RESUMEN
The purpose of the work was to study the nature of the Campylobacter spp. contamination during the processing of food products of plant and animal origin (raw poultry and beef meat, raw milk, leafy salads, sliced raw vegetables). In the study of 148 samples 50 strains of Campylobacter spp. (33.8%) were found. For the main phenotypic characteristics they were identified as C. jejuni spp. jejuni and C. jejuni spp. doylei (over 75%). The highest level of detection of campylobacteria (over 45%) was set for raw poultry, including the carcasses of chickens broilers, quails, turkeys and their semi-finished products. 19 of the 27 strains from poultry were identified as C. jejuni. Among the strains isolated from the environment, including swabs from equipment surfaces, 91% of the isolates were also presented by C. jejuni. It was found that the investigated foodstuffs were characterized by high levels of contamination with bacteria of the family Enterobacteriaceae, the content of which was comparable with the identified values of total viable bacteria (cfu). Salmonella was detected in 19% of the investigated poultry samples and in 14.3% of raw cow milk. In the study of swabs from surfaces of poultry processing equipment, the frequency of detection of Campylobacter strains was 38.7%, Salmonella - 12.9%. Most commonly Campylobacter and Salmonella were detected in the zones of primary processing of poultry: the frequency of isolation of Salmonella in slaughter corner was 25%, Campylobacter - 43%. When testing the swabs taken in the cooking zone of «fast food¼ restaurants Campylobacter and Salmonella were not detected. For studying the swabs from equipment surfaces and the environment for the presence of Campylobacter spp. a modified technique of sampling was developed. The method includes a comprehensive analysis in the test area with the use of three types of media for transportation and incubation of Campylobacter spp. (Preston broth with blood, Brucella broth, Cary-Blair medium), that increase the probability of detection of these pathogens.
Asunto(s)
Campylobacter , Comida Rápida/microbiología , Contaminación de Alimentos , Microbiología de Alimentos , Alimentos Crudos/microbiologíaRESUMEN
The Headache and Pain Clinic (HPC) is a unit of the Zürich Neurology Department, established in 1966. In the present study demographic features, clinical characteristics and medical management of primary and tertiary care patients were compared in two groups of 181 patients each, seen by general practitioners (GPs) or the HPC, respectively, for primary headaches in 1998. There was a preponderance of women and the socially underprivileged in both samples. Chronic headache was overrepresented in the HPC (44.7%). Loss of work for >2 months was found exclusively in the HPC (9.9%). Of the GP patients, 40% were using triptans and 26.5% in the HPC. One-third of both groups had had complementary and alternative medical treatment. Differences in management strategies reflected differences in headache severity and chronicity. Results indicated that remaining shortcomings of diagnosis and treatment of headache in primary care could be minimized by involving GPs in similar non-commercial studies.
Asunto(s)
Cefalea/epidemiología , Clínicas de Dolor/estadística & datos numéricos , Atención Primaria de Salud/estadística & datos numéricos , Demografía , Femenino , Cefalea/terapia , Humanos , Masculino , Persona de Mediana Edad , SuizaRESUMEN
In addition to the primary cell surface receptor CD4, CCR5 or another coreceptor is necessary for infections by human immunodeficiency virus type 1 (HIV-1), yet the mechanisms of coreceptor function and their stoichiometries in the infection pathway remain substantially unknown. To address these issues, we studied the effects of CCR5 concentrations on HIV-1 infections using wild-type CCR5 and two attenuated mutant CCR5s, one with the mutation Y14N at a critical tyrosine sulfation site in the amino terminus and one with the mutation G163R in extracellular loop 2. The Y14N mutation converted a YYT sequence at positions 14 to 16 to an NYT consensus site for N-linked glycosylation, and the mutant protein was shown to be glycosylated at that position. The relationships between HIV-1 infectivity values and CCR5 concentrations took the form of sigmoidal (S-shaped) curves, which were dramatically altered in different ways by these mutations. Both mutations shifted the curves by factors of approximately 30- to 150-fold along the CCR5 concentration axis, consistent with evidence that they reduce affinities of virus for the coreceptor. In addition, the Y14N mutation specifically reduced the maximum efficiencies of infection that could be obtained at saturating CCR5 concentrations. The sigmoidal curves for all R5 HIV-1 isolates were quantitatively consistent with a simple mathematical model, implying that CCR5s reversibly associate with cell surface HIV-1 in a concentration-dependent manner, that approximately four to six CCR5s assemble around the virus to form a complex needed for infection, and that both mutations inhibit assembly of this complex but only the Y14N mutation also significantly reduces its ability to successfully mediate HIV-1 infections. Although several alternative models would be compatible with our data, a common feature of these alternatives is the cooperation of multiple CCR5s in the HIV-1 infection pathway. This cooperativity will need to be considered in future studies to address in detail the mechanism of CCR5-mediated HIV-1 membrane fusion.
