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1.
Nat Med ; 25(9): 1402-1407, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501610

RESUMO

Natalizumab (NZM), a humanized monoclonal IgG4 antibody to α4 integrins, is used to treat patients with relapsing-remitting multiple sclerosis (MS)1,2, but in about 6% of the cases persistent neutralizing anti-drug antibodies (ADAs) are induced leading to therapy discontinuation3,4. To understand the basis of the ADA response and the mechanism of ADA-mediated neutralization, we performed an in-depth analysis of the B and T cell responses in two patients. By characterizing a large panel of NZM-specific monoclonal antibodies, we found that, in both patients, the response was polyclonal and targeted different epitopes of the NZM idiotype. The neutralizing activity was acquired through somatic mutations and correlated with a slow dissociation rate, a finding that was supported by structural data. Interestingly, in both patients, the analysis of the CD4+ T cell response, combined with mass spectrometry-based peptidomics, revealed a single immunodominant T cell epitope spanning the FR2-CDR2 region of the NZM light chain. Moreover, a CDR2-modified version of NZM was not recognized by T cells, while retaining binding to α4 integrins. Collectively, our integrated analysis identifies the basis of T-B collaboration that leads to ADA-mediated therapeutic resistance and delineates an approach to design novel deimmunized antibodies for autoimmune disease and cancer treatment.


Assuntos
Anticorpos Neutralizantes/administração & dosagem , Epitopos de Linfócito T/imunologia , Esclerose Múltipla/tratamento farmacológico , Natalizumab/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Neutralizantes/química , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Linfócitos B/efeitos dos fármacos , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Integrina alfa4/antagonistas & inibidores , Integrina alfa4/imunologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Conformação Proteica/efeitos dos fármacos , Linfócitos T/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
2.
Nat Protoc ; 14(8): 2571-2594, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31341290

RESUMO

RNase H-dependent PCR-enabled T-cell receptor sequencing (rhTCRseq) can be used to determine paired alpha/beta T-cell receptor (TCR) clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples. With the enhanced specificity of RNase H-dependent PCR (rhPCR), it achieves TCR-specific amplification and addition of dual-index barcodes in a single PCR step. For single cells, the protocol includes sorting of single cells into plates, generation of cDNA libraries, a TCR-specific amplification step, a second PCR on pooled sample to generate a sequencing library, and sequencing. In the bulk method, sorting and cDNA library steps are replaced with a reverse-transcriptase (RT) reaction that adds a unique molecular identifier (UMI) to each cDNA molecule to improve the accuracy of repertoire-frequency measurements. Compared to other methods for TCR sequencing, rhTCRseq has a streamlined workflow and the ability to analyze single cells in 384-well plates. Compared to TCR reconstruction from single-cell transcriptome sequencing data, it improves the success rate for obtaining paired alpha/beta information and ensures recovery of complete complementarity-determining region 3 (CDR3) sequences, a prerequisite for cloning/expression of discovered TCRs. Although it has lower throughput than droplet-based methods, rhTCRseq is well-suited to analysis of small sorted populations, especially when analysis of 96 or 384 single cells is sufficient to identify predominant T-cell clones. For single cells, sorting typically requires 2-4 h and can be performed days, or even months, before library construction and data processing, which takes ~4 d; the bulk RNA protocol takes ~3 d.


Assuntos
Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Células Cultivadas , Clonagem Molecular , Humanos , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Ribonuclease H/metabolismo , Linfócitos T/química , Linfócitos T/citologia
3.
Sensors (Basel) ; 19(13)2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31261919

RESUMO

T-cell resonators have been used lately for non-invasive blood glucose measurements for photoacoustic spectroscopy on skin samples. A resonator has a significant role in determining the strength of the measured signal and the overall sensitivity of the sensor. Here we present results of the measurement of the photoacoustic signal of such a T-cell resonator. The signal is also modelled using the amplitude mode expansion method, which is based on eigenmode expansion and the introduction of losses in the form of loss factors. The measurement reproduced almost all the calculated resonances from the numerical models with fairly good agreement. The cause of the differences between the measured and the simulated resonances are explained. In addition, the amplitude mode expansion simulation model is established as a faster and computationally less demanding photoacoustic simulation alternative to the viscothermal model. The resonance frequencies from the two models differ by less than 1.8%. It is noted that the relative height of the amplitudes from the two models depends on the location of the antinodes within the different parts of the resonator. The amplitude mode expansion model provides a quick simulation tool for the optimization and design of macro resonators.


