RESUMEN
In 2005, the French-speaking task force on pediatric critical and emergency care [Groupe Francophone de Réanimation et d'Urgences Pédiatriques (GFRUP)] issued recommendations on withholding and withdrawing treatments in pediatric critical care. Since then, the French Public Health Code, modified by the laws passed in 2005 and 2016 and by their enactment decrees, has established a legal framework for practice. Now, 15 years later, an update of these recommendations was needed to factor in the experience acquired by healthcare teams, new questions raised by practice surveys, the recommendations issued in the interval, the changes in legislation, and a few legal precedents. The objective of this article is to help pediatric critical care teams find the closest possible compromise between the ethical principles guiding the care offered to the child and the family and compliance with current regulations and laws.
Asunto(s)
Cuidados Críticos/normas , Unidades de Cuidado Intensivo Pediátrico/normas , Cuidados Paliativos/normas , Órdenes de Resucitación , Privación de Tratamiento , Servicios Médicos de Urgencia , Humanos , Sociedades MédicasRESUMEN
The gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay is a reference method for the ex vivo monitoring of antigen-specific T cells and a primary tool for assessing clinical trials of human immunodeficiency virus (HIV) or cancer vaccines. Four experienced laboratories in Paris compared their results with this method by exchanging frozen blood samples from eight HIV-seronegative and eight HIV-seropositive subjects. Each laboratory measured the IFN-gamma-producing cells specific for HIV, Epstein-Barr virus, cytomegalovirus, and influenza using the same set of peptides and the same ELISPOT reader but its own ELISPOT technique. The cutoff values for positive responses (50 or 100 spot-forming cells/10(6) peripheral blood mononuclear cells over background) were consistent with the binomial statistic criterion. The global qualitative concordance, as assessed by the kappa index, ranged from 0.38 to 0.92, that is, moderate to excellent, and was better for non-HIV 9-mer peptide pools than for HIV 15-mer peptide pools. The interlaboratory coefficient of variation for the frequency of virus-specific T cells was 18.7% (data are expressed on a log scale). Clustering analysis of HIV-positive subjects showed qualitative agreement for ELISPOT results from all four laboratories. Overall, the good interlaboratory qualitative concordance of IFN-gamma ELISPOT assays with only the peptide source and ELISPOT reader in common suggests that a qualitative comparison of interlaboratory findings is feasible. Nonetheless, a single set of standard operating procedures should be used in multicenter trials to improve standardization.
Asunto(s)
Infecciones por VIH/inmunología , Interferón gamma/inmunología , Linfocitos T/fisiología , Anticuerpos/análisis , Anticuerpos/uso terapéutico , Análisis por Conglomerados , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos de Linfocito T , Infecciones por VIH/patología , Infecciones por VIH/terapia , Infecciones por VIH/virología , Humanos , Péptidos/inmunología , Reproducibilidad de los Resultados , Estadísticas no Paramétricas , Linfocitos T/inmunologíaRESUMEN
Successful immunologic control of HIV infection is achieved only in rare individuals. Dendritic cells (DCs) are required for specific antigen presentation to naive T lymphocytes and for antiviral, type I interferon secretion. Two major blood DC populations are found: CD11c+ (myeloid) DCs, which secrete IL-12, and CD123+ (IL-3-receptor+) DCs (lymphoid), which secrete type I interferons in response to viral stimuli. The authors have previously found a decreased proportion of blood CD11c+ DCs in chronic HIV+ patients. In this study, 26 to 57 days after infection and before treatment, CD123+ and CD11c+ DC numbers were dramatically reduced in 13 HIV+ patients compared with 13 controls (P =.0002 and P =.001, respectively). After 6 to 12 months of highly active antiretroviral therapy, DC subpopulation average numbers remained low, but CD123+ DC numbers increased again in 5 of 13 patients. A strong correlation was found between this increase and CD4 T-cell count increase (P =.0009) and plasma viral load decrease (P =.009). Reduced DC numbers may participate in the functional impairment of HIV-specific CD4+ T cells and be responsible for the low type I interferon responsiveness already known in HIV infection. The restoration of DC numbers may be predictive of immune restoration and may be a goal for immunotherapy to enhance viral control in a larger proportion of patients.
