Assuntos
Betacoronavirus , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico por imagem , Pneumonia Viral/diagnóstico por imagem , COVID-19 , Teste para COVID-19 , Infecções por Coronavirus/diagnóstico , Humanos , Nasofaringe/virologia , Pandemias , SARS-CoV-2 , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: The number of circulating endothelial progenitor cells (EPCs) decreases on account of chronic kidney disease (CKD). METHODS: Twenty patients were enrolled in this prospective and randomised study in two parallel arms: conventional haemodialysis versus online haemodiafiltration. EPCs number and T-cell activation were analysed at baseline and monthly during a 4-month period of follow-up. RESULTS: CD38(bright) and HLA-DR+ expression among CD8 memory T cells were negatively associated with both CD34+ (r = -0.70, p = 0.0006) and CD34+ CD133+ (r = -0.62, p = 0.004) cell numbers. Conversely, a positive correlation was observed between CD34+ and CD34+ CD133+ cells with transferrin (r = 0.75, p = 0.0001 and r = 0.47, p = 0.04, respectively), and CD34+ CD133+ cells with transthyretin (r = 0.51, p = 0.02). No significant association was observed between dialysis modality and the evolution of the EPC number. CONCLUSIONS: Chronic T-cell activation may be a component of the malnutrition inflammation complex syndrome that adversely influences EPC mobilization in CKD patients.
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Células Progenitoras Endoteliais/metabolismo , Falência Renal Crônica/imunologia , Falência Renal Crônica/metabolismo , Ativação Linfocitária/imunologia , Desnutrição , Diálise Renal , Linfócitos T/imunologia , Biomarcadores , Contagem de Células , Feminino , Humanos , Imunofenotipagem , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/terapia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Estado Nutricional , Diálise Renal/efeitos adversos , Diálise Renal/métodos , Linfócitos T/metabolismoRESUMO
Individuals infected with HIV have higher circulating Epstein-Barr virus (EBV) DNA load compared to healthy carriers. This study investigated whether level of spontaneous immunoglobulin secreting cells, one of the major hallmarks of HIV infection, is associated with an increase of EBV DNA load in PBMCs and the spontaneous EBV lytic cycle ex vivo in patients infected with HIV. Spontaneous virus production by cells infected with EBV and EBV DNA loads in PBMCs from which CD8(+) T-cells were removed were measured in 20 HIV-aviremic and 14 HIV-viremic patients. The number of circulating immunoglobulin-secreting cells (Ig-SCs) and CD8(+) T-lymphocyte activation were also investigated. Patients with detectable HIV RNA in plasma exhibited higher spontaneous ex vivo EBV secretion and higher levels of EBV DNA in PBMCs than their aviremic counterparts. In the two groups observed, a positive correlation was found between PBMCs EBV DNA viral load and Ig-SCs, CD38(bright) expression on CD8(+) T-cells and EBV DNA load in cell culture supernatants. These findings suggest that B-cell polyclonal activation and B-cell terminal differentiation into Ig-SCs may fuel EBV DNA reservoir and promote EBV production ex vivo in patients infected with HIV.
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Células Produtoras de Anticorpos/imunologia , Infecções por Vírus Epstein-Barr/virologia , Infecções por HIV/complicações , Infecções por HIV/imunologia , Herpesvirus Humano 4/isolamento & purificação , Carga Viral , Adulto , Células Cultivadas , DNA Viral/análise , DNA Viral/genética , Feminino , HIV/isolamento & purificação , Humanos , Leucócitos Mononucleares/virologia , Masculino , Pessoa de Meia-Idade , RNA Viral/sangueRESUMO
Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P < 0.0001), but no significant correlation existed between EBV DNA levels in whole blood and enriched B-cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios.