Asunto(s)
VIH-1/patogenicidad , Receptores CCR5/metabolismo , Antígenos CD4/metabolismo , Quimiocina CCL4 , Citometría de Flujo/métodos , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Células HeLa , Humanos , Proteínas Inflamatorias de Macrófagos/metabolismo , Modelos Biológicos , Mutación , Radioinmunoensayo , Receptores CCR5/genéticaRESUMEN
Strains of human immunodeficiency virus type 1 (HIV-1) that use the coreceptor CXCR4 (X4 strains) become laboratory adapted (LA) when selected for ability to replicate in leukemic T cell lines such as H9. Compared with patient X4 viruses, the gp120-gp41 complexes of LA viruses have a constellation of common properties including enhanced affinities for CD4, greater sensitivities to inactivations by diverse antibodies and by soluble CD4, increased shedding of gp120, and improved abilities to infect HeLa-CD4 cell clones that contain only trace quantities of CD4. These common characteristics, which may result from a concerted structural rearrangement of the gp120-gp41 complexes, have made it difficult to identify a specific feature that is critical for laboratory adaptation. To test the hypothesis that replication of patient X4 HIV-1 is limited by the low CD4 concentration in H9 cells (7.0 x 10(3) CD4/cell), we constructed H9 derivatives that express at least 10 times more of this receptor. Interestingly, most patient X4 isolates readily grew in these derivative cells, and the resulting virus preparations retained the characteristics of primary viruses throughout multiple passages. In contrast, selection of the same viruses in the parental H9 cells resulted in outgrowth of LA derivatives. We conclude that a weak interaction of patient X4 HIV-1 isolates with CD4 is the primary factor that limits their replication in leukemic T cell lines.
Asunto(s)
Antígenos CD4/fisiología , VIH-1/fisiología , VIH-1/patogenicidad , Adaptación Fisiológica , Línea Celular , Variación Genética , Proteína gp120 de Envoltorio del VIH/fisiología , Proteína gp41 de Envoltorio del VIH/fisiología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Células HeLa , Humanos , Linfocitos T/inmunología , Linfocitos T/virología , Replicación ViralRESUMEN
BACKGROUND: Diabetic patients taking insulin often have suboptimal glucose control, and standard methods of health care delivery are ineffective in improving such control. This study was undertaken to determine if insulin adjustment according to advice provided by telephone by a diabetes nurse educator could lead to better glucose control, as indicated by level of glycated hemoglobin (HbA1c). METHODS: The authors conducted a prospective randomized trial involving 46 insulin-requiring diabetic patients who had poor glucose control (HbA1c of 0.085 or more). Eligible patients were those already taking insulin and receiving endocrinologist-directed care through a diabetes centre and whose most recent HbA1c level was 0.085 or higher. The patients were randomly assigned to receive standard care or to have regular telephone contact with a diabetes nurse educator for advice about adjustment of insulin therapy. RESULTS: At baseline there was no statistically significant difference between the 2 groups in terms of HbA1c level (mean [and standard deviation] for standard-care group 0.094 [0.008] and for intervention group 0.096 [0.010]), age, sex, type or duration of diabetes, duration of insulin therapy or complications. After 6 months, the mean HbA1c level in the standard-care group was 0.089 (0.010), which was not significantly different from the mean level at baseline. However, the mean HbA1c level in the intervention group had fallen to 0.078 (0.008), which was significantly lower than both the level at baseline for that group (p < 0.001) and the level for the standard-care group at 6 months (p < 0.01). INTERPRETATION: Insulin adjustment according to advice from a diabetes nurse educator is an effective method of improving glucose control in insulin-requiring diabetic patients.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/enfermería , Insulina/administración & dosificación , Relaciones Enfermero-Paciente , Educación del Paciente como Asunto , Glucemia/metabolismo , Colombia Británica , Distribución de Chi-Cuadrado , Diabetes Mellitus Tipo 1/sangre , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Personal de Enfermería en Hospital , Cooperación del Paciente , Estudios Prospectivos , Consulta Remota , TeléfonoRESUMEN
The differential susceptibilities of mouse strains to xenotropic and polytropic murine leukemia viruses (X-MLVs and P-MLVs, respectively) are poorly understood but may involve multiple mechanisms. Recent evidence has demonstrated that these viruses use a common cell surface receptor (the X-receptor) for infection of human cells. We describe the properties of X-receptor cDNAs with distinct sequences cloned from five laboratory and wild strains of mice and from hamsters and minks. Expression of these cDNAs in resistant cells conferred susceptibilities to the same viruses that naturally infect the animals from which the cDNAs were derived. Thus, a laboratory mouse (NIH Swiss) X-receptor conferred susceptibility to P-MLVs but not to X-MLVs, whereas those from humans, minks, and several wild mice (Mus dunni, SC-1 cells, and Mus spretus) mediated infections by both X-MLVs and P-MLVs. In contrast, X-receptors from the resistant mouse strain Mus castaneus and from hamsters were inactive as viral receptors. These results suggest that X-receptor polymorphisms are a primary cause of resistances of mice to members of the X-MLV/P-MLV family of retroviruses and are responsible for the xenotropism of X-MLVs in laboratory mice. By site-directed mutagenesis, we substituted sequences between the X-receptors of M. dunni and NIH Swiss mice. The NIH Swiss protein contains two key differences (K500E in presumptive extracellular loop 3 [ECL 3] and a T582 deletion in ECL 4) that are both required to block X-MLV infections. Accordingly, a single inverse mutation in the NIH Swiss protein conferred X-MLV susceptibility. Furthermore, expression of an X-MLV envelope glycoprotein in Chinese hamster ovary cells interfered efficiently with X-MLV and P-MLV infections mediated by X-receptors that contained K500 and/or T582 but had no effect on P-MLV infections mediated by X-receptors that lacked these amino acids. In contrast, moderate expression of a P-MLV (MCF247) envelope glycoprotein did not cause substantial interference, suggesting that X-MLV and P-MLV glycoproteins interfere nonreciprocally with X-receptor-mediated infections. We conclude that P-MLVs have become adapted to utilize X-receptors that lack K500 and T582. A penalty for this adaptation is a reduced ability to interfere with superinfection. Because failure of interference is a hallmark of several exceptionally pathogenic retroviruses, we propose that it contributes to P-MLV-induced diseases.
