RESUMEN
OBJECTIVE: To estimate the diagnostic value of tumor and immune related proteins in the discrimination between benign and malignant adnexal masses, and between different subgroups of tumors. METHODS: In this exploratory diagnostic study, 254 patients with an adnexal mass scheduled for surgery were consecutively enrolled at the University Hospitals Leuven (128 benign, 42 borderline, 22 stage I, 55 stage II-IV, and 7 secondary metastatic tumors). The quantification of 33 serum proteins was done preoperatively, using multiplex high throughput immunoassays (Luminex) and electrochemiluminescence immuno-assay (ECLIA). We calculated univariable areas under the Receiver Operating Characteristic Curves (AUCs). To discriminate malignant from benign tumors, multivariable ridge logistic regression with backward elimination was performed, using bootstrapping to validate the resulting AUCs. RESULTS: CA125 had the highest univariable AUC to discriminate malignant from benign tumors (0.85, 95% confidence interval 0.79-0.89). Combining CA125 with CA72.4 and HE4 increased the AUC to 0.87. For benign vs borderline tumors, CA125 had the highest univariable AUC (0.74). For borderline vs stage I malignancy, no proteins were promising. For stage I vs II-IV malignancy, CA125, HE4, CA72.4, CA15.3 and LAP had univariable AUCs ≥0.80. CONCLUSIONS: The results confirm the dominant role of CA125 for identifying malignancy, and suggest that other markers (HE4, CA72.4, CA15.3 and LAP) may help to distinguish between stage I and stage II-IV malignancies. However, further research is needed, also to investigate the added value over clinical and ultrasound predictors of malignancy, focusing on the differentiation between subtypes of malignancy.
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Antígeno Ca-125/sangre , Proteínas de la Membrana/sangre , Neoplasias Ováricas/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Carbohidratos Asociados a Tumores/sangre , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Antígeno Ca-125/inmunología , Diagnóstico Diferencial , Femenino , Humanos , Proteínas de la Membrana/inmunología , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/sangre , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/cirugía , Ovario/patología , Ovario/cirugía , Periodo Preoperatorio , Estudios Prospectivos , Curva ROC , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP/análisis , Adulto JovenRESUMEN
Lymphocytic airway inflammation is a major risk factor for chronic lung allograft dysfunction, for which there is no established treatment. We investigated whether azithromycin could control lymphocytic airway inflammation and improve allograft function. Fifteen lung transplant recipients demonstrating acute allograft dysfunction due to isolated lymphocytic airway inflammation were prospectively treated with azithromycin for at least 6 months (NCT01109160). Spirometry (FVC, FEV1 , FEF25-75 , Tiffeneau index) and FeNO were assessed before and up to 12 months after initiation of azithromycin. Radiologic features, local inflammation assessed on airway biopsy (rejection score, IL-17(+) cells/mm(2) lamina propria) and broncho-alveolar lavage fluid (total and differential cell counts, chemokine and cytokine levels); as well as systemic C-reactive protein levels were compared between baseline and after 3 months of treatment. Airflow improved and FeNO decreased to baseline levels after 1 month of azithromycin and were sustained thereafter. After 3 months of treatment, radiologic abnormalities, submucosal cellular inflammation, lavage protein levels of IL-1ß, IL-8/CXCL-8, IP-10/CXCL-10, RANTES/CCL5, MIP1-α/CCL3, MIP-1ß/CCL4, Eotaxin, PDGF-BB, total cell count, neutrophils and eosinophils, as well as plasma C-reactive protein levels all significantly decreased compared to baseline (p < 0.05). Administration of azithromycin was associated with suppression of posttransplant lymphocytic airway inflammation and clinical improvement in lung allograft function.
