RESUMEN
Although analytical treatment interruption is used as a strategy to test immunotherapeutic agents in HIV-infection, it may pose a risk for study participants. The potential risks of short-term interruption of antiretroviral therapy (ART) during treatment with an autologous dendritic cell immune-based therapy (AGS-004-001) were assessed using data from a subgroup of subjects in the strategies for management of antiretroviral therapy (SMART) study with matched eligibility criteria. A retrospective subgroup analysis of the SMART study population using the eligibility criteria and treatment stopping rules of AGS-004-001 study was analyzed. Key inclusion criteria for AGS-004-001 study were applied to the data collected from participants of the SMART study. There were 440 of 2,720 on the drug conservation arm and 436 of 2,752 on the viral suppression arm that matched the AGS-004-001 inclusion criteria and were used in the SMART subgroup analysis. In the first 16 weeks following randomization into the SMART study there were no deaths in either subgroup. There were two AIDS-related events in the drug conservation subgroup and one in the viral suppression subgroup, making the overall risk of AIDS-related events 2 per 100 person years (0.005%) and 1 per 100 person years (0.002%) in the two subgroups, respectively. There were 6/440 subjects (1.4%) in the drug conservation subgroup and 4/436 subjects (0.92%) in the viral suppression subgroup who experienced Grade 2 adverse events. These results demonstrated that analytical treatment interruption within the context of highly selective, closely monitored studies assessing the antiviral activity of immune-based agents should be an acceptable strategy for at least 16 weeks.
Asunto(s)
Infecciones por VIH/terapia , Inmunoterapia/métodos , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Medición de Riesgo , Privación de TratamientoRESUMEN
OBJECTIVES: Conflicting results have been reported regarding the ability of valproic acid (VPA) to reduce the size of HIV reservoirs in patients receiving suppressive highly active antiretroviral therapy (HAART). In a randomized multicentre, cross-over study, we assessed whether adding VPA to stable HAART could potentially reduce the size of the latent viral reservoir in CD4 T cells of chronically infected patients. METHODS: A total of 56 virologically suppressed patients were randomly assigned either to receive VPA plus HAART for 16 weeks followed by HAART alone for 32 weeks (arm 1; n = 27) or to receive HAART alone for 16 weeks and then VPA plus HAART for 32 weeks (arm 2; n = 29). VPA was administered at a dose of 500 mg twice a day (bid) and was adjusted to the therapeutic range. A quantitative culture assay was used to assess HIV reservoirs in CD4 T cells at baseline and at weeks 16 and 48. RESULTS: No significant reductions in the frequency of CD4 T cells harbouring replication-competent HIV after 16 and 32 weeks of VPA therapy were observed. In arm 1, median (range) values of IU per log(10) billion (IUPB) cells were 2.55 (range 1.20-4.20), 1.80 (range 1.0-4.70) and 2.70 (range 1.0-3.90; P = 0.87) for baseline, week 16 and week 48, respectively. In arm 2, median values of IUPB were 2.55 (range 1.20-4.65), 1.64 (range 1.0-3.94) and 2.51 (range 1.0-4.48; P = 0.50) for baseline, week 16 and week 48, respectively. CONCLUSIONS: Our study demonstrates that adding VPA to stable HAART does not reduce the latent HIV reservoir in virally suppressed patients.
Asunto(s)
Terapia Antirretroviral Altamente Activa , Linfocitos T CD4-Positivos/virología , Inhibidores Enzimáticos/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Ácido Valproico/uso terapéutico , Latencia del Virus/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Estudios Cruzados , Quimioterapia Combinada/métodos , Femenino , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Carga ViralRESUMEN
The rapid evolution of HIV-1 is a major obstacle to viral eradication. Early antiretroviral therapy (ART) during primary HIV-1 infection could limit viral diversity. Eighteen patients recently infected with HIV-1 were selected. Nine initiated ART soon after enrolment and nine remained untreated. Replication-competent (RC) viruses were quantified at baseline and after one year of follow-up. Viral diversity in the C2V5 envelope region was evaluated from plasma, peripheral blood mononuclear cells (PBMCs), and cell culture at both time points. The amount of RC virus in the treated group declined (median -5.42 infectious units per million [IUPM]) while it remained stable or increased in the untreated group (median +0.87 IUPM). At one year post infection, we observed a significant increase in diversity for the C2V5 (+0.150%) region, specifically in the hypervariable loops V4 (+0.73%) and V5 (+0.77%), in the untreated group. More importantly, viral diversity did not significantly increase in treated individuals during the first year post infection. Genetic diversity during primary infection remains low through the first year of infection. Early treatment could contribute to a decrease in RC viruses from PBMCs and to limitation of viral diversification in the viral reservoir. These findings may have relevance for the rational design of specific immunotherapeutic strategies.