Asunto(s)
Virus de la Leucemia Murina/metabolismo , Leucemia Experimental/virología , Polimorfismo Genético , Receptores Virales/genética , Infecciones por Retroviridae/virología , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Cricetinae , ADN Complementario/genética , Susceptibilidad a Enfermedades , Humanos , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/inmunología , Leucemia Experimental/inmunología , Leucemia Experimental/metabolismo , Ratones , Datos de Secuencia Molecular , Muridae , Mutagénesis Sitio-Dirigida , Receptores Acoplados a Proteínas G , Receptores Virales/química , Receptores Virales/metabolismo , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/metabolismo , Transfección , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virología , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Infections by human immunodeficiency virus type 1 (HIV-1) involve interactions of the viral envelope glycoprotein gp120 with CD4 and then with a coreceptor. R5 isolates of HIV-1 use CCR5 as a coreceptor, whereas X4 isolates use CXCR4. It is not known whether coreceptors merely trigger fusion of the viral and cellular membranes or whether they also influence the energetics of virus adsorption, the placement of the membrane fusion reaction, and the metabolism of adsorbed gp120. Surprisingly, the pathway for metabolism of adsorbed gp120 has not been investigated thoroughly in any cells. To address these issues, we used purified (125)I-gp120s derived from the R5 isolate BaL and from the X4 isolate IIIB as ligands for binding onto human cells that expressed CD4 alone or CD4 with a coreceptor. The gp120 preparations were active in forming ternary complexes with CD4 and the appropriate coreceptor. Moreover, the cellular quantities of CD4 and coreceptors were sufficient for efficient infections by the corresponding HIV-1 isolates. In these conditions, the kinetics and affinities of (125)I-gp120 adsorptions and their subsequent metabolisms were strongly dependent on CD4 but were not significantly influenced by CCR5 or CXCR4. After binding to CD4, the (125)I-gp120s slowly became resistant to extraction from the cell monolayers by pH 3.0 buffer, suggesting that they were endocytosed with half-times of 1-2 h. Within 20-30 min of endocytosis, the (125)I-gp120s were proteolytically degraded to small products that were shed into the media. The weak base chloroquine strongly inhibited (125)I-gp120 proteolysis and caused its intracellular accumulation, suggesting involvement of a low pH organelle. Results supporting these methods and conclusions were obtained by confocal immunofluorescence microscopy. We conclude that the energetics, kinetics, and pathways of (125)I-gp120 binding, endocytosis, and proteolysis are determined principally by CD4 rather than by coreceptors in cells that contain sufficient coreceptors for efficient infections. Therefore, the role of coreceptors in HIV-1 infections probably does not include steerage or subcellular localization of adsorbed virus.
Asunto(s)
Antígenos CD4/fisiología , Proteína gp120 de Envoltorio del VIH/metabolismo , Receptores del VIH/fisiología , Antígenos CD4/metabolismo , Endocitosis , VIH-1/fisiología , Células HeLa , Humanos , Hidrólisis , Inmunohistoquímica , Cinética , Fusión de Membrana , Microscopía Confocal , Unión Proteica , Receptores CCR5/metabolismo , Receptores CCR5/fisiología , Receptores CXCR4/metabolismo , Receptores CXCR4/fisiología , Receptores del VIH/metabolismoRESUMEN
Signal transductions by the dual-function CXCR4 and CCR5 chemokine receptors/HIV type 1 (HIV-1) coreceptors were electrophysiologically monitored in Xenopus laevis oocytes that also coexpressed the viral receptor CD4 and a G protein-coupled inward-rectifying K+ channel (Kir 3.1). Large Kir 3.1-dependent currents generated in response to the corresponding chemokines (SDF-1alpha for CXCR4 and MIP-1alpha; MIP-1beta and RANTES for CCR5) were blocked by pertussis toxin, suggesting involvement of inhibitory guanine nucleotide-binding proteins. Prolonged exposures to chemokines caused substantial but incomplete desensitization of responses with time constants of 5-7 min and recovery time constants of 12-19 min. CXCR4 and CCR5 exhibited heterologous desensitization in this oocyte system, suggesting possible inhibition of a common downstream step in their signaling pathways. In contrast to chemokines, perfusion with monomeric or oligomeric preparations of the glycoprotein of Mr 120, 000 (gp120) derived from several isolates of HIV-1 did not activate signaling by CXCR4 or CCR5 regardless of CD4 coexpression. However, adsorption of the gp120 from a T-cell-tropic virus resulted in CD4-dependent antagonism of CXCR4 response to SDF-1alpha, whereas gp120 from macrophage-tropic viruses caused CD4-dependent antagonism of CCR5 response to MIP-1alpha. These antagonisms could be partially overcome by high concentrations of chemokines and were specific for coreceptors of the corresponding HIV-1 isolates, suggesting that they resulted from direct interactions of gp120-CD4 complexes with coreceptors and that they did not involve the desensitization pathway. These results indicate that monomeric or oligomeric gp120s specifically antagonize CXCR4 and CCR5 signaling in response to chemokines, but they do not exclude the possibility that gp120s might also function as weak agonists in some cells. The gp120-mediated disruption of CXCR4 and CCR5 signaling may contribute to AIDS pathogenesis.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/farmacología , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Transducción de Señal/efectos de los fármacos , Animales , Antígenos CD4/fisiología , Quimiocinas/farmacología , Electrofisiología , Femenino , Humanos , Transducción de Señal/fisiología , Xenopus laevisRESUMEN
The principal side effect of the antimycobacterial agent ethambutol (EMB) is an optic neuropathy with clinical features very similar to a mitochondrial hereditary optic neuropathy (Leber's). The mechanism of EMB-induced optic neuropathy may be EMB's chelation of copper, thereby precluding normal cytochrome c oxidase activity and mitochondrial metabolism in the optic nerve. Before attempting to use therapeutic copper to replenish endogenous stores in an attempt to preclude EMB-induced optic neuropathy, we wished to determine whether EMB is still effective against mycobacteria in the presence of copper. EMB and copper, alone and in combination, were tested against six strains of Mycobacterium tuberculosis and five strains of Mycobacterium avium using a radiometric broth macrodilution assay. Copper did not effect EMB's antimicrobial actions against either species of mycobacteria. This in vitro study suggests that if copper were given to patients to prevent EMB-induced optic neuropathy, it would not compromise EMB's bacteriostatic properties.
