RESUMEN
First anti-HCV treatments, that include protease inhibitors in conjunction with IFN-α and Ribavirin, increase the sustained virological response (SVR) up to 80% in patients infected with HCV genotype 1. The effects of triple therapies on dendritic cell (DC) compartment have not been investigated. In this study we evaluated the effect of telaprevir-based triple therapy on DC phenotype and function, and their possible association with treatment outcome. HCV+ patients eligible for telaprevir-based therapy were enrolled, and circulating DC frequency, phenotype, and function were evaluated by flow-cytometry. The antiviral activity of plasmacytoid DC was also tested. In SVR patients, myeloid DC frequency transiently decreased, and returned to baseline level when telaprevir was stopped. Moreover, an up-regulation of CD80 and CD86 on mDC was observed in SVR patients as well as an improvement of IFN-α production by plasmacytoid DC, able to inhibit in vitro HCV replication.
Asunto(s)
Antivirales/uso terapéutico , Células Dendríticas/inmunología , Hepatitis C Crónica/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Anciano , Antígeno B7-1/inmunología , Antígeno B7-2/inmunología , Quimioterapia Combinada , Femenino , Hepatitis C Crónica/inmunología , Humanos , Interferón-alfa/inmunología , Interferón-alfa/uso terapéutico , Masculino , Persona de Mediana Edad , Polietilenglicoles/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Ribavirina/uso terapéutico , Respuesta Virológica Sostenida , Resultado del Tratamiento , Regulación hacia Arriba , Replicación ViralRESUMEN
West Nile virus (WNV) causes a severe central nervous system infection in humans, primarily in the elderly and immunocompromised subjects. Human γδ T-cells play a critical role in the immune response against viruses, and studies of WNV meningoencephalitis in laboratory mice described a role of γδ T-cells in the protective immune response. Aim of this study was to analyze the cytolytic and non-cytolytic antiviral activity of human Vδ2 T-cells against WNV replication. The anti-WNV activity of soluble factor released by zoledronic acid (ZA)-activated Vδ2 T-cell lines and the cytotoxic capability of Vδ2 T-cell lines against WNV-infected cells were tested in vitro. The activation of Vδ2 T-cell lines was able to inhibit WNV replication through the release of soluble factors. IFN-γ is massively released by activated Vδ2 T-cell lines and is involved in the anti-WNV activity. Moreover, the Vδ2 T-cell lines can efficiently kill WNV-infected cells possibly through perforin-mediated mechanism. Altogether, our results provide insight into the effector functions of human Vδ2 T-cells against WNV. The possibility to target these cells by ZA, a commercially available drug used in humans, could potentially offer a new immunotherapeutic strategy for WNV infection.
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Subgrupos de Linfocitos T/fisiología , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/inmunología , Animales , Línea Celular , Difosfonatos/farmacología , Humanos , Imidazoles/farmacología , Ratones , Ácido ZoledrónicoRESUMEN
BACKGROUND: Antiretroviral therapy has considerably reduced HIV disease progression, but complete eradication of HIV cannot actually be achieved. Moreover, prolonged use of protease inhibitors (PIs) causes profound changes in lipid metabolism with an increased risk of cardiovascular diseases. P-glycoprotein (P-gp) is expressed on many cell types, playing an important role in the efflux of drugs including PIs, limiting their intracellular concentration. Furthermore, several studies showed that P-gp is involved in lipid homeostasis and its activity is regulated by cholesterol. METHODS: THP-1 monocytes were used to study: (i) the influence of low-density lipoprotein (LDL) on P-gp expression and function, assessed by flow cytometry and quantitative real-time PCR analysis and measuring ritonavir and rhodamine-123 dye efflux, respectively; and (ii) the influence of ritonavir on cholesterol mobilization. The intracellular levels of ritonavir or cholesterol were measured by HPLC-UV and filipin staining, respectively. RESULTS: In THP-1 cells, LDL was able to yield an increase in both P-gp expression and activity. THP-1 cells treated with LDL showed a decrease in the intracellular ritonavir concentration in a dose-dependent manner. Notably, ritonavir induced reduced cholesterol mobilization in THP-1 cells, probably due to its inhibitory action on P-gp activity. CONCLUSIONS: Our data indicate a potential interplay between LDL and ritonavir mediated by P-gp. This interaction could influence both therapy effectiveness and cellular lipid metabolism, with important implications in the management of HIV patients treated with boosted PIs.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacocinética , Lipoproteínas LDL/metabolismo , Ritonavir/metabolismo , Ritonavir/farmacocinética , Línea Celular , Cromatografía Líquida de Alta Presión , Citosol/química , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Monocitos , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrofotometría UltravioletaRESUMEN
Hepatitis C virus (HCV) standard of care (SOC) therapy is not effective in a large percentage of patients and its efficacy may be evaluated only after several weeks. The aim of this work was to set up an in vitro liver culture assay able to preemptively predict SOC outcome by using residual liver samples from HCV patients. The in vitro response to SOC was found associated with the in vivo treatment outcome with a concordance of 100%. A wider clinical trial on a larger patient group is necessary to fully evaluate the impact of this procedure on the clinical management of untreated HCV patients.
