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1.
Cells ; 9(1)2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31947623

RESUMO

Androgen receptor (AR) signaling remains crucial in castration-resistant prostate cancer (CRPC). Since it is also essential in immune cells, we studied whether the expression of AR full-length (ARFL) and its splicing variant ARV7 in peripheral blood mononuclear cells (PBMC) predicts systemic treatment response in mCRPC in comparison with circulating-tumor cells (CTC). We measured ARFL and ARV7 mRNA in PBMC and CTC from patients prior to receiving abiraterone (AA), enzalutamide (E), or taxanes by a pre-amplification plus quantitative reverse-transcription PCR. They were also tested in LNCaP-ARV7-transfected and in 22RV1 docetaxel-resistant (22RV1DR) cells. We studied 171 PBMC from 136 patients and from 24 non-cancer controls, and 47 CTC from 22 patients. High PBMC ARV7 levels correlated with worse AA/E and better taxane response. In taxane-treated patients high PBMC ARFL also correlated with longer progression-free survival (PFS). High ARV7 and ARFL expression were independently associated with better biochemical-PFS. Conversely, high CTC ARV7 and ARFL correlated with shorter radiological-PFS and overall survival, respectively. High ARV7 in 22RV1DR and LNCaP-ARV7 cells correlated with taxane resistance. In conclusion, ARFL and ARV7 at PBMC or CTC have a different predictive role in the taxane response, suggesting a potential influence of the AR pathway from PBMC in such response modulation.

2.
Proc Natl Acad Sci U S A ; 116(17): 8463-8470, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30962374

RESUMO

There are adaptive T-cell and antibody autoimmune responses to myelin-derived peptides in multiple sclerosis (MS) and to aquaporin-4 (AQP4) in neuromyelitis optica spectrum disorders (NMOSDs). Strategies aimed at antigen-specific tolerance to these autoantigens are thus indicated for these diseases. One approach involves induction of tolerance with engineered dendritic cells (tolDCs) loaded with specific antigens. We conducted an in-human phase 1b clinical trial testing increasing concentrations of autologous tolDCs loaded with peptides from various myelin proteins and from AQP4. We tested this approach in 12 patients, 8 with MS and 4 with NMOSD. The primary end point was the safety and tolerability, while secondary end points were clinical outcomes (relapses and disability), imaging (MRI and optical coherence tomography), and immunological responses. Therapy with tolDCs was well tolerated, without serious adverse events and with no therapy-related reactions. Patients remained stable clinically in terms of relapse, disability, and in various measurements using imaging. We observed a significant increase in the production of IL-10 levels in PBMCs stimulated with the peptides as well as an increase in the frequency of a regulatory T cell, known as Tr1, by week 12 of follow-up. In this phase 1b trial, we concluded that the i.v. administration of peptide-loaded dendritic cells is safe and feasible. Elicitation of specific IL-10 production by peptide-specific T cells in MS and NMOSD patients indicates that a key element in antigen specific tolerance is activated with this approach. The results warrant further clinical testing in larger trials.

3.
Mol Ther Methods Clin Dev ; 12: 134-144, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30623002

RESUMO

Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdc scid Il2rd tm1Wjl /SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients.

4.
J Crohns Colitis ; 13(5): 634-647, 2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-30521002

RESUMO

BACKGROUND AND AIMS: Recent studies have shown the efficacy of autologous haematopoietic stem cell transplantation [HSCT] in severely refractory Crohn's disease [CD] patients. HSCT is thought to eliminate auto-reactive cells; however, no specific studies of immune reconstitution in CD patients are available. METHODS: We followed a group of CD patients [n = 18] receiving autologous HSCT, with 50% of them achieving endoscopic drug-free remission. To elucidate the mechanisms driving efficacy, we monitored changes after HSCT in blood and intestine immune-cell composition. CD patients [n = 22] receiving anti-tumour necrosis factor [TNF]-α were included for comparison. RESULTS: Severe immune ablation followed by HSCT induced dramatic changes in both peripheral blood T and B cells in all patients regardless of the efficacy of the treatment. Endoscopic remission at week 52 following HSCT was associated with significant intestinal transcriptional changes. A comparison of the remission signature with that of anti-TNFα identified both common and unique genes in the HSCT-induced response. Based on deconvolution analysis of intestinal biopsy transcriptome data, we show that response to HSCT, but not to anti-TNFα, is associated with an expansion of naïve B-cells, as seen in blood, and a decrease in the memory resting T-cell content. As expected, endoscopic remission, in response to both HSCT and anti-TNFα, led to a significant reduction in intestinal neutrophil and M1 macrophage content. CONCLUSIONS: Peripheral blood immune remodelling after HSCT does not predict efficacy. In contrast, a profound intestinal T-cell depletion that is maintained long after transplant is associated with mucosal healing following HSCT, but not anti-TNFα.