Asunto(s)
Antirretrovirales/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , Enfermedad Aguda , Adulto , Evolución Molecular , Femenino , Variación Genética , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/sangre , VIH-1/efectos de los fármacos , Humanos , Leucocitos Mononucleares/virología , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/genética , Estudios Prospectivos , Estadísticas no ParamétricasRESUMEN
Endothelial progenitor cells (EPCs) consist of two different subpopulations named early (eEPCs) and late EPCs (lEPCs) that are derived from CD14(+) and CD14(-) circulating cells, respectively. These cells are regularly cultured over fibronectin-coated surfaces in endothelial basal medium (EBM)-2 supplemented with insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). We have developed a new and simplified method for culturing human EPCs obtained from peripheral blood and tested their ability to preserve cardiac function following infarction. We first demonstrated that eEPCs derived from human peripheral blood mononuclear cells (PBMCs) and cultured in EBM-2 medium supplemented with autologous serum (10%) over fibronectin-coated surfaces (10 µg/ml) in the presence of IGF-1 (50 ng/ml) only, have a secretome similar to eEPCs cultured under regular conditions with IGF-1, VEGF, EGF, and FGF. Our data also indicate that IGF-1 modulates PBMC secretome in a dose-dependent manner. In another series of experiments, we showed that PBMCs cultured in suspension in bags (S-PBMCs) in basal medium supplemented with fibronectin and IGF-1 secrete significant amounts of stem cell factor (SCF, 31.3 ± 3.1 pg/ml)), hepatocyte growth factor (HGF, 438.6 ± 41.4 pg/ml), soluble tumor necrosis factor receptor 1 (sTNFR1, 127.1 ± 9.9 pg/ml), VEGF (139.3 ± 9.6 pg/ml), and IGF-1 (147.2 ± 46.1 pg/ml) but very low levels of TNF-α (13.4 ± 2.5 pg/ml). S-PBMCs injected intravenously into NOD SCID mice migrated to the injured myocardium, reduced cardiac fibrosis, enhanced angiogenesis, and preserved cardiac function after myocardial infarction (MI) in a manner similar to eEPCs cultured under standard conditions. In conclusion, we show in this study a refined and optimized method for culturing eEPCs. Our data indicate that S-PBMCs are composed of several cell populations including eEPCs and that they secrete high amounts of antiapoptotic, anti-inflammatory, and proangiogenic factors capable of preserving cardiac function following MI.