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Antivirales/administración & dosificación , Monitoreo de Drogas/métodos , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Hígado/virología , Adulto , Anciano , Biopsia , Femenino , Hepacivirus/genética , Hepacivirus/aislamiento & purificación , Hepacivirus/fisiología , Hepatitis C/virología , Humanos , Técnicas In Vitro , Hígado/efectos de los fármacos , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Antigen-specific γδ T cells represent an early innate defense known to play an important role in anti-mycobacterial immunity. We have investigated the immune functions of Vγ9Vδ2 T cells from Broncho-Alveolar lavages (BAC) samples of active TB patients. We observed that BAC Vγ9Vδ2 T cells presented a strong down-modulation of CD3 expression compared with Vγ9Vδ2 T cells from peripheral blood. Furthermore, Vγ9Vδ2 T cells mainly showed a central memory phenotype, expressed high levels of NK inhibitory receptors and TEMRA cells showed low expression of CD16 compared to circulating Vγ9Vδ2 T cells. Interestingly, the ability of BAC Vγ9Vδ2 T cells to respond to antigen stimulation was dramatically reduced, differently from blood counterpart. These observations indicate that γδ T cell functions are specifically impaired in situ by active TB, suggesting that the alveolar ambient during tuberculosis may affect resident γδ T cells in comparison to circulating cells.
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Pulmón/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Tuberculosis/inmunología , Adolescente , Adulto , Líquido del Lavado Bronquioalveolar/inmunología , Complejo CD3/inmunología , Complejo CD3/metabolismo , Femenino , Citometría de Flujo , Humanos , Memoria Inmunológica/inmunología , Inmunofenotipificación , Interferón gamma/inmunología , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Pulmón/metabolismo , Pulmón/patología , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Receptores de Células Asesinas Naturales/inmunología , Receptores de Células Asesinas Naturales/metabolismo , Linfocitos T/metabolismo , Tuberculosis/sangre , Tuberculosis/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adulto JovenRESUMEN
In this work we evaluated plasmacytoid (pDC) and myeloid dendritic (mDC) cells activation before and during anti-HCV treatment in HCV+/HIV+ individuals. HCV+/HIV+ patients received Peg-IFN-α2b subcutaneously for 28 days, followed by oral weight-based ribavirin. DCs activation was evaluated by flow cytometry. Baseline pDC CD80 and CD86 expression was correlated with HIV, but not with HCV viral load. A transient decrease of HIV RNA was found not associated with DC activation. When patients were grouped according to early/sustained virological response (EVR/SVR) to anti-HCV treatment, baseline pDC CD80 and CD86 expression was higher in non-EVR and non-SVR compared to EVR and SVR. Moreover, in responder patients CD80 and CD86 were upregulated by IFN-α. Our data suggest a correlation between DCs activation and response to therapy. These findings could be helpful to better understand the mediators of IFN-α action in HCV+/HIV+ patients and to explore possible exploitation of this knowledge to improve therapeutic response.
