RESUMEN
Aminooxypentane (AOP)-RANTES is a potent inhibitor of nonsyncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) isolates. Although classical chemotactic responses are not induced in primary leukocytes by AOP-RANTES, recent studies suggest that a remnant of cell signaling occurs upon binding of receptor to this compound. We have detected a breakthrough of NSI/R5 replication from the inhibitory effects of high AOP-RANTES concentrations (<100 nM). A stimulation of different primary syncytium-inducing (SI), CXCR4-tropic (X4) HIV-1 isolates was also observed in the presence of AOP-RANTES. This stimulation was also observed after 110 h in PCR and RT-PCR for minus-strand strong-stop DNA and unspliced and multiply spliced RNA, respectively. However, there was significant variability between different SI/X4 or NSI/R5 HIV-1 isolates with regard to this AOP-RANTES-mediated stimulation or breakthrough, respectively. To further define the mechanism(s) responsible for this AOP-RANTES effect, we performed detailed retroviral replication studies with an NSI/R5 (B-92BR021) and SI/X4 (D-92UG021) HIV-1 isolate in the presence of the drug. Treatment of peripheral blood mononuclear cells with 125 nM AOP-RANTES and virus did not alter coreceptor expression, HIV-1 entry, reverse transcription, or mRNA transcription from the long terminal repeat, but it did result in increased HIV-1 integration. This AOP-RANTES-mediated increase in HIV-1 integration was diminished by treatment with pertussis toxin. Phosphorylation of the mitogen-activated protein kinase (MAPK) isoforms, extracellular signal-regulated kinase 1 (ERK1) and ERK2, was increased in a CD4(+) CCR5(+) U87 cell line treated with AOP-RANTES or with an NSI/R5 HIV-1 isolate. These findings suggest that AOP-RANTES may induce a MAPK/ERK signal transduction pathway upon binding to a G-protein-coupled receptor. MAPK/ERK1 and -2 appear to phosphorylate the HIV-1 preintegration complex, a step necessary for nuclear translocation and successful integration.
Asunto(s)
Fármacos Anti-VIH/farmacología , Quimiocina CCL5/farmacología , VIH-1/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/metabolismo , VIH-1/fisiología , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/virología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Provirus/genética , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Transcripción Genética , Integración ViralRESUMEN
UNLABELLED: An 131I labeled trivalent antigen binding construct, formed from 3 Fab' fragments of murine anti-CEA monoclonal antibody (Mab) 35, has shown favorable biokinetics in animal studies. OBJECTIVES: The aim of this study was to evaluate biodistribution and tumor uptake of 131I-F(ab')3 in patients and its potential utility for radioimmunotherapy of CEA expressing tumors. PATIENTS AND METHODS: Six patients (5 M, 1 F; age 62 +/- 13 y) with liver metastases of colorectal cancer, scheduled for hepatic surgery were studied by 2-3 whole body scans immediately post infusion of 111-137 MBq of 131I labeled Mab 35 F(ab')3 and up to 72 h. Circulating CEA ranged from 1.2 to 1930 ng/ml. We evaluated plasma and whole body clearance, activity accumulation by post-surgical ex-vivo tissue measurement in primary tumor (T) and metastases (M), and calculated M to blood (M/B) and M to liver (M/L) ratios. RESULTS: All known tumor sites were detected by immunoscintigraphy and confirmed at surgery. Whole body effective T1/2 calculated in two patients was 51.5 h and 55.6 h respectively. Effective serum T1/2 was mono-exponential in 3 patients (short observation interval) with 20.9 +/- 7 h and bi-exponential in three with alpha T1/2 of 6.3 +/- 1 h and beta T1/2 of 38.6 +/- 5 h. In a patient with concomitant colic and hepatic lesions uptake of primary tumor was 0.0071% injected dose per gram of tissue (%ID/g) and mean metastases activity was 0.0275 %ID/g at 48 h. In the 3 patients who had surgery at 48 h, mean uptake in metastases and normal liver was 0.0182 %ID/g and 0.0021 %ID/g, respectively (M/L 8.67). In the single subject followed until 7 days post infusion, residual activity in liver metastases was 10 times higher than in normal parenchyma. CONCLUSIONS: Tumor uptake and tumor to blood ratio, as well as serum clearance of the triconstruct are similar to those observed with intact iodinated anti-CEA antibodies. In the patient studied for 7 days the tumor residence time was favorable. Further improvements, however, need to be obtained before considering this approach for radioimmunotherapy.
