RESUMEN
Diabetes-related foot disease remains a common problem. For wounds, classic teaching recommends the treatment of any infection, offloading the wound and ensuring a good blood supply, as well as ensuring that the other modifiable risk factors are addressed and optimized. There remain, however, several questions about these and other aspects of the care of diabetes-related foot disease. Some of these questions are addressed in the present report; in particular, the impact of newer technologies in the identification of any organisms present in a wound, as well as the use of novel approaches to treat infections. The use of new remote sensing technology to identify people at risk of developing foot ulceration is also considered, in an attempt to allow early intervention and prevention of foot ulcers. The psychological impact of foot disease is often overlooked, but with an increasing number of publications on the subject, the cause-and-effect role that psychology plays in foot disease, such as ulcers and Charcot neuroarthropathy, is considered. Finally, because of heterogeneity in diabetic foot studies, comparing results is difficult. A recently published document focusing on ensuring a standardized way of reporting foot disease trials is discussed.
Asunto(s)
Pie Diabético/prevención & control , Pie Diabético/terapia , Medicina Basada en la Evidencia , Salud Global , Infección de Heridas/terapia , Investigación Biomédica/métodos , Investigación Biomédica/tendencias , Terapia Combinada , Congresos como Asunto , Pie Diabético/etiología , Pie Diabético/microbiología , Medicina Basada en la Evidencia/tendencias , Humanos , Reino Unido , Infección de Heridas/etiología , Infección de Heridas/microbiología , Infección de Heridas/prevención & controlRESUMEN
Currently available chromogenic and fluorogenic substrates for endogenous thrombin potential (ETP) measurement are cleaved by both free (active) and alpha-2-macroglobulin-bound (inactive) thrombin, leading to an overestimation of ETP. Commercial methods for ETP measurement determine this using a mathematical algorithm, which assumes the contribution of alpha-2-macroglobulin to the ETP. This limits application of such methods to populations where variation in alpha-2-macroglobulin concentrations is observed, primarily children. This study examined the contribution of alpha-2-macroglobulin-bound thrombin to the ETP measurement in neonates, children and adults, to determine whether automated methods are appropriate for use in neonates and children.
Asunto(s)
Pruebas de Coagulación Sanguínea , Desarrollo Infantil , Trombina/metabolismo , alfa-Macroglobulinas/metabolismo , Adulto , Algoritmos , Área Bajo la Curva , Coagulación Sanguínea/fisiología , Estudios de Casos y Controles , Niño , Humanos , Recién Nacido , Sensibilidad y Especificidad , Trombina/análisisRESUMEN
Thromboembolic disease (TE) has been described as the new epidemic of tertiary paediatrics, and no where is this more evident than in the neonatal population. As survival of premature and sick newborns has improved, the frequency of complications associated with intensive supportive therapy and monitoring has increased. Clinically significant thrombosis is emerging as one of the more common complications associated with improved neonatal outcome. The long-term implications of neonatal thrombosis are only just being realised. This systematic review will consider the epidemiology, diagnostic strategies, and outcome for both arterial and venous TE in neonates. The role of inherited thrombophilic abnormalities, and the evidence for anticoagulation therapy will also be considered. The lack of high level evidence in determining optimum therapy is obvious. Further research regarding diagnostic strategies, and optimal therapies is urgently needed.