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Células Sanguíneas/citología , Técnicas de Cultivo de Célula/métodos , Células Endoteliales/citología , Células Endoteliales/trasplante , Isquemia/terapia , Enfermedades Vasculares/terapia , Inductores de la Angiogénesis/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula/economía , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Fibronectinas/farmacología , Pruebas de Función Cardíaca/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Inyecciones Intravenosas , Factor I del Crecimiento Similar a la Insulina/farmacología , Isquemia/complicaciones , Isquemia/fisiopatología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Infarto del Miocardio/complicaciones , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/terapia , Trasplante de Células Madre , Enfermedades Vasculares/complicaciones , Enfermedades Vasculares/fisiopatologíaRESUMEN
In this study, we have investigated the hypothesis that previously reported beneficial effect of peripheral blood mononuclear cells cultured under angiogenic conditions on cardiovascular function following ischemia is not limited to EPCs but also to monocytes contained therein. We first purified and analyzed the phenotype and secretome of human and murine blood monocytes cultured under angiogenic conditions (named MDs for monocyte derivatives) and tested their effect in a mouse model of myocardial infarction (MI). FACS analysis of MDs shows that these cells express mature endothelial cell markers and that their proliferative capacity is virtually absent, consistent with their end-differentiated monocytic ontogeny. MDs secreted significant levels of HGF, IGF-1, MCP-1, and sTNFR-1 relative to their monocyte precursors. MDs were unable to form vascular networks in vitro when cultured on matrix coated flasks. Treatment of murine HL-1 cardiomyocyte cell line with MD-conditioned medium reduced their death induced by TNF-alpha, staurosporine, and oxidative stress, and this effect was dependent upon MD-derived sTNFR-1, HGF, and IGF-1. We further demonstrate that MD secretome promoted endothelial cell proliferation and capacity to form vessels in vitro and this was dependent upon MD-derived MCP-1, HGF, and IGF-1. Echocardiography analysis showed that MD myocardial implantation improved left ventricle fractional shortening of mouse hearts following MI and was associated with reduced myocardial fibrosis and enhancement of angiogenesis. Transplanted MDs and their secretome participate in preserving functional myocardium after ischemic insult and attenuate pathological remodeling.
Asunto(s)
Monocitos/metabolismo , Células Musculares/citología , Infarto del Miocardio/terapia , Neovascularización Fisiológica , Animales , Apoptosis , Células Cultivadas , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Citometría de Flujo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Monocitos/trasplante , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Estaurosporina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Remodelación VentricularRESUMEN
AIMS: To analyze preapheresis blood CD34+ cells and corresponding apheresis products in order to investigate whether peripheral blood CD34+ cell counts correlate with peripheral blood progenitor cell (PBPC) yields and to determine the optimal timing for starting PBPC collections in our clinical setting. MATERIAL AND METHODS: Thirty-eight patients with hematological malignancies and undergoing varied mobilization regimens were enrolled. White blood cell counts (WBC), CD34+ cells and granulocyte-macrophage colony-forming units (GM-CFU) enumeration were performed on blood samples taken immediately prior to each apheresis procedure and in the corresponding PBPC collection. RESULTS: A total of 61 apheresis procedures were performed, with a median of two collections per patient (range 1-3). The number of CD34+ cell/ml in the preapheresis blood correlated closely with CD34+ cells/kg and, to a lesser degree, with GM-CFU/kg in the apheresis products (r = 0.81 and r = 0.67, respectively, P < 0.0001). WBC showed significant but poor correlation with CD34+ cells/kg and GM-CFU/kg (r = 0.43 and r = 0.45, respectively, P = 0.004). A significant correlation was also found between CD34+ cells/kg and GM-CFU/kg in PBPC collections (r = 0.62, P < 0.0001). Linear regression analysis indicated that the minimum threshold of 2 x 10(6) CD34+ cells/kg might be attained with a single apheresis if the CD34+ cells/ml in the peripheral blood measured prior to apheresis on the day of collection is > or =26 x 10(3) CD34+ cells/ml. CONCLUSION: Collectively, these data demonstrate that circulating CD34+ cells is more useful than GM-CFU or WBC for predicting the optimal timing of PBPC harvests.