Asunto(s)
Adenocarcinoma/secundario , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Antineoplásicos/uso terapéutico , Antígenos de Neoplasias/inmunología , Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/radioterapia , Inmunoconjugados/uso terapéutico , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Radioisótopos de Yodo/farmacocinética , Neoplasias Hepáticas/secundario , Radioinmunodetección , Radioinmunoterapia , Adenocarcinoma/sangre , Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/inmunología , Adenocarcinoma/radioterapia , Adenocarcinoma/cirugía , Anciano , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/sangre , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/inmunología , Terapia Combinada , Femenino , Semivida , Humanos , Fragmentos Fab de Inmunoglobulinas/sangre , Radioisótopos de Yodo/sangre , Radioisótopos de Yodo/uso terapéutico , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/cirugía , Masculino , Tasa de Depuración Metabólica , Ratones , Persona de Mediana Edad , Distribución TisularRESUMEN
Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1(Ba-L) infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08-4.77%). HIV-1-infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1-1 microg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1(Ba-L) (an R5 HIV-1 strain) more efficiently infected LC-T cell cocultures when compared with HIV-1(IIIB) (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1.
Asunto(s)
Fármacos Anti-VIH/farmacología , Quimiocina CCL5/análogos & derivados , Células Epiteliales/virología , Infecciones por VIH/transmisión , VIH-1 , Células de Langerhans/virología , Linfocitos T CD4-Positivos/virología , Movimiento Celular , Quimiocina CCL5/farmacología , Técnicas de Cocultivo , HumanosRESUMEN
We studied the combined anti-human immunodeficiency virus type 1 (HIV-1) effects of a derivative of stroma-derived factor 1beta (SDF-1beta), Met-SDF-1beta, and a modified form of RANTES, aminooxypentane (AOP)-RANTES. The antiviral agents were tested singly or in combination at 95 and 99% virus inhibitory concentrations. Clinical R5 and X4 HIV-1 isolates were used. AOP-RANTES inhibited R5 but not X4 viruses, whereas Met-SDF-1beta had the opposite effect. Combinations of these compounds inhibited mixed infections with R5 and X4 viruses (95 to 99%), whereas single drugs were less inhibitory (32 to 61%). Combinations of R5 and X4 inhibitors are promising and deserve further evaluation.
Asunto(s)
Fármacos Anti-VIH/farmacología , Antagonistas de los Receptores CCR5 , Quimiocina CCL5/farmacología , Citocinas/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1 , Receptores CXCR4/antagonistas & inhibidores , Secuencia de Aminoácidos , Fármacos Anti-VIH/uso terapéutico , Línea Celular , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/uso terapéutico , Quimiocina CXCL12 , Citocinas/química , Citocinas/uso terapéutico , Genoma Viral , Infecciones por VIH/virología , VIH-1/genética , Humanos , Datos de Secuencia MolecularRESUMEN
Aminooxypentane (AOP)-RANTES efficiently and specifically blocks entry of non-syncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) into host cells. Inhibition appears to be mediated by increased intracellular retention of the CCR5 coreceptor- AOP-RANTES complex and/or competitive binding of AOP-RANTES with NSI R5 HIV-1 isolates for CCR5. Although AOP-RANTES and other beta-chemokine analogs are potent inhibitors, the extreme heterogeneity of the HIV-1 envelope glycoproteins (gp120 and gp41) and variable coreceptor usage may affect the susceptibility of variant HIV-1 strains to these drugs. Using the same peripheral blood mononuclear cells (PBMC) with all isolates, we observed a significant variation in AOP-RANTES inhibition of 13 primary NSI R5 isolates; 50% inhibitory concentrations (IC(50)) ranged from 0.04 nM with HIV-1(A-92RW009) to 1.3 nM with HIV-1(B-BaL). Experiments performed on the same isolate (HIV-1(B-BaL)) with PBMC from different donors revealed no isolate-specific variation in AOP-RANTES IC(50) values but did show a considerable difference in virus replication efficiency. Exclusive entry via the CCR5 coreceptor by these NSI R5 isolates suggests that variable inhibition by AOP-RANTES is not due to alternative coreceptor usage but rather differential CCR5 binding. Analysis of the envelope V3 loop sequence linked a threonine or arginine at position 319 (numbering based on the HXB2 genome) with AOP-RANTES resistance. With the exception of one isolate, A319 was associated with increased sensitivity to AOP-RANTES inhibition. Distribution of AOP-RANTES IC(50) values with these isolates has promoted ongoing screens for new CCR5 agonists that show broad inhibition of HIV-1 variants.