Asunto(s)
Células Dendríticas/patología , Infecciones por VIH/sangre , Integrina alfaXbeta2/análisis , Receptores de Interleucina-3/análisis , Adulto , Terapia Antirretroviral Altamente Activa , Recuento de Células Sanguíneas , Recuento de Linfocito CD4 , Células Dendríticas/metabolismo , Femenino , Estudios de Seguimiento , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1 , Humanos , Interferón-alfa/sangre , Interferón-alfa/deficiencia , Interferón-alfa/metabolismo , Interleucina-12/metabolismo , Subunidad alfa del Receptor de Interleucina-3 , Linfocitos/metabolismo , Linfocitos/patología , Masculino , Persona de Mediana Edad , Células Mieloides/metabolismo , Células Mieloides/patología , ARN Viral/sangre , Carga ViralRESUMEN
OBJECTIVE: An assessment of the impact of one year potent antiretroviral treatment initiated during primary HIV infection on the cell-associated viral burden. DESIGN AND METHODS: Proviral HIV-1 DNA was quantified in serial peripheral blood mononuclear cell (PBMC) samples from 19 patients enrolled in the French prospective PRIMO Cohort for whom plasma HIV RNA was suppressed to undetectable levels after one year of triple therapy; that is, plasma HIV-1 RNA was maintained below 200 copies/ml. Results were compared with those observed in 19 patients with chronic HIV-1 infection presenting the same degree of virus suppression after 12 months of treatment. RESULTS: At study entry, PRIMO subjects presented heterogeneous levels of proviral HIV-1 DNA: 2-3.92 log10 copies/10(6) PBMC and plasma HIV RNA: 2.3-6.5 log10 copies/ml. One year of effective highly active antiretroviral therapy (HAART) resulted in a median diminution of proviral DNA of -0.78 log10/10(6) PBMC in PRIMO subjects. The median decline in chronic-phase patients was -0.32 for those who were pre-treated and -0.52 for those previously naive of treatment. CONCLUSION: The decline in cell-associated HIV DNA observed throughout one year treatment indicated that HAART reduces the proviral HIV-DNA load more effectively when initiated during the primary rather than the chronic phase of HIV infection. These findings therefore tend to lend support to the early initiation of treatment. Nevertheless, heterogeneous baseline values observed for CD4 cell count, plasma HIV RNA and proviral HIV DNA in PRIMO subjects, raise the question of whether treatment should be delayed in some to spare early adverse effects of HAART.
Asunto(s)
Terapia Antirretroviral Altamente Activa , ADN Viral/sangre , Infecciones por VIH/tratamiento farmacológico , VIH-1 , Provirus , Recuento de Linfocito CD4 , Estudios de Cohortes , Infecciones por VIH/virología , Humanos , Estudios Prospectivos , ARN Viral/biosíntesis , ARN Viral/sangre , Carga Viral , Replicación ViralRESUMEN
To assess prospectively the influence of the control of viral replication on the frequency of cytokine-producing T cells, and to correlate these changes with immune activation, we conducted a 15-month follow-up study of IFN-gamma- and IL-2-producing CD4+ and CD8+ T cells at a single-cell level in 12 previously untreated patients receiving highly active antiretroviral therapy (HAART). At baseline we observed a strikingly high proportion of IFN-gamma-producing CD8+ T cells. The treatment-induced decrease in the proportion of IFN-gamma-producing CD8+ T cells ran parallel to the decrease in HLA-DR+ and CD38+CD8+ T cell subsets and was associated with the reduction in HIV RNA level. IL-2-producing cells were mainly CD4+. As a consequence of CD4+ T cell loss, the number of IL-2-producing CD4+ T cells was lower in patients than in control subjects (52 vs. 171 cells/microl), but the proportion of these cells was unchanged (22.4 vs. 19.3). During therapy the proportion of CD4+ IL-2-producing cells was initially stable and then fell markedly at month 5, followed by a gradual return to previous values. The reduction in viral load was associated with the fall in the proportion of CD4+ activated subsets. Intracellular cytokine assays are a new approach to the assessment of T cell function in HIV infection. Our results suggest that the functional capacity of CD4+ T cells is probably less severely altered than previously thought on the basis of conventional assays. CD8+ T cells exhibit an increased capacity to produce IFN-gamma that is associated with an increase in activation marker expression. These alterations decrease partially and in parallel under treatment.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/biosíntesis , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Subgrupos de Linfocitos T/inmunología , Adulto , Didanosina/uso terapéutico , Quimioterapia Combinada , Femenino , Citometría de Flujo , Estudios de Seguimiento , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Masculino , Estudios Prospectivos , ARN Viral/sangre , Ritonavir/uso terapéutico , Estavudina/uso terapéutico , Carga ViralRESUMEN