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Azitromicina/uso terapéutico , Bronquitis/tratamiento farmacológico , Rechazo de Injerto/tratamiento farmacológico , Trasplante de Pulmón/efectos adversos , Linfocitos/efectos de los fármacos , Neumonía/tratamiento farmacológico , Complicaciones Posoperatorias , Adolescente , Adulto , Antibacterianos/uso terapéutico , Bronquitis/etiología , Lavado Broncoalveolar , Proteína C-Reactiva , Citocinas/metabolismo , Femenino , Estudios de Seguimiento , Rechazo de Injerto/etiología , Supervivencia de Injerto , Humanos , Enfermedades Pulmonares/complicaciones , Enfermedades Pulmonares/cirugía , Linfocitos/patología , Masculino , Persona de Mediana Edad , Neumonía/etiología , Pronóstico , Estudios Prospectivos , Pruebas de Función Respiratoria , Estudios Retrospectivos , Espirometría , Trasplante Homólogo , Adulto JovenRESUMEN
BACKGROUND: At present, the only way to conclusively diagnose endometriosis is laparoscopic inspection, preferably with histological confirmation. This contributes to the delay in the diagnosis of endometriosis which is 6-11 years. So far non-invasive diagnostic approaches such as ultrasound (US), MRI or blood tests do not have sufficient diagnostic power. Our aim was to develop and validate a non-invasive diagnostic test with a high sensitivity (80% or more) for symptomatic endometriosis patients, without US evidence of endometriosis, since this is the group most in need of a non-invasive test. METHODS: A total of 28 inflammatory and non-inflammatory plasma biomarkers were measured in 353 EDTA plasma samples collected at surgery from 121 controls without endometriosis at laparoscopy and from 232 women with endometriosis (minimal-mild n = 148; moderate-severe n = 84), including 175 women without preoperative US evidence of endometriosis. Surgery was done during menstrual (n = 83), follicular (n = 135) and luteal (n = 135) phases of the menstrual cycle. For analysis, the data were randomly divided into an independent training (n = 235) and a test (n = 118) data set. Statistical analysis was done using univariate and multivariate (logistic regression and least squares support vector machines (LS-SVM) approaches in training- and test data set separately to validate our findings. RESULTS: In the training set, two models of four biomarkers (Model 1: annexin V, VEGF, CA-125 and glycodelin; Model 2: annexin V, VEGF, CA-125 and sICAM-1) analysed in plasma, obtained during the menstrual phase, could predict US-negative endometriosis with a high sensitivity (81-90%) and an acceptable specificity (68-81%). The same two models predicted US-negative endometriosis in the independent validation test set with a high sensitivity (82%) and an acceptable specificity (63-75%). CONCLUSIONS: In plasma samples obtained during menstruation, multivariate analysis of four biomarkers (annexin V, VEGF, CA-125 and sICAM-1/or glycodelin) enabled the diagnosis of endometriosis undetectable by US with a sensitivity of 81-90% and a specificity of 63-81% in independent training- and test data set. The next step is to apply these models for preoperative prediction of endometriosis in an independent set of patients with infertility and/or pain without US evidence of endometriosis, scheduled for laparoscopy.
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Biomarcadores/metabolismo , Endometriosis/sangre , Endometriosis/diagnóstico , Adulto , Estudios de Casos y Controles , Ácido Edético/metabolismo , Femenino , Humanos , Inflamación , Laparoscopía , Análisis de los Mínimos Cuadrados , Ciclo Menstrual , Persona de Mediana Edad , Modelos Estadísticos , Curva ROC , Análisis de Regresión , Sensibilidad y EspecificidadRESUMEN
The bicyclam AMD3100 (formula weight 830) blocks HIV-1 entry and membrane fusion via the CXCR4 co-receptor, but not via CCR5. AMD3100 prevents monoclonal antibody 12G5 from binding to CXCR4, but has no effect on binding of monoclonal antibody 2D7 to CCR5. It also inhibits binding of the CXC-chemokine, SDF-1alpha, to CXCR4 and subsequent signal transduction, but does not itself cause signaling and has no effect on RANTES signaling via CCR5. Thus, AMD3100 prevents CXCR4 functioning as both a HIV-1 co-receptor and a CXC-chemokine receptor. Development of small molecule inhibitors of HIV-1 entry is feasible.
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Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD4-Positivos/virología , Quimiocinas CXC , VIH-1/fisiología , Compuestos Heterocíclicos/farmacología , Receptores CXCR4/fisiología , Anticuerpos Monoclonales/farmacología , Bencilaminas , Antígenos CD4/inmunología , Antígenos CD4/fisiología , Linfocitos T CD4-Positivos/efectos de los fármacos , Calcio/metabolismo , Carbacol/farmacología , Fusión Celular , Línea Celular , Células Cultivadas , Quimiocina CCL5/farmacología , Quimiocina CXCL12 , Ciclamas , Citocinas/metabolismo , Citocinas/farmacología , Proteína gp120 de Envoltorio del VIH/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/efectos de los fármacos , Humanos , Interleucina-2/farmacología , Cinética , Fusión de Membrana/efectos de los fármacos , Receptores CCR5/fisiología , Receptores CXCR4/efectos de los fármacos , Receptores CXCR4/inmunología , Transducción de Señal/efectos de los fármacos , Somatostatina/farmacologíaRESUMEN
Injecting human peripheral blood mononuclear cells into severe combined immunodeficient (SCID) mice results in long-term engraftment of human lymphocytes, of which > 98% are phenotypically mature, activated T cells. Here we have characterized the human T cells that populate such hu-PBL-SCID chimeras. We report that these human T cells do not mobilize Ca2+ after CD3 stimulation, i.e., their T cell receptor (TCR)-mediated signal transduction is deficient. Chimera-derived human T cells do not secrete lymphokines or undergo blastogenesis after CD3 stimulation, but proliferate in response to interleukin 2 (IL-2), defining the chimera derived human T cells as anergic. Anergy was seen in both the CD4+ and the CD8+ subpopulations. We established human T cell lines from chimeras. These T cells retained their anergic state for 1-2 mo in culture, after which they simultaneously regained the ability to mobilize Ca2+, secrete lymphokines, and to undergo blastogenesis following stimulation via the TCR. Once regaining proliferative responsiveness to CD3 stimulation, these CD4+ T cell lines displayed anti-SCID mouse reactivity and showed no specificity for recall antigens. All CD3-responsive CD4+ T cell clones obtained from such lines were SCID mouse specific, recognizing native major histocompatibility complex class II products on the murine cells. In contrast, chimera-derived human CD8+ cell lines and clones did not display detectable anti-mouse reactivity. The data show that the human T cell system in long term hu-PBL-SCID chimeras is nonfunctional due to both anergy and the limitation of the CD4+ repertoire to xenoreactive clones. The data suggest that long-term hu-PBL-SCID chimerism represents an atypical graft-versus-host reaction in which the human effector T cells become anergic in the murine environment.