Asunto(s)
Fármacos Anti-VIH/farmacología , Quimiocina CCL5/análogos & derivados , VIH-1/efectos de los fármacos , Receptores CCR5/metabolismo , Secuencia de Aminoácidos , Línea Celular , Quimiocina CCL5/farmacología , Farmacorresistencia Microbiana , Células Gigantes/fisiología , Proteína gp120 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/genética , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , Concentración 50 Inhibidora , Leucocitos Mononucleares/virología , Datos de Secuencia Molecular , Fragmentos de Péptidos/genéticaRESUMEN
Modification of the amino terminus of regulated on activated normal T-cell expressed (RANTES) has been shown to have a significant effect on biological activity and produces proteins with antagonist properties. Two amino-terminally modified RANTES proteins, Met-RANTES and aminooxypentane-RANTES (AOP-RANTES), exhibit differential inhibitory properties on both monocyte and eosinophil chemotaxis. We have investigated their binding properties as well as their ability to activate the RANTES receptors CCR1, CCR3, and CCR5 in cell lines overexpressing these receptors. We show that Met-RANTES has weak activity in eliciting a calcium response in Chinese hamster ovary cells expressing CCR1, CCR3, and CCR5, whereas AOP-RANTES has full agonist activity on CCR5 but is less effective on CCR3 and CCR1. Their ability to induce chemotaxis of the murine pre-B lymphoma cell line, L1.2, transfected with the same receptors, consolidates these results. Monocytes have detectable mRNA for CCR1, CCR2, CCR3, CCR4, and CCR5, and they respond to the ligands for these receptors in chemotaxis but not always in calcium mobilization. AOP-RANTES does not induce calcium mobilization in circulating monocytes but is able to do so as these cells acquire the macrophage phenotype, which coincides with a concomitant up-regulation of CCR5. We have also tested the ability of both modified proteins to induce chemotaxis of freshly isolated monocytes and eosinophils. Cells from most donors do not respond, but occasionally cells from a particular donor do respond, particularly to AOP-RANTES. We therefore hypothesize that the occasional activity of AOP-RANTES to induce leukocyte chemotaxis is due to donor to donor variation of receptor expression.
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Fármacos Anti-VIH/farmacología , Quimiocina CCL5/análogos & derivados , Receptores de Quimiocina/efectos de los fármacos , Animales , Unión Competitiva , Quimiocina CCL5/farmacología , Quimiotaxis/efectos de los fármacos , Cricetinae , Regulación hacia Abajo , VIH-1/patogenicidad , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Receptores CCR1 , Receptores CCR3 , Receptores CCR5/metabolismo , Receptores de Quimiocina/metabolismo , Propiedades de SuperficieAsunto(s)
Fármacos Anti-VIH/farmacología , Quimiocina CCL5/análogos & derivados , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Quimiocina CCL5/farmacología , Relación Dosis-Respuesta a Droga , VIH-1/aislamiento & purificación , VIH-1/fisiología , HumanosRESUMEN
Studies have demonstrated that the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta suppress human immunodeficiency type 1 (HIV-1) replication in vitro. Infection with HIV-1 requires expression of CD4 antigen and the chemokine receptor CXCR4 (X4) or CCR5 (R5) on the surface of target cells. The engagement of these receptors with the viral surface proteins is essential for the membrane fusion process. This study investigated the anti-HIV-1 activity of a derivative of RANTES, the CCR5 antagonist aminooxypentane (AOP)-RANTES, on R5 HIV-1 isolates in peripheral blood mononuclear cells. In drug exposure experiments, AOP-RANTES efficiently inhibited viral replication of HIV-1 R5 strains, with a viral breakthrough observed after the withdrawal of the compound. The HIV-1-specific proliferative capacity was maintained under all conditions when compared with controls. An increase in IFN-gamma production accompanied by a parallel decrease in the generation of IL-10 was observed following the in vitro exposure of cells to AOP-RANTES in the presence of three of four HIV-1 R5 isolates. These experiments confirmed that the chemokine receptor antagonist AOP-RANTES was effective as an inhibitor of HIV-1 R5 strain infectivity in peripheral blood mononuclear cells. The capacity of this compound to maintain HIV-1-specific proliferative activity with a shift toward a type 1 cytokine profile makes this compound a unique molecule, one adopting an immunological pathway to limit HIV-1 infection.
