RESUMEN
Overexpression of a membrane glycoprotein (P170) represents the most common multidrug resistance (MDR) mechanism in cancer therapy. Specific autoantibodies to extracellular loops 1, 2 and 4 of murine P170 are elicited in mice using palmitoylated synthetic peptides reconstituted in liposomes with or without Lipid A and resuspended in alum. IgM antibodies are detected 14 days following the first injection and IgG1 become predominant after the third challenge. Animals do not show any autoimmunity symptoms or induced toxicity up to 18 months after the immunization. Previous immunizations of mice using liposomes with mdr1 peptides efficiently improve chemotherapy with doxorubicin and vinblastine against P388 R cells with a 77% increase of survival half time in the immunized group. Sera from immunized mice are also effective in reducing cellular resistance to vinblastine and doxorubicin in vitro. Taken together these data suggest that this immunization approach might have potential clinical applications.
Asunto(s)
Resistencia a Múltiples Medicamentos/inmunología , Resistencia a Antineoplásicos/inmunología , Inmunoterapia , Neoplasias/terapia , Animales , Autoanticuerpos/inmunología , Línea Celular Tumoral , Inmunización , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/farmacología , Inmunoglobulina M/administración & dosificación , Inmunoglobulina M/farmacología , Leucemia P388/tratamiento farmacológico , Liposomas , Ratones , Neoplasias/tratamiento farmacológicoRESUMEN
Overexpression of the membrane glycoprotein (P170) represents the most common multidrug resistance (MDR) mechanism in cancer therapy. Specific auto-antibodies to extracellular loops 1, 2 and 4 of murine P170 were elicited in mice using palmitoylated synthetic peptides reconstituted in liposomes, with or without Lipid A, and resuspended in alum. IgM antibodies were detected 14 days following the first injection and IgG1 became predominant after the third challenge. Animals did not show any auto-immune symptoms or induced toxicity up to 18 months after the immunisation. Previous immunisations of mice using liposomes with MDR1 peptides increases the efficacy of chemotherapy treatments with doxorubicin and vinblastine against P388 R cells with increase of 77% in the survival half time in the immunised group. Sera from the immunised mice were also effective in reducing cellular resistance to vinblastine and doxorubicin in vitro. Taken together, these data suggest that this immunisation approach might have potential clinical applications.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Resistencia a Múltiples Medicamentos/inmunología , Resistencia a Antineoplásicos/inmunología , Glicoproteínas/inmunología , Linfoma/terapia , Subfamilia B de Transportador de Casetes de Unión a ATP , Animales , Autoanticuerpos/inmunología , Doxorrubicina/uso terapéutico , Femenino , Glicoproteínas/uso terapéutico , Lípido A/inmunología , Liposomas/inmunología , Linfoma/inmunología , Ratones , Vinblastina/uso terapéuticoRESUMEN
Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1beta (IL-1beta). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1beta-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2-4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.
Asunto(s)
Sistemas de Liberación de Medicamentos , Endotelio Vascular/efectos de los fármacos , Interleucina-1/administración & dosificación , Enfermedades Cardiovasculares/tratamiento farmacológico , Sistema Cardiovascular/efectos de los fármacos , Sistema Cardiovascular/inmunología , Células Cultivadas , Portadores de Fármacos , Selectina E/inmunología , Endotelio Vascular/inmunología , Humanos , Liposomas , Proteínas Recombinantes/administración & dosificaciónRESUMEN
OBJECTIVE: To investigate the initial longterm effect of inhaled NO on hypoxemia in ARDS patients. DESIGN: Retrospective study. PATIENTS: Nine hypoxemic patients with ARDS (Murray Lung Injury Score, LIS, 2.8 +/- 0.3), treated with conventional mechanical ventilation. INTERVENTIONS: Continuous NO inhalation was started after a test of inhaled NO efficacy on gas exchange and hemodynamics. Long term effects of inhaled NO were evaluated daily in terms of arterial oxygenation and methemoglobin formation. RESULTS: The initial NO inhalation increased the PaO2/FiO2 from 141 +/- 64 mmHg to 216 +/- 70 mmHg (p < 0.0001) and decreased the mean pulmonary pressure from 38 +/- 7 mmHg to 32 +/- 5 mmHg (p < 0.01), the pulmonary venous admixture from 29 +/- 10% to 20 +/- 8% (p < 0.01) and the pulmonary vascular resistance from 325 +/- 97 dyne.s.cm-5 to 238 +/- 48 dyne.s.cm-5 (p < 0.01). Daily withdrawal of inhaled NO, which was administered for 14 +/- 16 days at 8 +/- 2 ppm, was associated with a decrease in PaO2/FiO2 by 61 +/- 32 mmHg (p < 0.0001). During prolonged NO inhalation the FiO2 was decreased, on average, by 0.34 +/- 0.19 (p < 0.01), the positive end-expiratory pressure by 4 +/- 2 cmH2O (p < 0.01) and the peak inspiratory pressure by 7 +/- 4 cmH2O (p < 0.01). Three patients died during the ICU stay. CONCLUSIONS: Our results confirm the interest for inhaled NO as an additional approach for the treatment of hypoxemia in ARDS. Inhaled NO seems to allow for a better control of gas exchange, rather than for a rapid reduction of the ventilatory support.