Assuntos
Linfócitos B/virologia , Sangue/virologia , Técnicas de Laboratório Clínico/métodos , DNA Viral/isolamento & purificação , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/virologia , Carga Viral , Técnicas de Cultura de Células , Meios de Cultura , Monitoramento de Medicamentos/métodos , Infecções por HIV/complicações , Humanos , Linfoma de Células B/virologia , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Virologia/métodosRESUMO
A high prevalence of monoclonal gammopathy (MG) has been observed in HIV-infected patients. We explored the conditions associated with long-term persistence of serum monoclonal protein (M protein) in HIV-infected patients on antiretroviral therapy (ART). Of 21 patients with MG, M protein disappeared in 12 patients (58%) over 5 years of ART. Higher level of serum γ-globulin and higher percentages of circulating plasmablasts and plasma cells were observed in patients with persistent MG compared with patients with transient MG. MG persistence was associated with the cumulative time of detectable plasma HIV RNA after ART initiation, detection of Epstein-Barr virus (EBV) DNA in plasma, and a high level of EBV DNA in B cells. Poor control of HIV replication and detectable EBV replication in plasma were both associated with long-term MG persistence in patients on ART. In the case of viral control, MG associated with HIV infection is usually transient.
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Antirretrovirais/uso terapêutico , Terapia Antirretroviral de Alta Atividade , DNA Viral/antagonistas & inibidores , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por HIV/tratamento farmacológico , Paraproteinemias/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Adulto , Coinfecção , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Glicoproteínas/sangue , Glicoproteínas/genética , Glicoproteínas/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Herpesvirus Humano 4/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Paraproteinemias/imunologia , Paraproteinemias/virologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Plasmócitos/virologia , Fatores de Tempo , Carga Viral/efeitos dos fármacos , gama-Globulinas/genética , gama-Globulinas/imunologiaRESUMO
BACKGROUND: Despite the use of combined antiretroviral therapy, HIV-infected individuals have a higher risk of developing B-cell lymphoma compared to the general population. We aim to explore whether lymphocyte activation, increase in Th1 response as well as markers of EBV reactivation, may precede lymphoma diagnosis. METHODS: Thirteen cases and 26 controls matched on CD4(+) T-cell count and HIV plasma viral load were identified. Samples were collected 0 to 5 years prior to B-cell lymphoma diagnosis. Seven out of 13 (54 %) and 16/26 (61.5 %) of cases and controls were receiving antiretroviral therapy at the time of sampling, respectively. CD8(+) T-cell activation and Th1 cytokine concentrations were measured before lymphoma onset, together with IgG antibodies directed against viral capsid antigen (VCA) and serum levels of EBV DNA. RESULTS: A higher level of CD8(+) T-cell activation was observed in patients developing lymphoma. Four out of seven Th1 cytokine serum concentrations were significantly higher in patients with lymphoma than in the control group: IL-2R, IL-12p40/70, IFN-γ-inducible protein 10 (IP-10) and monokine induced by IFN-γ (MIG). Anti-VCA IgG level were significantly higher in cases than in controls. Four cases (30 %) but no controls had detectable EBV DNA in serum. CONCLUSION: A higher level of T-cell activation, Th1 cytokine serum concentration and markers of EBV replication, preceded B-cell lymphoma diagnosis. This may suggest that viral antigen stimulation is associated with the genesis of lymphoma in HIV-infected patients.