Asunto(s)
Fármacos Anti-VIH/farmacología , Quimiocina CCL5/análogos & derivados , VIH-1/efectos de los fármacos , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , División Celular/efectos de los fármacos , Quimiocina CCL5/farmacología , Humanos , Factores de TiempoRESUMEN
The natural ligands for the CCR5 chemokine receptor, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES (regulated on T-cell activation, normal T-cell expressed and secreted), are known to inhibit human immunodeficiency virus (HIV) entry, and N-terminally modified RANTES analogues are more potent than native RANTES in blocking infection. However, potent CCR5 blocking agents may select for HIV-1 variants that use alternative coreceptors at less than fully inhibitory concentrations. In this study, two N-terminal chemical modifications of RANTES produced by total synthesis, aminooxypentane (AOP)-RANTES[2-68] and N-nonanoyl (NNY)-RANTES[2-68], were tested for their ability to prevent HIV-1 infection and to select for coreceptor switch variants in the human peripheral blood lymphocyte-SCID mouse model. Mice were infected with a CCR5-using HIV-1 isolate that requires only one or two amino acid substitutions to use CXCR4 as a coreceptor. Even though it achieved lower circulating concentrations than AOP-RANTES (75 to 96 pM as opposed to 460 pM under our experimental conditions), NNY-RANTES was more effective in preventing HIV-1 infection. However, in a subset of treated mice, these levels of NNY-RANTES rapidly selected viruses with mutations in the V3 loop of envelope that altered coreceptor usage. These results reinforce the case for using agents that block all significant HIV-1 coreceptors for effective therapy.
Asunto(s)
Fármacos Anti-VIH/farmacología , Quimiocina CCL5/análogos & derivados , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Secuencia de Aminoácidos , Animales , Fármacos Anti-VIH/síntesis química , Quimiocina CCL5/síntesis química , Quimiocina CCL5/farmacología , Modelos Animales de Enfermedad , Variación Genética , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , Humanos , Ratones , Ratones SCID , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat hepatocytes by determination of [3H]glycogen from d-[6-3H]glucose. The dose-response curve of insulin in this assay exhibited an EC50 of 0.42 (+/-0.04) nM, which is comparable to the dissociation constant of insulin from its receptor in hepatocytes. We used this assay to examine possible residual insulin-like activity of the four major fragments formed upon insulin degradation by insulin protease. Fragments A1-13B1-9, A1-14B1-9,and A14-21B14-30 showed no measurable activity. Although preparations of fragment A14-21B10-30 displayed dose-dependent agonist activity with an EC50 of 380 (+/-40) nM, we conclude that this was due to an insulin-like impurity since the chemically synthesized fragment showed no such activity. In summary, this bioassay demonstrates the action of insulin on glycogen formation in hepatocytes and provides a rapid and sensitive measurement of insulin-like activity which could facilitate screening studies.