Asunto(s)
Óxido Nitroso/uso terapéutico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Adolescente , Adulto , Anciano , Preescolar , Femenino , Humanos , Hipoxia/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Síndrome de Dificultad Respiratoria/fisiopatología , Estudios RetrospectivosRESUMEN
Human erythrocytes bearing electroinserted full-length CD4 (RBC-CD4) can bind and fuse with a laboratory strain of human immunodeficiency virus type 1 (HIV-1) or with T cells infected by HIV-1. Here we show that RBC-CD4 neutralize primary HIV-1 strains in an assay of cocultivation of peripheral blood mononuclear cells (PBMC) from HIV-1-infected persons with uninfected PBMC. RBC-CD4 inhibited viral p24 core antigen accumulation in these cocultures up to 10,000-fold compared with RBC alone. Viral p24 accumulation was inhibited equally well when measured in culture supernatants or in call extracts. The inhibition was dose-dependent and long-lived. Two types of recombinant CD4 tested in parallel were largely ineffective. The neutralization of primary HIV-1 by RBC-CD4 in vitro was demonstrated in PBMC cultures from 21 of a total of 23 patients tested at two independent sites. RBC-CD4 may offer a route to blocking HIV-1 infection in vivo.
Asunto(s)
Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/virología , Eritrocitos/virología , VIH-1/fisiología , Síndrome de Inmunodeficiencia Adquirida/sangre , Unión Competitiva , Antígenos CD4/genética , Células Cultivadas , Técnicas de Cocultivo , Electroporación , Membrana Eritrocítica/metabolismo , Proteína p24 del Núcleo del VIH/análisis , Humanos , Cinética , Proteínas Recombinantes/metabolismo , Replicación ViralRESUMEN
This study shows that the lateral mobility of CD4, an important plasma-membrane immune receptor, can be modulated by intracellular application of an anti-CD4 antibody. For this purpose, (i) full-length CD4 and a truncated CD4 mutant, lacking a 32-residue-long C-terminal intracellularly exposed domain, were expressed in Spodoptera frugiperda (Sf9) insect cells, (ii) a monoclonal antibody, C6, with specificity for the C-terminal domain was generated, and (iii) a versatile apparatus for fluorescence microphotolysis (FM) studies was constructed. By these means it was found that the commercial anti-CD4 antibody Leu3a-PE, in contrast with several other anti-CD4 antibodies, could be used as a fluorescent label of CD4 without interfering greatly with CD4 mobility. Labelled by Leu3a-PE, full-length CD4 had a lateral diffusion coefficient of D = (4.7 +/- 1.9) x 10(-10) cm2/s and a mobile fraction of fm = 80 +/- 16% (room temperature). Within experimental accuracy the truncated CD4 had the same mobility as full-length CD4. Introduction of the C6 antibody into Sf9 cells by microinjection or by fusion with C6-loaded liposomes decreased the mobility of full-length CD4 (fm = 40%) but not of truncated CD4 (fm = 80%). Treatment of Sf9 cells with phorbol ester also reduced the mobility of full-length CD4 (fm = 50%) but not truncated CD4 (fm = 90%). A calmodulin inhibitor but not a protein kinase C (PKC) inhibitor abolished the phorbol ester effect.