Assuntos
Técnicas Biossensoriais , Glucose/isolamento & purificação , Técnicas Fotoacústicas , Linfócitos T/metabolismo , Simulação por Computador , Glucose/metabolismo , Humanos , Sistemas de Infusão de Insulina , Linfócitos T/química
4.
J Clin Exp Hematop ; 59(1): 1-16, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30918139

RESUMO

The microenvironment influences the behavior of follicular lymphoma (FL) but the specific roles of the immunomodulatory BTLA and TNFRSF14 (HVEM) are unknown. Therefore, we examined their immunohistochemical expression in the intrafollicular, interfollicular and total histological compartments in 106 FL cases (57M/49F; median age 57-years), and in nine relapsed-FL with transformation to DLBCL (tFL). BTLA expression pattern was of follicular T-helper cells (TFH) in the intrafollicular and of T-cells in the interfollicular compartments. The mantle zones were BTLA+ in 35.6% of the cases with similar distribution of IgD. TNFRSF14 expression pattern was of neoplastic B lymphocytes (centroblasts) and "tingible body macrophages". At diagnosis, the averages of total BTLA and TNFRSF14-positive cells were 19.2%±12.4STD (range, 0.6%-58.2%) and 46.7 cells/HPF (1-286.5), respectively. No differences were seen between low-grade vs. high-grade FL but tFL was characterized by low BTLA and high TNFRSF14 expression. High BTLA correlated with good overall survival (OS) (total-BTLA, Hazard Risk=0.479, P=0.022) and with high PD-1 and FOXP3+Tregs. High TNFRSF14 correlated with poor OS and progression-free survival (PFS) (total-TNFRSF14, HR=3.9 and 3.2, respectively, P<0.0001), with unfavorable clinical variables and higher risk of transformation (OR=5.3). Multivariate analysis including BTLA, TNFRSF14 and FLIPI showed that TNFRSF14 and FLIPI maintained prognostic value for OS and TNFRSF14 for PFS. In the GSE16131 FL series, high TNFRSF14 gene expression correlated with worse prognosis and GSEA showed that NFkB pathway was associated with the "High-TNFRSF14/dead-phenotype".In conclusion, the BTLA-TNFRSF14 immune modulation pathway seems to play a role in the pathobiology and prognosis of FL.


Assuntos
Linfoma Folicular/diagnóstico , Linfoma Difuso de Grandes Células B/diagnóstico , Receptores Imunológicos/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/química , Linfócitos B/patologia , Transformação Celular Neoplásica , Feminino , Humanos , Fatores Imunológicos , Linfoma Folicular/mortalidade , Linfoma Difuso de Grandes Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida , Linfócitos T/química
5.
Molecules ; 24(5)2019 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-30832312

RESUMO

The Class I Major Histocompatibility Complex (MHC) is a central protein in immunology as it binds to intracellular peptides and displays them at the cell surface for recognition by T-cells. The structural analysis of bound peptide-MHC complexes (pMHCs) holds the promise of interpretable and general binding prediction (i.e., testing whether a given peptide binds to a given MHC). However, structural analysis is limited in part by the difficulty in modelling pMHCs given the size and flexibility of the peptides that can be presented by MHCs. This article describes APE-Gen (Anchored Peptide-MHC Ensemble Generator), a fast method for generating ensembles of bound pMHC conformations. APE-Gen generates an ensemble of bound conformations by iterated rounds of (i) anchoring the ends of a given peptide near known pockets in the binding site of the MHC, (ii) sampling peptide backbone conformations with loop modelling, and then (iii) performing energy minimization to fix steric clashes, accumulating conformations at each round. APE-Gen takes only minutes on a standard desktop to generate tens of bound conformations, and we show the ability of APE-Gen to sample conformations found in X-ray crystallography even when only sequence information is used as input. APE-Gen has the potential to be useful for its scalability (i.e., modelling thousands of pMHCs or even non-canonical longer peptides) and for its use as a flexible search tool. We demonstrate an example for studying cross-reactivity.