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Glucógeno/biosíntesis , Insulina/análisis , Hígado/metabolismo , Animales , Bioensayo/métodos , Células Cultivadas , Glucosa/metabolismo , Insulisina/metabolismo , Masculino , Fragmentos de Péptidos/análisis , Ratas , Ratas Wistar , Sensibilidad y Especificidad , Factores de Tiempo , Tritio/metabolismoRESUMEN
To circumvent radionuclide accumulation in nontarget tissues when employing metallic radionuclides for radioimmunoscintigraphy or radioimmunotherapy, we have investigated the effect of the chelating agent deferroxamine (DFO) on the biodistribution of 67Ga following its administration attached to intact monoclonal antibody MAb35 and its F(ab')2 fragment. Following administration of 67Ga-labeled MAb35, DFO accelerated whole-body elimination of 67Ga and reduced its accumulation in several normal tissues, including liver, spleen and kidney. No reduction in tumor accumulation of 67Ga was observed. Following administration of 67Ga-labeled F(ab')2 fragment, kidney accumulation was higher than with the intact antibody (29% and 4% ID/g, respectively) and blood levels lower (0.69% and 5% ID/g, respectively). Again, no alteration in tumor accumulation of 67Ga was seen following DFO, although liver, kidney and blood levels were reduced and whole-body elimination accelerated.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Deferoxamina/farmacología , Radioisótopos de Galio/farmacocinética , Radioisótopos de Galio/uso terapéutico , Animales , Quelantes , Neoplasias del Colon/metabolismo , Neoplasias del Colon/radioterapia , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Riñón/metabolismo , Cinética , Ratones , Ratones Desnudos , Radioinmunodetección , Radioinmunoterapia , Distribución Tisular/efectos de los fármacos , Trasplante HeterólogoRESUMEN
Escherichia coli stimulates neutrophil migration across human uroepithelial cell layers. This study investigated the role of the neutrophil chemokine interleukin-8 (IL-8) in this process. E. coli and IL-1alpha stimulated urinary tract epithelial layers to secrete IL-8 and induced transepithelial neutrophil migration. Anti-IL-8 antibody reduced neutrophil migration across epithelial cell layers, indicating a central role for this chemokine in the migration process. Furthermore, addition of recombinant IL-8 to unstimulated cell layers was sufficient to induce migration. The IL-8 dependence of neutrophil migration was maintained after removal of soluble IL-8 by washing of the cell layers. Flow cytometry analysis with fluorescein isothiocyanate-labelled IL-8 confirmed IL-8's ability to bind to the epithelial cell surface. Indirect immunofluorescence with confocal laser scanning microscopy showed that IL-8 associated with the epithelial cell layers. Prior incubation of neutrophils with antibodies to IL-8 receptor A (IL-8RA) reduced neutrophil migration. Anti-IL-8 RB antibody had no effect on neutrophil migration. These results demonstrate that IL-8 plays a key role in E. coli- or IL-1alpha-induced transuroepithelial migration and suggest that epithelial cell-produced IL-8 interacts with IL-8RA on the neutrophil surface.
Asunto(s)
Antígenos CD/fisiología , Escherichia coli/fisiología , Interleucina-8/fisiología , Neutrófilos/fisiología , Receptores de Interleucina/fisiología , Sistema Urinario/citología , Línea Celular , Movimiento Celular , Células Epiteliales , Humanos , Interleucina-1/farmacología , Receptores de Interleucina-8ARESUMEN
We describe the application of a stable isotope dilution assay (IDA) to determine precise insulin, C-peptide, and proinsulin levels in blood by extraction from serum and quantitation by mass spectrometry using analogues of each target protein labeled with stable isotopes. Insulin and C-peptide levels were also determined by immunoassay, which gave consistently higher results than by IDA, the relative difference being larger at low concentrations. Insulin, C-peptide, and proinsulin levels were all shown by IDA to be higher in type II diabetics than in non-diabetics, with mean values rising from 22 (+/- 2) to 92 (+/- 8), 335 (+/- 11) to 821 (+/- 24), and 6 (+/- 1) to 37 (+/- 3) pM, respectively. Interestingly, the ratio between IDA and immunoassay values for insulin levels increased from 1.3 in non-diabetics to 1.7 in type II diabetics. The ratio between proinsulin and insulin levels by IDA increased from 0.24 in non-diabetics to 0.36 in type II diabetics, whereas the ratio between C-peptide and insulin levels by IDA decreased from 17.6 to 10.7. This disproportionate change in protein levels between different types of individuals has implications for the metabolism of insulin in the diabetics studied (type II) and suggests that C-peptide levels are not always a reliable guide as to pancreatic insulin secretion. In addition, levels of the 33-residue C-peptide (partially trimmed form) were shown to be less than 10% that of the fully trimmed 31-residue C-peptide levels, and we tested IDA in a clinical context by two post-pancreatic graft studies. IDA was shown to give direct, positive identification of the target protein with unrivaled accuracy, avoiding many of the problems associated with present methodology for protein determination.