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Linfocitos T CD4-Positivos/inmunología , Quimera/inmunología , Ratones SCID/inmunología , Adulto , Animales , Complejo CD3/inmunología , Linfocitos T CD8-positivos/inmunología , Calcio/metabolismo , Línea Celular , Citocinas/biosíntesis , Femenino , Reacción Injerto-Huésped , Humanos , Interleucina-2/farmacología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana EdadRESUMEN
Bicyclams are a novel class of antiviral compounds that are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. Surprisingly, however, when the prototype compound AMD3100 was tested against M-tropic virus strains such as BaL, ADA, JR-CSF, and SF-162 in human peripheral blood mononuclear cells, the compound was completely inactive. Because of the specific and potent inhibitory effect of AMD3100 on T-tropic viruses, but not M-tropic viruses, it was verified that AMD3100 interacts with the CXC-chemokine receptor CXCR4, the main coreceptor used by T-tropic viruses. AMD3100 dose dependently inhibited the binding of a specific CXCR4 monoclonal antibody to SUP-T1 cells as measured by flow cytometry. It did not inhibit the binding of the biotinylated CC-chemokine macrophage inflammatory protein (MIP) 1alpha or MIP-1beta, ligands for the chemokine receptor CCR5 (the main coreceptor for M-tropic viruses). In addition, AMD3100 completely blocked (a) the Ca2+ flux at 100 ng/ml in lymphocytic SUP-T1 and monocytic THP-1 cells, and (b) the chemotactic responses of THP-1 cells induced by stromal cell-derived factor 1alpha, the natural ligand for CXCR4. Finally, AMD3100 had no effect on the Ca2+ flux induced by the CC-chemokines MIP-1alpha, regulated on activation normal T cell expressed and secreted (RANTES; also a ligand for CCR5), or monocyte chemoattractant protein 3 (a ligand for CCR1 and CCR2b), nor was it able to induce Ca2+ fluxes by itself. The bicyclams are, to our knowledge, the first low molecular weight anti-HIV agents shown to act as potent and selective CXCR4 antagonists.
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Quimiocinas CXC , VIH/inmunología , VIH/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Linfocitos T/virología , Fármacos Anti-VIH/farmacología , Especificidad de Anticuerpos , Bencilaminas , Sitios de Unión de Anticuerpos/efectos de los fármacos , Unión Competitiva/efectos de los fármacos , Calcio/antagonistas & inhibidores , Calcio/metabolismo , Línea Celular , Quimiocina CXCL12 , Quimiotaxis de Leucocito/efectos de los fármacos , Ciclamas , Citocinas/farmacología , Relación Dosis-Respuesta a Droga , VIH/efectos de los fármacos , VIH-2/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Receptores CXCR4/inmunologíaRESUMEN
In this article, we report that the human fetal thymus contains CD34bright cells (< 0.01% of total thymocytes) with a phenotype that resembles that of multipotent hematopoietic progenitors in the fetal bone marrow. CD34bright thymocytes were CD33-/dull and were negative for CD38, CD2, and CD5 as well as for the lineage markers CD3, CD4, and CD8 (T cells), CD19 and CD20 (B cells), CD56 (NK cells), glycophorin (erythrocytes), and CD14 (monocytes). In addition, total CD34+ lineage negative (lin-) thymocytes contained a low number of primitive myeloid progenitor cells, thus suggesting that the different hematopoietic lineages present in the thymus may be derived from primitive hematopoietic progenitor cells seeding the thymus. To investigate whether the thymus is permissive for the development of non-T cells, human fetal organ culture (FTOC) assays were performed by microinjecting sorted CD34+lin- fetal liver cells into fragments of HLA-mismatched fetal thymus. Sequential phenotypic analysis of the FTOC-derived progeny of CD34+lin- cells indicated that the differentiation into T cells was preceded by a wave of myeloid differentiation into CD14+CD11b+CD4dull cells. Donor-derived B cells (CD19+CD20+) were also generated, which produced immunoglobulins (IgG and IgM) when cultured under appropriate conditions, as well as functional CD56+CD3- NK cells, which efficiently killed K562 target cells in cytotoxicity assays. These results demonstrate that the microinjection of fetal liver hematopoietic progenitors into fetal thymic organ fragments results in multilineage differentiation in vitro.