Assuntos
Doença de Crohn/terapia , Transplante de Células-Tronco Hematopoéticas , Contagem de Linfócitos , Adulto , Linfócitos B , Doença de Crohn/imunologia , Feminino , Humanos , Macrófagos , Masculino , Neutrófilos , Linfócitos T , Resultado do Tratamento
5.
Front Immunol ; 9: 1169, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29904379

RESUMO

The identification of activated T-lymphocytes restricted to myelin-derived immunogenic peptides in multiple sclerosis (MS) and aquaporin-4 water channel in neuromyelitis optica (NMO) in the blood of patients opened the possibility for developing highly selective and disease-specific therapeutic approaches. Antigen presenting cells and in particular dendritic cells (DCs) represent a strategy to inhibit pro-inflammatory T helper cells. DCs are located in peripheral and lymphoid tissues and are essential for homeostasis of T cell-dependent immune responses. The expression of a particular set of receptors involved in pathogen recognition confers to DCs the property to initiate immune responses. However, in the absence of danger signals different DC subsets have been revealed to induce active tolerance by inducing regulatory T cells, inhibiting pro-inflammatory T helper cells responses or both. Interestingly, several protocols to generate clinical-grade tolerogenic DC (Tol-DC) in vitro have been described, offering the possibility to restore the homeostasis to central nervous system-related antigens. In this review, we discuss about different DC subsets and their role in tolerance induction, the different protocols to generate Tol-DCs and preclinical studies in animal models as well as describe recent characterization of Tol-DCs for clinical application in autoimmune diseases and in particular in MS and NMO patients. In addition, we discuss the clinical trials ongoing based on Tol-DCs to treat different autoimmune diseases.


Assuntos
Células Dendríticas/imunologia , Imunoterapia/métodos , Esclerose Múltipla/terapia , Neuromielite Óptica/terapia , Linfócitos T Auxiliares-Indutores/imunologia , Alarminas/metabolismo , Animais , Apresentação do Antígeno , Aquaporina 4/imunologia , Autoantígenos/imunologia , Células Dendríticas/transplante , Humanos , Tolerância Imunológica , Imunoterapia/tendências , Ativação Linfocitária , Bainha de Mielina/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T
7.
Front Oncol ; 8: 127, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29755954

RESUMO

Background and objective: Diffuse intrinsic pontine glioma (DIPG) is a lethal brainstem tumor in children. Dendritic cells (DCs) have T-cell stimulatory capacity and, therefore, potential antitumor activity for disease control. DCs vaccines have been shown to reactivate tumor-specific T cells in both clinical and preclinical settings. We designed a phase Ib immunotherapy (IT) clinical trial with the use of autologous dendritic cells (ADCs) pulsed with an allogeneic tumors cell-lines lysate in patients with newly diagnosed DIPG after irradiation (radiation therapy). Methods: Nine patients with newly diagnosed DIPG met enrollment criteria. Autologous dendritic cell vaccines (ADCV) were prepared from monocytes obtained by leukapheresis. Five ADCV doses were administered intradermally during induction phase. In the absence of tumor progression, patients received three boosts of tumor lysate every 3 months during the maintenance phase. Results: Vaccine fabrication was feasible in all patients included in the study. Non-specific KLH (9/9 patients) and specific (8/9 patients) antitumor response was identified by immunologic studies in peripheral blood mononuclear cells (PBMC). Immunological responses were also confirmed in the T lymphocytes isolated from the cerebrospinal fluid (CSF) of two patients. Vaccine administration resulted safe in all patients treated with this schema. Conclusion: These preliminary results demonstrate that ADCV preparation is feasible, safe, and generate a DIPG-specific immune response detected in PBMC and CSF. This strategy shows a promising backbone for future schemas of combination IT.