Asunto(s)
Péptido C/sangre , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 2/sangre , Insulina/sangre , Proinsulina/sangre , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Inmunoensayo , Masculino , Persona de Mediana Edad , Trasplante de PáncreasRESUMEN
The chemokine receptors CXCR4 and CCR5 have recently been shown to act as coreceptors, in concert with CD4, for human immunodeficiency virus-type 1 (HIV-1) infection. RANTES and other chemokines that interact with CCR5 and block infection of peripheral blood mononuclear cell cultures inhibit infection of primary macrophages inefficiently at best. If used to treat HIV-1-infected individuals, these chemokines could fail to influence HIV replication in nonlymphocyte compartments while promoting unwanted inflammatory side effects. A derivative of RANTES that was created by chemical modification of the amino terminus, aminooxypentane (AOP)-RANTES, did not induce chemotaxis and was a subnanomolar antagonist of CCR5 function in monocytes. It potently inhibited infection of diverse cell types (including macrophages and lymphocytes) by nonsyncytium-inducing, macrophage-tropic HIV-1 strains. Thus, activation of cells by chemokines is not a prerequisite for the inhibition of viral uptake and replication. Chemokine receptor antagonists like AOP-RANTES that achieve full receptor occupancy at nanomolar concentrations are strong candidates for the therapy of HIV-1-infected individuals.
Asunto(s)
VIH-1/efectos de los fármacos , Macrófagos/virología , Receptores de Quimiocina , Receptores de Citocinas/antagonistas & inhibidores , Receptores del VIH/antagonistas & inhibidores , Linfocitos T/virología , Animales , Unión Competitiva , Antígenos CD4/metabolismo , Gatos , Línea Celular , Células Cultivadas , Quimiocina CCL4 , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacología , Quimiotaxis de Leucocito , VIH-1/fisiología , Células HeLa , Humanos , Proteínas Inflamatorias de Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Receptores CCR5 , Receptores de Citocinas/metabolismo , Receptores del VIH/metabolismo , Linfocitos T/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Chemokines are chemotactic proteins which play a central role in immune and inflammatory responses. Chemokine receptors are members of the seven transmembrane G-protein coupled family and have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. To study chemokine endocytosis in detail we have used novel site-specific chemistry to make a fluorescently labeled CC-chemokine agonist (rhodamine-MIP-1alpha) and antagonist (NBD-RANTES). We have also generated a CHO cell line stably expressing a hemagglutinin-tagged version of the CC-chemokine receptor 1 (CCR1), and using these reagents we have examined the receptor-mediated endocytosis of CC-chemokines by confocal microscopy. Our studies reveal that the agonist was internalized and accumulated in transferrin receptor-positive endosomes whereas the antagonist failed to internalize. However, receptor-bound antagonist could be induced to internalize by co-administration of agonist. Analysis of receptor redistribution following chemokine addition confirmed that sequestration was induced by agonists but not by antagonists.
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Quimiocinas/metabolismo , Endocitosis , Receptores de Quimiocina , Receptores de Citocinas/metabolismo , 4-Cloro-7-nitrobenzofurazano , Quimiocina CCL4 , Quimiocina CCL5/metabolismo , Colorantes Fluorescentes , VIH , Humanos , Proteínas Inflamatorias de Macrófagos/metabolismo , Receptores CCR8 , Receptores de Transferrina/análisis , RodaminasAsunto(s)
Quimiocinas/síntesis química , Quimiocinas/farmacología , Colorantes Fluorescentes , Receptores de Quimiocina/análisis , Animales , Unión Competitiva , Células CHO , Membrana Celular/metabolismo , Quimiocina CCL5/metabolismo , Quimiocinas/química , Quimiocinas/metabolismo , Quimiotaxis de Leucocito , Cricetinae , Citometría de Flujo , Expresión Génica , Humanos , Interleucina-8/análogos & derivados , Interleucina-8/metabolismo , Microscopía Confocal , Unión Proteica , Receptores CCR1 , Receptores de Quimiocina/metabolismo , Proteínas Recombinantes/metabolismo , Bases de SchiffRESUMEN
Insulin levels in humans were measured by a new assay, the isotope dilution assay (IDA), based on stable isotope dilution mass spectrometry. A known amount of a deuterated analog of insulin was used as an internal standard and added to the serum samples before sample processing. After isolation by immunoaffinity chromatography and solid phase extraction, followed by a purification step on reversed-phase microbore high-performance liquid chromatography (HPLC), the insulin-containing fraction was analyzed by mass spectrometry. The relative intensity of the signals due to insulin and its deuterated analog in the mass spectrum was used to determine the concentration of insulin in the sample. Using serum samples of 0.5-2.0 ml, we were able to measure insulin levels in the range of 3-1700 pmol/l in several clinical samples from type II diabetic patients. The basal level of endogenous insulin was also determined in two normal subjects and found to be approximately 20 pmol/l. Insulin secretion was followed after the ingestion of 75 g glucose in one healthy volunteer. Finally, the determination of the insulin level of one hemolyzed post-mortem blood sample, for which immunoassays gave inconsistent results, was performed to help forensic investigations. Our results showed a good correlation with standard immunoassay data, except in six samples where much lower values were obtained by our stable isotope dilution assay, suggesting an overestimation of insulin levels by immunoassay in some cases. As it is not subject to immunological interferences by insulin-related compounds, this new assay has a major clinical advantage in that it avoids confusions related to hyperinsulinemia.