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD4/metabolismo , Membrana Celular/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Baculoviridae/genética , Antígenos CD4/genética , Antígenos CD4/inmunología , Calmodulina/antagonistas & inhibidores , Línea Celular , Humanos , Liposomas , Microinyecciones , Microscopía Fluorescente , Microscopía de Contraste de Fase , Datos de Secuencia Molecular , Mutagénesis/genética , Proteína Quinasa C/antagonistas & inhibidores , Spodoptera , Sulfonamidas/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Intrinsically, or after exposure to chemotherapeutic drugs, many cancer cells overexpress a class of high molecular weight membrane glycoproteins associated with the multidrug resistance (mdr) of these cells. This report describes an immunization protocol eliciting autoantibodies specific to extracellular epitopes of the murine mdr 1 P-glycoprotein (Pgp). Synthetic peptides with the sequences of extracellular loops of murine Pgp were covalently coupled with four palmitic acid moieties per peptide molecule. These "lipopeptides" were reconstituted in the bilayer of liposomes containing lipid A and used to immunize mice. Antibodies against the lipopeptides corresponding to loop 2 and 4 were elicited in sera of immunized mice. They reacted specifically with extracellular epitopes of the naturally occurring murine Pgp. After interaction with resistant cancer cells, the antibodies induced an average 50% increase in cellular accumulation of doxorubicin and Bodipy-verapamil. In the presence of these antibodies the resistance of L1210 mdr cells was reduced from an LD50 of 4 x 10(-5) M to 5 x 10(-7) M doxorubicin.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/inmunología , Autoanticuerpos/sangre , Inmunización , Lipoproteínas/inmunología , Liposomas , Secuencia de Aminoácidos , Animales , Autoanticuerpos/inmunología , Autoanticuerpos/farmacología , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Resistencia a Medicamentos , Epítopos/inmunología , Leucemia L1210 , Lípido A , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Ácido Palmítico , Ácidos Palmíticos/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Células Tumorales CultivadasRESUMEN
Membrane recombinant CD4 was electroinserted into the plasma membrane of red blood cells (RBCs) from four HIV patients. CD4 had been labeled with 125I before electroinsertion. The RBCs-CD4-125I were labeled with 51Cr and autotransfused to the donor patients. The hematological indexes and the P50 value of the RBCs were not modified by the electroinsertion of CD4. The life span of the RBCs was not affected by electroinsertion of CD4 (t1/2 approximately 30 days), whereas the exposed CD4 showed a kinetics of disappearance characterized by two half-life times: a short one (t1/2 approximately 1 day) and a long one approximately equal to that of the RBCs. No side effects or anti-CD4 immune responses were observed in patients over a period of 28 days. The RBC-CD4 entity appears to be long-lived and has no adverse effect in HIV patients.
Asunto(s)
Antígenos CD4/inmunología , Senescencia Celular , Membrana Eritrocítica/inmunología , Eritrocitos/fisiología , Infecciones por VIH/sangre , VIH-1 , Adulto , Transfusión de Sangre Autóloga , Estudios de Factibilidad , Semivida , Humanos , Masculino , Persona de Mediana Edad , Proteínas RecombinantesRESUMEN
Concentration-dependent electroinsertion of recombinant human membrane CD4 in human erythrocytes shows a saturation at an average of about 3,500 inserted CD4 epitopes per cell, detectable by flow cytometry. The erythrocyte recovery drops to 10% at this high level of electroinsertion. Experimentally an optimum for cell recovery and insertion rate was found at about 2,500 CD4 epitopes per red blood cell. In vitro stability assay by flow cytometry indicated a temperature- and medium-dependent decrease in the number of CD4 epitopes inserted per cell. This decrease is biphasic, with an exponential part during the first 24 h after electroinsertion followed by a much slower linear decay.