Assuntos
Linfoma de Burkitt/imunologia , Citocinas/metabolismo , Doença de Hodgkin/imunologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Células Th1/virologia , Regulação para Cima/imunologia , Adulto , Linfoma de Burkitt/diagnóstico , Linfoma de Burkitt/virologia , Estudos de Casos e Controles , Comorbidade , Citocinas/biossíntese , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/virologia , Humanos , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Retrospectivos , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/virologia , Células Th1/metabolismoRESUMO
OBJECTIVES: To evaluate the potential implication of circulating endothelial cells (CECs) in complications following endovascular treatment (EVT) of unruptured intracranial aneurysms. CECs characterized as CD146(+)/CD105(+)/CD45(-)/DAPI(+) were considered to originate from an altered endothelial cell layer of the vessel wall. STUDY DESIGN: In 15 patients, CECs were characterized and enumerated by the CellTracks(®) System in blood samples from: (1) femoral artery (FA), (2) internal carotid artery (ICA) before (ICA1) and after procedure (ICA2), and (3) a peripheral vein before (PV1) and after EVT (PV2). Ischemic brain events were assessed using diffusion weighted imaging (DWI-MRI) before and 24h after EVT. RESULTS: In ICA1, the median number of single CECs and clusters of 2-5 CECs were higher than in FA, ICA2, PV1 and PV2 samples (P<0.001). Clusters >5 cells, sometimes >50µm, were mainly observed in ICA1 and never in PV1, PV2 or PV samples from ten healthy subjects. This distribution of CECs suggested femoral and ICA injury by the devices used, leading to endothelium shearing and desquamation of CECs. All patients discharged on day two (NIHSS score=0), however silent ischemic brain lesions were observed in 9/15 (60%). CONCLUSIONS: EVT detaches single and clusters of CECs from wall arteries that may be implicated in silent ischemic brain lesions genesis. Enumeration of CECs associated with DWI-MRI might represent an interesting strategy for monitoring and optimizing endovascular devices, and further limit EVT-related complications.
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Imagem de Difusão por Ressonância Magnética/métodos , Células Endoteliais/imunologia , Endotélio Vascular/imunologia , Procedimentos Endovasculares/efeitos adversos , Aneurisma Intracraniano/imunologia , Aneurisma Intracraniano/terapia , Adulto , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Estatísticas não ParamétricasRESUMO
Immune activation is a main driver of AIDS- and non-AIDS-linked morbidities in the course of HIV-1 infection. As CCR5, the main HIV-1 co-receptor, is not only a chemokine receptor but also a co-activation molecule expressed at the surface of T cells, it could be directly involved in this immune activation. To test this hypothesis, we measured by flow cytometry the mean number of CCR5 molecules at the surface of non-activated CD4(+) T cells (CCR5 density), which determines the intensity of CCR5 signalling, and the percentage of CD8(+) T cells over-expressing CD38 (CD38 expression), a major marker of immune activation, in the blood of 67 HIV-1-infected, non-treated individuals. CCR5 density was correlated with CD38 expression independently of viral load (P=0.016). CCR5 density remained unchanged after highly active anti-retroviral therapy (HAART) introduction or cessation, whereas CD38 expression decreased and increased, respectively. Moreover, pre-therapeutic CCR5 density was highly predictive (r=0.736, P<10(-4) ) of residual CD38 over-expression after 9 months of HAART. Hence, CCR5 might play an immunological role in HIV-1 infection as a driver of immune activation. This could explain why CCR5 antagonists may have an inhibitory effect on immune activation.
Assuntos
ADP-Ribosil Ciclase 1/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1 , Ativação Linfocitária , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Humanos , Receptores de Morte Celular/metabolismo , Carga ViralRESUMO
The circulating endothelial cell population characterized by Damani et al. might be contaminated by endothelium damage after femoral catheterization.
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Movimento Celular , Células Endoteliais , Infarto do Miocárdio/patologia , Feminino , Humanos , MasculinoRESUMO
By compensating for the relative immaturity of the neonatal immune system, breast milk and breast-feeding prevent deaths in children. Nevertheless, transmission of HIV-1 through breast-feeding is responsible for more than half of new pediatric HIV infections. Recent studies of possible HIV-1 reservoirs in breast milk shed new light on features that influence HIV-1 transmission through breast-feeding. The particular characteristics of breast milk CD4(+) T cells that distinguish them from circulating blood lymphocytes (high frequency of cell activation and expression of memory and mucosal homing markers) facilitate the establishment of HIV-1 replication. Breast milk also contains a plethora of factors with anti-infectious, immunomodulatory, or anti-inflammatory properties that can regulate both viral replication and infant susceptibility. In addition, CD8(+) T lymphocytes, macrophages, and epithelial cells in breast milk can alter the dynamics of HIV-1 transmission. Even during efficient antiretroviral therapy, a residual stable, CD4(+) T cell-associated reservoir of HIV-1 is persistently present in breast milk, a likely source of infection. Only prophylactic treatment in infants--ideally with a long-acting drug, administered for the entire duration of breast-feeding--is likely to protect HIV-exposed babies against all forms of HIV transmission from breast milk, including cell-to-cell viral transfer.