Asunto(s)
Antivirales/uso terapéutico , Células Dendríticas/inmunología , Infecciones por VIH/tratamiento farmacológico , Hepatitis C Crónica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Polietilenglicoles/uso terapéutico , Células Dendríticas/efectos de los fármacos , Quimioterapia Combinada , Infecciones por VIH/complicaciones , Infecciones por VIH/inmunología , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/inmunología , Humanos , Interferón alfa-2 , Proteínas Recombinantes , Estudios Retrospectivos , Ribavirina/uso terapéutico , Resultado del Tratamiento , Carga Viral/efectos de los fármacosRESUMEN
BACKGROUND: When to initiate antiretroviral therapy in HIV infected patients is a difficult clinical decision. Actually, it is still a matter of discussion whether early highly active antiretroviral therapy (HAART) during primary HIV infection may influence the dynamics of the viral rebound, in case of therapy interruption, and overall the main disease course. METHODS: In this article we use a computational model and clinical data to identify the role of HAART timing on the residual capability to control HIV rebound after treatment suspension. Analyses of clinical data from three groups of patients initiating HAART respectively before seroconversion (very early), during the acute phase (early) and in the chronic phase (late), evidence differences arising from the very early events of the viral infection. RESULTS: The computational model allows a fine grain assessment of the impact of HAART timing on the disease outcome, from acute to chronic HIV-1 infection. Both patients' data and computer simulations reveal that HAART timing may indeed affect the HIV control capability after treatment discontinuation. In particular, we find a median time to viral rebound that is significantly longer in very early than in late patients. CONCLUSIONS: A timing threshold is identified, corresponding to approximately three weeks post-infection, after which the capability to control HIV replication is lost. Conversely, HAART initiation occurring within three weeks from the infection could allow to preserve a significant control capability. This time could be related to the global triggering of uncontrolled immune activation, affecting residual immune competence preservation and HIV reservoir establishment.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Terapia Antirretroviral Altamente Activa/métodos , Infecciones por VIH/tratamiento farmacológico , Simulación por Computador , Femenino , Humanos , Masculino , Factores de Tiempo , Resultado del Tratamiento , Carga ViralRESUMEN
Several subsets of alphabeta regulatory T cells (Tregs) have been described and studied intensively, but the potential regulatory role of gammadelta T cells remains largely unclear. Lymphocytes expressing gammadelta TCR are involved in both innate and adaptive immune responses, and their major adult human peripheral blood subset (Vgamma9Vdelta2) displays a broad reactivity against microbial agents and tumors. In this study we report that gammadelta T lymphocytes with regulatory functions (Vdelta2 Tregs) are induced in vitro in the presence of specific Ag stimulation and cytokines (TGF-beta1 and IL-15). These cells express FOXP3 and, similarly as alphabeta Tregs, suppress the proliferation of anti-CD3/anti-CD28 stimulated-PBMC. Phenotypic and functional analyses of Vdelta2 Tregs will very likely improve our understanding about the role of gammadelta T cells in the pathogenesis of autoimmune, infectious, and neoplastic diseases.
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Presentación de Antígeno , Factores de Transcripción Forkhead , Interleucina-15/fisiología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/fisiología , Proliferación Celular , Células Cultivadas , Citocinas , Humanos , Leucocitos Mononucleares/citología , Receptores de Antígenos de Linfocitos T gamma-deltaRESUMEN
Vgamma9Vdelta2 T cells display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. The activation of Vgamma9Vdelta2 T cells by aminobisphosphonate drugs such as zoledronic acid (ZOL) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells. The aim of this work was to evaluate the ability of soluble factors released by ZOL-activated Vgamma9Vdelta2 T cells to induce granulocyte activation. We showed that soluble factors released by ZOL-stimulated Vgamma9Vdelta2 T cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. Proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated Vgamma9Vdelta2 T cells. Moreover, MCP-2 depletion by neutralizing Ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. Altogether, these data show a Vgamma9Vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of Vgamma9Vdelta2 T-lymphocytes in orchestrating the immune response. In conclusion, an immune modulating strategy targeting Vgamma9Vdelta2 T cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases.
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Quimiocina CCL8/metabolismo , Granulocitos/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta , Subgrupos de Linfocitos T/inmunología , Efecto Espectador/efectos de los fármacos , Efecto Espectador/inmunología , Degranulación de la Célula/inmunología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Células Cultivadas , Quimiocina CCL8/fisiología , Técnicas de Cocultivo , Difosfonatos/farmacología , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Humanos , Imidazoles/farmacología , Activación de Linfocitos/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/metabolismo , Ácido Zoledrónico , alfa-Defensinas/metabolismoRESUMEN
Protective immunity against mycobacterial infections such as Mycobacterium tuberculosis is mediated by interactions between specific T cells and activated antigen presenting cells. To date, many aspects of mycobacterial immunity have shown that innate cells could be the key elements that substantially may influence the subsequent adaptive host response. During the early phases of infection, innate lymphocyte subsets play a pivotal role in this context. Here we summarize the findings of recent investigations on γδ T lymphocytes and their role in tuberculosis immunity.