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Antígenos CD/análisis , Feto/inmunología , Células Madre Hematopoyéticas/inmunología , Linfocitos T/inmunología , Timo/citología , Antígenos CD34 , Linfocitos B/fisiología , Diferenciación Celular , Células Cultivadas , Femenino , Humanos , Hígado/inmunología , Técnicas de Cultivo de Órganos , EmbarazoRESUMEN
Astrocytes actively participate in synaptic integration by releasing transmitter (glutamate) via a calcium-regulated, exocytosis-like process. Here we show that this process follows activation of the receptor CXCR4 by the chemokine stromal cell-derived factor 1 (SDF-1). An extraordinary feature of the ensuing signaling cascade is the rapid extracellular release of tumor necrosis factor-alpha (TNFalpha). Autocrine/paracrine TNFalpha-dependent signaling leading to prostaglandin (PG) formation not only controls glutamate release and astrocyte communication, but also causes their derangement when activated microglia cooperate to dramatically enhance release of the cytokine in response to CXCR4 stimulation. We demonstrate that altered glial communication has direct neuropathological consequences and that agents interfering with CXCR4-dependent astrocyte-microglia signaling prevent neuronal apoptosis induced by the HIV-1 coat glycoprotein, gp120IIIB. Our results identify a new pathway for glia-glia and glia-neuron communication that is relevant to both normal brain function and neurodegenerative diseases.
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Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Microglía/fisiología , Receptores CXCR4/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis , Astrocitos/fisiología , Western Blotting , Calcio/metabolismo , Comunicación Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Dinoprost/metabolismo , Espacio Extracelular/metabolismo , Proteína gp120 de Envoltorio del VIH/farmacología , Humanos , Inmunohistoquímica , Técnicas In Vitro , Inyecciones Intraventriculares , Ratones , Ratones Mutantes , Neocórtex/citología , Neocórtex/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Transducción de SeñalRESUMEN
Many HIV-1 isolates at the late stage of disease are capable of using both CXCR4 and CCR5 in transfected cell lines, and are thus termed dual-tropic. Here we asked whether these dual-tropic variants also use both coreceptors for productive infection in a natural human lymphoid tissue microenvironment, and whether use of a particular coreceptor is associated with viral cytopathicity. We used 3 cloned dual-tropic HIV-1 variants, 89.6 and its chimeras 89-v345.SF and 89-v345.FL, which use both CCR5 and CXCR4 in transfected cell lines. In human lymphoid tissue ex vivo, one variant preferentially used CCR5, another preferentially used CXCR4, and a third appeared to be a true dual-tropic variant. The 2 latter variants severely depleted CD4(+) T cells, whereas cytopathicity of the virus that used CCR5 only in lymphoid tissue was mild and confined to CCR5(+)/CD4(+) T cells. Thus, (a) HIV-1 coreceptor usage in vitro cannot be unconditionally extrapolated to natural microenvironment of human lymphoid tissue; (b) dual-tropic viruses are not homogeneous in their coreceptor usage in lymphoid tissue, but probably comprise a continuum between the 2 polar variants that use CXCR4 or CCR5 exclusively; and (c) cytopathicity toward the general CD4(+) T cell population in lymphoid tissue is associated with the use of CXCR4.