Assuntos
Antígenos de Histocompatibilidade Classe I/química , Complexos Multiproteicos/química , Peptídeos/química , Linfócitos T/química , Sítios de Ligação , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe I/imunologia , Modelos Moleculares , Complexos Multiproteicos/imunologia , Peptídeos/imunologia , Ligação Proteica , Conformação Proteica , Linfócitos T/imunologia
6.
Clin Microbiol Infect ; 25(6): 753-758, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30292792

RESUMO

INTRODUCTION: Although solid organ transplant (SOT) recipients with pretransplant serology for cytomegalovirus (CMV-R+) are considered at intermediate risk for CMV infection post transplantation, CMV infection remains a major cause of morbidity in this population. We prospectively characterized whether having pretransplant CMV-specific cellular immunity is independently associated with controlling infection after transplantation in R + SOT recipients. METHODS: A prospective cohort of consecutive R + SOT recipients that received pre-emptive treatment for CMV infection was monitored after transplantation and variables were recorded during the follow-up. The cytomegalovirus-specific T-cell immune response was characterized by intracellular cytokine staining and viral loads determined using real-time PCR. RESULTS: One hundred and thirty-five R + SOT recipients were included (67 kidney, 64 liver, four liver-kidney). Only one-third of the patients (42; 31.85%) had CMV-specific T-cell immunity (CD8+CD69+INF-γ+ T cells >0.25%) before transplantation. Patients with negative pretransplant immunity had more CMV infection (49, 52.7% vs. 15, 35.7%; p 0.07) and received more antiviral therapy than those with immunity (32, 34.4% vs. 6, 14.3%, p 0.016). Having CMV specific immunity was an independent factor for protection from developing viraemia ≥2000 IU/mL (OR 0.276, 95% CI 0.105-0.725, p < 0.01) and lower administration of treatment (OR 0.398, 95% CI 0.175-0.905, p 0.028). Only patients with no pretransplant CMV-specific T-cell response were diagnosed with CMV-disease (8, 8.6% vs. 0, 0%, p 0.05). DISCUSSION: Our results show that having a pretransplant CMV specific T-cell response may be associated with a lower rate of CMV viraemia and less antiviral treatment after transplantation; however, more prospective studies are needed to confirm these findings.


Assuntos
Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Citomegalovirus/imunologia , Transplante de Órgãos/efeitos adversos , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Citocinas/análise , Citomegalovirus/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Coloração e Rotulagem , Linfócitos T/química , Carga Viral , Adulto Jovem
7.
Clin Lab Med ; 38(4): 595-605, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30420055

RESUMO

HLA epitope matching provides a better approach to stratify patients at risk of developing antibody-mediated rejection compared with counting HLA mismatches. However, several immunologic parameters are not incorporated into these algorithms used to assess HLA epitopes, raising questions about the predictive value of these programs. Therefore, it is imperative to obtain more 3D structural data of antibody-antigen binding to "train" these computer algorithms. Also, mechanistic studies should be performed to prove these theoretic "epitopes." Most important, more information is needed to ensure these predictive computer algorithms are equitable and safe to use in clinical diagnostics before wide-scale implementation.


Assuntos
Epitopos , Antígenos HLA , Teste de Histocompatibilidade , Transplante , Algoritmos , Linfócitos B/química , Linfócitos B/imunologia , Biologia Computacional , Humanos , Linfócitos T/química , Linfócitos T/imunologia
8.
Clin Epigenetics ; 10(1): 138, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30400990

RESUMO

BACKGROUND: Cancer treatments have substantially improved childhood cancer survival but are accompanied by long-term complications, notably chronic inflammatory diseases. We hypothesize that cancer treatments could lead to long-term epigenetic changes in immune cells, resulting in increased prevalence of inflammatory diseases in cancer survivors. RESULTS: To test this hypothesis, we established the epigenetic and transcriptomic profiles of immune cells from 44 childhood cancer survivors (CCS, > 16 years old) on full remission (> 5 years) who had received chemotherapy alone or in combination with total body irradiation (TBI) and hematopoietic stem cell transplant (HSCT). We found that more than 10 years post-treatment, CCS treated with TBI/HSCT showed an altered DNA methylation signature in T cell, particularly at genes controlling immune and inflammatory processes and oxidative stress. DNA methylation remodeling in T cell was partially associated with chronic expression changes of nearby genes, increased frequency of type 1 cytokine-producing T cell, elevated systemic levels of these cytokines, and over-activation of related signaling pathways. Survivors exposed to TBI/HSCT were further characterized by an Epigenetic-Aging-Signature of T cell consistent with accelerated epigenetic aging. To investigate the potential contribution of irradiation to these changes, we established two cell culture models. We identified that radiation partially recapitulated the immune changes observed in survivors through a bystander effect that could be mediated by circulating factors. CONCLUSION: Cancer treatments, in particular TBI/HSCT, are associated with long-term immune disturbances. We propose that epigenetic remodeling of immune cells following cancer therapy augments inflammatory- and age-related diseases, including metabolic complications, in childhood cancer survivors.