Asunto(s)
Antígenos CD34/sangre , Eliminación de Componentes Sanguíneos , Granulocitos/citología , Recuento de Leucocitos , Macrófagos/citología , Adulto , Anciano , Femenino , Movilización de Célula Madre Hematopoyética , Trasplante de Células Madre Hematopoyéticas , Humanos , Linfoma no Hodgkin/terapia , Masculino , Persona de Mediana Edad , Mieloma Múltiple/terapiaRESUMEN
Interleukin (IL)-7 and its receptor (IL-7Ralpha) play important roles in regulating lymphopoiesis. Previous studies have reported that human immunodeficiency virus-1 (HIV-1) viraemia affects the expression of IL-7Ralpha, but its effects on CD4+ and CD8+ T cell memory subsets have not been studied. Using eight-colour flow cytometry, we compared the immunophenotypic patterns of CD4+ and CD8+ T cell subsets expressing IL-7Ralpha and activation markers, as well as circulating IL-7 levels, in three well-defined groups of HIV-1-infected subjects: successfully treated, viraemic and long-term non-progressor (LTNP). Compared with successfully treated and LTNP subjects, viraemic patients had reduced expression of IL-7Ralpha on both CD4+ and CD8+ T cells, particularly on central and effector memory T cell compartments, and substantially elevated expression of activation markers on CD8+ T cell subsets. Circulating IL-7 levels were correlated negatively with the number of CD4+ and CD8+ T cell subsets expressing IL-7Ralpha; these associations were stronger with CD4+ T cell subsets and mainly with central and effector memory cells. The expression of activation markers on CD4+ and CD8+ cell T subsets was not related to circulating IL-7 levels. A strong negative correlation was observed between central memory CD4+ or CD8+ T cells expressing IL-7Ralpha and those expressing activation markers, independently of IL-7 levels. Collectively, these results provide further insight on the role of unsuppressed viral load in disrupting the IL-7/IL-7Ralpha system and contributing to HIV-1 disease progression.
Asunto(s)
Antígenos Virales/sangre , Infecciones por VIH/inmunología , VIH-1/inmunología , Subunidad alfa del Receptor de Interleucina-7/sangre , Subgrupos de Linfocitos T/inmunología , Recuento de Linfocito CD4 , Linfocitos T CD8-positivos/inmunología , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Infecciones por VIH/virología , Sobrevivientes de VIH a Largo Plazo , VIH-1/aislamiento & purificación , Humanos , Inmunofenotipificación , Interleucina-7/sangre , Activación de Linfocitos/inmunología , Masculino , Carga ViralRESUMEN
Previous studies on patients who develop drug resistant HIV-1 variants have shown that continued use of failing regimens might provide clinical benefit. However, the effect of long-term exposure to drug resistant variants may lead to emergence of compensatory mutations that may jeopardize this effect. In this study, we assess associations among type and number of drug resistant mutations, viral load and disease progression in patients with long-term follow up. Patients with genotypic testing performed at the time of treatment failure were enrolled. Comparison of viral load and CD4 cell count between different resistance groups was performed using analysis of variance. Multiple linear regression analysis was performed to assess the simultaneous effects of the presence of particular mutations and their accumulation on viral load. Data from 475 patients who were followed for a median of 43 months from October 1999 to July 2005 were studied. A "V shape" relationship was observed between the number of mutations and viral load. Specifically, in patients harboring up to five mutations, viral load was reduced by 0.8 log/copies when compared to wild-type variants. However, with more than six mutations viral load progressively increased. Certain reverse transcriptase mutations such as M184V/I, K70R, V108I, and protease mutations such as L33FIV, M84V, and M36I were associated with reduced viral load. Together, these findings suggest that long-term maintenance of a sub-optimal antiretroviral regimen may have deleterious consequences for the patient.
Asunto(s)
Antirretrovirales/farmacología , Antirretrovirales/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Adulto , Anciano , Canadá , Estudios de Cohortes , Estudios Transversales , Progresión de la Enfermedad , Farmacorresistencia Viral , Femenino , Genes Virales , Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Mutación , Especificidad de la Especie , Factores de Tiempo , Insuficiencia del Tratamiento , Carga ViralRESUMEN
Early absolute lymphocyte count (ALC) has become an important end point for engraftment in patients undergoing autologous peripheral stem cell transplantation (APSCT). In this retrospective study, we evaluate the prognostic significance of early recovery of ALC ( > or = 0.5 cells x 10(9)/l on or before day 15) following APSCT in predicting transplant outcome in 72 patients with lymphoproliferative disorders, including non-Hodgkin's lymphoma (n = 30), Hodgkin's lymphoma (n = 8) and multiple myeloma (n = 34). The median quantities of CD34+ stem cells and lymphocytes infused were 4.97 x 10(6)/kg (range 0.64-11.7) and 11.3 x 10(7)/kg (range 1.11-110) respectively. After a median follow-up of 18 months (range 2-68), 28 patients had experienced a relapse and 16 had died. Of the 72 patients, 27 (37%) demonstrated early recovery of ALC. Early recovery of ALC was strongly associated with long-term overall and disease-free survival in patients aged less than 50 years (P < 0.001). In both univariate and multivariate survival analyses, a shorter time from diagnosis to APSCT was associated with early recovery of ALC (P = 0.03). These findings indicate that early recovery of ALC may contribute to longer survival in younger patients with lymphoproliferative disorders. A shorter time from diagnosis to APSCT may favor recovery of ALC independent of the infused stem cell or lymphocyte doses.