Asunto(s)
Antituberculosos/farmacología , Cobre/farmacología , Etambutol/farmacología , Neuropatía Óptica Isquémica/prevención & control , Antituberculosos/administración & dosificación , Antituberculosos/efectos adversos , Contraindicaciones , Cobre/administración & dosificación , Interacciones Farmacológicas , Etambutol/administración & dosificación , Etambutol/efectos adversos , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium avium/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Neuropatía Óptica Isquémica/inducido químicamenteRESUMEN
CCR5, a receptor for the CC chemokines RANTES, Mip1alpha, and Mip1beta, has been identified as a coreceptor for infections by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). To study its structure and function, we isolated cDNA clones of human, African green monkey (AGM), and NIH/Swiss mouse CCR5s, and we quantitatively analyzed infections by macrophage-tropic HIV-1 and SIVmac251 after transfecting human HeLa-CD4 cells with the CCR5 expression vectors. The AGM and NIH/Swiss mouse CCR5 proteins are 97.7 to 98.3% and 79.8% identical to the human protein, respectively. In addition, we analyzed site-directed mutants and chimeras of these CCR5s. Cell surface expression of CCR5 proteins was monitored by using a specific rabbit antiserum and by binding the chemokine [125I]Mip1beta. Our major results were as follows. (i) Two distinct AGM CCR5 sequences were reproducibly found in DNA from CV-1 cells. The AGM clone 1 CCR5 protein differs from that of clone 2 by two substitutions, Y14N in the amino-terminal extracellular region and L352F at the carboxyl terminus. Interestingly, AGM clone 1 CCR5 was inactive as a coreceptor for all tested macrophage-tropic isolates of HIV-1, whereas AGM clone 2 CCR5 was active. As shown by chimera studies and site-directed mutagenesis, the Y14N substitution in AGM clone 1 CCR5 was solely responsible for blocking HIV-1 infections. In contrast, both AGM CCR5 clones were active coreceptors for SIVmac251. Studies of DNA samples from other AGMs indicated frequent additional CCR5 polymorphisms, and we cloned an AGM clone 2 variant with a Q93R substitution in the extracellular loop 1 from one heterozygote. This variant CCR5 was active as a coreceptor for SIVmac251 but was only weakly active for macrophage-tropic isolates of HIV-1. In addition, SIVmac251 appeared to be dependent on the extracellular amino terminus and loop 2 regions of human CCR5 for maximal infection. Our results suggest major differences in the interactions of SIVmac251 and macrophage-tropic HIV-1 isolates with 19, N13, and Y14 in the amino terminus; with Q93 in extracellular loop 1; and with extracellular loop 2 of human CCR5. (ii) The NIH/Swiss mouse CCR5 protein differs at multiple positions from sequences recently reported for other inbred strains of mice. This CCR5 was inactive as a coreceptor for HIV-1 and SIVmac251. Studies of chimeras that contained different portions of NIH/Swiss mouse CCR5 substituted into human CCR5, as well as the reciprocal chimeras, indicated that the amino-terminal region and extracellular loops 1 and 2 of human CCR5 contribute to its coreceptor activity for macrophage-tropic isolates of HIV-1. Specific differences with previous CCR5 chimera results occurred because the NIH/Swiss mouse CCR5 contains a unique substitution corresponding to P183L in extracellular loop 2 that is nonpermissive for coreceptor activity. We conclude that diverse CCR5 sequences occur in AGMs and mice, that SIVmac251 and macrophage-tropic HIV-1 isolates interact differently with specific CCR5 amino acids, and that multiple regions of human CCR5 contribute to its coreceptor functions. In addition, we have identified naturally occurring amino acid polymorphisms in three extracellular regions of CCR5 (Y14N, Q93R, and P183L) that do not interfere with cell surface expression or Mip1beta binding but prevent infections by macrophage-tropic isolates of HIV-1. In contrast to previous evidence, these results suggest that CCR5 contains critical sites that are essential for HIV-1 infections.