8.
J Leukoc Biol ; 102(3): 881-895, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28630103

RESUMO

Dendritic cells (DCs) are APCs essential in regulating the immune response. PGE2, produced during inflammation, has a pivotal role in the maturation of DCs and, therefore, is vital for the immune response. The large variety of biologic functions governed by PGE2 is mediated by its signaling through 4 distinct E-type prostanoid (EP) receptors. Immunogenic DCs express EP2 and EP4, which mediate the PGE2 signaling. However, the expression and function of EP receptors in human tolerogenic DCs (tol-DCs), which present an inhibitory phenotype, have not yet, to our knowledge, been assessed. To clarify the role of EP receptors in tol-DCs, we examined the expression of different EP receptors and their effect using selective agonists in human cells. We find that EP2 and EP3 expression are up-regulated in in vitro-generated tol-DCs compared with mature DCs (mDCs). Activation of EP2-EP4 has a direct effect on the surface expression of costimulatory molecules and maturation receptors, such as CD80, CD83, and CD86 or MHCII and CCR7 in tol-DCs, the latter being exclusively modulated by PGE2-EP4 signaling. Importantly, we find that EP2 and EP3 receptors are involved in tolerance induction through IL-10 production by tol-DCs. These results are in sharp contrast with the inflammatory role of EP4 Moreover, we show that DCs generated in the presence of agonists for EP receptors, induce naive T cell differentiation toward polarized Th1/Th17 cells. Given the differential effects of EP receptors, our results suggest that EP receptor agonist/antagonists might become relevant novel drug templates to modulate immune response.


Assuntos
Células Dendríticas/imunologia , Dinoprostona/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Imunossupressores/farmacologia , Receptores de Prostaglandina E Subtipo EP2/imunologia , Receptores de Prostaglandina E Subtipo EP3/imunologia , Antígenos CD/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Interleucina-10/imunologia , Receptores CCR7/imunologia , Células Th1/imunologia , Células Th17/imunologia
9.
Neurol Neuroimmunol Neuroinflamm ; 3(5): e276, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27648463

RESUMO

Neuromyelitis optica (NMO) and spectrum disorder (NMO/SD) represent a vexing process and its clinical variants appear to have at their pathogenic core the loss of immune tolerance to the aquaporin-4 water channel protein. This process results in a characteristic pattern of astrocyte dysfunction, loss, and demyelination that predominantly affects the spinal cord and optic nerves. Although several empirical therapies are currently used in the treatment of NMO/SD, none has been proven effective in prospective, adequately powered, randomized trials. Furthermore, most of the current therapies subject patients to long-term immunologic suppression that can cause serious infections and development of cancers. The following is the first of a 2-part description of several key immune mechanisms in NMO/SD that might be amenable to therapeutic restoration of immune tolerance. It is intended to provide a roadmap for how potential immune tolerance restorative techniques might be applied to patients with NMO/SD. This initial installment provides a background rationale underlying attempts at immune tolerization. It provides specific examples of innovative approaches that have emerged recently as a consequence of technical advances. In several autoimmune diseases, these strategies have been reduced to practice. Therefore, in theory, the identification of aquaporin-4 as the dominant autoantigen makes NMO/SD an ideal candidate for the development of tolerizing therapies or cures for this increasingly recognized disease.

10.
Neurol Neuroimmunol Neuroinflamm ; 3(5): e277, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27648464

RESUMO

Neuromyelitis optica spectrum disorder (NMO/SD) and its clinical variants have at their core the loss of immune tolerance to aquaporin-4 and perhaps other autoantigens. The characteristic phenotype is disruption of astrocyte function and demyelination of spinal cord, optic nerves, and particular brain regions. In this second of a 2-part article, we present further perspectives regarding the pathogenesis of NMO/SD and how this disease might be amenable to emerging technologies aimed at restoring immune tolerance to disease-implicated self-antigens. NMO/SD appears to be particularly well-suited for these strategies since aquaporin-4 has already been identified as the dominant autoantigen. The recent technical advances in reintroducing immune tolerance in experimental models of disease as well as in humans should encourage quantum leaps in this area that may prove productive for novel therapy. In this part of the article series, the potential for regulatory T and B cells is brought into focus, as are new approaches to oral tolerization. Finally, a roadmap is provided to help identify potential issues in clinical development and guide applications in tolerization therapy to solving NMO/SD through the use of emerging technologies. Each of these perspectives is intended to shine new light on potential cures for NMO/SD and other autoimmune diseases, while sparing normal host defense mechanisms.