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Recuento de Linfocitos , Trastornos Linfoproliferativos/cirugía , Trasplante de Células Madre de Sangre Periférica/estadística & datos numéricos , Adulto , Anciano , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Supervivencia de Injerto , Movilización de Célula Madre Hematopoyética , Enfermedad de Hodgkin/mortalidad , Enfermedad de Hodgkin/cirugía , Humanos , Estimación de Kaplan-Meier , Linfoma no Hodgkin/mortalidad , Linfoma no Hodgkin/cirugía , Trastornos Linfoproliferativos/mortalidad , Masculino , Persona de Mediana Edad , Mieloma Múltiple/mortalidad , Mieloma Múltiple/cirugía , Pronóstico , Recurrencia , Estudios Retrospectivos , Acondicionamiento Pretrasplante , Trasplante Autólogo , Resultado del TratamientoRESUMEN
OBJECTIVES: Interleukin-7 (IL-7), RANTES (regulated on activation, normal T cell expressed and secreted), stromal cell-derived factor-1 (SDF-1) and transforming growth factor-beta (TGF-beta) appear to share certain biological properties in vitro and all are involved in HIV-1 disease progression. Our earlier observations indicated that IL-7 levels decrease upon CD4 T-cell recovery and represent a new, independent predictor of virological response. Here, we examine associations among circulating levels of IL-7, RANTES, SDF-1 and TGF-beta in hopes of gaining insight into their contribution to the predictive value of IL-7. METHODS: Levels of IL-7, RANTES, SDF-1 and TGF-beta, and immune and viral parameters were assessed in HIV-1-infected patients. RESULTS: Cross-sectional (n=148) and longitudinal (n=36) analyses showed that levels of IL-7, but not RANTES, SDF-1 or TGF-beta, were increased in HIV-1-infected adults compared with those of healthy controls. In the cross-sectional study, levels of IL-7 were correlated with RANTES (r=0.31, P=0.002) and TGF-beta (r=0.53, P<0.001) but not with SDF-1 (r=0.12, P=0.22), and these associations were more pronounced in patients with CD4 T-cell counts >200 cells/microL. In contrast to IL-7, levels of RANTES, SDF-1 and TGF-beta were not correlated with CD4 T-cell counts. Longitudinal analysis revealed a marked decline in IL-7 levels accompanied by an increase in CD4 T-cell count following antiretroviral therapy (ART), but no changes in RANTES, SDF-1 or TGF-beta levels. Multivariate regression analysis showed no influence of baseline RANTES, SDF-1 or TGF-beta levels on the value of IL-7 as a predictor of virological response at 48 weeks. CONCLUSIONS: Collectively, these results indicate that changes in IL-7 levels did not induce changes in RANTES, SDF-1 or TGF-beta. Furthermore, they indicate that RANTES, SDF-1 or TGF-beta levels do not explain the predictor value of IL-7 in patients receiving ART.