Asunto(s)
Chlorocebus aethiops/genética , VIH-1/crecimiento & desarrollo , Receptores CCR5/genética , Virus de la Inmunodeficiencia de los Simios/crecimiento & desarrollo , Células 3T3 , Secuencia de Aminoácidos , Animales , Genes , Células HeLa , Humanos , Técnicas Inmunológicas , Macrófagos/virología , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Polimorfismo Genético , Conejos , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
We have used a focal infectivity method to quantitatively analyze the CD4, CXCR-4, and CCR-5 dependencies for infections by diverse primary patient (PR) and laboratory-adapted (LA) isolates of human immunodeficiency virus type 1 (HIV-1). Infectivities of T-cell-tropic viruses were analyzed in a panel of HeLa-CD4 cell clones that have distinct quantities of CD4 and in human astroglioma U87MG-CD4 cells that express a large quantity of CD4 and become highly susceptible to infection after transfection with a CXCR-4 expression vector. The latter analysis indicated that PR as well as LA T-cell-tropic viruses efficiently employ CXCR-4 as a coreceptor in an optimal human cell line that contains abundant CD4. Previous uncertainties regarding coreceptor usage by PR T-cell-tropic HIV-1 isolates may therefore have derived from the assay conditions. As reported previously, unrelated LA and PR T-cell-tropic HIV-1 isolates differ in infectivities for the HeLa-CD4 clonal panel, with LA viruses infecting all clones equally and PR viruses infecting the clones in proportion to cellular CD4 quantities (D. Kabat, S. L. Kozak, K. Wherly, and B. Chesebro, J. Virol. 68:2570-2577, 1994). To analyze the basis for this difference, we used the HeLa-CD4 panel to compare a molecularly cloned T-cell-tropic PR virus (ELI1) with six of its variants that grow to different extents in CD4-positive leukemic cell lines and that differ only at specific positions in their gp120 and gp41 envelope glycoproteins. All mutations in gp120 or gp41 that contributed to laboratory adaptation preferentially enhanced infectivity for cells that had little CD4 and thereby decreased the CD4 dependencies of the infections. There was a close correlation between abilities of T-cell-tropic ELI viruses to grow in an expanded repertoire of leukemic cell lines, the reduced CD4 dependencies of their infections of the HeLa-CD4 panel, and their sensitivities to inactivation by soluble CD4 (sCD4). Since all of the ELI viruses can efficiently use CXCR-4 as a coreceptor, we conclude that an increase in viral affinity for CD4 rather than a switch in coreceptor specificity is principally responsible for laboratory adaption of T-cell-tropic HIV-1. Syncytium-inducing activities of the ELI viruses, especially when analyzed on cells with low amounts of CD4, were also highly correlated with their laboratory-adapted properties. Results with macrophage-tropic HIV-1 were strikingly different in both coreceptor and CD4 dependencies. When assayed in HeLa-CD4 cells transfected with an expression vector for CCR-5, macrophage-tropic HIV-1 isolates that had been molecularly cloned shortly after removal from patients were equally infectious for cells that had low or high CD4 quantities. Moreover, despite their substantial infectivities for cells that had only a trace of CD4, macrophage-tropic isolates were relatively resistant to inactivation by sCD4. We conclude that T-cell-tropic PR viruses bind weakly to CD4 and preferentially infect cells that coexpress CXCR-4 and large amounts of CD4. Their laboratory adaptation involves corresponding increases in affinities for CD4 and in abilities to infect cells that have relatively little CD4. In contrast, macrophage-tropic HIV-1 appears to interact weakly with CD4 although it can infect cells that coexpress CCR-5 and small quantities of CD4. We propose that cooperative binding of macrophage-tropic HIV-1 onto CCR-5 and CD4 may enhance virus adsorption and infectivity for cells that have only a trace of CD4.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , Antígenos CD4/inmunología , VIH-1 , Células HeLa/virología , Proteínas de la Membrana/inmunología , Receptores de Citocinas/inmunología , Receptores del VIH/inmunología , Subgrupos de Linfocitos T/virología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Proteína gp120 de Envoltorio del VIH/genética , Células HeLa/inmunología , Humanos , Mutación , Receptores CCR5 , Receptores CXCR4RESUMEN
Recent evidence suggests that primary patient isolates of T-cell-tropic human immunodeficiency virus type 1 (HIV-1 ) have lower affinities for CD4 than their laboratory-adapted derivatives, that this may partly result from tighter gp120-gp41 bonds that constrain the CD4 binding sites of the primary viruses, and that selection for increased CD4 affinity may be the principal factor in laboratory adaptation of HIV-1 (S. L. Kozak, E. J. Platt, N. Madani, F. E. Ferro, Jr., K. Peden, and D. Kabat, J. Virol. 71:873-882, 1997). These conclusions were based on studies with a panel of HeLa-CD4 cell clones that differ in CD4 levels over a broad range, with laboratory-adapted viruses infecting all clones with equal efficiencies and primary T-cell-tropic viruses infecting the clones in proportion to cellular CD4 levels. Additionally, all of the primary and laboratory-adapted T-cell-tropic viruses efficiently used CXCR-4 (fusin) as a coreceptor. To test these conclusions by an independent approach, we studied mutations in the laboratory-adapted virus LAV/IIIB that alter the CD)4 binding region of gp120 and specifically reduce CD4 affinities of free gp 120 by 85 to 98% (U. Olshevsky et al., J. Virol. 