Assuntos
Envelhecimento/genética , Metilação de DNA , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Linfócitos T/imunologia , Adolescente , Sobreviventes de Câncer , Criança , Pré-Escolar , Epigênese Genética , Feminino , Humanos , Lactente , Recém-Nascido , Células Jurkat , Estresse Oxidativo , Linfócitos T/química
9.
Lab Chip ; 18(24): 3733-3749, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30397689

RESUMO

Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell activation upon recognition of target tumor cells. Notably, our platform includes a tracking system for each clone as well as a sorting procedure with 100% specificity validated by downstream single cell reverse-transcription PCR and sequencing of TCR chains. Our TCR screening prototype will facilitate immunotherapeutic screening and development of T cell therapies.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Receptores de Antígenos de Linfócitos T , Análise de Célula Única , Linfócitos T/química , Linfócitos T/citologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Desenho de Equipamento , Humanos , Imunoterapia Adotiva , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Linfócitos T/metabolismo , Linfócitos T/transplante
10.
ACS Nano ; 12(12): 11871-11880, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30421608

RESUMO

Understanding the binding of nanoparticles to receptors on biomembranes is critical to the development and screening of therapeutic materials. A prevailing understanding is that multivalent ligand-receptor binding leads to slower and confined translational motion of nanoparticles. In contrast, we report in this study distinct types of rotational dynamics of nanoparticles during their seemingly similar translational confinements in ligand-receptor binding. Our nanoparticles are fluorescently anisotropic and camouflaged with T cell membranes. As they bind to ligands on planar lipid bilayers, the particles transition from back-and-forth rocking motion to circling and eventually confined circling motion, while "hopping" between translational confinements. Both rotational and translational motions of the nanoparticles become more confined at higher ligand density. The time-dependent changes in particle rotation reveal different stages in the progression of multivalent binding between the cell-membrane coated nanoparticles and their ligands. Our work also demonstrates the promise of using combined rotational and translational single particle tracking to resolve biological interactions that could be "hidden" in translational measurements alone.


Assuntos
Membrana Celular/química , Bicamadas Lipídicas/química , Nanopartículas/química , Complexo Receptor-CD3 de Antígeno de Linfócitos T/química , Membrana Celular/metabolismo , Difusão , Polarização de Fluorescência , Corantes Fluorescentes/química , Humanos , Células Jurkat , Cinética , Ligantes , Imagem Óptica/métodos , Poliestirenos/química , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Propriedades de Superfície , Linfócitos T/química , Linfócitos T/metabolismo
11.
J Acquir Immune Defic Syndr ; 79(3): 399-406, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30312276

RESUMO

BACKGROUND: HIV infection is associated with premature aging, and mitochondrial integrity is compromised during the aging process. Because mitochondrial toxicity is a consequence of antiretroviral therapies (ARTs), we hypothesized HIV and long-term ART would correlate with immunosenescence and mitochondrial DNA (mtDNA) pathology. SETTING: Thirteen older HIV-infected individuals (aged >40 years) with virologic suppression (stratified by duration of ART) were compared with 10 uninfected controls well-matched for age. METHODS: Peripheral blood T-cells were immunophenotyped to measure immune activation, proliferation, and immunosenescence in subsets. mtDNA copies per cell and the relative abundance of mtDNA carrying the "common deletion" (RACD) were quantified by droplet digital polymerase chain reaction. RESULTS: Immune activation was higher in HIV-infected individuals than HIV-uninfected individuals in mature CD4 T-cell subsets (CD4TTM P = 0.025, CD4TEM P = 0.0020) regardless of ART duration. Cell populations from uninfected individuals were more likely to be more senescent populations in mature CD4 T-cell subsets (TTM P = 0.017), and CD8 (CD8TEMRA+ P = 0.0026). No differences were observed in mtDNA or RACD levels in any CD4 T-cell subsets, while CD8TSCM of infected individuals trended to have more mtDNA (P = 0.057) and reduced RACD (P = 0.0025). CONCLUSIONS: HIV-infected individuals demonstrated increased immune activation, but reduced senescence in more mature T-cell subsets. Increased mtDNA content and lower RACD in CD8TSCM suggest immune activation driven turnover of these cells in HIV-infected persons.