Asunto(s)
Enfermedades del Recién Nacido , Tromboembolia , Trombosis de la Vena , Cateterismo Periférico , Catéteres de Permanencia , Niño , Factor V/genética , Femenino , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Humanos , Recién Nacido , Enfermedades del Recién Nacido/etiología , Enfermedades del Recién Nacido/terapia , Masculino , Deficiencia de Proteína C/genética , Deficiencia de Proteína S/genética , Tromboembolia/etiología , Tromboembolia/terapia , Terapia Trombolítica/métodos , Trombosis de la Vena/etiología , Trombosis de la Vena/terapiaAsunto(s)
Mutación Puntual , Protrombina/genética , Niño , Humanos , Proyectos de Investigación , Factores de Riesgo , Trombosis/etiologíaRESUMEN
OBJECTIVE: To establish the percentage prevalence of hypovitaminosis D in chronically ill or disabled children in Melbourne, Australia. METHODOLOGY: A group of inpatients at the Royal Children's Hospital, Melbourne, Victoria, as identified by the primary unit, were sampled to measure serum vitamin D and parameters of bone turnover. A second group of disabled children (outpatients) were also measured to establish vitamin D status. RESULTS: Of the total population, 54.9% were found to have low serum 25 hydroxy (25OH) vitamin D levels. Of the inpatient group, 25.4% were vitamin D deficient (<30 nM/L), and 27.1% were vitamin D insufficient (30-50 nM/L). The mean 25OH vitamin D was 52.1 nM/L. Of the outpatient group, 15.4% were vitamin D deficient, whilst 42.3% were found to be insufficient. The mean vitamin D level was 41.2 nM/L. No difference attributable to intellectual versus physical disability was found. Anticonvulsant use and ambulatory status was not predictive of vitamin D status in the children examined. Of the total population, 0.05% were found to have secondary hyperparathyroidism. The mean 25OH vitamin D level of this subgroup was 30.6 nM/L. Dark skin tone was found to be significantly associated with hypovitaminosis D (P = 0.001), where all five children with dark skin tone were found to have serum 25OH vitamin D levels <50 nM/L. Of the seven disabled children (outpatients) found to be iron deficient, four had coexistent hypovitaminosis D. CONCLUSION: The percentage prevalence of hypovitaminosis D is high in both chronically ill, and physically/intellectually disabled children in Melbourne, Australia. Increased vigilance and recognition of this deficiency state is needed as an important health prevention strategy.
Asunto(s)
Niños con Discapacidad , Deficiencia de Vitamina D/epidemiología , Niño , Enfermedad Crónica , Femenino , Humanos , Pacientes Internos , Masculino , Prevalencia , Victoria/epidemiología , Deficiencia de Vitamina D/sangreRESUMEN
Simian and human immunodeficiency virus type 1 (SIV and HIV-1) Nef proteins are thought to use different molecular surfaces to mediate the protein-protein interactions required for their otherwise similar functions. This genetically separable function suggests convergent evolution of primate lentiviruses and/or structural differences between human and nonhuman primate cellular target proteins. However, such comparative molecular analyses have not been undertaken so far using the respective natural host-derived cellular targets. We cloned simian Src family kinase Hck and analyzed structurally and biochemically its interaction with SIV Nef.
Asunto(s)
Productos del Gen nef/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Clonación Molecular , Evolución Molecular , Productos del Gen nef/genética , VIH-1/metabolismo , Humanos , Macaca fascicularis , Ratones , Datos de Secuencia Molecular , Filogenia , Proteínas Tirosina Quinasas/química , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-hck , Análisis de Secuencia de ADN , Virus de la Inmunodeficiencia de los Simios/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
OBJECTIVE: HIV-1 infection impairs a number of macrophage effector functions, but the mechanism is unknown. We studied the role of HIV-1 Nef in modulating phagocytosis by human monocytes and monocyte-derived macrophages (MDM). DESIGN AND METHODS: Using a flow cytometric assay, phagocytosis of Mycobacterium avium complex (MAC) by monocytes in whole blood of Sydney Blood Bank Cohort (SBBC) members infected with a nef-deleted (Delta nef) strain of HIV-1 was compared with that of monocytes from uninfected or wild-type (WT) HIV-infected subjects. The specific impact of Nef on phagocytosis by MDM was determined by either infecting cells in vitro with Delta nef strains of HIV-1 or electroporating Nef into uninfected MDM. RESULTS: MAC phagocytic capacity of monocytes from SBBC members was equivalent to that of cells from uninfected individuals (P = 0.81); it was greater than that of cells from individuals infected with WT HIV-1 (P < 0.0001), irrespective of CD4 counts and HIV viral load. In contrast, in vitro infection of MDM with either Delta nef or WT strains of HIV-1 resulted in similar levels of HIV replication and equivalent impairment of phagocytosis via Fc gamma and complement receptors. Electroporation of Nef into MDM did not alter phagocytic capacity. CONCLUSIONS: This study provides evidence demonstrating the complex indirect effect of Nef on phagocytosis by peripheral blood monocytes (infrequently infected with HIV-1) in vivo. Conversely, the fact that MDM infected with either Delta nef or WT HIV-1 in vitro (high multiplicity of infection) show comparably impaired phagocytosis, indicates that HIV-1 infection of macrophages can directly impair function, independent of Nef.