64:5701-5707, 1990). These mutations reduced virus titers to widely varying extents that ranged from severalfold to several orders of magnitude and converted infectivities on the HeLa-CD4 panel from CD4 independency to a high degree of CD4 dependency that resembled the behavior of primary patient viruses. The relative infectivities of the mutants correlated closely with their sensitivities to inactivation by soluble CD4 but did not correlate with the relative CD4 affinities of their free gp120s. Most of the mutations did not substantially alter envelope glycoprotein synthesis, processing, expression on cell surfaces, incorporation into virions, or rates of gp120 shedding from virions. However, one mutation (D457R) caused a decrease in gp160 processing by approximately 80%. The fact that several mutations increased rates of spontaneous viral inactivation (especially D368P) suggests that HIV-1 life spans may be determined by structural stabilities of viral envelope glycoproteins. All of the wild-type and mutant viruses were only slowly and inefficiently adsorbed onto cultured CD4-positive cells at 37 degrees C, and the gradual declines in viral titers in the media were caused almost exclusively by spontaneous inactivation rather than by adsorption. The extreme inefficiency with which infectious HIV-1 is able to infect cultured susceptible CD4-positive cells in standard assay conditions casts doubt on previous inferences that the vast majority of retrovirions produced in cultures are noninfectious. Apparent infectivity of T-cell-tropic HIV-1 in culture is limited by productive associations with CD4 and is influenced in an interdependent manner by CD4 affinities of viral gp120-gp41 complexes and quantities of cell surface CD4.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , Antígenos CD4/inmunología , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Sitios de Unión/genética , Sitios de Unión/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , VIH-1/patogenicidad , Células HeLa , Humanos , Mutación , Virulencia/genéticaRESUMEN
The cell surface receptor for ecotropic host-range (infection limited to mice or rats) murine leukemia viruses (MuLVs) is the widely expressed system y+ transporter for cationic amino acids (CAT-1). Like other retroviruses, ecotropic MuLV infection eliminates virus-binding sites from cell surfaces and results in complete interference to superinfection. Surprisingly, infection causes only partial (ca 40 to 60%) loss of mouse CAT-1 transporter activity. The NIH/Swiss mouse CAT-1 (mCAT-1) contains 622 amino acids with 14 hydrophobic potential membrane-spanning sequences, and it is known that the third extracellular loop from the amino terminus is required for virus binding. Although loop 3 is hypervariable in different species and mouse strains, consistent with its proposed role in virus-host coevolution, loop 3 sequences of both susceptible and resistant species contain consensus sites for N-linked glycosylation. Both of the consensus sites in loop 3 of mCAT-1 are known to be glycosylated and to contain oligosaccharides with diverse sizes (J. W. Kim and J. M. Cunningham, J. Biol. Chem. 268:16316-16320, 1993). We confirmed by several lines of evidence that N-linked glycosylation occludes a potentially functional virus-binding site in the CAT-1 protein of hamsters, thus contributing to resistance of that species. To study the role of receptor glycosylation in animals susceptible to infection, we eliminated loop 3 glycosylation sites by mutagenesis of an mCAT-1 cDNA clone, and we expressed wild-type and mutant receptors in mink fibroblasts and Xenopus oocytes. These receptors had indistinguishable transport properties, as determined by kinetic and voltage-jump electrophysiological studies of arginine uptake in oocytes and by analyses Of L-[3H]arginine uptake in mink cells. Bindings of ecotropic envelope glycoprotein gp7O to the accessible receptor sites on surfaces of mink cells expressing wild-type or mutant mCAT-1 were not significantly different in kinetics or in equilibrium affinities (i.e., K(D) approximately 3.7 X 10(-10) to 7.5 X 10(-10) M). However, when values were normalized to the same levels of mCAT-1 transporter expression, cells with wild-type glycosylated mCAT-1 had only approximately 50% as many sites for gp70 binding as cells with unglycosylated mCAT-1. Although infection with ecotropic MuLV had no effect on activity of the mink CAT-1 transporter that does not bind virus, it caused partial down-modulation of wild-type mCAT-1 and complete down-modulation of unglycosylated mutant mCAT-1. These results suggest that N-linked glycosylation causes wild-type mCAT-1 heterogeneity and that a significant proportion is inaccessible to virus. In part because only the interactive fraction of mCAT-1 can be down-modulated, infected murine cells conserve an amino acid transport capability that supports their viability.