Asunto(s)
Antígenos CD4/inmunología , Membrana Eritrocítica/inmunología , Antígenos CD4/sangre , Células Cultivadas , Estabilidad de Medicamentos , Electroporación , Epítopos/sangre , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas Recombinantes/inmunologíaRESUMEN
Application of an electrical pulse field at a strength slightly below the value required for electroporation to a suspension of red blood cells in the presence of membrane xenoproteins leads to the insertion of those proteins in the erythrocyte plasma membrane. This observation is extended to nucleated cells. In the presence of glycophorin A, application of such pulses leads to the insertion of 10(4)-10(5) molecules of glycophorin A per cell in CEM-CM3, Hela S3, and bovine CD8+ T cells. Electroinserted glycophorin A is detected by flow cytometry using anti-glycophorin monoclonal antibodies. The survival of the cells subjected to electroinsertion was 55% for CEM-CM3 cells, 69% for Hela S3 cells, and 65% for CD8+ T cells. Cells cultured after electroinsertion lost the electroinserted glycophorin A, with two different rates, by a temperature and cell type-dependent mechanism. During the first 2 h after electroinsertion, the CD8+ T cells lost 12.5% of the inserted glycophorin A per h, the CEM-CM3 cells lost 7.7% per h, whereas the Hela S3 cells lost only 0.8% of the inserted protein per h. After 2 h, the rate increased substantially, to 41.7% per h for the CD8+ T cells, 13.5% for the CEM-CM3 cells, and 8.9% for the Hela S3 cells. Cytochalasin D efficiently inhibited the disappearance of electroinserted glycophorin A during the first 2 h after electroinsertion only.
Asunto(s)
Electroporación , Células Eucariotas/metabolismo , Glicoforinas/administración & dosificación , Animales , Bovinos , Citocalasina D/farmacología , Dimetilsulfóxido/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células HeLa/metabolismo , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Subgrupos de Linfocitos T/metabolismo , Células Tumorales CultivadasAsunto(s)
Membrana Eritrocítica/metabolismo , Proteínas/administración & dosificación , Animales , Proteínas Sanguíneas/administración & dosificación , Línea Celular , Electricidad , Electroporación , Membrana Eritrocítica/ultraestructura , Humanos , Proteínas Recombinantes/administración & dosificaciónRESUMEN
The baculovirus-insect cell system is reliable in expressing a variety of recombinant proteins. A recombinant baculovirus encoding the full length human CD4 has been used to infect Spodoptera frugiperda 9 cells in 6-L-airlift fermentors. The procedured described in this report permitted a 6.5-fold enhancement of rCD4 expression as compared to standard procedures previously published. The increase of rCD4 expression on the cell surface was achieved by using the following steps: (1) Optimal seeding density of 0.8 x 10(6) cells/mL used to multiply cells at a maximum exponential growth of 4.5 x 10(6); (2) high multiplicity of infection (MOI) of 580 PFU/cell; (3) addition of medium at time of infection. In addition to full-length rCD4, a "short" rCD4 with largely deleted cytoplasmic sequence (last 31 C-terminal amino acids) was also efficiently expressed.
RESUMEN
Electroinsertion, a novel method of implanting xeno-proteins in red blood cell plasma membranes is applied to the insertion of human glycophorin in mouse red blood cells. The procedures yield erythrocytes with approx. 10(4) glycophorin molecules per cell, displaying the patching phenomenon when reacted with antiglycophorin monoclonal antibodies. Insertion of FITC-labeled glycophorin and subsequent quenching of FITC fluorescence with anti FITC antibody indicated that 70% of the inserted molecules were 'correctly' oriented, displaying the epitopes shown by glycophorin in human red blood cells. Moreover, insertion of FITC glycophorin in the red blood cell membranes yielded, under the fluorescence microscope continuous fluorescence which became patchy after reaction with anti-glycophorin monoclonal antibodies. When injected in mice, biotinylated RBC-Glyc+ were shown to have the same life-span as normal mouse red blood cells, i.e. t1/2 approximately 12 days. Cytofluorometric assay of human glycophorin inserted in the red blood cell showed the same life-span for the inserted protein as for the red blood cell, with the protein fully 'functional', i.e. capable of binding antibodies. Human glycophorin inserted in mouse erythrocyte did not elicit any immune response in mice, whereas the same concentrations of free glycophorin injected i.v. were highly immunogenic.