Assuntos
DNA Mitocondrial/análise , Infecções por HIV/patologia , Imunofenotipagem , Linfócitos T/química , Linfócitos T/imunologia , Adulto , Idoso , Proliferação de Células , Senescência Celular , DNA Mitocondrial/genética , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Reação em Cadeia da Polimerase
12.
Nat Chem Biol ; 14(10): 934-942, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30224695

RESUMO

T cell receptor cross-reactivity allows a fixed T cell repertoire to respond to a much larger universe of potential antigens. Recent work has emphasized the importance of peptide structural and chemical homology, as opposed to sequence similarity, in T cell receptor cross-reactivity. Surprisingly, though, T cell receptors can also cross-react between ligands with little physiochemical commonalities. Studying the clinically relevant receptor DMF5, we demonstrate that cross-recognition of such divergent antigens can occur through mechanisms that involve heretofore unanticipated rearrangements in the peptide and presenting MHC protein, including binding-induced peptide register shifts and extensions from MHC peptide binding grooves. Moreover, cross-reactivity can proceed even when such dramatic rearrangements do not translate into structural or chemical molecular mimicry. Beyond demonstrating new principles of T cell receptor cross-reactivity, our results have implications for efforts to predict and control T cell specificity and cross-reactivity and highlight challenges associated with predicting T cell reactivities.


Assuntos
Oligopeptídeos/química , Receptores de Antígenos de Linfócitos T/química , Antígenos/química , Autoimunidade , Reações Cruzadas , Cristalografia por Raios X , Epitopos/química , Humanos , Cinética , Ligantes , Mimetismo Molecular , Ligação Proteica , Domínios Proteicos , Retroviridae , Ressonância de Plasmônio de Superfície , Linfócitos T/química
13.
J Theor Biol ; 459: 36-44, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30240578

RESUMO

We consider a dynamic framework frequently used to model gene regulatory and signal transduction networks: monotonic ODEs that are composed of Hill functions. We derive conditions under which activity or inactivity in one system variable induces and sustains activity or inactivity in another. Cycles of such influences correspond to positive feedback loops that are self-sustaining and control-robust, in the sense that these feedback loops "trap" the system in a region of state space from which it cannot exit, even if the other system variables are externally controlled. To demonstrate the utility of this result, we consider prototypical examples of bistability and hysteresis in gene regulatory networks, and analyze a T-cell signal transduction ODE model from the literature.


Assuntos
Retroalimentação Fisiológica , Redes Reguladoras de Genes/fisiologia , Modelos Biológicos , Animais , Humanos , Transdução de Sinais , Linfócitos T/química , Linfócitos T/fisiologia
14.
HLA ; 92(6): 403-407, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30239163

RESUMO

Development of effective immunotherapy for chemoresistant malignancies can be advanced by studies in spontaneous cancer models, such as the dog. A crucial first step, T-cell epitope discovery, can be assisted by determination of binding motifs of common dog leukocyte antigen (DLA) class Ia allotypes. Boxers are popular, inbred dogs with increased risks of relevant target cancers and restricted MHC diversity. We sought to identify the motif of DLA-88*034:01, a breed-dominant allotype, to assist peptide prediction from tumor antigens. Mass spectrometry of eluted peptides showed a preference for nonamers with conserved amino acid preferences: basic at position (P)1; hydrophobic at P2; acidic at P4; histidine at P6; and phenylalanine at P9. This data should expedite finding epitopes restricted by this DLA-88 allotype.