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Quimiocina CCL5/inmunología , Quimiocinas CXC/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Interleucina-7/inmunología , Inhibidores de Proteasas/uso terapéutico , Factor de Crecimiento Transformador beta/inmunología , Adulto , Anciano , Recuento de Linfocito CD4/métodos , Quimiocina CCL5/sangre , Quimiocina CXCL12 , Quimiocinas CXC/sangre , Estudios Transversales , Progresión de la Enfermedad , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Humanos , Interleucina-7/sangre , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Factor de Crecimiento Transformador beta/sangre , Carga ViralAsunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Interleucina-7/sangre , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Interleucina-15/sangre , Masculino , Persona de Mediana Edad , Factor de Crecimiento Transformador beta/sangre , Carga ViralRESUMEN
OBJECTIVES: To examine the relationship between levels of the T-cell regulatory cytokine interleukin-7 (IL-7) and CD4 cell counts during immune reconstitution and to assess its prognostic value in advanced HIV-1-infected patients receiving lopinavir/ritonavir-based therapy. METHODS: Thirty-six HIV-1-infected adults who completed 48 weeks of follow-up visits were included in this prospective study. Patients having failed two or more antiretroviral therapy regimens were treated with lopinavir/ritonavir-based therapy. An enzyme-linked immunosorbent assay was used to determine IL-7 plasma levels, flow cytometry was used to analyse cell surface antigens, and polymerase chain reaction was used to quantify plasma HIV-1. RESULTS: Pretreatment IL-7 levels were elevated in all patients (mean 11.0 pg/mL) and were negatively correlated with CD4 cell counts and age (r=-0.59, P<0.001 and r=-0.57, P<0.001, respectively). During the course of treatment, IL-7 levels decreased by 34% while CD4 cell numbers progressively increased by 88%. Multivariate regression analysis showed that only pretreatment IL-7 levels predicted viral load at 48 weeks when controlling for baseline CD4 cell counts, viral load and patient demographics. CONCLUSIONS: These findings are consistent with regulation of T-cell recovery by IL-7, and suggest that IL-7 measurements might be used to predict virological response.
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Infecciones por VIH/tratamiento farmacológico , Interleucina-7/sangre , Pirimidinonas/uso terapéutico , Ritonavir/uso terapéutico , Adulto , Fármacos Anti-VIH/uso terapéutico , Femenino , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , Humanos , Lopinavir , Recuento de Linfocitos , Masculino , Estudios Prospectivos , Linfocitos T/metabolismo , Carga ViralRESUMEN
In order to assess immune responses during HIV-1 therapeutic immunization, a large number of blood mononuclear cells (PBMC) are needed. Clinical tolerance and safety, as well as changes in immunological and virological parameters, were assessed, following leukapheresis in HIV-1 infected subjects with CD4(+) cell count >200 x 10(6)/l. PBMC were collected using a Fenwal CS3000 cell separator in 29 subjects with mean CD4(+) cell counts of 503 x 10(6)/l (range 172-1,119) and viral load of 2.5 log(10) copies/ml (range <1.7-5.4). Twenty-four (83%) subjects were on antiretroviral therapy while 5 (17%) were untreated. The blood volume processed was 7 L over a period of 3 hours. A mean value (+/- standard error) of 82 +/- 26 x 10(9)/l lymphocytes was collected by a single apheresis in a mean volume of 200 +/- 1.8 ml, containing 9.0 +/- 1.3 x 10(9)/l CD4(+) and 10.2 +/- 1.3 x 10(9)/l CD8(+) cells. The leukapheresis procedures were well tolerated and no immediate or delayed side effects were observed within 90 days of follow-up. No changes from blood pre-leukapheresis values were detected for white blood cells, lymphocytes, monocytes, CD8(+), CD34(+), naive and memory CD4(+) cell counts immediately after, 1 h, 7 days, or within 90 days after leukapheresis. However, absolute CD4(+) cell counts and percentage significantly increased from pre-leukapheresis values after 1 h (530 +/- 43 vs. 700 +/- 75 cell x 10(6)/l; 32.6 +/- 1.6 vs. 36.9 +/- 1.9%; P < 0.001 for both paired t-tests) before returning to pre-leukapheresis levels on day 7. No significant changes in viral load from pre-leukapheresis levels in treated or untreated subjects were detected at any time points. We conclude that leukapheresis in HIV-1 infected subjects with CD4(+) cell counts >200 x 10(6)/l is safe and induces a transient increase in the absolute and percentage of CD4(+) cell count without enhancing viral replication.