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Aleitamento Materno/efeitos adversos , HIV-1/patogenicidade , Leite Humano/virologia , Feminino , Humanos , Transmissão Vertical de Doenças InfecciosasRESUMO
In a low-incidence setting, health care workers (HCW) are at a higher risk of tuberculosis than the general population. The suboptimal sensitivity of the QuantiFERON-TB Gold In-Tube (QFT) test remains a critical issue when identifying occupational latent tuberculosis infection (LTBI) in HCW. The aim of this study was to identify additional biomarkers in order to overcome the limits of gamma interferon (IFN-γ) release assays (IGRAs) and improve the performance of LTBI diagnosis within this population. Seventy Bacille Calmette-Guérin-vaccinated HCW regularly exposed to Mycobacterium tuberculosis were grouped according to QFT results into an LTBI-positive group (positive QFT, n = 8), an LTBI-negative group (normal QFT and negative tuberculin skin test [TST], n = 21), and an undetermined group (subpositive QFT and/or positive TST, n = 41). The secretion of 22 cytokines in response to QFT-specific stimulation was quantified using a multiparameter-based immunoassay. As a result, thresholds discriminating LTBI-positive from LTBI-negative HCW were established by comparing areas under the receiver operating characteristic curves for interleukin-2 (IL-2), IL-15, IFN-γ-induced protein 10 (IP-10), and the monokine induced by IFN-γ (MIG), which are biomarkers differentially secreted by the two groups. The combination of IL-15 and MIG provided a sensitivity of 100% and a specificity of 94.1% in distinguishing LTBI-positive from LTBI-negative HCW. When using IL-15 and MIG among the undetermined group, 6/45 HCW could be classified in the LTBI-positive group. The use of additional biomarkers after IGRA screening could improve the diagnosis of LTBI. The performance of these biomarkers and their use in combination with TST and/or QFT, as well as the cost-effectiveness of such a diagnostic strategy, should be evaluated in further larger clinical trials.
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Técnicas de Laboratório Clínico/métodos , Citocinas/análise , Pessoal de Saúde , Tuberculose Latente/diagnóstico , Mycobacterium tuberculosis/imunologia , Adulto , Biomarcadores/análise , Feminino , Humanos , Imunoensaio/métodos , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Detection of circulating tumor cells (CTCs) in the peripheral blood is a rapidly developing research field with clear clinical implications for the staging and monitoring of cancer patients. Current CTC assays, including the US Food and Drug Administration-cleared CellSearch® system, typically use markers [e.g., cytokeratins (CKs), the transmembrane protein EpCAM (epithelial cell adhesion molecule)] that are expressed on normal and malignant epithelial cells but not on the surrounding normal leukocytes. METHODS: We enrolled 53 patients with benign colon diseases (e.g., diverticulosis, benign polyps, Crohn disease, ulcerative rectocolitis, colonic endometriosis) and analyzed their peripheral blood with 2 previously validated CTC assays: the epithelial immunospot (EPISPOT) assay and the CellSearch system. The EPISPOT assay detects only viable, CK19-releasing CTCs that were enriched by depletion of CD45(+) leukocytes, whereas the CellSearch system detects CK-positive CTCs after positive EpCAM-based immunomagnetic enrichment. RESULTS: In patients with benign colon diseases, positive events that met the criteria for "tumor cells" were detected with both the CellSearch system (11.3%) and the CK19-EPISPOT assay (18.9%), whereas no positive events were detected in samples from healthy volunteers. Positive events were detected most frequently in patients with diverticulosis and Crohn disease. All positive events lacked expression of CD45, a common leukocyte antigen. CONCLUSIONS: These results indicate that patients with benign inflammatory colon diseases in particular can harbor viable circulating epithelial cells that are detectable with current CTC assays. This finding points to the need for further molecular characterization of circulating epithelial cells and has important implications for the use of CTC testing.