Assuntos
Aminoácidos/química , Epitopos/química , Antígenos de Histocompatibilidade Classe I/química , Oligopeptídeos/química , Motivos de Aminoácidos , Aminoácidos/imunologia , Animais , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Cães , Epitopos/genética , Epitopos/imunologia , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Ligação Proteica , Linfócitos T/química , Linfócitos T/imunologia , Espectrometria de Massas em Tandem
15.
Methods Mol Biol ; 1813: 317-326, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30097878

RESUMO

Mouse T cells express the toxin-related ecto-ADP-ribosyltransferase ARTC2 that catalyzes the posttranslational ADP-ribosylation of cell surface proteins by transferring the ADP-ribose group of its substrate nicotinamide adenine dinucleotide (NAD+) to arginine residues of its target proteins. One well known target of ARTC2 is the ATP-gated P2X7 ion channel. ADP-ribosylation of P2X7 induces gating of the channel, calcium influx, ecto-domain shedding, phosphatidylserine externalization, and finally cell death. Previous studies have shown that the ARTC2 substrate NAD+ is released during T cell preparation. Since P2X7 is differentially expressed among T cell subpopulations, preparation-related ADP-ribosylation has a strong impact on the vitality of T cells that express high levels of P2X7. With this chapter we provide a protocol to monitor the consequences of preparation-related P2X7 ADP-ribosylation on T cells using regulatory T cells as generic T cell subpopulation known to express high levels of P2X7. However, this protocol could be easily adapted to other T cell populations.


Assuntos
ADP Ribose Transferases/química , Biologia Molecular/métodos , Linfócitos T/enzimologia , ADP Ribose Transferases/genética , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/genética , Animais , Arginina/química , Camundongos , NAD/química , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Linfócitos T/química
16.
Mol Cancer Res ; 16(12): 1902-1911, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30093564

RESUMO

Uveal melanoma progression can be predicted by gene expression profiles enabling a clear subdivision between tumors with a good (class I) and a poor (class II) prognosis. Poor prognosis uveal melanoma can be subdivided by expression of immune-related genes; however, it is unclear whether this subclassification is justified; therefore, T cells in uveal melanoma specimens were quantified using a digital PCR approach. Absolute T-cell quantification revealed that T-cell influx is present in all uveal melanomas associated with a poor prognosis. However, this infiltrate is only accompanied by differential immune-related gene expression profiles in uveal melanoma with the highest T-cell infiltrate. Molecular deconvolution of the immune profile revealed that a large proportion of the T-cell-related gene expression signature does not originate from lymphocytes but is derived from other immune cells, especially macrophages. Expression of the lymphocyte-homing chemokine CXCL10 by activated macrophages correlated with T-cell infiltration and thereby explains the correlation of T-cell numbers and macrophages. This was validated by in situ analysis of CXCL10 in uveal melanoma tissue with high T-cell counts. Surprisingly, CXCL10 or any of the other genes in the activated macrophage-cluster was correlated with reduced survival due to uveal melanoma metastasis. This effect was independent of the T-cell infiltrate, which reveals a role for activated macrophages in metastasis formation independent of their role in tumor inflammation. IMPLICATIONS: The current report uses an innovative digital PCR method to study the immune environment and demonstrates that absolute T-cell quantification and expression profiles can dissect disparate immune components.


Assuntos
Perfilação da Expressão Gênica/métodos , Macrófagos/patologia , Melanoma/genética , Linfócitos T/citologia , Neoplasias Uveais/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimiocina CXCL10/genética , Progressão da Doença , Feminino , Redes Reguladoras de Genes , Humanos , Linfócitos do Interstício Tumoral , Macrófagos/imunologia , Masculino , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Aprendizado de Máquina Supervisionado , Análise de Sobrevida , Linfócitos T/química , Linfócitos T/patologia , Microambiente Tumoral , Neoplasias Uveais/imunologia , Neoplasias Uveais/patologia , Adulto Jovem
17.
Diagn Pathol ; 13(1): 62, 2018 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-30153845

RESUMO

BACKGROUND: Breast carcinoma with osteoclast-like giant cells (OGCs) is infrequent, being most reported cased described as ductal invasive carcinomas. Invasive pleomorphic lobular carcinoma (PLC) is a distinct morphological variant of invasive lobular carcinoma characterized by higher nuclear atypia and pleomorphism than the classical type. In the best of our knowledge, a PLC with OGCs has not been previously reported. CASE PRESENTATION: We report the case of a 72-year-old woman presenting with a pleomorphic tumor of the left breast with a dense infiltration by OGCs and T lymphocytes with a 10:1 predominance of CD8+ over CD4+ cells. The diagnosis of a lymphoid or mesenchymal neoplasia was excluded after demonstrating keratin expression by the neoplastic cells. The absence of E-cadherin expression and the morphological features were consistent with the diagnosis PLC with OGCs. In addition, we demonstrated the deleterious mutation C.del866C in CDH1gene, but no mutations in any of the other 33 genes analyzed by next generation sequencing. CONCLUSIONS: Breast carcinoma with stromal osteoclast-like giant cells is a very rare tumor, for that reason, the use of the cytologic features and growth patterns in combination with immunohistochemically studies is mandatory for a correct diagnosis of lobular carcinoma. In addition, further studies are necessary to clarify the influence of OGCs in the prognosis of these patients.


