of systemic immunosuppression in HGGs. Metronomic cyclophosphamide in combination with an active immunization alleviates the latter and the combined treatment allows induction of a high rate of anti-glioma immune responses.


Assuntos
Neoplasias Encefálicas/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Glioma/imunologia , Imunossupressão , Imunoterapia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Vacinas Anticâncer/administração & dosagem , Estudos de Casos e Controles , Células Cultivadas , Criança , Células Dendríticas/imunologia , Feminino , Glioma/patologia , Glioma/terapia , Humanos , Tolerância Imunológica , Depleção Linfocítica , Masculino , Pessoa de Meia-Idade , Adulto Jovem
9.
Oncoimmunology ; 5(7): e1188245, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27622043

RESUMO

Interleukin 12 (IL12) is a key inflammatory cytokine critically influencing Th1/Tc1-T-cell responses at the time of initial antigen encounter. Therefore, it may be exploited for cancer immunotherapy. Here, we investigated how IL12, and other inflammatory cytokines, shape effector functions of human T-cells. Using a defined culture system, we followed the gradual differentiation and function of antigen-specific CD8(+) T cells from their initial activation as naïve T cells through their expansion phase as early memory cells to full differentiation as clonally expanded effector T cells. The addition of IL12 8 days after the initial priming event initiated two mechanistically separate events: First, IL12 sensitized the T-cell receptor (TCR) for antigen-specific activation, leading to an approximately 10-fold increase in peptide sensitivity and, in consequence, enhanced tumor cell killing. Secondly, IL12 enabled TCR/HLA-independent activation and cytotoxicity: this "non-specific" effect was mediated by the NK cell receptor DNAM1 (CD226) and dependent on ligand expression of the target cells. This IL12 regulated, DNAM1-mediated killing is dependent on src-kinases as well as on PTPRC (CD45) activity. Thus, besides enhancing TCR-mediated activation, we here identified for the first time a second IL12 mediated mechanism leading to activation of a receptor-dependent killing pathway via DNAM1.

10.
Cytotherapy ; 18(9): 1178-86, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27421737

RESUMO

BACKGROUND AIMS: Atypical rhabdoid/teratoid tumors (AT/RT) are the most common brain tumors in infants and associated with a dismal prognosis. Although intensification of first-line therapy has resulted in improvement of overall survival, novel treatment strategies are needed. Because immunotherapy has resulted in remarkable results in several adult tumor entities, incorporation of immunotherapy into AT/RT treatment offers a novel alternative. METHODS: We retrospectively analyzed data from 7 AT/RT patients from five countries treated within the HGG-Immuno Consortium. Two patients were ≤1 year and 4 patients were ≤2 years of age at diagnosis. All received immunotherapy with autologous, tumor-lysate-loaded dendritic cells (DCs) on a compassionate use basis using a schedule of three to four weekly DC vaccinations with up to 2 × 10(7) DCs per vaccine, followed by three lysate boosts each 1 month apart. RESULTS: Monocyte collections (median age at apheresis 31.5, range 20-143 months) and vaccinations were uneventful without any severe adverse event related to the vaccine, demonstrating feasibility and safety in this very young age group. Two children received immunotherapy during their primary and the remaining five during second- or third-line therapy. Three of seven patients survived long term with a follow-up of 143, 138 and 46 months, with at least two of them harboring somatic mutations. One long-term survivor was vaccinated during primary treatment and the other two after first or second relapse/progression. Two analyzed patients showed positive CD8(+) T-cell responses after vaccination. DISCUSSION: Our data demonstrate that anti-tumor immunotherapy with autologous DCs is feasible and safe in young children with ATRTs and that this approach warrants further investigation in controlled clinical trials.


Assuntos
Neoplasias Encefálicas/terapia , Vacinas Anticâncer/uso terapêutico , Imunoterapia/métodos , Tumor Rabdoide/terapia , Neoplasias Encefálicas/imunologia , Criança , Pré-Escolar , Ensaios de Uso Compassivo , Células Dendríticas/imunologia , Células Dendríticas/transplante , Feminino , Humanos , Lactente , Masculino , Monitorização Imunológica/métodos , Prognóstico , Estudos Retrospectivos , Tumor Rabdoide/imunologia , Inquéritos e Questionários , Resultado do Tratamento
11.
BMC Cancer ; 16: 115, 2016 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-26883117