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Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus/sangre , Insulina/sangre , Insulina/metabolismo , Obesidad , Adulto , Animales , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Deuterio , Sobredosis de Droga , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Humanos , Hiperinsulinismo , Inmunoensayo/métodos , Insulina/envenenamiento , Secreción de Insulina , Marcaje Isotópico/métodos , Masculino , Espectrometría de Masas/métodos , Persona de Mediana Edad , Valores de Referencia , Sensibilidad y Especificidad , PorcinosRESUMEN
In an attempt to improve the pharmacokinetic behavior of an antitumor radioimmunoconjugate, we have prepared a trivalent antigen-binding construct formed from three Fab' fragments derived from the parent murine monoclonal antibody (MAb) 35 directed against the carcinoembryonic antigen. The construct was generated by a novel approach using polyoxime chemistry. This approach leads to a homogeneous construct, as judged by SDS-PAGE and by mass spectrometry, which was found to retain full immunoreactivity. A comparison of the monovalent, divalent, and trivalent F(ab')n materials in vitro revealed the expected trend of increasing association constant with increasing valency. The in vivo biodistribution of the 125I-labeled trivalent construct was studied in xenograft-bearing nude mice. Absolute tumor accumulation seen with the trivalent construct (10.8% injected dose/g) was lower than that seen with the intact MAb35 (15.2% injected dose/g). This finding and the more rapid loss of activity from tumor are presumably the consequence of the quicker blood clearance of the trivalent material. However, the construct showed tumor:blood ratios up to 10-fold higher than those seen for the parent antibody, and ratios of tumor:normal tissue accumulation were generally greatly improved. These improvements were achieved despite only modest reduction in maximum tumor accumulation when compared to the parent MAb35, and this augurs well for an improved potential for this novel construct as an agent for radioimmunotherapy and radioimmunoscintigraphy.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Antígeno Carcinoembrionario/análisis , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Animales , Antígeno Carcinoembrionario/inmunología , Femenino , Indicadores y Reactivos , Radioisótopos de Yodo/farmacocinética , Ratones , Ratones Desnudos , Oximas , Radioinmunodetección/métodos , Radioinmunoterapia/métodos , Factores de Tiempo , Distribución Tisular , Trasplante HeterólogoRESUMEN
Extension of recombinant human RANTES by a single residue at the amino terminus is sufficient to produce a potent and selective antagonist. RANTES is a proinflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases. When mature RANTES was expressed heterologously in Escherichia coli, the amino-terminal initiating methionine was not removed by the endogenous amino peptidases. This methionylated protein was fully folded but completely inactive in RANTES bioassays of calcium mobilization and chemotaxis of the promonocytic cell line THP-1. However, when assayed as an antagonist of both RANTES and macrophage inflammatory polypeptide-1 alpha (MIP-1 alpha) in these assays, the methionylated RANTES (Met-RANTES) inhibited the actions of both chemokines. T cell chemotaxis was similarly inhibited. The antagonistic effect was selective since Met-RANTES had no effect on interleukin-8- or monocyte chemotractant protein-1-induced responses in these cells. Met-RANTES can compete with both [125I]RANTES and [125I]IMP-1 alpha binding to THP-1 cells or to stably transfected HEK cells recombinantly expressing their common receptor, CC-CKR-1. These data show that the integrity of the amino terminus of RANTES is crucial to receptor binding and cellular activation.