Asunto(s)
Genes nef , Infecciones por VIH/inmunología , VIH-1/genética , Macrófagos/inmunología , Monocitos/inmunología , Fagocitosis , Recuento de Linfocito CD4 , Estudios de Cohortes , Electroporación , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Eliminación de Gen , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/patogenicidad , Humanos , Immunoblotting , Técnicas In Vitro , Macrófagos/virología , Monocitos/virología , Carga ViralAsunto(s)
Productos del Gen nef/fisiología , VIH-1/patogenicidad , Secuencia de Aminoácidos , Animales , Antígenos CD4/análisis , Secuencia Conservada , Regulación hacia Abajo , Productos del Gen nef/química , Productos del Gen nef/genética , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Modelos Animales , Datos de Secuencia Molecular , Receptores de Interleucina-2/análisis , Transducción de Señal , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Interferons are important in regulating cell growth and differentiation, immune function and initiating anti-viral responses. While the pleotrophic actions of interferons have been well documented, the molecular mechanisms underpinning their biological effects have not been fully characterized. IFI 16 is a member of the interferon-inducible HIN-200 family of nuclear proteins, which we have recently shown can function as a potent transcriptional repressor. A murine member of the HIN-200 family, p202, can indirectly interact with p53 via the p53 binding protein (p53bp) and inhibit p53-mediated transcriptional activation. The binding activity of p202 to p53bp was shown to require the conserved MFHATVAT motif present in all 200 amino acid repeat regions of HIN-200 proteins. Given that IFI 16 contains two MFHATVAT motifs, we sought to determine whether IFI 16 may form a complex with p53 and if so to ascertain the functional significance of this interaction. We demonstrate that IFI 16 can directly bind to the C-terminal region of p53 and augment p53-mediated transcriptional activation without altering the steady state levels of p53. Thus, in addition to its ability to directly regulate gene expression, IFI 16 can also modulate the transcription function of other cellular transcription factors. These findings demonstrate a possible link between gene induction following interferon stimulation and p53-mediated cellular events.
Asunto(s)
Interferones/fisiología , Proteínas Nucleares/metabolismo , Fosfoproteínas , Proteínas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Línea Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , ADN/genética , ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Genes Reporteros/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Pruebas de Precipitina , Unión Proteica , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia/genética , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Regulación hacia ArribaRESUMEN
The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases.
Asunto(s)
Productos del Gen nef/metabolismo , VIH-1/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Activación Enzimática , Productos del Gen nef/genética , Humanos , Células Jurkat , Datos de Secuencia Molecular , Fosfotransferasas/metabolismo , Proteínas Proto-Oncogénicas c-hck , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Dominios Homologos srcRESUMEN
In determining levels of expression of HIV-1 Nef protein within the central nervous system (CNS) we assessed antibody responses to the protein both peripherally and in CNS. Antibodies to Nef were not detected within the CNS despite detection of antibodies to both gp41 and Nef in peripheral blood and representative virus isolates derived from CNS and peripheral blood (PB) samples containing full length nef sequence and virus-infected cells expressing Nef protein. We conclude from this that expression of Nef within the CNS is such that little or no antibody production occurs and that these differences indicate that Nef protein may not be directly contributing to the AIDS dementia complex. Expression of Nef protein in PHA-activated peripheral blood mononuclear cells from CNS derived isolates was different to that of coincidental PB derived isolates in that partial surface expression was observed for the latter. The results suggest that antigenic presentation of Nef within the CNS is anomalous and that Nef protein expression, at least for the limited number of in vitro derived isolates tested, has a different localization pattern.