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Doenças do Colo/patologia , Células Epiteliais/patologia , Células Neoplásicas Circulantes/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/imunologia , Estudos de Casos e Controles , Moléculas de Adesão Celular/sangue , Moléculas de Adesão Celular/imunologia , Contagem de Células , Doenças do Colo/sangue , Molécula de Adesão da Célula Epitelial , Reações Falso-Positivas , Feminino , Humanos , Imunoensaio , Inflamação/sangue , Inflamação/patologia , Antígenos Comuns de Leucócito/sangue , Masculino , Pessoa de Meia-Idade , Coloração e Rotulagem , Adulto JovemRESUMO
The early steps of differentiation of human B cells into plasma cells are poorly known. We report a transitional population of CD20(low/-)CD38(-) preplasmablasts along differentiation of human memory B cells into plasma cells in vitro. Preplasmablasts lack documented B cell or plasma cell (CD20, CD38, and CD138) markers, express CD30 and IL-6R, and secrete Igs at a weaker level than do plasmablasts or plasma cells. These preplasmablasts further differentiate into CD20(-)CD38(high)CD138(-) plasmablasts and then CD20(-)CD38(high)CD138(+) plasma cells. Preplasmablasts were fully characterized in terms of whole genome transcriptome profiling and phenotype. Preplasmablasts coexpress B and plasma cell transcription factors, but at a reduced level compared with B cells, plasmablasts, or plasma cells. They express the unspliced form of XBP1 mRNA mainly, whereas plasmablasts and plasma cells express essentially the spliced form. An in vivo counterpart (CD19(+)CD20(low/-)CD38(-)IL-6R(+) cells) of in vitro-generated preplasmablasts could be detected in human lymph nodes (0.06% of CD19(+) cells) and tonsils (0.05% of CD19(+) cells). An open access "B to Plasma Cell Atlas," which makes it possible to interrogate gene expression in the process of B cell to plasma cell differentiation, is provided. Taken together, our findings show the existence of a transitional preplasmablast population using an in vitro model of plasma cell generation and of its in vivo counterpart in various lymphoid tissues.
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Linfócitos B/citologia , Diferenciação Celular/imunologia , Plasmócitos/citologia , Linfócitos B/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunofenotipagem , Análise de Sequência com Séries de Oligonucleotídeos , Plasmócitos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The concept of HIV-1/HSV-negative immunosynergy has recently come to light, which leads us to explore the impact of HIV-1 infection on HSV-specific T-cell immunity. METHODS: : A combination of interferon (IFN)-γ ELISpot and Luminex-based multicytokine profiling assays was used to compare, in a cross-sectional study, the HSV-specific CD4 and CD8 T-cell responses between 20 HIV-1/HSV-coinfected and 12 HIV-1-uninfected/HSV-infected individuals after in vitro restimulation with HSV glycoprotein D (gD) peptide epitopes. RESULTS: In response to CD4 and CD8 gD peptide epitopes, mean value (±standard errors of the mean) of the different IFN-γ-secreting T cells (ISC) means was significantly reduced in HIV-1/HSV-coinfected individuals (70 ISC ± 10 and 60 ISC ± 8/10 cells) compared with HIV-1-uninfected/HSV-infected individuals (280 ISC ± 25 and 234 ISC ± 23/10 cells, both P < 0.001). After stimulation with the immunodominant CD4 gD and CD8 gD peptide epitopes, the Th1 cytokine and CCR5 ligand secretions were decreased in the HIV-1-infected group although Th17 cytokines increased. The mean concentration of interleukin (IL)-2, IFN-γ, the IFN-γ-induced protein 10 kDa, and the monokine induced by IFN-γ was correlated to the mean concentration of macrophage inflammatory proteins (MIP-1α, MIP-1ß), RANTES and Eotaxin (ρ = 0.56, P = 0.02 and ρ = 0.52, P = 0.03). CONCLUSIONS: HIV-1 infection impairs both the number and function of HSV-specific T cells. The downregulation of Th1 cytokines and CCR5 ligands in HIV-1/HSV-coinfected individuals may further facilitate both HSV reactivations and HIV-1 replication.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Citocinas/metabolismo , Infecções por HIV/imunologia , HIV-1 , Simplexvirus/imunologia , Adulto , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Regulação para Baixo , Epitopos/imunologia , Feminino , Infecções por HIV/complicações , Infecções por HIV/virologia , Herpes Simples/complicações , Herpes Simples/imunologia , Humanos , Ligantes , Masculino , Pessoa de Meia-Idade , Receptores CCR5/metabolismo , Células Th1/imunologia , Proteínas Virais/imunologiaRESUMO