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Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Receptores del VIH/metabolismo , Fármacos Anti-VIH/farmacología , Bencilaminas , Relación CD4-CD8 , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Quimiocina CCL5/farmacología , Quimiocinas/fisiología , Técnicas de Cocultivo , Ciclamas , Efecto Citopatogénico Viral , Genes env , Proteína p24 del Núcleo del VIH/biosíntesis , VIH-1/clasificación , VIH-1/genética , VIH-1/patogenicidad , Compuestos Heterocíclicos/farmacología , Humanos , Activación de Linfocitos , Sustancias Macromoleculares , Macrófagos/virología , Fusión de Membrana , Modelos Biológicos , Especificidad de Órganos , Tonsila Palatina/citología , Tonsila Palatina/virología , Receptores CCR5/efectos de los fármacos , Receptores CXCR4/efectos de los fármacos , Subgrupos de Linfocitos T/virología , Transfección , VirulenciaRESUMEN
LD78alpha and LD78beta are 2 highly related nonallelic genes that code for different isoforms of the human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). Two molecular forms of natural LD78beta (7.778 and 7.793 kDa) were identified from conditioned media of stimulated peripheral blood mononuclear cells. Although LD78alpha and LD78beta only differ in 3 amino acids, both LD78beta variants were 100-fold more potent chemoattractants for mouse lymphocytes than was LD78alpha. On the contrary, LD78beta was only 2-fold more efficient than LD78alpha in chemoattracting human lymphocytes and monocytes. Using CC chemokine receptor-transfected cells, both molecular forms of LD78beta proved to be much more potent than LD78alpha in inducing an intracellular calcium rise through CCR5. Compared with LD78alpha and RANTES, this preferential binding of LD78beta to CCR5 resulted in a 10- to 50-fold higher potency in inhibiting infection of peripheral blood mononuclear cells by CCR5-using (R5) HIV-1 strains. To date, LD78beta is the most potent chemokine for inhibiting HIV-1 infection, and can be considered as a potentially important drug candidate for the treatment of infection with R5 HIV-1 strains.
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VIH-1/efectos de los fármacos , Proteínas Inflamatorias de Macrófagos/genética , Proteínas Inflamatorias de Macrófagos/farmacología , Receptores CCR5/agonistas , Animales , Línea Celular , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/farmacología , Infecciones por VIH/prevención & control , Infecciones por VIH/terapia , VIH-1/patogenicidad , Humanos , Técnicas In Vitro , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Proteínas Inflamatorias de Macrófagos/aislamiento & purificación , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/farmacología , Receptores CCR5/genética , TransfecciónRESUMEN
A series of novel compounds 3a-j and 6a-j with primaquine and hydroxyl or halogen substituted benzene moieties bridged by urea or bis-urea functionalities were designed, synthesized and evaluated for biological activity. The title compounds were prepared using benzotriazole as the synthon, through several synthetic steps. 3-[3,5-Bis(trifluoromethyl)phenyl]-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3j) was the most active urea and 1-[({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]-3-[3-(trifluoromethyl)phenyl]urea (6h) the most active bis-urea derivative in antiproliferative screening in vitro against eight tested cancer cell lines. Urea derivatives 3a-g with hydroxy group or one halogen atom showed moderate antiproliferative effects against all the tested cell lines, but stronger activity against breast carcinoma MCF-7 cell line, while trifluoromethyl derivatives 3h-j showed antiproliferative effects against all the tested cell lines in low micromolar range. Finally, bis-ureas with hydroxy and fluoro substituents 6a-d showed extreme selectivity and chloro or bromo derivatives 6e-g high selectivity against MCF-7 cells (IC50 0.1-2.6 µM). p-Fluoro derivative 6d, namely 3-(4-fluorophenyl)-1-[({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]urea, is the most promising compound. Further biological experiments showed that 6d affected cell cycle and induced cell death of MCF-7 cell line. Due to its high activity against MCF-7 cell line (IC50 0.31 µM), extreme selectivity and full agreement with the Lipinski's and Gelovani's rules for prospective small molecular drugs, 6d may be considered as a lead compound in development of breast carcinoma drugs. Urea 3b and almost all bis-ureas showed high antioxidant activity in DPPH assay, but urea derivatives were more active in lipid peroxidation test. Only few compounds exhibited weak inhibition of soybean lipoxygenase. Compound 3j exhibited the strongest antimicrobial activity in susceptibility assay in vitro (MIC = 1.6-12.5 µg ml-1).
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Apoptosis/efectos de los fármacos , Benceno/química , Neoplasias de la Mama/tratamiento farmacológico , Halógenos/química , Primaquina/síntesis química , Primaquina/farmacología , Urea/síntesis química , Antioxidantes/síntesis química , Antioxidantes/química , Antioxidantes/farmacología , Bacterias/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Células MCF-7 , Pruebas de Sensibilidad Microbiana , Primaquina/química , Urea/química , Urea/farmacologíaRESUMEN
The chemokine stromal-cell-derived factor-1alpha (SDF-1alpha) chemoattracts lymphocytes and CD34+ haematopoietic progenitors and is the ligand for CXCR4 (CXC chemokine receptor 4), the main co-receptor for T-tropic HIV-1 strains. SDF-1alpha was NH2-terminally cleaved to SDF-1alpha(3-68) by dipeptidyl-peptidase IV (CD26/DPP IV), which is present in blood in soluble and membrane-bound form. SDF-1alpha(3-68) lost both lymphocyte chemotactic and CXCR4-signaling properties. However, SDF-1alpha(3-68) still desensitized the SDF-1alpha(1-68)-induced Ca2+ response. In contrast to CD26/DPP IV-processed RANTES(3-68), SDF-1alpha(3-68) had diminished potency to inhibit HIV-1 infection. Thus, CD26/DPP IV impairs the inflammatory and haematopoietic potency of chemokines but plays a dual role in AIDS.