Asunto(s)
Complejo SIDA Demencia/fisiopatología , Anticuerpos Antivirales/inmunología , Regulación Viral de la Expresión Génica , Productos del Gen nef/genética , VIH-1/aislamiento & purificación , Complejo SIDA Demencia/inmunología , Complejo SIDA Demencia/virología , Secuencia de Aminoácidos , Encéfalo/fisiopatología , Encéfalo/virología , Clonación Molecular , Epítopos , Productos del Gen nef/líquido cefalorraquídeo , Productos del Gen nef/inmunología , Proteína gp41 de Envoltorio del VIH/líquido cefalorraquídeo , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Datos de Secuencia Molecular , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
OBJECTIVE: To investigate whether members of a transfusion-linked cohort (the Sydney Bloodbank Cohort) infected with a nef-deleted strain of HIV-1 could be differentiated from individuals infected with wild-type strains of HIV-1 by characterizing the Nef antibody response of cohort members. DESIGN: Retrospective and prospective analysis of the nef gene sequence and the antibody response to Nef peptides in HIV-infected subjects. METHODS: Plasma was obtained from all individuals of the Sydney cohort, and from a variety of HIV-1-infected and uninfected controls. Antibodies recognizing full-length recombinant HIV-1NL43 Nef protein and synthetic peptide analogues were assessed by enzyme-linked immunosorbent assay. RESULTS: All 34 individuals infected with wild-type HIV-1 had antibodies reacting with full-length Nef protein as well as with a series of synthetic peptides (6-23-mers) spanning most of the Nef protein of HIV-1NL43. Although the HIV-1 quasispecies infecting the Sydney cohort had a consensus deletion of the nef gene corresponding to amino-acids 165-206, HIV-1 strains from individual members of the cohort had additional deletions comprising up to 80% of the nef gene. Members of the cohort had antibodies to peptides homologous to all regions of the Nef protein tested, except for a single peptide (amino-acids 162-177) that lies within the consensus nef deletion for the cohort quasispecies. CONCLUSION: These data show that nef-deleted strains of HIV-1 can be detected serologically. In the Sydney cohort, detection of antibodies to all regions of Nef tested, except that corresponding to amino-acids 162-177, suggests that observed deletions outside this domain occurred after this virus had infected these subjects and stimulated an immune response. A Nef peptide serological assay may be useful for identifying further examples of individuals infected with nef-deleted, attenuated HIV-1 quasispecies and for assessing the evolution of those variants in vivo.
Asunto(s)
Serodiagnóstico del SIDA/métodos , Genes nef/genética , Anticuerpos Anti-VIH/sangre , VIH-1/inmunología , Eliminación de Secuencia/inmunología , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática/métodos , Mapeo Epitopo , Evolución Molecular , Productos del Gen nef , Seropositividad para VIH/inmunología , Seropositividad para VIH/virología , VIH-1/genética , Humanos , Péptidos/síntesis química , Estudios Prospectivos , Proteínas Recombinantes , Estudios Retrospectivos , Eliminación de Secuencia/genética , Sobrevivientes , Victoria , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaAsunto(s)
Linfocitos T CD4-Positivos/inmunología , Productos del Gen nef/fisiología , VIH-1/fisiología , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/virología , Línea Celular , Humanos , Receptores de Interleucina-2/metabolismo , Transducción de Señal , Linfocitos T/virología , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Productos del Gen nef/metabolismo , VIH-1/metabolismo , Linfocitos T/virología , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Productos del Gen nef/genética , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfocitos T/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
This study examined the relationship between scores on the Harris-Lingoes MMPI-2 subscales for Depression and the categories of Exner's Rorschach Depression Index (DEPI) for 53 clients of a counselling agency. Scores on the subscale Mental Dullness related to the Depression Index as a whole and to the category Blends < 4. The subscale Subjective Depression also related to the category Blends < 4.