RESUMO

BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children and can be divided in different molecular subgroups. Patients whose tumor is classified as a Group 3 tumor have a dismal prognosis. However only very few tumor models are available for this subgroup. METHODS: We established a robust orthotopic xenograft model with a cell line derived from the malignant pleural effusions of a child suffering from a Group 3 medulloblastoma. RESULTS: Besides classical characteristics of this tumor subgroup, the cells display cancer stem cell characteristics including neurosphere formation, multilineage differentiation, CD133/CD15 expression, high ALDH-activity and high tumorigenicity in immunocompromised mice with xenografts exactly recapitulating the original tumor architecture. CONCLUSIONS: This model using unmanipulated, human medulloblastoma cells will enable translational research, specifically focused on Group 3 medulloblastoma.


Assuntos
Meduloblastoma/patologia , Neoplasias Experimentais/patologia , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Feminino , Humanos , Lactente , Masculino , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Pediatr Blood Cancer ; 62(11): 2033-5, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26153219

RESUMO

Acute Graft-versus-Host-Disease (GvHD) is a potentially life-threatening complication after allogeneic stem cell transplantation. If not treated early and adequately, the complex immunological mechanisms may lead to a self-perpetuating cycle of alloreactivity, which is then associated with a high mortality. Here we assessed the cytokine profile on a daily basis in a patient with grade 4 skin GvHD, demonstrating a signature resembling cytokine-release-syndrome. After multimodal immunosuppressive intervention, including treatment with the IL6 receptor-blocking antibody tocilizumab, the severe clinical symptoms unexpectedly resolved within 48 hr.


Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Doença Enxerto-Hospedeiro/tratamento farmacológico , Imunossupressão , Dermatopatias/tratamento farmacológico , Aloenxertos , Criança , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Masculino , Dermatopatias/imunologia , Dermatopatias/patologia , Transplante de Células-Tronco , Síndrome
13.
Br J Haematol ; 169(5): 711-8, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25817915

RESUMO

Patients with Langerhans cell histiocytosis (LCH) refractory to conventional chemotherapy have a poor outcome. There are currently two promising treatment strategies for high-risk patients: the first involves the combination of 2-chlorodeoxyadenosine and cytarabine; the other approach is allogeneic haematopoietic stem cell transplantation (HSCT). Here we evaluated 87 patients with high-risk LCH who were transplanted between 1990 and 2013. Prior to the year 2000, most patients underwent HSCT following myeloablative conditioning (MAC): only 5 of 20 patients (25%) survived with a high rate (55%) of transplant-related mortality (TRM). After the year 2000 an increasing number of patients underwent HSCT with reduced intensity conditioning (RIC): 49/67 (73%) patients survived, however, the improved survival was not overtly achieved by the introduction of RIC regimens with similar 3-year probability of survival after MAC (77%) and RIC transplantation (71%). There was no significant difference in TRM by conditioning regimen intensity but relapse rates were higher after RIC compared to MAC regimens (28% vs. 8%, P = 0·02), although most patients relapsing after RIC transplantation could be salvaged with further chemotherapy. HSCT may be a curative approach in 3 out of 4 patients with high risk LCH refractory to chemotherapy: the optimal choice of HSCT conditioning remains uncertain.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Histiocitose de Células de Langerhans/terapia , Condicionamento Pré-Transplante , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Histiocitose de Células de Langerhans/mortalidade , Histiocitose de Células de Langerhans/patologia , Humanos , Lactente , Masculino , Estudos Retrospectivos , Transplante Homólogo , Resultado do Tratamento , Adulto Jovem
14.
Mol Med ; 21: 185-96, 2015 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-25811991

RESUMO

Parent-of-origin imprints have been implicated in the regulation of neural differentiation and brain development. Previously we have shown that, despite the lack of a paternal genome, human parthenogenetic (PG) embryonic stem cells (hESCs) can form proliferating neural stem cells (NSCs) that are capable of differentiation into physiologically functional neurons while maintaining allele-specific expression of imprinted genes. Since biparental ("normal") hESC-derived NSCs (N NSCs) are targeted by immune cells, we characterized the immunogenicity of PG NSCs. Flow cytometry and immunocytochemistry revealed that both N NSCs and PG NSCs exhibited surface expression of human leukocyte antigen (HLA) class I but not HLA-DR molecules. Functional analyses using an in vitro mixed lymphocyte reaction assay resulted in less proliferation of peripheral blood mononuclear cells (PBMC) with PG compared with N NSCs. In addition, natural killer (NK) cells cytolyzed PG less than N NSCs. At a molecular level, expression analyses of immune regulatory factors revealed higher HLA-G levels in PG compared with N NSCs. In line with this finding, MIR152, which represses HLA-G expression, is less transcribed in PG compared with N cells. Blockage of HLA-G receptors ILT2 and KIR2DL4 on natural killer cell leukemia (NKL) cells increased cytolysis of PG NSCs. Together this indicates that PG NSCs have unique immunological properties due to elevated HLA-G expression.