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Mycobacterium tuberculosis/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología , Tuberculosis/inmunología , Inmunidad Adaptativa , Animales , Humanos , Inmunidad Innata , Memoria Inmunológica , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Tuberculosis/microbiologíaRESUMEN
Multicompartment nanoscopic carriers can be easily assembled by inducing the aggregation of anionic "hybrid" niosomes by means of cationic biocompatible polyelectrolytes. The resulting vesicle clusters, whose size and overall net charge can be easily controlled by varying the polyelectrolyte-to-particle charge ratio, show an interesting potential for multidrug delivery. In this article we provide strong evidence for their effective use in vitro as multicompartment vectors selectively directed toward monocyte/macrophage cells, showing that the monocyte/macrophage-mediated activation of Tγδ lymphocytes induced by zoledronic acid is enhanced by a factor 10(3) when the zoledronic acid is intracellularly delivered through these carriers. Furthermore, the multicompartment É-polylysine niosome clusters, with their intrinsic selectivity toward macrophages, appear particularly suitable for implementing therapeutic strategies against chronically infected macrophages. FROM THE CLINICAL EDITOR: É-polylysine niosome clusters, with their intrinsic selectivity toward macrophages, offer the potential for multidrug delivery. The effectiveness of aminobisphosphonate zoledronate is demonstrated to enhance the recruitment of Tγδ lymphocytes by macrophages by 2 orders of magnitude, suggesting a new therapeutic strategy for addressing pathologies featuring chronically infected macrophages.
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Conservadores de la Densidad Ósea/administración & dosificación , Difosfonatos/administración & dosificación , Imidazoles/administración & dosificación , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/metabolismo , Conservadores de la Densidad Ósea/metabolismo , Difosfonatos/metabolismo , Humanos , Imidazoles/metabolismo , Leucocitos/metabolismo , Liposomas , Macrófagos/metabolismo , Nanomedicina , Polilisina/química , Polilisina/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Ácido ZoledrónicoRESUMEN
BACKGROUND: Pandemic A/H1N1v influenza is characterized by a mild clinical course. However, a small subset of patients develops a rapidly progressive course caused by primary viral pneumonia or secondary bacterial infections that, in many cases, lead to death due to respiratory failure. The aim of the present study was to analyze the involvement of the immune response in the clinical presentation of H1N1v influenza. METHODS: The differentiation and functional capability of T cells from H1N1v-infected patients presenting with either mild disease (n=22) or severe or fatal disease (n=6) were compared. Moreover, plasma cytokines and chemokines were quantified. RESULTS: T cells from H1N1v-infected patients presenting with a severe clinical course resulted in impaired effector cell differentiation and failed to respond to mitogenic stimulation. T cell anergy was strictly associated with a severe acute phase of infection, but T cells could be restored in patients able to recover. Of interest, massive expression of CD95 marker was found on anergic T cells, suggesting an apoptosis-related mechanism. Finally, lower plasma levels of interferon-alpha and monocyte chemoattractant protein-1 were found in patients with a worse clinical course of influenza, suggesting impaired production of these cytokines. CONCLUSIONS: Our results show a strict association between host immune competence and the severity of the clinical course of H1N1v infection. By monitoring host functional response, patients with an enhanced risk of developing influenza-associated severe complications could be identified in a timely manner.
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Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/inmunología , Gripe Humana/fisiopatología , Linfocitos T/inmunología , Linfocitos T/patología , Adolescente , Adulto , Diferenciación Celular , Niño , Preescolar , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/mortalidad , Gripe Humana/virología , Interferón-alfa/sangre , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Adulto Joven , Receptor fas/metabolismoAsunto(s)
Antirretrovirales/uso terapéutico , Terapia Antirretroviral Altamente Activa/métodos , Infecciones por VIH/tratamiento farmacológico , Células Madre Hematopoyéticas/fisiología , Linfopoyesis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Humanos , Masculino , Prevención Secundaria , Subgrupos de Linfocitos T/inmunología , Adulto JovenRESUMEN
INTRODUCTION: Amino-bisphosphonates are potent activators of human gammadelta T cells. The aim of our study was to evaluate the immunomodulating properties of a single-dose of zoledronic acid (ZA) on gammadelta T cells in a select group of disease-free breast cancer patients with osteopenia. MATERIALS AND METHODS: Blood samples were obtained, from 23 patients, before and 7, 28, 56, 90 and 180 days after a single-dose (4 mg) of ZA and analyzed by flow cyometry. RESULTS: A significant decrease of the different gammadelta T cell subsets was observed: Naïve (CD3+/Vdelta2+/CD45RA+/CD27+) after 180 days (P < 0.01); Central Memory (CD3+/Vdelta2+/CD45RA-CD27+) after 28 (P < 0.05), 90 (P < 0.01) and 180 days (P < 0.01); and Effector Memory (CD3+/Vdelta2+/CD45RA-/CD27-) after 56 (P < 0.01) and 90 (P < 0.05) days. Based on the observed gammadelta T cells kinetics patients could be divided in two groups: "responders" that showed a significant decrease in total numbers of gammadelta T cells and "non-responders" that showed no significant change. However, in vitro phosphoantigen stimulation of patients cells did not show significant differences in terms of IFN-gamma response by Vdelta2 T cells. CONCLUSION: We describe for the first time a long-lasting activation of effector subsets of gammadelta T cells in disease-free breast cancer patients after a single-dose of ZA. Our results highlight the need to further investigate the clinical significance of the immunomodulating properties of N-BPs.