Asunto(s)
Trastorno Depresivo/diagnóstico , MMPI/estadística & datos numéricos , Prueba de Rorschach/estadística & datos numéricos , Adaptación Psicológica , Nivel de Alerta , Consejo , Trastorno Depresivo/psicología , Humanos , Solución de Problemas , Psicometría , Reproducibilidad de los ResultadosRESUMEN
A blood donor infected with human immunodeficiency virus-type 1 (HIV-1) and a cohort of six blood or blood product recipients infected from this donor remain free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10 to 14 years after infection. HIV-1 sequences from either virus isolates or patient peripheral blood mononuclear cells had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the long terminal repeat (LTR). Full-length sequencing of one isolate genome and amplification of selected HIV-1 genome regions from other cohort members revealed no other abnormalities of obvious functional significance. These data show that survival after HIV infection can be determined by the HIV genome and support the importance of nef or the U3 region of the LTR in determining the pathogenicity of HIV-1.
Asunto(s)
Donantes de Sangre , Genes nef , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH , VIH-1/genética , VIH-1/patogenicidad , Adulto , Anciano , Composición de Base , Secuencia de Bases , Transfusión Sanguínea , Recuento de Linfocito CD4 , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Reordenamiento Génico , Genoma Viral , Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , VIH-1/fisiología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Familia de Multigenes , Eliminación de Secuencia , Virulencia , Replicación ViralRESUMEN
Human immunodeficiency virus type 1 (HIV-1) Nef protein causes the loss of cell surface CD4 and interleukin-2 (IL-2) receptor (Tac) from peripheral blood mononuclear cells (PBMC) and CD4+ T-cell lines. As both CD4 and the IL-2 receptor play crucial roles in antigen-driven helper T-cell signalling and T-cell proliferation, respectively, the role of Nef in the viral life cycle may be to perturb signalling pathways emanating from these receptors. However, the intracellular targets for Nef that result in receptor down-regulation are unknown. Using a recombinant glutathione S-transferase-full-length 27 kDa Nef (Nef27) fusion protein, produced in Escherichia coli by translation from the first start codon of HIV-1 nef clone pNL4-3, as an affinity reagent to probe cytoplasmic extracts of MT-2 cells and PBMC, we have shown interaction with at least seven host cell protein species ranging from 24 to 75 kDa. Immunoblotting identified four of these proteins as p56lck, CD4, p53, and p44mapk/erk1, all of which are intimately involved in intracellular signalling. To assess the relevance of these interactions and further define the biochemical activity of Nef in signal transduction pathways, highly purified Nef27 protein was introduced directly into PBMC by electroporation. Nef27-treated PBMC showed reduced proliferative responsiveness to exogenous recombinant IL-2. Normally, stimulation of T-cells by IL-2 or phorbol 12-myristate 13-acetate provokes both augmentation of p56lck activity and corresponding posttranslational modification of p56lck. These changes were also inhibited by treatment of PBMC with Nef, suggesting that Nef interferes with activation of p56lck and as a consequence of signalling via the IL-2 receptor. Further evidence for Nef interfering with cell proliferation was the decreased production of the proto-oncogene c-myb, which is required for cell cycle progression, in Nef-treated MT-2 cells. In contrast to the binding characteristics and biological effects of Nef27, the alternate 25-kDa isoform of Nef (Nef25) produced by translation from the second start codon of HIV nef pNL4-3 (57 nucleotide residues downstream) was shown to interact with only three cellular proteins of approximately 26, 28, and 56 kDa from PBMC and MT-2 cells, one of which was identified as p56lck. Also, proliferation and posttranslational modification of p56lck in response to IL-2 stimulation were not profoundly affected by treatment of PBMC with Nef25 compared with Nef27.