BACKGROUND: Acute infectious mononucleosis (AIM) is generally associated with a large EBV B cell reservoir cells and an intense B-cell polyclonal activation whereas the number of quiescent EBV-infected memory B cells in chronically EBV-infected healthy controls is very low. OBJECTIVES: To evaluate the extent and functionality of ex vivo B-cell polyclonal activation, quantify the EBV DNA integrated in B cells, enumerate the functional EBV DNA reservoir in B cells and circulating B cells spontaneously secreting EBV antigens in AIM. STUDY DESIGN: Circulating B cells and B cells differentiating into plamablasts and plasma cells, early (BZLF1)- and late viral antigen (gp350)-secreting-cells (SCs) were enumerated in six AIM patients and seven healthy EBV carriers. RESULTS: In vitro B-cell polyclonal activation induced 8000-24,000 BZLF1- and 1000-3000gp350-SCs/10(6) B cells, respectively. These data suggest that only 11.1-19.5% of cells expressing BZLF1 synthesized gp350 and so completed the EBV-lytic cycle. Furthermore, circulating spontaneous BZLF1- and gp350-SCs that reflect ongoing viral replication were rare (20-120 and 10-30/10(6) B cells, respectively), and their low numbers contrasted with the high levels of circulating plasma cells (1.1-10.2% of CD19(+) B cells). CONCLUSION: The in vivo terminal-B-cell differentiation into plasma cells could unmask EBV B-cell reservoir to specific cytotoxic T-cell response and combined with a predominant abortive functional-EBV-reservoir, strongly contribute to rapid decay of cellular EBV reservoir in AIM.
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Linfócitos B/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Mononucleose Infecciosa/imunologia , Ativação Linfocitária/imunologia , Doença Aguda , Antígenos Virais/imunologia , Linfócitos B/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/metabolismo , Humanos , Mononucleose Infecciosa/diagnóstico , Mononucleose Infecciosa/patologia , Linfócitos T Citotóxicos/imunologia , Transativadores/análise , Transativadores/biossíntese , Transativadores/metabolismo , Proteínas Virais/análise , Proteínas Virais/biossíntese , Proteínas Virais/metabolismo , alfa-Macroglobulinas/imunologiaRESUMO
BACKGROUND: Transmission of human immunodeficiency virus type 1 (HIV-1) through breast-feeding may involve both cell-free and cell-associated virus. This latter viral reservoir remains, however, to be fully explored. CD4+ T cell-associated virus production in breast milk was therefore investigated. METHODS: The ex vivo spontaneous production of HIV-1 antigen and HIV-1 RNA by CD4+ T cells was measured in paired blood and breast milk samples from 15 HIV-1 infected women treated or not with antiretroviral drugs. Spontaneous antigen secreting cells (HIV-1-AgSCs) from breast milk and blood were enumerated by an ELISpot assay, and cell-associated HIV-1 RNA was quantified by real-time PCR in supernatants of CD4+ T cells cultured for 18 hours without addition of polyclonal activators. RESULTS: Among the CD4+ T cells present in breast milk, memory cells expressing high levels of cell-surface activation markers were predominant. Spontaneous HIV-1-AgSCs were detected and enumerated in the breast milk of all 15 women, with a median number of 13.0 and 9.5 HIV-1- AgSCs/106 CD4+ T cells in aviremic (n = 7) and viremic (n = 8) women, respectively. Cell- associated HIV-1 RNA was detected in cell-free supernatants from 4/7 aviremic and 5/8 viremic individuals at median levels of 190 and 245 copies/ml, respectively. CONCLUSIONS: Activated CD4+ T cells producing HIV-1 are detected in the breast milk of untreated individuals as well as those receiving highly active antiretroviral therapy. This finding strongly suggests that HIV-1 replication occurs in latently infected CD4+ T cells that, upon spontaneous activation, revert to productively infected cells. These cells might be responsible for a residual breast milk transmission despite maternal highly active antiretroviral therapy.