11.
PeerJ ; 4: e2300, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27635311

RESUMO

Cellular therapies with tolerogenic antigen-presenting cells (tolAPC) show great promise for the treatment of autoimmune diseases and for the prevention of destructive immune responses after transplantation. The methodologies for generating tolAPC vary greatly between different laboratories, making it difficult to compare data from different studies; thus constituting a major hurdle for the development of standardised tolAPC therapeutic products. Here we describe an initiative by members of the tolAPC field to generate a minimum information model for tolAPC (MITAP), providing a reporting framework that will make differences and similarities between tolAPC products transparent. In this way, MITAP constitutes a first but important step towards the production of standardised and reproducible tolAPC for clinical application.

12.
Int J Pharm ; 511(2): 785-93, 2016 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-27477102

RESUMO

The design of innovative strategies to selectively target cells, such antigen-presenting cells and dendritic cells, in vivo to induce immune tolerance is gaining interest and relevance for the treatment of immune-mediated diseases. A novel loaded-nanosystem strategy to generate tolerogenic dendritic cells (tol-DCs) was evaluated. Hence budesonide (BDS) was encapsulated in multiwalled polyurethane-polyurea nanoparticles (PUUa NPs-BDS) based on self-stratified polymers by hydrophobic interactions at the oil-water interface. DCs treated with encapsulated BDS presented a prominent downregulation of costimulatory molecules (CD80, CD83 and MHCII) and upregulation of inhibitory receptors. Moreover, DCs treated with these PUUa NPs-BDS also secreted large amounts of IL-10, a crucial anti-inflammatory cytokine to induce tolerance, and inhibited T lymphocyte activation in a specific manner compared to those cells generated with free BDS. These results demonstrate that PUUa NPs-BDS are a highly specific and efficient system through which to induce DCs with a tolerogenic profile. Given the capacity of PUUa NPs-BDS, this delivery system has a clear advantage for translation to in vivo studies.


Assuntos
Budesonida/farmacologia , Células Dendríticas/imunologia , Tolerância Imunológica/efeitos dos fármacos , Nanopartículas/química , Polímeros/química , Poliuretanos/química , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Budesonida/química , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Imunoglobulinas/metabolismo , Interleucina-10/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Nanopartículas/ultraestrutura , Proteínas Nucleares/metabolismo , Tamanho da Partícula , Linfócitos T/efeitos dos fármacos , Transativadores/metabolismo
13.
Eur J Cancer ; 64: 167-74, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27428073

RESUMO

BACKGROUND: Autologous tumour lysate dendritic cell vaccine (ADC) has T-cell stimulatory capacity and, therefore, potential antitumour activity. We designed a phase II randomised trial of ADC + best supportive care (BSC) (experimental arm [EA]) compared with BSC (control arm [CA]), in pre-treated metastatic colorectal cancer (mCRC) patients. PATIENTS AND METHODS: Patients with progressive mCRC, at least to two chemotherapy regimens and Eastern Cooperative Oncology Group performance status (ECOG PS) 0-2, were randomised to EA versus CA. Stratification criteria: ECOG PS (0-1 versus 2) and lactate dehydrogenase (ULN). EA was administered subcutaneously till progressive disease. Primary end-point was progression-free survival (PFS) at 4 months. RESULTS: Fifty-two patients were included (28 EA/24 CA). An interim analysis recommended early termination for futility. No objective radiological response was observed in EA. Median PFS in EA was 2.7 months (95% confidence interval [CI], 2.3-3.2 months) versus 2.3 months (95% CI, 2.1-2.5 months) in CA (p = 0.628). Median overall survival (OS) was 6.2 months (95% CI, 4.4-7.9 months) in EA versus 4.7 months (95% CI, 2.3-7 months) in CA (p = 0.41). No ADC-related adverse events were reported. Immunization induces tumour-specific T-cell response in 21 of 25 (84%) patients. Responder patients have an OS of 7.3 months (95% CI, 5.2-9.4 months) versus 3.8 months (95% CI, 0.6-6.9 months) in non-responders; p = 0.026). CONCLUSION: Our randomised clinical trial comparing ADC + BSC versus BSC in mCRC demonstrates that ADC generates a tumour-specific immune response but not benefit on PFS and OS. Our results do not support the use of ADC alone, in a phase III trial.