Assuntos
Neoplasias da Mama/patologia , Carcinoma Lobular/patologia , Células Gigantes/patologia , Osteoclastos/patologia , Idoso , Antígenos CD/análise , Antígenos CD/genética , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias da Mama/química , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Caderinas/análise , Caderinas/genética , Carcinoma Lobular/química , Carcinoma Lobular/genética , Carcinoma Lobular/terapia , Quimioterapia Adjuvante , Análise Mutacional de DNA , Feminino , Células Gigantes/química , Humanos , Imuno-Histoquímica , Queratinas/análise , Linfócitos do Interstício Tumoral/química , Linfócitos do Interstício Tumoral/patologia , Mastectomia , Mutação , Osteoclastos/química , Linfócitos T/química , Linfócitos T/patologia , Resultado do Tratamento
18.
Saudi J Kidney Dis Transpl ; 29(4): 893-901, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30152427

RESUMO

Little was known about the relationships between the T lymphocytes (CD3+), expression of glucocorticoid receptors (GCR) and the response to GC treatment in children with the idiopathic nephrotic syndrome (INS). Our objective was to determine the relation between steroid responsiveness and GCR expression in T lymphocytes. The present study was carried out on 80 children with new-onset INS admitted in Pediatric Nephrology Units of Zagazig and Tanta University Hospitals and on 40 healthy children of the same age and sex who served as control group. The Subjects were subdivided into three groups as follows: Group 1 with 40 healthy children of comparable age and sex served as control group; Group 2 consisted of 60 patients diagnosed with INS with early response to steroid therapy [early responder (ER)] and Group 3 with 20 patients diagnosed with INS with late response to steroid therapy [late responder (LR)]. They were subjected to history taking, focusing on the pattern of response to steroids (ERs), clinical examination, routine laboratory investigations and the GCR/CD3% relationship. 75% of newly diagnosed INS cases were ER whereas 25% were LR. GCR/CD3% was significantly lower in LR group in comparison with ER and control groups, with a significant negative correlation between time of steroid responsiveness and GCR/CD3%. LR group showed lower GCR expression in T lymphocytes before starting therapy which may mean that GCR expression could be part of a pathophysiological mechanism of steroid responsiveness in these children and can be used as a useful diagnostic marker to predict steroid responsiveness in patients with INS.


Assuntos
Síndrome Nefrótica , Receptores de Glucocorticoides/análise , Esteroides/uso terapêutico , Linfócitos T/química , Estudos de Casos e Controles , Pré-Escolar , Monitoramento de Medicamentos , Feminino , Humanos , Masculino , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/epidemiologia , Síndrome Nefrótica/metabolismo , Receptores de Glucocorticoides/metabolismo , Linfócitos T/metabolismo
19.
Theranostics ; 8(15): 4199-4209, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30128047