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Infecciones por VIH/inmunología , Leucaféresis/normas , Adulto , Recolección de Muestras de Sangre/métodos , Recuento de Linfocito CD4 , Relación CD4-CD8 , Femenino , Infecciones por VIH/terapia , Infecciones por VIH/virología , VIH-1/genética , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Recuento de Plaquetas , ARN Viral/sangreRESUMEN
OBJECTIVE: This study was designed to identify the 58-kDa inner ear protein against which the sera of some patients with idiopathic, progressive sensorineural hearing loss or Ménière's disease strongly react. BACKGROUND: We and other groups have previously demonstrated that a 58-kDa protein extracted from guinea pig or bovine inner ear tissue is a target of antibodies in serum samples from some patients with autoimmune inner ear diseases. METHODS: After separation of inner ear proteins by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the bands corresponding to 58 kDa were localized and excised from the gel. The concentrated protein was then digested with trypsin, and the peptide fragments were separated by high-pressure liquid chromatography. Three fractions were subjected to amino acid sequencing by the classic Edman degradation. RESULTS: The sequence of a stretch of 14 amino acids of the first fragment was identical to that of amino acids 526 to 539 of the COCH5B2 protein. The sequences of 11 and 10 amino acids of the second and third fragments, respectively, also were identical to residues 417 to 427 and 396 to 405 of the COCH5B2 protein. These data, together with two-dimensional gel electrophoresis followed by Western blot experiments, confirmed that the 58-kDa inner ear protein is the COCH5B2 protein. DISCUSSION: These findings indicate that the 58-kDa target protein of antibodies in serum samples of patients with autoimmune inner ear diseases is the COCH5B2 protein, a molecule that is highly and specifically expressed in the cochlea and vestibule.
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Anticuerpos/inmunología , Oído Interno/inmunología , Oído Interno/metabolismo , Pérdida Auditiva Sensorineural/inmunología , Enfermedad de Meniere/inmunología , Enfermedad de Meniere/metabolismo , Proteínas/inmunología , Proteínas/metabolismo , Animales , Western Blotting , Cromatografía Líquida de Alta Presión/métodos , Electroforesis en Gel de Agar/métodos , Femenino , Cobayas , MasculinoRESUMEN
Immunological mechanisms are thought to play an important role in the pathogenesis of some cochleo-vestibular diseases. This study attempts to present further evidence of autoantibodies reactive against guinea pig inner ear proteins found in patients with autoimmune inner ear diseases (AIED) and specifically identifies the main target antigens of these antibodies. Sera from 110 patients with a clinical diagnosis of either rapidly progressive sensorineural hearing loss (n = 32). Ménière's disease (n = 41), sudden deafness (n = 6) or other aetiologies of hearing loss (n = 11) were screened by the Western blot technique. Forty-four percent of the patients' sera had antibodies to several inner ear proteins, of which the 30, 42 and 68 kDa proteins were found to be the most reactive. These highly reactive proteins were identified by gas-phase micro sequencing after digestion with trypsin and separation of peptide fragments by high-performance liquid chromatography. A partial sequence of each protein was determined. These data, together with those obtained from 2-dimensional gel electrophoresis followed by Western blotting, demonstrated that the 30 and 42 kDa inner ear proteins are the major peripheral myelin protein P0 and the beta-actin protein, respectively, while sequence analysis indicated that the 68 kDa protein is novel. These findings further support the hypothesis that several populations of antibodies may contribute to the enhanced immunological activity of AIED patients. They also add a new dimension to our knowledge of AIED and may open new avenues in the development of simple serological assays, which are easier to perform and more rapid than Western blotting.