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Fármacos Anti-VIH/farmacología , Quimiocinas CXC/farmacología , Dipeptidil Peptidasa 4/metabolismo , Fragmentos de Péptidos/farmacología , Procesamiento Proteico-Postraduccional , Fármacos Anti-VIH/metabolismo , Quimiocina CXCL12 , Quimiocinas , Quimiocinas CXC/metabolismo , Quimiotaxis de Leucocito , Relación Dosis-Respuesta a Droga , VIH-1/efectos de los fármacos , Humanos , Linfocitos , Receptores CXCR4/metabolismo , Transducción de SeñalRESUMEN
Multinucleated giant cell (syncytium) formation induced by the interaction between the gp120 glycoprotein expressed on the surface of cells infected with human immunodeficiency virus type (HIV-1) and the CD4 receptor of uninfected CD4-positive (CD4+) cells may play an important role in the depletion of T4 lymphocytes in acquired immune deficiency syndrome (AIDS) patients. Using a double fluorescence cell-staining technique and analysis of the cells by the fluorescence-activated cell sorter (FACS), we have demonstrated that giant cell formation between persistently HIV-1-infected HUT-78 cells and uninfected MOLT-4 cells results in a selective destruction of the uninfected CD4+ MOLT-4 cells. Apparently, bystander CD4+ cells may serve as targets for the killing effect of the HIV-1-infected cells, and this killing effect is preceded by fusion between the target (uninfected) and aggressor (infected) cells. Pentosan polysulfate, dextran sulfate, and various other sulfated polysaccharides, but not heparin, have proved to inhibit this cell fusion process and hence protect the target CD4+ cells against destruction by the killer HIV-1-infected cells. Azidothymidine does not interfere with this process. Assuming that fusion between HIV-infected and uninfected CD4+ cells is a crucial event in the pathogenesis of AIDs, any compounds that specifically interfere with this process may be therapeutically advantageous in the treatment of this disease.
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Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Dextranos/farmacología , Células Gigantes/efectos de los fármacos , VIH-1/aislamiento & purificación , Heparina/farmacología , Poliéster Pentosan Sulfúrico/farmacología , Polisacáridos/farmacología , Replicación Viral/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida/microbiología , Síndrome de Inmunodeficiencia Adquirida/patología , Antígenos CD4 , Línea Celular , Separación Celular , Células Cultivadas , Células Clonales , Sulfato de Dextran , Citometría de Flujo , Células Gigantes/patología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Cultivo de VirusRESUMEN
The first step in the replicative cycle of human immunodeficiency virus type 1 (HIV-1) is binding of the virions to the cellular CD4 receptor. This process may be considered as an important target for chemotherapeutic agents against acquired immune deficiency syndrome (AIDS). A method has now been devised whereby virion binding to the cell membrane was visualized by an indirect immunofluorescence assay using human anti-HIV-1 serum, rabbit anti-human-IG-F(ab')2-fluorescein isothiocyanate, and flow cytometry. Heparin, dextran sulfate, and pentosan polysulfate suppressed HIV-1 binding to MT-4 cells at concentrations that protected the cells against HIV-1 cytopathogenicity. Dextran and dermatan sulfate, two compounds that are inactive against HIV-1, had no inhibitory effect on the binding of HIV-1 to the cells. The potent and selective HIV-1 inhibitor azidothymidine (AZT) did not affect virus binding to the cells, whereas suramin partially blocked HIV-1 binding to the cells at concentrations that fully protected MT-4 cells against destruction of HIV-1. Our immunofluorescence assay thus demonstrated that suramin not only acts as an inhibitor of reverse transcriptase but also interferes with virus-cell binding. Also, Evans blue, an anionic dye structurally related to suramin, partially inhibited HIV-1 attachment to the cells. The present method permits a quantitative determination of the inhibitory effect of anti-HIV-1 agents on virion-cell binding.