Asunto(s)
Proteínas Portadoras/metabolismo , Virus de la Leucemia Murina/metabolismo , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animales , Proteínas Portadoras/genética , Línea Celular , Cricetinae , Regulación hacia Abajo , Glicosilación , Humanos , Virus de la Leucemia Murina/patogenicidad , Ratones , Datos de Secuencia Molecular , Mutación , Ratas , Análisis de Secuencia , Virulencia , XenopusRESUMEN
Chinese hamster ovary (CHO) cells are naturally resistant to infection by amphotropic and ecotropic murine retroviruses, but they become susceptible after expressing corresponding receptors rRAM-1 and mCAT-1, respectively, and they then form abundant syncytia when exposed to these viruses. The fusogenic activities of CHO cell clones increase much more strongly with levels of receptor expression than do their susceptibilities to infection, suggesting that the assembly of receptor clusters may limit syncytium formation. However, other cell lines are not fusogenic, even if they express larger amounts of receptors. Our results suggest that a factor that is relatively abundant or active in CHO cells may functionally interact with rRAM-1 and mCAT-1 in a pathway that enables receptor-bearing membranes to fuse with membranes that contain viral envelope glycoproteins. In the case of CHO/rRAM-1 cells, syncytia form at foci of amphotropic 4070A virus infection by fusion-from-within of infected with uninfected cells. This fusogenic propensity is a sole property of the uninfected CHO/rRAM-1 cells, which fuse in cocultures with any cells infected with 4070A virus. With CHO/mCAT-1 cells, fusogenicity is even greater and involves fusion-from-without by ecotropic virion particles. In contrast to infection, which behaves as expected for a process limited by ecotropic virus attachment to single receptors, fusion-from-without increases dramatically for cells that express the highest levels of mCAT-1. We propose that infection and syncytium formation are limited at distinct steps of a common pathway that requires virus binding to a single receptor, assembly of multivalent virus-receptor complexes, structural changes in viral envelope glycoproteins, and membrane fusion. The limiting step in syncytium formation is a cellular process that depends on receptor clustering and is relatively active in CHO cells.
Asunto(s)
Virus de la Leucemia Murina/fisiología , Fusión de Membrana , Proteínas de Transporte de Fosfato , Receptores Virales/fisiología , Simportadores , Animales , Células CHO , Cricetinae , Proteínas Cotransportadoras de Sodio-FosfatoRESUMEN
A rat cDNA (rRam-1), which was cloned on the basis that it enables Chinese hamster ovary (CHO) cells to be infected by amphotropic host range murine retroviruses, was recently found to encode a widely expressed Na(+)-phosphate symporter (M. P. Kavanaugh, D. G. Miller, W. Zhang, W. Law, S. L. Kozak, D. Kabat, and A. D. Miller, Proc. Natl. Acad. Sci. USA 91:7071-7075, 1994). CHO cells express the hamster homolog of Ram-1 but are resistant to amphotropic retroviruses. Although the amphotropic envelope glycoprotein gp70 bound weakly onto control CHO cells, CHO/rRam-1 cells had novel high-affinity binding sites, and the resulting strongly adsorbed gp70 was only slowly removed from cell surfaces, with a half-life of greater than 6 h. CHO/rRam-1 cells were also specifically and efficiently killed by exposure to amphotropic gp70 followed by antiserum to gp70 in the presence of complement. Infection with an appropriately pseudotyped form of amphotropic retrovirus 4070A did not perturb control CHO cells or inhibit their phosphate transport. In contrast, 4070A infection of CHO/rRam-1 cells caused major alterations including cell-cell fusions, a specific 40% down-modulation of the rRam-1 component of phosphate transport, and complete interference to super-infection by amphotropic viruses. The 4070A virus-infected CHO/rRam-1 cells retained a substantial cell surface pool of rRam-1 that functioned as a phosphate transporter but not as a viral receptor. We conclude that amphotropic gp70 binds more strongly to rRam-1 than to the homologous hamster protein and that this stable attachment is necessary for infection, interference, membrane fusion, and pathogenesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Productos del Gen env/metabolismo , Virus de la Leucemia Murina/metabolismo , Fosfatos/metabolismo , Receptores Virales/metabolismo , Células 3T3 , Animales , Células CHO , Proteínas Portadoras/genética , Clonación Molecular , Cricetinae , Efecto Citopatogénico Viral , Regulación hacia Abajo , Productos del Gen env/biosíntesis , Virus de la Leucemia Murina/patogenicidad , Ratones , Ratones Endogámicos BALB C , Proteínas de Unión a Fosfato , RatasRESUMEN
Ping-pong amplification is an efficient process by which helper-free retrovirions replicate in cocultures of cell lines that package retroviruses into distinct host-range envelopes [11]. Transfection of a retroviral vector DNA into these cocultures results in massive virus production, with potentially endless cross-infection between different types of packaging cells. Because the helper-free virus spreads efficiently throughout the coculture, it is unnecessary to use dominant selectable marker genes, and the retroviral vectors can be simplified and optimized for expressing a single gene of interest. The most efficient ping-pong vector, pSFF, derived from the Friend erythroleukemia virus, has been used for high-level expression of several genes that could not be expressed with commonly employed two-gene retroviral vectors. Contrary to previous claims, problems of vector recombination are not inherent to ping-pong methods. Indeed, the pSFF vector has not formed replication-competent recombinants as shown by stringent assays. Here we review these methods, characterize the ping-pong process using the human erythropoietin gene as a model, and describe a new vector (pSFY) designed for enhanced expression in T lymphocytes. Factors that limit tissue-specific expression are reviewed.