Asunto(s)
Membrana Eritrocítica/metabolismo , Glicoforinas/metabolismo , Animales , Anticuerpos Monoclonales , Formación de Anticuerpos , Estimulación Eléctrica , Citometría de Flujo , Glicoforinas/inmunología , Semivida , Humanos , Ratones , Ratones Endogámicos BALB C , Especificidad de la EspecieRESUMEN
gp120, the coat glycoprotein of the human immunodeficiency virus type 1 (HIV1) binds to a molecule on the surface of a class of T-lymphocytes, CD4, which is also the receptor for major histocompatibility complex class II (MHCII). To study the events that follow the interaction of gp120 with CD4, we have incorporated CD4 into lipid bilayers and recorded the electrical changes which occur after the addition of gp120. Interaction of gp120 to CD4-containing bilayers induces multistate ion-permeable channels with a maximum conductance of 380-400 picosiemens. When CD4+ bilayers were preexposed to either MHCII or to OKT4A antibody, no channels were formed after the addition of gp120. These results indicate that CD(4+)-containing bilayers bind gp120, MHCII, and OKT4A, that binding of gp120 produces ion-permeable channels, and that CD4+ bilayers can be used to assay for gp120 in the solution bathing the bilayer.
Asunto(s)
Antígenos CD4/inmunología , Cápside/metabolismo , VIH-1/metabolismo , Membrana Dobles de Lípidos , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Canales Iónicos/fisiología , Potenciales de la MembranaRESUMEN
Recombinant full-length CD4 expressed in Spodoptera frugiperda 9 cells with the baculovirus system was electroinserted in erythrocyte (RBC) membranes. Of the inserted CD4, 70% was "correctly" oriented as shown by fluorescence quenching experiments with fluorescein-labeled CD4. The inserted CD4 displayed the same epitopes as the naturally occurring CD4 in human T4 cells. Double-labeling experiments (125I-CD4 and 51Cr-RBC) showed that the half-life of CD4 electroinserted in RBC membrane in rabbits was approximately 7 days. Using the fluorescence dequenching technique with octadecylrhodamine B-labeled human immunodeficiency virus (HIV)-1, we showed fusion of the HIV envelope with the plasma membrane of RBC-CD4, whereas no such fusion could be detected with RBC. The dequenching efficiency of RBC-CD4 is the same as that of CEM cells. Exposure to anti-CD4 monoclonal antibody OKT4A, which binds to the CD4 region that attaches to envelope glycoprotein gp120, caused a significant decrease in the dequenching of fluorescence. In vitro infectivity studies showed that preincubation of HIV-1 with RBC-CD4 reduced by 80-90% the appearance of HIV antigens in target cells, the amount of viral reverse transcriptase, and the amount of p24 core antigen produced by the target cells. RBC-CD4, but not RBCs, aggregated with chronically HIV-1-infected T cells and caused formation of giant cells. These data show that the RBC-CD4 reagent is relatively long lived in circulation and efficient in attaching to HIV-1 and HIV-infected cells, and thus it may have value as a therapeutic agent against AIDS.
Asunto(s)
Antígenos CD4/sangre , Membrana Eritrocítica/inmunología , VIH/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD4/inmunología , Agregación Celular , Electricidad , Membrana Eritrocítica/microbiología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , VIH-1/fisiología , Semivida , Humanos , Fusión de Membrana , Conejos , Proteínas Recombinantes , Linfocitos T/inmunología , Linfocitos T/microbiologíaRESUMEN
A specific, rapid, and sensitive method for the detection of CD4 in solution was developed using pairs of fluorescently stained monoclonal antibodies which do not cross-compete. The assay is quantitated by flow cytometry using Simply Cellular microbeads (SC beads) as the primary support for the first anti-CD4 mAb. This method uses the standard conditions for anti-CD4 monoclonal antibody binding, washing, detection, and quantitation by flow cytometry of the CD4 antigen either bound to the SC beads or expressed on the cell surface. The monoclonal antibody used (Leu 3a PE) is the standard reference used to evaluate the CD4 concentration. This method differs from ELISA techniques, which need an antigen standard curve and thus can be influenced by the quality and source of the antigen. This type of assay is also a procedure which enables determination of the level of oligomerization of the bound antigen. It can be used for any antigen to which monoclonal antibodies recognizing at least two distinct epitopes are available. The use of soluble or full-length CD4 derivatives as potential therapeutic agents against AIDS, would benefit from a precise quantitation of the CD4 molecules which still have their proper tertiary structure.