Asunto(s)
Antivirales/farmacología , Citometría de Flujo , VIH-1/efectos de los fármacos , Receptores Virales/metabolismo , Linfocitos T/microbiología , Virión/metabolismo , Antígenos de Diferenciación de Linfocitos T/análisis , Dermatán Sulfato/farmacología , Sulfato de Dextran , Dextranos/farmacología , Anticuerpos Anti-VIH/análisis , Heparina/farmacología , Humanos , Poliéster Pentosan Sulfúrico/farmacología , Receptores del VIH , Suramina/farmacología , Linfocitos T/inmunología , Zidovudina/farmacologíaRESUMEN
Polyhydroxycarboxylates (MW: 3,800-14,000) derived from phenolic (PDP) compounds have been found to inhibit the cytopathicity of HIV-1 and HIV-2 in MT-4 cells at concentrations that are not toxic to the host cells. The PDP compounds also inhibited syncytium formation in cocultures of MOLT-4 cells with HIV-1- or HIV-2-infected HUT-78 cells. They also interfered with the binding of OKT4A/leu3a monoclonal antibody (mAb) to the CD4 receptor, the binding of anti-gp120 mAb to HIV-1 gp120, and attachment of HIV-1 virions to MT-4 cells. The anti-HIV activity in this series of compounds can be ascribed to inhibition of the gp120-CD4 interaction and seems to depend on the presence of the anionic carboxylate groups. Their mechanism of action is similar to that of the heterogeneous polymer aurintricarboxylic acid (ATA).
Asunto(s)
Antivirales/farmacología , Ácidos Carboxílicos/química , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Fenoles/química , Replicación Viral/efectos de los fármacos , Adsorción , Anticuerpos Monoclonales/inmunología , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Ácidos Carboxílicos/farmacología , Células Cultivadas , Células Gigantes/microbiología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , VIH-2/crecimiento & desarrollo , VIH-2/inmunología , Fenoles/farmacología , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Several cholic acid derivatives such as taurolithocholic acid, lithocholic acid 3-sulfate, taurolithocholic acid 3-sulfate, and glycolithocholic acid 3-sulfate were shown to inhibit selectively the replication of human immunodeficiency virus type 1 (HIV-1) in vitro. These compounds completely protected MT-4 cells against HIV-1-induced cytopathogenicity at a concentration of 100 micrograms/ml, whereas no toxicity for the host cells was observed at 200 micrograms/ml. They also inhibited HIV-1 antigen expression in HIV-1-infected CEM cells. The bile acids (cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid) did not show any inhibitory effect on HIV-1 replication at concentrations that were not toxic to the host (MT-4) cells. From a structure-function analysis of a number of cholic acid derivatives, the presence of either a sulfonate (as in the tauro conjugates) or a sulfate group as well as the "litho" configuration appeared to be necessary for the expression of anti-HIV-1 activity. The active cholic acid derivatives did not directly inactivate the virus particles at the concentrations that were not toxic to the host cells. Lithocholic acid 3-sulfate, taurolithocholic acid 3-sulfate, and glycolithocholic acid 3-sulfate, but not taurolithocholic acid, partially inhibited virus adsorption to MT-4 cells. These three compounds were also inhibitory to the reverse transcriptase activity associated with HIV-1.
Asunto(s)
Ácidos Cólicos/farmacología , VIH-1/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Adsorción , Antígenos Virales/análisis , Linfocitos T CD4-Positivos , Línea Celular , Membrana Celular/metabolismo , VIH-1/enzimología , VIH-1/inmunología , VIH-1/fisiología , Humanos , Pruebas de Neutralización , ADN Polimerasa Dirigida por ARN/análisisRESUMEN
Infection of human T4 lymphocyte MT-4 cells with human immunodeficiency virus (HIV) results in cell death 4-5 days after infection. We have now developed a highly sensitive and rapid procedure for estimating the cytopathic effect of HIV in MT-4 cells. This method is based on fluorometric as well as flow cytometric evaluation of HIV-infected MT-4 cultures. By the use of fluorescein diacetate (FDA), a non-fluorescent diacetyl fluorescein ester that becomes fluorescent upon hydrolysis by esterases present in the cytoplasm of viable cells, as few as 100-200 viable MT-4 cells can be accurately determined. Applying this new method to HIV-infected MT-4 cell cultures treated with differing concentrations of the potent anti-HIV agent azidothymidine (AZT), we obtained a virus-inhibitory dose-response comparable to those obtained by the conventional (labour-intensive and time-consuming) methods. The FDA-based cell viability assay appears particularly suited for the rapid, reliable and sensitive evaluation of potential anti-AIDS agents in cell culture.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Citometría de Flujo , VIH/efectos de los fármacos , Zidovudina/farmacología , Síndrome de Inmunodeficiencia Adquirida/microbiología , Síndrome de Inmunodeficiencia Adquirida/patología , Linfocitos T CD4-Positivos/microbiología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Efecto Citopatogénico Viral/efectos de los fármacos , Fijadores , Citometría de Flujo/métodos , Fluoresceínas , Humanos , Recuento de LeucocitosRESUMEN
A series of polyoxometalates have been synthesized and evaluated for their inhibitory effects on HIV-1(III(B)) and HIV-1(ROD) replication in MT-4 cells. All compounds showed activity against HIV-1 and HIV-2, but the antiviral potency of the heteropolytungstates varied considerably depending on their chemical structure. The antiviral activity of single, double, and triple Keggin-type of compounds against HIV-1(III(B)) replication was comparable (IC(50): 0.4-0.5 microgram/mL), whereas HIV-2(ROD) appeared to become less sensitive with the increasing number of Keggin structures per compound. The same trend was observed for single and double Dawson structures. Some of these compounds were examined for their inhibitory effect on the replication of HIV-1(RF) and SIV(MAC(251)) in MT-4 cells. Their anti-HIV-1(RF) and anti-SIV(MAC(251)) potencies were comparable to those for the HIV-1(III(B)) or HIV-2(ROD) strain, respectively. The polyoxometalates represent a class of polyanionic compounds, which block the binding of the envelope glycoprotein gp120 of HIV to CD4(+) cells. The compounds interfered with the binding of anti-CD4 mAb to the OKT4A/Leu3a epitope of the CD4 receptor, compound 24 being the most active in this regard, and inhibited the binding of anti-gp120 mAb to infected MT-4 cells. None of the polyoxometalates inhibited the binding of a specific CXCR4 mAb to SUP-T1 cells, suggesting that they do not interact with CXCR4, the main co-receptor for T-tropic HIV strains, and thus act as virus binding, and not as fusion, inhibitors.