RESUMO

B7-H3 is a transmembrane protein widely expressed in a variety of cancers and has been shown to play a role in anti-tumor immunity. This study aims to develop a molecular imaging probe to identify B7-H3 expression in tumors and to develop 89Zr-DS-5573a as a theranostic that could aid patient selection in clinical Phase I studies. Methods: The anti-B7-H3 humanised monoclonal antibody DS-5573a was labeled with zirconium-89 (89Zr-), and assessed for radiochemical purity, immunoreactivity (Lindmo analysis), antigen binding affinity (Scatchard analysis), and serum stability in vitro. In vivo biodistribution and imaging studies were performed with positron emission tomography and magnetic resonance imaging (PET/MRI) studies to identify and quantitate 89Zr-DS-5573a tumor uptake in a B7-H3-positive breast cancer model (MDA-MB-231) and a B7-H3-negative murine colon cancer model (CT26). Imaging and biodistribution studies were also performed in MDA-MB-231 tumor-bearing SCID mice in the absence and presence of therapeutic DS-5573a antibody dose (3 mg/kg DS-5573a). Results:89Zr-DS-5573a showed high and specific binding to B7-H3-expressing MDA-MB-231 cells (immunoreactivity on day 0, 75.0 ± 2.9%), and low binding to B7-H3-negative CT26 cells (immunoreactivity on day 0, 10.85 ± 0.11%) in vitro. 89Zr-DS-5573a demonstrated good serum stability in vitro with 57.2 ± 2.0% of immunoreactivity remaining on day 7. In vivo biodistribution studies showed high uptake of 89Zr-DS-5573a in B7-H3-expressing MDA-MB-231 tumor-bearing mice, achieving 32.32 ± 6.55 %ID/g on day 7 post injection in BALB/c nu/nu mice and 25.76 ± 1.79 %ID/g in SCID mice, with minimal evidence of non-specific uptake in normal tissues, and excellent tumor localization on PET/MRI. In a combined imaging/therapy study, receptor saturation was demonstrated in tumors responding to therapy. Conclusion:89Zr-DS-5573a demonstrates specific and prolonged targeting of B7-H3-expressing tumors in vivo. Saturation of binding sites was demonstrated in tumors responding to DS-5573a therapy. These results indicate that 89Zr-DS-5573a has potential to target B7-H3-expressing tumors in cancer patients. Furthermore 89Zr-DS-5573a has the potential to provide important insights into T cell biology through its specific binding to B7-H3.


Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antígenos B7/análise , Neoplasias da Mama/diagnóstico , Neoplasias do Colo/diagnóstico , Imagem Molecular/métodos , Radioisótopos/administração & dosagem , Linfócitos T/química , Zircônio/administração & dosagem , Animais , Modelos Animais de Doenças , Humanos , Imagem por Ressonância Magnética/métodos , Camundongos Endogâmicos BALB C , Camundongos SCID , Tomografia por Emissão de Pósitrons/métodos , Nanomedicina Teranóstica/métodos
20.
J Infect Dis ; 218(4): 624-632, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-29986086

RESUMO

Background: There are contradictory data about the influence that hepatitis C virus (HCV) has on immune activation and inflammation in patients coinfected with human immunodeficiency virus (HIV) and HCV. Methods: HIV/HCV-coinfected patients receiving antiretroviral treatment who achieved a sustained virological response with interferon-free regimens were consecutively enrolled in a prospective study. The following factors were assessed before, immediately after the end of, and 1 month after the end of therapy: expression of HLA-DR/CD38, PD-1, and CD57 on CD4+ and CD8+ T-cells; measurement of the total HIV DNA load in peripheral blood mononuclear cells; and determination of plasma levels of soluble CD14 (sCD14), lipopolysaccharide (LPS), 16S ribosomal DNA (rDNA), interleukin 6 (IL-6), D-dimers, and high-sensitivity C-reactive protein (hsCRP). Results: Ninety-seven patients were consecutively included. At the end of therapy and 1 month later, there were significant reductions in the expression of HLA-DR and CD38 in CD4+ and CD8+ T cells, as well as levels of proviral HIV DNA, sCD14, LPS, 16S rDNA, and D-dimer (P < .001). By contrast, the expression of PD-1 and CD57 in CD4+ and CD8+ T cells and levels of IL-6 and hsCRP did not change. The improvement in levels of immune activation markers, proviral HIV DNA, and microbial translocation markers did not translate into an increased CD4+ T-cell count or increased ratio of the CD4+ T-cell count to the CD8+ T-cell count. Conclusions: HCV eradication in HIV/HCV-coinfected patients results in significant decreases in levels of immune activation markers, proviral HIV DNA load, microbial translocation markers, and D-dimers. These findings support the use of HCV treatment for all HIV/HCV-coinfected patients, even those with low-grade fibrosis.


Assuntos
Antivirais/uso terapêutico , Translocação Bacteriana , Coinfecção/patologia , Infecções por HIV/patologia , HIV/isolamento & purificação , Hepatite C Crônica/tratamento farmacológico , Carga Viral , Biomarcadores/análise , Coinfecção/virologia , Feminino , HIV/imunologia , Infecções por HIV/complicações , Infecções por HIV/virologia , Hepatite C Crônica/complicações , Humanos , Fatores Imunológicos/análise , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Resposta Viral Sustentada , Linfócitos T/química , Linfócitos T/imunologia , Resultado do Tratamento
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