Assuntos
Diferenciação Celular , Citotoxicidade Imunológica , Células-Tronco Embrionárias/citologia , Expressão Gênica , Antígenos HLA-G/genética , Células Matadoras Naturais/imunologia , Células-Tronco Neurais/imunologia , Células-Tronco Neurais/metabolismo , Apoptose/genética , Apoptose/imunologia , Linhagem Celular , Regulação da Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Antígenos HLA-G/imunologia , Antígenos HLA-G/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , MicroRNAs/genética , Células-Tronco Neurais/citologia
15.
Br J Haematol ; 169(1): 90-102, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25495919

RESUMO

Children with B cell malignancies refractory to standard therapy are known to have a poor prognosis and very limited treatment options. Here, we report on the treatment and follow-up of ten patients diagnosed with relapsed or refractory mature B-cell Non Hodgkin Lymphoma (B-NHL), Burkitt leukaemia (B-AL) or pre B-acute lymphoblastic leukaemia (pre B-ALL). All children were treated with FBTA05 (now designated Lymphomun), an anti-CD3 x anti-CD20 trifunctional bispecific antibody (trAb) in compassionate use. Within individual treatment schedules, Lymphomun was applied (a) after allogeneic stem cell transplantation (allo-SCT, n = 6) to induce sustained long-term remission, or (b) stand alone prior to subsequent chemotherapy to eradicate residual disease before allo-SCT (n = 4). Nine of ten children displayed a clinical response: three stable diseases (SD), one partial remission (PR) and five induced or sustained complete remissions (CR). Five of these nine responders died during follow-up. The other patients still maintain CR with a current overall survival of 874-1424 days (median: 1150 days). In conclusion, despite the dismal clinical prognosis of children refractory to standard therapy, immunotherapy with Lymphomun resulted in a favourable clinical outcome in this cohort of refractory paediatric patients.


Assuntos
Anticorpos Biespecíficos/administração & dosagem , Linfoma de Burkitt/terapia , Imunoterapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Transplante de Células-Tronco , Adolescente , Aloenxertos , Anticorpos Biespecíficos/efeitos adversos , Linfoma de Burkitt/mortalidade , Linfoma de Burkitt/patologia , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Masculino , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Taxa de Sobrevida
16.
BMC Hematol ; 14(1): 18, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25317335

RESUMO

BACKGROUND: Reliable central venous access (CVC) is essential for hematology-oncology patients since frequent puncture of peripheral veins-e.g., for chemotherapy, antibiotic administration, repeated blood sampling, and monitoring-can cause unacceptable pain and psychological trauma, as well as severe side effects in cases of extravasation of chemotherapy drugs. However, CVC lines still carry major risk factors, including thrombosis, infection (e.g., entry site, tunnel, and luminal infections), and catheter dislocation, leakage, or breakage. METHODS: Here we performed a retrospective database analysis to determine the incidence of CVC-associated thrombosis in a single-center cohort of 448 pediatric oncologic patients, and to analyze whether any subgroup of patients was at increased risk and thus might benefit from prophylactic anticoagulation. RESULTS: Of the 448 patients, 269 consecutive patients received a CVC, and 55 of these 269 patients (20%) also had a thrombosis. Of these 55 patients, 43 had at least one CVC-associated thrombosis (total number of CVC-associated thrombosis: n = 52). Among all patients, the median duration of CVC exposure was 464 days. Regarding exposure time, no significant difference was found between patients with and without CVC-associated thrombosis. Subclavia catheters and advanced tumor stages seem to be the main risk factors for the development of CVC-associated thrombosis, whereas pharmacologic prophylaxis did not seem to have a relevant impact on the rate of thrombosis. CONCLUSIONS: We conclude that pediatric surgeons and oncologists should pay close attention to ensuring optimal and accurate CVC placement, as this appears the most effective tool to minimize CVC-associated complications.