Assuntos
Vacinas Anticâncer/administração & dosagem , Neoplasias Colorretais/terapia , Células Dendríticas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/secundário , Feminino , Humanos , Imunoterapia/métodos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de Sobrevida
15.
Gastroenterology ; 151(3): 489-500.e3, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27267052

RESUMO

BACKGROUND & AIMS: Crohn's disease (CD) has been associated with an altered immune response to commensal microbiota, mostly based on increased seroreactivity to microbial proteins. Although T cells are believed to contribute to the development of CD, little is known about the antigens involved. We investigated the antigen-specificity of T cells isolated from patients with CD. METHODS: We isolated peripheral blood mononuclear cells from 65 patients with CD and 45 healthy individuals (controls). We investigated T-cell reactivity to commensal microbial antigens using proliferation assays (based on thymidine incorporation and carboxyfluorescein succinimidyl ester dilution). Gene expression patterns were determined using microarray and real-time polymerase chain reaction analyses. Cytokines, chemokines, and antibodies were measured by enzyme-linked immunosorbent assay, flow cytometry, or multiplex cytokine assays. Intestinal crypts were obtained from surgical resection specimens of 7 individuals without inflammatory bowel disease. We examined the effects of commensal-specific CD4(+) T cells on primary intestinal epithelial cells from these samples. RESULTS: The bacterial proteins FlaX, A4-fla2, and YidX increased proliferation of CD4(+) T cells isolated from peripheral blood of patients with CD compared with controls. In blood samples from controls, CD4(+) T cells specific for FlaX, A4-fla2, or YidX had a T-helper (Th)1 phenotype; a larger proportion of CD4(+) T cells specific for these proteins in patients with CD had a Th17 phenotype or produced Th1 and Th17 cytokines. When supernatants collected from commensal-specific CD4(+) T cells from patients with CD were applied to healthy intestinal epithelial cells, the epithelial cells increased the expression of the chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL8 and the CC chemokine ligand 20 (CCL20). CONCLUSIONS: A larger proportion of commensal-specific CD4(+) T cells from patients with CD have a Th17 phenotype or produce Th1 and Th17 cytokines, compared with T cells from controls; this might contribute to intestinal inflammation in patients with CD. These cells might be targeted for treatment of CD. The transcriptional data of commensal-specific CD4(+) T cells from healthy individuals and CD patients have been deposited in the Gene Expression Omnibus at the National Center for Biotechnology Information (accession no: GSE70469).


Assuntos
Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Doença de Crohn/imunologia , Simbiose/imunologia , Células Th17/imunologia , Adulto , Anticorpos/sangue , Linfócitos T CD4-Positivos/microbiologia , Estudos de Casos e Controles , Quimiocinas/sangue , Doença de Crohn/microbiologia , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Microbioma Gastrointestinal/imunologia , Humanos , Mucosa Intestinal/imunologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real
16.
Clin Transl Immunology ; 5(1): e61, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26900474

RESUMO

HLDA10 is the Tenth Human Leukocyte Differentiation Antigen (HLDA) Workshop. The HLDA Workshops provide a mechanism to allocate cluster of differentiation (CD) nomenclature by engaging in interlaboratory studies. As the host laboratory, we invited researchers from national and international academic and commercial institutions to submit monoclonal antibodies (mAbs) to human leukocyte surface membrane molecules, particularly those that recognised molecules on human myeloid cell populations and dendritic cells (DCs). These mAbs were tested for activity and then distributed as a blinded panel to 15 international laboratories to test on different leukocyte populations. These populations included blood DCs, skin-derived DCs, tonsil leukocytes, monocyte-derived DCs, CD34-derived DCs, macrophage populations and diagnostic acute myeloid leukaemia and lymphoma samples. Each laboratory was provided with enough mAb to perform five repeat experiments. Here, we summarise the reactivity of different mAb to 68 different cell-surface molecules expressed by human myeloid and DC populations. Submitted mAbs to some of the molecules were further validated to collate data required to designate a formal CD number. This collaborative process provides the broader scientific community with an invaluable data set validating mAbs to leukocyte-surface molecules.