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Autoanticuerpos/análisis , Enfermedades Autoinmunes/inmunología , Oído Interno/inmunología , Enfermedades del Laberinto/inmunología , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Cobayas , Pérdida Auditiva Sensorineural/inmunología , Pérdida Auditiva Súbita/inmunología , Humanos , Enfermedad de Meniere/inmunología , Proteínas/inmunologíaRESUMEN
The etiology of Ménière's disease (MD) is still unknown, but it is likely to be multifactorial, one of the factors being an immunological causation. Antifood allergens as well as anti-baker's yeast antibodies are humoral factors that may be linked with allergenic disorders and other autoimmune conditions. To determine their possible role in MD activity, we investigated 29 MD sera for the presence of antibodies against gliadin, beta-lactoglobulin, albumin, ovalbumin, soya, and Dermatophagoides pteronyssinus and Saccharomyces cerevisiae strains using an ELISA technique. The patients were compared with 29 healthy individuals matched for sex and age. A serum was regarded as positive if the absorbance was two standard deviations higher than values obtained with sera from healthy subjects. Historical data, including factors which the patients believed to provoke their Ménière's symptoms, were obtained from patients' questionnaires. MD patients showed no significant symptoms of allergenic disorders suggesting allergies when compared to controls (p > 0.05). IgG and IgA antibody levels were not significantly raised in MD patients as compared with healthy controls (p > 0.05) for gliadin, beta-lactoglobulin, soya, albumin, ovalbumin, and D. pteronyssinus and S. cerevisiae strains. These data do not convincingly support a hypothesis of increased serum levels of antifood antibodies in patients with MD, as very few patients were antibody positive.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad a los Alimentos/inmunología , Alimentos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Enfermedad de Meniere/inmunología , Adulto , Anciano , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Encuestas y CuestionariosRESUMEN
Recent data have focused on the peripheral nerve myelin glycoprotein P0 as a putative autoantigen involved in the autoimmune etiology of some cases of Meniere's disease, idiopathic sensorineural hearing loss and sudden deafness. To determine whether antibodies to myelin P0 can alter cochlear function, 13 healthy guinea pigs were immunized with purified porcine myelin P0 while 10 controls were injected with saline water. The animals were then evaluated for evidence of evolving inner ear disease using immunological, electrophysiological and morphological methods. Twenty-six experimental ears were tested weekly with a brainstem auditory evoked potential technique for a period of 4 months and were compared to 20 control ears. Uniformly, all P0-sensitized guinea pigs showed antibodies to myelin protein P0 as evidenced by ELISA. Clinical signs of inflammatory demyelination were not discernible in P0-sensitized guinea pigs and all the animals were qualitatively normal. No significant increase of evoked potential thresholds was found in the P0-sensitized animals when compared to controls (P>0.05). Peak latencies of waves I, II, III, IV and V and inter-peak latencies in P0-sensitized guinea pigs did not significantly differ from those of controls (P>0.05). Histological sections of inner ear and peripheral nerves were free of disease in both groups. These findings indicate that the sole presence of antibodies to myelin P0 in the sera of guinea pigs or patients suspected of having autoimmune inner ear diseases is unlikely to elicit auditory abnormalities and that additional factors are necessary for the pathogenic development of these disorders.
Asunto(s)
Trastornos de la Audición/inmunología , Inmunización , Proteína P0 de la Mielina/inmunología , Animales , Anticuerpos/análisis , Umbral Auditivo/fisiología , Cobayas , Tiempo de Reacción/fisiología , Ganglio Espiral de la Cóclea/patología , Porcinos , Hueso Temporal/patologíaRESUMEN
The aim of this paper is to provide a concise portrayal of medical applications of a new fuzzy classification method called PROAFTN, which uses a multicriteria decision aid approach. The review summarises and discusses medical applications of the proposed method in acute leukemia, astrocytic and bladder tumours. Although still an investigative method, the preliminary results are very encouraging and demonstrate the potential performances of this procedure for solving medical classification problems.
Asunto(s)
Inteligencia Artificial , Toma de Decisiones Asistida por Computador , Lógica Difusa , Astrocitoma/diagnóstico , Neoplasias Encefálicas/diagnóstico , Medicina Clínica , Diagnóstico Diferencial , Humanos , Leucemia/diagnóstico , Neoplasias de la Vejiga Urinaria/diagnósticoRESUMEN
We recently developed a new multicriteria classification method called PROAFTN to help medical diagnosis. The aim of this paper is to present the application of the proposed method for the diagnosis of acute leukemia (AL). The implementation of PROAFTN was carried out on cytological data of 108 cases of AL, using the classification rules of French, American and British hematologists, and was then applied on an independent test set of 83 cases of AL. Forty-seven features, obtained by examining patients' bone-marrow smears with light microscope, defined each case. In order to determine the percentage of correct classifications for each subtype of AL, we compared our results with the clinical diagnosis given previously by the hematologist. 96.4% of the cases were correctly classified by the program, suggesting that the method yields good results in terms of discrimination between AL subtypes.