Asunto(s)
Expresión Génica , Vectores Genéticos/genética , Retroviridae/genética , Transfección , Animales , Eritropoyetina/genética , Humanos , Ratones , Virus de la Leucemia Murina de Moloney/genética , Provirus/genética , Provirus/fisiología , Secuencias Repetitivas de Ácidos Nucleicos/genética , Retroviridae/fisiología , Virus Formadores de Foco en el Bazo/genética , Virus Formadores de Foco en el Bazo/fisiología , Células Tumorales CultivadasRESUMEN
Although the Friend virus-encoded membrane glycoprotein (gp55) activates erythropoietin receptors (EpoR) to cause erythroblastosis only in certain inbred strains of mice but not in other species, mutant viruses can overcome aspects of mouse resistance. Thus, mice homozygous for the resistance allele of the Fv-2 gene are unaffected by gp55 but are susceptible to mutant glycoproteins that have partial deletions in their ecotropic domains. These and other results have suggested that proteins coded for by polymorphic Fv-2 alleles might directly or indirectly interact with EpoR and that changes in gp55 can overcome this defense. A new viral mutant with an exceptionally large deletion in its ecotropic domain is now also shown to overcome Fv-2rr resistance. In all cases, the glycoproteins that activate EpoR are processed to cell surfaces as disulfide-bonded dimers. To initiate analysis of nonmurine resistances, we expressed human EpoR and mouse EpoR in the interleukin 3-dependent mouse cell line BaF3 and compared the abilities of Friend virus-encoded glycoproteins to convert these cells to growth factor independence. Human EpoR was activated in these cells by erythropoietin but was resistant to gp55. However, human EpoR was efficiently activated in these cells by the same viral mutants that overcome Fv-2rr resistance in mice. By construction and analysis of human-mouse EpoR chimeras, we obtained evidence that the cytosolic domain of human EpoR contributes to its resistance to gp55 and that this resistance is mediated by accessory cellular factors. Aspects of host resistance in both murine and nonmurine species are targeted specifically against the ecotropic domain of gp55.
Asunto(s)
Leucemia Eritroblástica Aguda/etiología , Receptores de Eritropoyetina/fisiología , Virus Formadores de Foco en el Bazo/genética , Proteínas del Envoltorio Viral/fisiología , Animales , Productos del Gen env/metabolismo , Ratones , Ratones Endogámicos DBA , Mutación , Virus Formadores de Foco en el Bazo/patogenicidad , Proteínas del Envoltorio Viral/genéticaRESUMEN
Cell surface receptors for gibbon ape leukemia virus (Glvr-1) and murine amphotropic retrovirus (Ram-1) are distinct but related proteins having multiple membrane-spanning regions. Distant homology with a putative phosphate permease of Neurospora crassa suggested that these receptors might serve transport functions. By expression in Xenopus laevis oocytes and in mammalian cells, we have identified Glvr-1 and Ram-1 as sodium-dependent phosphate symporters. Two-electrode voltage-clamp analysis indicates net cation influx, suggesting that phosphate is transported with excess sodium ions. Phosphate uptake was reduced by > 50% in mouse fibroblasts expressing amphotropic envelope glycoprotein, which binds to Ram-1, indicating that Ram-1 is a major phosphate transporter in these cells. RNA analysis shows wide but distinct tissue distributions, with Glvr-1 expression being highest in bone marrow and Ram-1 in heart. Overexpression of Ram-1 severely repressed Glvr-1 synthesis in fibroblasts, suggesting that transporter expression may be controlled by net phosphate accumulation. Accordingly, depletion of extracellular phosphate increased Ram-1 and Glvr-1 expression 3- to 5-fold. These results suggest simple methods to modulate retroviral receptor expression, with possible applications to human gene therapy.
Asunto(s)
Proteínas Portadoras/metabolismo , Virus de la Leucemia del Gibón/metabolismo , Virus de la Leucemia Murina/metabolismo , Proteínas de Transporte de Fosfato , Fosfatos/metabolismo , Receptores Virales/metabolismo , Simportadores , Células 3T3 , Animales , Transporte Biológico , Línea Celular , Expresión Génica , Humanos , Ratones , Oocitos , Proteínas de Unión a Fosfato , Ratas , Receptores Virales/genética , Proteínas Recombinantes , Sodio/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III , Xenopus laevisRESUMEN
In this prospective, double-blind, randomized study of women undergoing elective cesarean birth, the hypothesis that epidural butorphanol in various doses could effectively reduce or eliminate the side effects caused by epidural morphine was tested. Patients were randomly assigned to one of four groups. All received a standard epidural anesthetic and 20 min after delivery each received 3 mg epidural morphine with either 1 mg butorphanol (Group A), 2 mg butorphanol (Group B), 3 mg butorphanol (Group C), or 3 mL normal saline (Group D). Patient evaluations were made preoperatively and 2, 8, and 24 h after delivery. These consisted of visual analog scores for pain, satisfaction, nausea, itch, and somnolence. At each evaluation, a CO2 challenge test, using portable equipment, was performed. Data from 71 patients were analyzed and all four groups were comparable in terms of age, height, weight, level of sensory block, and volume of local anesthetic used. There were no significant differences among groups in terms of pain, satisfaction, nausea, or pruritus. Groups A, B, and C had significantly higher somnolence scores at 8 h compared to Group D (P < 0.001). There were no significant differences among groups in CO2 challenge test data at any point during the study, but overall a reduced sensitivity to CO2 after opioid administration was observed across all groups. There were no clinically significant incidents of respiratory depression. Epidural butorphanol, in doses of 1-3 mg, failed to reduce the side effects from 3 mg epidural morphine given after cesarean birth. Patients who received epidural butorphanol reported significantly higher levels of somnolence.