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Productos del Gen nef/metabolismo , VIH-1/patogenicidad , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos , Linfocitos T/inmunología , Secuencia de Aminoácidos , Antígenos CD4/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Productos del Gen nef/farmacología , Humanos , Técnicas In Vitro , Interleucina-2/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Proteína Quinasa 3 Activada por Mitógenos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-myb , Acetato de Tetradecanoilforbol/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
The production of interferon-alpha (IFN-alpha) by normal human peripheral blood mononuclear cells (PBMNC) was studied using polyclonal antipeptide antibodies designed to react either with all IFN-alpha subtypes or with individual subtypes IFN-alpha 2 or IFN-alpha 4. In this study, we demonstrate the detection of intracellular IFN-alpha in PBMNC using immunofluorescence staining and flow-cytometric analysis. Virtually all cells of the PBMNC population were shown to produce IFN-alpha reactive with all three antisera after stimulation with Sendai virus. The immunofluorescence studies also demonstrated that IFN-alpha is produced by PBMNC in the absence of known viral stimulation but is not secreted in detectable levels. Double-labeling with specific monoclonal antibodies to T and B lymphocytes confirmed that the entire populations of these two cell types produce IFN-alpha, both constitutively and after virus induction. Polymorphonuclear cells (PMNC) isolated from Ficoll-Paque pellets were also shown to contain intracellular IFN-alpha, both before and after virus induction. The finding that all PBMNC produce IFN-alpha constitutively suggests that IFN-alpha may have important regulatory functions in situations other than during overt viral infections.
Asunto(s)
Interferón-alfa/biosíntesis , Leucocitos Mononucleares/metabolismo , Linfocitos B/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunofenotipificación , Activación de Linfocitos , Virus de la Parainfluenza 1 Humana/metabolismo , Linfocitos T/metabolismoRESUMEN
Localised interferon-alpha production was investigated in hepatitis C patients entered into a trial of interferon-alpha-2a therapy. Antibodies capable of reacting specifically with interferon-alpha-2, interferon-alpha-4 or with all interferon-alpha subtypes were used as immunohistochemical and immunofluorescence probes to study interferon-alpha production in liver biopsy tissue, and peripheral blood mononuclear cells prior to and after stimulation with Sendai virus. Measurement of cytoplasmic interferon-alpha, specifically interferon-alpha-2 and interferon-alpha-4, in peripheral blood mononuclear cells isolated from the hepatitis C patients and of total interferon-alpha secreted into culture supernatants by these cells showed interferon-alpha production similar to that of peripheral blood mononuclear cells isolated from normal individuals. Interferon-alpha-positive cells were observed in the infiltrating mononuclear cells of the liver biopsy tissue obtained from 8 of the 14 patients. Lymphocytes, fibroblasts, Kupffer cells, polymorphonuclear cells and monocytes stained positive for interferon-alpha, and specifically interferon-alpha-4, in all of the eight patients. The cytoplasm of hepatocytes also stained weakly positive in three of these patients. Interferon-alpha positive cells showed a good correlation with the degree of histological damage observed in the liver biopsies but not with presence of antibodies towards hepatitis C virus or levels of serum alanine aminotransferase measured prior to interferon-alpha-2a therapy. Interestingly, response to therapy seemed linked to local interferon-alpha production status. Those patients who responded best to therapy displayed no or only low levels of interferon-alpha positive cells in liver biopsy tissue. Thus patients with a lower activation of their endogenous interferon-alpha system may benefit from administration of exogenous interferon-alpha.