17.
PLoS One ; 11(1): e0146361, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26757431

RESUMO

BACKGROUND: During pregnancy, syncytiotrophoblast vesicles contribute to maternal tolerance towards the fetus, but also to pathologies such as pre-eclampsia. The aim of the study was to address whether Plasmodium falciparum and HIV infections in pregnancy affect the secretion, microRNA content and function of trophoblast microparticles. METHODS: Microparticles were isolated and characterized from 122 peripheral plasmas of Mozambican pregnant women, malaria- and/or HIV-infected and non-infected. Expression of placenta-related microRNAs in microparticles was analysed by qPCR and the effect of circulating microparticles on dendritic cells assessed by phenotype analysis and cytokine/chemokine measurement. RESULTS: Concentrations of total and trophoblast microparticles detected by flow cytometry were higher in HIV-positive (P = 0.005 and P = 0.030, respectively) compared to non-infected mothers, as well as in women delivering low birthweight newborns (P = 0.032 and P = 0.021, respectively). miR-517c was overexpressed in mothers with placental malaria (P = 0.034), compared to non-infected. Microparticles from HIV-positive induced a higher expression of MHCII (P = 0.021) and lower production of MCP1 (P = 0.008) than microparticles from non-infected women. CONCLUSIONS: In summary, alterations in total and trophoblast microparticles associated with malaria and HIV in pregnant women may have an immunopathogenic role. The potential for placental-derived vesicles and microRNAs as biomarkers of adverse outcomes during pregnancy and malaria infection should be confirmed in future studies.


Assuntos
Infecções por HIV/metabolismo , Malária Falciparum/metabolismo , MicroRNAs/metabolismo , Placenta/metabolismo , Plasmodium falciparum/fisiologia , Adulto , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Quimiocinas/metabolismo , Demografia , Células Dendríticas/metabolismo , Feminino , Humanos , Recém-Nascido , Malária Falciparum/patologia , Fenótipo , Gravidez , Resultado da Gravidez , Trofoblastos/metabolismo , Adulto Jovem
18.
J Crohns Colitis ; 9(12): 1071-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26303633

RESUMO

BACKGROUND AND AIMS: Ex vivo-generated autologous tolerogenic dendritic cells [tolDCs] can restore immune tolerance in experimental colitis. The aim of this study was to determine the safety and tolerability of administration of autologous tolDCs in refractory Crohn's disease [CD] patients. METHODS: A phase-I, single-centre, sequential-cohorts, dose-range study was designed. Stable tolDCs were generated ex vivo from monocytes following a previously developed protocol, and administered by sonography-guided intraperitoneal injection. Six sequential refractory-CD cohorts were established: the first three cohorts received a single intraperitoneal injection of tolDCs at escalating doses [2 x 10(6)/5 x 10(6)/10 x 10(6)]; and the last three cohorts received three biweekly intraperitoneal injections at same escalating doses. Safety was sequentially evaluated. Patients were assessed from week 0 to 12 and followed up for 1-year period for safety. RESULTS: Nine patients were included. No adverse effects were detected during tolDC injection or follow-up. Three patients withdrew from the study due to CD worsening. Crohn's Disease Activity Index [CDAI] decreased from 274 [60] {mean (standard deviation [SD])} to 222 [113] [p = 0.3]; one [11%] patient reached clinical remission [CDAI < 150] and two [22%] clinical response [CDAI decrease ≥ 100]. Crohn's Disease Endoscopic Index of Severity [CDEIS] decreased from 18 [5] to 13 [8] [p = 0.4]; lesions improved markedly in three patients [33%]. Quality of life (inflammatory bowel disease questionnaire [IBDQ]) changed from 125 [27] to 131 [38] [p = 0.7]; remission [IBDQ at Week 12 ≥ 170] was reached in one [11%] case and response [IBDQ score increase ≥ 16] in two [22%]. CONCLUSIONS: Intraperitoneal administration of autologous tolDCs appears safe and feasible in refractory CD patients. Further studies should be developed to test clinical benefit, determine the optimal administration route and dose, and monitor the immune responses; See [www.eudract.ema.europa.eu, EudraCT number 2007-003469-42; www.aemps.gob.es number PEI 08-049].