HIV-specific CD8(+) T cells play a major role in the control of virus during HIV primary infection (PI) but do not completely prevent viral replication. We used IFN-gamma enzyme-linked immunospot assay and intracellular staining to characterize the ex vivo CD8(+) T-cell responses to a large variety of HIV epitopic peptides in 24 subjects with early HIV PI. We observed HIV-specific responses in 71% of subjects. Gag and Nef peptides were more frequently recognized than Env and Pol peptides. The number of peptides recognized was low (median 2, range 0-6). In contrast, a much broader response was observed in 30 asymptomatic subjects with chronic infection: all were responders with a median of 5 peptides recognized (range 1-13). The frequency of HIV-specific CD8(+) T cells among PBMC for a given peptide was of the same order of magnitude in both groups. The proportion of HIV-specific CD8(+)CD28(-) terminally differentiated T cells was much lower in PI than at the chronic stage of infection. The weakness of the immune response during HIV PI could partially account for the failure to control HIV. These findings have potential importance for defining immunotherapeutic strategies and establishing the goals for effective vaccination.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Infecciones por VIH/inmunología , Seropositividad para VIH/inmunología , VIH-1/fisiología , Adulto , Anciano , Especificidad de Anticuerpos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Productos del Gen env/inmunología , Productos del Gen gag/inmunología , Productos del Gen nef/inmunología , Productos del Gen pol/inmunología , Anticuerpos Anti-VIH/inmunología , Humanos , Interferón gamma/biosíntesis , Masculino , Persona de Mediana Edad , Valores de Referencia , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
To investigate the temporal relationship between CD8+ lymphocyte phenotypic alterations, the CD4+ T cell decline, and plasma HIV RNA levels during the natural history of HIV infection, 33 treatment-naive HIV-infected patients with > or =400 CD4+ cells/microl were studied prospectively for 3 years. During the study period, 20 patients remained untreated, and only 6 received more than 6 months of therapy. A significant relationship was found between changes in plasma HIV RNA and changes in the proportion of CD38+CD8+ cells. Conversely, the number of CD4+ T cells lost per year was strongly related to the increase in the proportion of CD28-CD8+ T cells. A strong relationship between mean yearly changes in CD4+ T cell numbers and changes in HIV RNA was also observed. CD4+ T cell changes were associated with changes in both viral load and CD8+ T cell activation. These results provide support for the use of both virologic and immunologic parameters for prognosis and management during HIV infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1 , Activación de Linfocitos , Carga Viral , Adulto , Recuento de Linfocito CD4 , Citometría de Flujo , Infecciones por VIH/virología , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Subgrupos de Linfocitos TRESUMEN
The ex vivo antiviral CD8(+) repertoires of 34 human immunodeficiency virus (HIV)-seropositive patients with various CD4(+) T-cell counts and virus loads were analyzed by gamma interferon enzyme-linked immunospot assay, using peptides derived from HIV type 1 and Epstein-Barr virus (EBV). Most patients recognized many HIV peptides, with markedly high frequencies, in association with all the HLA class I molecules tested. We found no correlation between the intensity of anti-HIV CD8(+) responses and the CD4(+) counts or virus load. In contrast, the polyclonality of anti-HIV CD8(+) responses was positively correlated with the CD4(+) counts. The anti-EBV responses were significantly less intense than the anti-HIV responses and were positively correlated with the CD4(+) counts. Longitudinal follow-up of several patients revealed the remarkable stability of the anti-HIV and anti-EBV CD8(+) responses in two patients with stable CD4(+) counts, while both antiviral responses decreased in two patients with obvious progression toward disease. Last, highly active antiretroviral therapy induced marked decreases in the number of anti-HIV CD8(+) T cells, while the anti-EBV responses increased. These findings emphasize the magnitude of the ex vivo HIV-specific CD8(+) responses at all stages of HIV infection and suggest that the CD8(+) hyperlymphocytosis commonly observed in HIV infection is driven mainly by virus replication, through intense, continuous activation of HIV-specific CD8(+) T cells until ultimate progression toward disease. Nevertheless, highly polyclonal anti-HIV CD8(+) responses may be associated with a better clinical status. Our data also suggest that a decrease of anti-EBV CD8(+) responses may occur with depletion of CD4(+) T cells, but this could be restored by highly active antiretroviral treatment.