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Neoplasias de la Mama/tratamiento farmacológico , Difosfonatos/uso terapéutico , Imidazoles/uso terapéutico , Receptores de Antígenos de Linfocitos T alfa-beta/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/inmunología , Femenino , Humanos , Subgrupos Linfocitarios/efectos de los fármacos , Persona de Mediana Edad , Estadificación de Neoplasias , Linfocitos T/inmunología , Ácido ZoledrónicoAsunto(s)
Coinfección/inmunología , Células Dendríticas/inmunología , Infecciones por VIH/inmunología , VIH/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Interleucinas/genética , Coinfección/genética , Coinfección/virología , Células Dendríticas/virología , Genotipo , VIH/genética , Infecciones por VIH/genética , Infecciones por VIH/virología , Hepacivirus/genética , Hepatitis C/genética , Hepatitis C/virología , Humanos , Interferones , Interleucinas/inmunologíaRESUMEN
In the pre-highly active antiretroviral therapy (HAART) era, clinical trials showed that interferon (IFN) treatment was able to delay AIDS progression and prolong survival. Along with HAART, ancillary use of IFN during primary infection and before HAART therapy initiation has been effective. Also endogenous IFN may positively affect the progression of HIV infection, as suggested in GB virus type C (GBV-C) coinfected patients. In this pilot study, we tried to prevent rebound of HIV replication in patients who interrupted HAART by covering the drug-free time with administration of pegylated IFN (PEGIFN). Twenty-four HIV-hepatitis C virus (HCV) patients who started IFN treatment for liver disease, after variable time from having interrupted HAART, were enrolled. HIV RNA was determined during a 2-month period. In patients who interrupted HAART at variable times before initiating IFN and, therefore, had experienced a complete viral rebound, IFN caused a significant reduction of viral load lasting at least 4 weeks. Moreover, 3 of the 4 patients who started IFN concomitantly with the HAART discontinuation showed complete control of viral rebound, delaying the resumption of viral replication for more than 2 weeks. These preliminary findings suggest that a structured therapy interruptions (STI) strategy may be feasible provided that IFN is administered during the drug-free times, possibly delaying drug resistance, lessening toxicity, reducing costs, and prolonging survival.
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Terapia Antirretroviral Altamente Activa , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH/efectos de los fármacos , Interferones/farmacología , Carga Viral , Adulto , Recuento de Linfocito CD4 , Estudios de Casos y Controles , Femenino , Humanos , Interferones/inmunología , Masculino , Persona de Mediana Edad , ARN Viral/sangreAsunto(s)
Fármacos Anti-VIH/administración & dosificación , Terapia Antirretroviral Altamente Activa/métodos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Subgrupos de Linfocitos T/inmunología , Antígenos CD/análisis , Linfocitos T CD4-Positivos/química , Linfocitos T CD8-positivos/química , Homeostasis , Humanos , Factores de TiempoRESUMEN
We report the results of an external quality control program, including 17 Italian centers involved in the care of patients infected by HIV, to evaluate CD4 T cell count proficiency and reproducibility. The centers received two commercial stabilized blood preparations, one with "normal" and one with "low" CD4 T cell content. The centers were asked to process the samples two times, 1 week apart, with the same procedure used for samples from HIV patients. Most centers showed a good performance of CD4 frequency and absolute count determinations. In particular, the "low" sample was correctly analyzed by all centers; only two underestimated the "normal" sample CD4 frequency, and only one underestimated the CD4 absolute count by >100 CD4 cells/microl. Overall, our data suggest that most Italian laboratories provide reliable and reproducible results in evaluating CD4 T cells in HIV(+) samples.
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Recuento de Linfocito CD4 , Infecciones por VIH/sangre , Infecciones por VIH/terapia , Humanos , Italia , Laboratorios de Hospital/normas , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.