Assuntos
Doença de Crohn/terapia , Células Dendríticas/transplante , Adolescente , Adulto , Idoso , Doença de Crohn/imunologia , Feminino , Seguimentos , Humanos , Injeções Intraperitoneais , Masculino , Pessoa de Meia-Idade , Transplante Autólogo , Resultado do Tratamento , Adulto Jovem
19.
J Leukoc Biol ; 97(4): 751-60, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25624460

RESUMO

The aim of this study was to test the hypothesis whether MERTK, which is up-regulated in human DCs treated with immunosuppressive agents, is directly involved in modulating T cell activation. MERTK is a member of the TAM family and contributes to regulating innate immune response to ACs by inhibiting DC activation in animal models. However, whether MERTK interacts directly with T cells has not been addressed. Here, we show that MERTK is highly expressed on dex-induced human tol-DCs and participates in their tolerogenic effect. Neutralization of MERTK in allogenic MLR, as well as autologous DC-T cell cultures, leads to increased T cell proliferation and IFN-γ production. Additionally, we identify a previously unrecognized noncell-autonomous regulatory function of MERTK expressed on DCs. Mer-Fc protein, used to mimic MERTK on DCs, suppresses naïve and antigen-specific memory T cell activation. This mechanism is mediated by the neutralization of the MERTK ligand PROS1. We find that MERTK and PROS1 are expressed in human T cells upon TCR activation and drive an autocrine proproliferative mechanism. Collectively, these results suggest that MERTK on DCs controls T cell activation and expansion through the competition for PROS1 interaction with MERTK in the T cells. In conclusion, this report identified MERTK as a potent suppressor of T cell response.


Assuntos
Linfócitos T CD4-Positivos/enzimologia , Células Dendríticas/enzimologia , Tolerância Imunológica/fisiologia , Ativação Linfocitária/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Antígenos de Bactérias/imunologia , Comunicação Autócrina , Proteínas Sanguíneas/fisiologia , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Divisão Celular , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Humanos , Memória Imunológica , Testes de Liberação de Interferon-gama , Teste de Cultura Mista de Linfócitos , Monócitos/citologia , Especificidade do Receptor de Antígeno de Linfócitos T , Regulação para Cima/efeitos dos fármacos , c-Mer Tirosina Quinase
20.
Mol Immunol ; 63(2): 343-54, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25282042

RESUMO

Cellular therapies using immune cells and in particular dendritic cells (DCs) are being increasingly applied in clinical trials and vaccines. Their success partially depends on accurate delivery of cells to target organs or migration to lymph nodes. Delivery and subsequent migration of cells to regional lymph nodes is essential for effective stimulation of the immune system. Thus, the design of an optimal DC therapy would be improved by optimizing technologies for monitoring DC trafficking. Magnetic resonance imaging (MRI) represents a powerful tool for non-invasive imaging of DC migration in vivo. Domestic pigs share similarities with humans and represent an excellent animal model for immunological studies. The aim of this study was to investigate the possibility using pigs as models for DC tracking in vivo. Porcine monocyte derived DC (MoDC) culture with superparamagnetic iron oxide (SPIO) particles was standardized on the basis of SPIO concentration and culture viability. Phenotype, cytokine production and mixed lymphocyte reaction assay confirmed that porcine SPIO-MoDC culture were similar to mock MoDCs and fully functional in vivo. Alike, similar patterns were obtained in human MoDCs. After subcutaneous inoculation in pigs, porcine SPIO-MoDC migration to regional lymph nodes was detected by MRI and confirmed by Perls staining of draining lymph nodes. Moreover, after one dose of virus-like particles-pulsed MoDCs specific local and systemic responses were confirmed using ELISPOT IFN-γ in pigs. In summary, the results in this work showed that after one single subcutaneous dose of pulsed MoDCs, pigs were able to elicit specific local and systemic immune responses. Additionally, the dynamic imaging of MRI-based DC tracking was shown using SPIO particles. This proof-of-principle study shows the potential of using pigs as a suitable animal model to test DC trafficking with the aim of improving cellular therapies.


Assuntos
Rastreamento de Células/métodos , Células Dendríticas/imunologia , Monócitos/citologia , Animais , Forma Celular , Sobrevivência Celular , Células Dendríticas/citologia , Dextranos/metabolismo , ELISPOT , Humanos , Imunização , Imuno-Histoquímica , Interferon gama/metabolismo , Linfonodos/patologia , Imagem por Ressonância Magnética , Nanopartículas de Magnetita , Fenótipo , Coloração e Rotulagem , Sus scrofa
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