RESUMEN
Electroinsertion is a novel technique of protein implantation in cell membranes using electrical pulses, of field strength between 1.3 kV/cm and 2.1 kV/cm and up to 1 ms duration. The full length recombinant CD4 receptor could thus be inserted in human and murine red blood cell (RBC) membranes. 100% of the RBC subjected to this procedure were shown to expose different CD4 epitopes after electroinsertion. An average of 5000 epitopes per cell has been detected by immunofluorescence assay using flow cytometry and whole cell ELISA. CD4 electroinserted in red blood cell membranes showed upon reaction with monoclonal antibody significant patching similar to that observed in T4 cells expressing CD4. Furthermore, the fluorescent enhancement coming from accumulation of immune complex phycoerythrin-antiphycoerythrin was similar for both native CD4 on T4 cells or CD4 electroinserted into erythrocyte membrane. Attempts to electroinsert proteins without a membrane spanning sequence have consistently failed, suggesting that adsorption is not responsible for the observed phenomena.
Asunto(s)
Antígenos CD/administración & dosificación , Antígenos CD4/administración & dosificación , Membrana Eritrocítica , Anticuerpos Monoclonales/inmunología , Antígenos CD4/inmunología , Electricidad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas In Vitro , Proteínas RecombinantesAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/terapia , Antígenos CD4/uso terapéutico , Biotecnología , Antígenos CD4/administración & dosificación , Antígenos CD4/aislamiento & purificación , Agregación Celular/inmunología , Portadores de Fármacos , Membrana Eritrocítica/inmunología , Humanos , Inmunoterapia , LiposomasRESUMEN
The CD4 molecule was reconstituted into the bilayers of large liposomes. Fluorescence microscopy and electron microscopy showed that these liposomes interact with HIV-infected H9-HT cells, delivering their contents to the cell interior. Liposomes bearing CD4 did not interact in this way with noninfected H9-HT cells nor did liposomes without CD4 interact significantly with HIV-infected cells. From electron micrographs, it appeared that HIV binds to liposomes bearing CD4; no attachment of virions to liposomes without CD4 was observed.
Asunto(s)
Antígenos CD4/inmunología , VIH/metabolismo , Liposomas/metabolismo , Línea Celular , Epítopos/inmunología , Técnica de Fractura por Congelación , VIH/inmunología , VIH/ultraestructura , Humanos , Liposomas/inmunología , Microscopía Electrónica , Microscopía Fluorescente , Virión/inmunología , Virión/metabolismo , Virión/ultraestructuraRESUMEN
CD4 is an integral membrane glycoprotein that acts as the cellular receptor for human immunodeficiency virus (HIV). A cDNA encoding full-length CD4 was inserted into the genome of Autographa californica nuclear polyhedrosis virus under transcriptional regulation of the viral polyhedrin gene promoter. The recombinant virus was used to infect insect cells, which resulted in the abundant expression of CD4 as evaluated by flow cytometry and immunoblot analysis. Recombinant CD4 expressed on the surface of infected insect cells was immunologically indistinguishable from human CD4 when using 11 different anti-CD4 monoclonal antibodies. The extraction of infected cells by phase-transition separation with Triton X-114 followed by immunoaffinity chromatography yielded a single protein detected by NaDodSO4/PAGE using silver staining. N-terminal sequence analysis of the purified recombinant protein showed that CD4 produced in Sf9 cells is efficiently cleaved from the precursor protein. Immunoblot analysis under nondenaturing conditions showed that the purified protein reacted with the anti-CD4 monoclonal antibody Leu-3a. The potential use of the recombinant membrane-associated CD4 in anti-HIV therapy is discussed.