17.
Cytotherapy ; 16(7): 946-64, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24831836

RESUMO

BACKGROUND AND AIMS: One of the major challenges of dendritic cell (DC) vaccination is the establishment of harmonized DC production protocols. Here, we report the transfer and validation of a successfully used open DC manufacturing method into a closed system, good manufacturing practice (GMP)-compatible protocol. METHODS: All production steps (lysate generation, monocyte selection, DC culture and cryopreservation) were standardized and validated. RESULTS: Tumor lysate was characterized by histology, mechanically homogenized and avitalized. This preparation yielded a median of 58 ± 21 µg protein per milligram of tumor tissue. Avitality was determined by trypan blue staining and confirmed in an adenosine triphosphate release assay. Patient monocytes were isolated by elutriation or CD14 selection, which yielded equivalent results. DCs were subsequently differentiated in Teflon bags for an optimum of 7 days in CellGro medium supplemented with interleukin (IL)-4 and granulocyte macrophage colony stimulating factor and then matured for 48 h in tumor necrosis factor-α and IL-1ß after pulsing with tumor lysate. This protocol resulted in robust and reproducible upregulation of DC maturation markers such as cluster of differentiation (CD)80, CD83, CD86, human leukocyte antigen-DR and DC-SIGN. Functionality of these DCs was shown by directed migration toward C-C motif chemokine ligand 19/21, positive T-cell stimulatory capacity and the ability to prime antigen-specific T cells from naive CD8(+) T cells. Phenotype stability, vitality and functionality of DCs after cryopreservation, thawing and washing showed no significant loss of function. Comparison of clinical data from 146 patients having received vaccinations with plate-adherence versus GMP-grade DCs showed no inferiority of the latter. CONCLUSIONS: Our robust, validated and approved protocol for DC manufacturing forms the basis for a harmonized procedure to produce cancer vaccines, which paves the way for larger multi-center clinical trials.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Células Dendríticas/imunologia , Glioma/terapia , Vacinação , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Técnicas de Cultura de Células , Células Dendríticas/patologia , Glioma/imunologia , Glioma/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Leucaférese , Monócitos
18.
Lancet ; 383(9915): 436-48, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24161820

RESUMO

BACKGROUND: In chronic granulomatous disease allogeneic haemopoietic stem-cell transplantation (HSCT) in adolescents and young adults and patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality. We examined the effect of a reduced-intensity conditioning regimen designed to enhance myeloid engraftment and reduce organ toxicity in these patients. METHODS: This prospective study was done at 16 centres in ten countries worldwide. Patients aged 0-40 years with chronic granulomatous disease were assessed and enrolled at the discretion of individual centres. Reduced-intensity conditioning consisted of high-dose fludarabine (30 mg/m(2) [infants <9 kg 1·2 mg/kg]; one dose per day on days -8 to -3), serotherapy (anti-thymocyte globulin [10 mg/kg, one dose per day on days -4 to -1; or thymoglobuline 2·5 mg/kg, one dose per day on days -5 to -3]; or low-dose alemtuzumab [<1 mg/kg on days -8 to -6]), and low-dose (50-72% of myeloablative dose) or targeted busulfan administration (recommended cumulative area under the curve: 45-65 mg/L × h). Busulfan was administered mainly intravenously and exceptionally orally from days -5 to -3. Intravenous busulfan was dosed according to weight-based recommendations and was administered in most centres (ten) twice daily over 4 h. Unmanipulated bone marrow or peripheral blood stem cells from HLA-matched related-donors or HLA-9/10 or HLA-10/10 matched unrelated-donors were infused. The primary endpoints were overall survival and event-free survival (EFS), probabilities of overall survival and EFS at 2 years, incidence of acute and chronic GVHD, achievement of at least 90% myeloid donor chimerism, and incidence of graft failure after at least 6 months of follow-up. FINDINGS: 56 patients (median age 12·7 years; IQR 6·8-17·3) with chronic granulomatous disease were enrolled from June 15, 2003, to Dec 15, 2012. 42 patients (75%) had high-risk features (ie, intractable infections and autoinflammation), 25 (45%) were adolescents and young adults (age 14-39 years). 21 HLA-matched related-donor and 35 HLA-matched unrelated-donor transplants were done. Median time to engraftment was 19 days (IQR 16-22) for neutrophils and 21 days (IQR 16-25) for platelets. At median follow-up of 21 months (IQR 13-35) overall survival was 93% (52 of 56) and EFS was 89% (50 of 56). The 2-year probability of overall survival was 96% (95% CI 86·46-99·09) and of EFS was 91% (79·78-96·17). Graft-failure occurred in 5% (three of 56) of patients. The cumulative incidence of acute GVHD of grade III-IV was 4% (two of 56) and of chronic graft-versus-host disease was 7% (four of 56). Stable (≥90%) myeloid donor chimerism was documented in 52 (93%) surviving patients. INTERPRETATION: This reduced-intensity conditioning regimen is safe and efficacious in high-risk patients with chronic granulomatous disease. FUNDING: None.