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Linfócitos T CD4-Positivos/virologia , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/fisiologia , Leite Humano/citologia , Leite Humano/virologia , Fármacos Anti-HIV/uso terapêutico , ELISPOT , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/crescimento & desenvolvimento , HIV-1/isolamento & purificação , Humanos , Técnicas In Vitro , Gravidez , RNA Viral/biossíntese , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/biossíntese , Proteínas Virais/imunologia , Ativação ViralRESUMO
Bronchoalveolar lavage fluid (BALF) provides a source of mucosal CD4(+) T cells. We investigated the physiological properties of T lymphocytes from BALF and blood and their role on the dynamic of HIV-1 replication among AIDS patients with active lung infections. Pulmonary CD4(+) T cells consist mainly of effector memory cells (CD45RO(+) and CCR7(-)) with increased expression of activation markers (HLA-DR(+) and CD69(+)) when compared to the blood counterpart. We observed a high frequency of BALF cells capable of secreting HIV-1-Ags suggesting that the local lung environment may support favorable conditions for CD4(+) T lymphocytes harboring HIV-1 DNA to initiate the viral cycle. Nevertheless, the high number of IFN-γ-producing cells and the predominance of Th1 immune response in the lung could limit the secretion of HIV-1 RNA. In conclusion, the capacity of activated CD4(+) T cells to produce HIV-1 is driven by both the level and quality of cellular activation in the lung.
Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , HIV-1/fisiologia , Pulmão/virologia , Ativação Linfocitária/imunologia , Infecções Respiratórias/imunologia , Replicação Viral/imunologia , Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/imunologia , Antígenos CD/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/virologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Cicloeximida/farmacologia , DNA Viral/metabolismo , Antígenos HIV/metabolismo , HIV-1/isolamento & purificação , Humanos , Imunofenotipagem , Interferon gama/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Pulmão/imunologia , Pulmão/metabolismo , RNA Viral/metabolismo , Receptores CCR5 , Receptores CXCR4 , Infecções Respiratórias/etiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Subpopulações de Linfócitos T/virologia , Carga Viral/imunologia , Tropismo Viral/genética , Replicação Viral/efeitos dos fármacosRESUMO
INTRODUCTION: We evaluated whether CK19, one of the main cytoskeleton proteins of epithelial cells, is released as full-length protein from viable tumor cells and whether this property is relevant for metastatic progression in breast cancer patients. METHODS: EPISPOT (EPithelial ImmunoSPOT) assays were performed to analyze the release of full-length CK19 by carcinoma cells of various origins, and the sequence of CK19 was analyzed with mass spectrometry. Additional functional experiments with cycloheximide, Brefeldin A, or vincristine were done to analyze the biology of the CK19-release. CK19-EPISPOT was used to detect disseminated tumor cells in bone marrow (BM) of 45 breast cancer patients who were then followed up over a median of 6 years. RESULTS: CK19 was expressed and released by colorectal (HT-29, HCT116, Caco-2) and breast (MCF-7, SKBR3, and MDA-MB-231) cancer cell lines. The CK19-EPISPOT was more sensitive than the CK19-ELISA. Dual fluorescent EPISPOT with antibodies against different CK19 epitopes showed the release of the full-length CK19, which was confirmed by mass spectrometry. Functional experiments indicated that CK19 release was an active process and not simply the consequence of cell death. CK19-releasing cells (RCs) were detectable in BM of 44% to 70% of breast cancer patients. This incidence and the number of CK19-RCs were correlated to the presence of overt metastases, and patients with CK19-RCs had a reduced survival as compared with patients without these cells (P = 0.025, log-rank test; P = 0.0019, hazard ratio, 4.7; multivariate analysis). CONCLUSIONS: Full-length CK19 is released by viable epithelial tumor cells, and CK19-RCs might constitute a biologically active subset of breast cancer cells with high metastatic properties.