Asunto(s)
Fármacos Anti-VIH/síntesis química , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Óxidos/química , Tungsteno/química , Animales , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Anticuerpos Monoclonales/metabolismo , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Línea Celular , Epítopos , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Ratones , Óxidos/farmacología , Relación Estructura-Actividad , Tungsteno/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Several compounds corresponding to fragments of the schematic representation of the polymeric structure of aurintricarboxylic acid (ATA) have been prepared and tested for prevention of the cytopathic effect of HIV-1 and HIV-2 in MT-4 cell culture and HIV-1 in CEM cell culture. Both the triphenylcarbinol 3 as well as the triphenylmethane 5 were found to afford protection against the cytopathogenicity of HIV-2 in MT-4 cells and HIV-1 in CEM cells, but they were inactive against HIV-1 in MT-4 cells. Both substances were also found to inhibit syncytium formation when MOLT-4 cells were cocultured with HIV-2-infected HUT-78 cells, but were inactive in this assay against HIV-1-infected cells. When observed, the activity is generally moderate in degree of protection and requires concentrations in the 10(-4) molar range. In contrast to ATA, both of these substances were inactive when tested for prevention of the binding of the OKT4A monoclonal antibody to the CD4 receptor and also for inhibition of HIV-1 reverse transcriptase. These substances therefore appear act by a mechanism that is distinct from that of polymeric ATA. Several active and inactive structural analogues of 3 and 5 were also synthesized. The anti-HIV activity in this series seems to depend on the presence of anionic carboxylate groups, since the methyl esters 4, 6, and 12 were uniformly inactive. The diphenylmethanes 8, 14, 18, and 19 also reproducibly inhibited the cytopathic effect of HIV-1 in CEM cell culture.
Asunto(s)
Antivirales/síntesis química , Ácido Aurintricarboxílico/análogos & derivados , Ácido Aurintricarboxílico/síntesis química , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Ácido Aurintricarboxílico/química , Ácido Aurintricarboxílico/farmacología , Antígenos CD4/metabolismo , Línea Celular , VIH-1/enzimología , VIH-2/enzimología , Humanos , Indicadores y Reactivos , Estructura Molecular , Peso Molecular , Inhibidores de la Transcriptasa Inversa , Relación Estructura-ActividadRESUMEN
In order to increase the ratio of anti-HIV activity to anticoagulant activity, glycosaminoglycan derivatives selectively substituted at OH and/or COOH groups were prepared. Standard heparin, heparin fragments, or dermatan sulfate were converted to their tributylammonium or tetrabutylammonium salts. Their selective O-acylation to various (controlled) degrees was carried out in a homogeneous way in N,N-dimethylformamide using carboxylic acid anhydrides and 4-(dimethylamino)pyridine as catalyst. Esterification of the COOH groups was performed by the addition of alkyl halide to an N,N-dimethylformamide solution of glycosaminoglycan tetrabutylammonium salts. The in vitro anticoagulant activity, the activity against HIV-1 and HIV-2 cytopathicity, the cytotoxicity, and the activity on the induction of giant cell formation were determined. O-acylation (O-butyrylation or O-hexanoylation) of the heparin fragments obtained by periodate depolymerization (compounds 2d and 2e), and their esters (compounds 7i and 7j), yielded products with very low anticoagulant effects in vitro, yet potent activity against both HIV-1 and HIV-2 induced cytopathicity, and low, if any, cytotoxicity. As compared to other anionic polysaccharides, these acylated derivatives are more active as inhibitors of HIV-induced giant-cell formation. Their anti-HIV activity is related to the degree of O-acylation and is mainly due to the inhibition of virus adsorption to the target cells.