Assuntos
Doença Granulomatosa Crônica/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Condicionamento Pré-Transplante/métodos , Adolescente , Adulto , Alemtuzumab , Anticorpos Monoclonais Humanizados/administração & dosagem , Soro Antilinfocitário/administração & dosagem , Bussulfano/administração & dosagem , Criança , Pré-Escolar , Quimioterapia Combinada , Sobrevivência de Enxerto/efeitos dos fármacos , Doença Enxerto-Hospedeiro/prevenção & controle , Antígenos HLA , Humanos , Imunossupressores/administração & dosagem , Lactente , Estudos Prospectivos , Quimeras de Transplante/fisiologia , Resultado do Tratamento , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados , Adulto Jovem
19.
Leuk Lymphoma ; 55(4): 870-5, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23786458

RESUMO

Chemotherapy for childhood acute lymphoblastic leukemia (ALL) is a highly effective treatment, but at the same time causes significant suppression of the patient's immunity. Immune reconstitution was studied in a homogeneous cohort of 48 children with standard or medium risk ALL treated according to the ALL-Berlin-Frankfurt-Münster (BFM) protocol. Whereas the T-cell compartment was only moderately affected and recovered to normal levels quickly after treatment cessation, B-cells were significantly reduced during and after therapy. In particular, the naive B-cell compartment declined. Even 5 years after the end of therapy, B-cell distribution was disturbed and patients showed an ongoing reconstitution. Thus, even standard regimens for chemotherapy cause severe B-cell depletion that resolves only gradually.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Subpopulações de Linfócitos B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Adolescente , Asparaginase/uso terapêutico , Fator Ativador de Células B/metabolismo , Subpopulações de Linfócitos B/metabolismo , Biomarcadores/metabolismo , Criança , Pré-Escolar , Daunorrubicina/uso terapêutico , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Imunofenotipagem , Lactente , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Prednisona/uso terapêutico , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Resultado do Tratamento , Vincristina/uso terapêutico
20.
Blood ; 122(7): 1203-13, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23836556

RESUMO

Src-kinase inhibitors hold great potential as targeted therapy against malignant cells. However, such inhibitors may also affect nonmalignant cells and cause pronounced off-target effects. We investigated the role of the dual kinase inhibitor dasatinib on human myeloid cells. Dasatinib is clinically used for the treatment of bcr/abl⁺ leukemias because it blocks the mutated tyrosine kinase abl. To understand its effect on the development of antigen-specific T-cell responses, we assessed antigen-specific priming of human, naïve T cells. In surprising contrast to the direct inhibition of T-cell activation by dasatinib, pretreatment of maturing dendritic cells (DCs) with dasatinib strongly enhanced their stimulatory activity. This effect strictly depended on the activating DC stimulus and led to enhanced interleukin 12 (IL-12) production and T-cell responses of higher functional avidity. Src-kinase inhibitors, and not conventional tyrosine kinase inhibitors, increased IL-12 production in several cell types of myeloid origin, such as monocytes and classical or nonclassical DCs. Interestingly, only human cells, but not mouse or macaques DCs, were affected. These data highlight the potential immunostimulatory capacity of a group of novel drugs, src-kinase inhibitors, thereby opening new opportunities for chemoimmunotherapy. These data also provide evidence for a regulatory role of src kinases in the activation of myeloid cells.


Assuntos
Células Dendríticas/efeitos dos fármacos , Interleucina-12/metabolismo , Células Mieloides/efeitos dos fármacos , Pirimidinas/farmacologia , Linfócitos T/efeitos dos fármacos , Tiazóis/farmacologia , Receptores Toll-Like/metabolismo , Quinases da Família src/antagonistas & inibidores , Animais , Células Cultivadas , Dasatinibe , Células Dendríticas/imunologia , Células Dendríticas/patologia , Citometria de Fluxo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Macaca mulatta , Camundongos , Células Mieloides/imunologia , Células Mieloides/patologia , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia
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