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Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Queratina-19/biossíntese , Adulto , Idoso , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Células Epiteliais/metabolismo , Epitopos , Feminino , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Metástase NeoplásicaRESUMO
While secretory Abs have been extensively explored in human breast milk, the existence, features, and functions of B lymphocytes remain largely unexplored in this compartment. We analyzed breast milk and blood lymphocytes from 21 lactating women, including 12 HIV-1-infected mothers. Breast milk B cells displayed a phenotype of class-switched memory B cells, with few IgD(+) memory and naive B cells. We observed that breast milk B lymphocytes bore a unique profile of adhesion molecules (CD44(+), CD62L(-), alpha(4)beta(7)(+/-), alpha(4)beta(1)(+)). Higher percentages of activated B cells (CD38(+)), large-sized B cells, plasmablasts, and plasma cells (CD19(+), CD20(low/-), CD27(high), CD138(+)) were found as compared with blood. This indicates that a significant proportion of breast milk B cells underwent terminal plasma cell differentiation. We also observed a higher frequency of cells secreting Ig spontaneously in breast milk. Among these cells, IgG-secreting cells predominated over IgA-secreting cells as measured by Ig ELISPOT assays. Specific Ab-secreting cells were investigated following polyclonal activation using the CD40L ligation. Finally, the detection of anti-HIV-1-secreting cells demonstrates the existence of B cells specific to HIV-1 Ag in breast milk from HIV-1-infected women. Breast milk B cells display a phenotype strikingly different from blood, are primed to secrete Abs, and have a mucosal homing profile similar to B cells located in gut-associated lymphoid tissue.
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Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Memória Imunológica , Leite Humano/imunologia , Anticorpos/análise , Moléculas de Adesão Celular/análise , Feminino , Anticorpos Anti-HIV/análise , Infecções por HIV , Humanos , Isotipos de Imunoglobulinas/análise , Ativação Linfocitária , Leite Humano/virologia , PlasmócitosRESUMO
PURPOSE: Circulating cell-free DNA in the blood of cancer patients harbors tumor-specific aberrations. Here, we investigated whether this DNA might also reflect the presence of circulating tumor cells (CTC). EXPERIMENTAL DESIGN: To identify the source of cell-free DNA in blood, plasma derived from 81 patients with prostate cancer was examined for CTCs and cell-free DNA. An epithelial immunospot assay was applied for detection of CTCs, and a PCR-based fluorescence microsatellite analysis with a panel of 14 polymorphic markers was used for detection of allelic imbalances (AI). RESULTS: The plasma DNA levels significantly correlated with the diagnosis subgroups of localized (stage M0, n = 69) and metastasized prostate cancer (stage M1, n = 12; P = 0.03) and with the tumor stage of these patients (P < 0.005). AI was found on cell-free DNA in plasma from 45.0% and 58.5% of M0 and M1 patients, respectively. Detection of CTCs showed that 71.0% or 92.0% of the M0 and M1 patients harbored 1 to 40 CTCs in their blood, respectively. The occurrence of CTCs correlated with tumor stage (P < 0.03) and increasing Gleason scores (P = 0.04). Notably, significant associations of the number of CTCs with the AI frequencies at the markers D8S137 (P = 0.03), D9S171 (P = 0.04), and D17S855 (P = 0.02) encoding the cytoskeletal protein dematin, the inhibitor of the cyclin-dependent kinase CDKN2/p16 and BRCA1, respectively, were observed. CONCLUSIONS: These findings show, for the first time, a relationship between the occurrence of CTCs and circulating tumor-associated DNA in blood, which, therefore, might become a valuable new source for monitoring metastatic progression in cancer patients.