Asunto(s)
Antivirales/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Seropositividad para VIH/inmunología , VIH-1/inmunología , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 4/inmunología , Infecciones Tumorales por Virus/inmunología , Células Cultivadas , Quimioterapia Combinada , Seropositividad para VIH/sangre , Seropositividad para VIH/tratamiento farmacológico , Humanos , Interferón gamma/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Estudios Retrospectivos , Carga ViralRESUMEN
The CD8+CD28- cell population in the blood of HIV-infected individuals is considerably expanded. Yet the cause of this expansion is not clear. The recent demonstration of identical TCR-rearranged genes in CD8+CD28+ and CD8+CD28- expanded T cells of HIV-seropositive patients supports the hypothesis that these two subpopulations are phenotypic variants of the same lineage. To further elucidate the precise relationship between them, we measured the fraction of CD28+ and CD28- T cell subsets in IFN-gamma-producing CD8+ T cells by intracellular staining and cytofluorometry as a functional test for ex vivo recognition of epitopes derived from HIV-1, Epstein-Barr virus (EBV) and influenza virus. HIV-specific CD8+ T cells were predominantly CD28 in all the eight HIV-seropositive subjects tested. In contrast, the anti-EBV and anti-influenza CD8+ T cells were mainly CD28+ in these patients as well as in HIV-seronegative individuals. This supports the notion that the CD8 CD28 hyperlymphocytosis observed in HIV infection is due mainly to chronic activation and differentiation of HIV-specific memory CD8+CD28+ T cells into terminally differentiated CD8+CD28-lymphocytes, because of intense HIV-1 replication and without any important bystander activation. This clarification of the mechanisms underlying the CD8+CD28- expansion in HIV-induced pathogenesis may have important therapeutic implications.
Asunto(s)
Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/inmunología , Seropositividad para VIH/inmunología , VIH-1/inmunología , Subgrupos de Linfocitos T/inmunología , Estudios de Casos y Controles , Epítopos , Seronegatividad para VIH/inmunología , Herpesvirus Humano 4/inmunología , Humanos , Memoria Inmunológica , Técnicas In Vitro , Interferón gamma/biosíntesis , Activación de Linfocitos , Recuento de Linfocitos , Orthomyxoviridae/inmunologíaRESUMEN
Schistosoma mansoni infection in mice is associated with a switch from a Th1 to a Th2-type cytokine response. The role of Th1 and Th2 responses in immune dysregulations associated with AIDS and murine AIDS (MAIDS) is controversial, but a Th2 bias could be associated with disease progression, raising the hypothesis that helminth infections might accelerate the retroviral disease progression. Here, we used the murine model of AIDS to evaluate the course of the viral disease during co-infection with S. mansoni. C57BL/6 mice were infected with S. mansoni cercariae 8 weeks before intravenous challenge with the LP-BM5 retroviral complex. MAIDS did not progress faster in co-infected mice, in terms of spleen and inguinal lymphadenopathy size, ecotropic virus titres in the spleen, or in vitro proliferative responses to mitogen. Th2 cytokine production was not enhanced in co-infected animals, except for an isolated increase in IL-4 production 21 weeks after LP-BM5 infection. Co-infected animals had significantly lower lymph node and spleen weights than mice infected with LP-BM5 only. MAIDS did not influence the granulomatous response to S. mansoni in the liver of co-infected mice. Finally, infection with S. mansoni neither enhanced Th2 cytokine production nor accelerated MAIDS progression in animals subsequently challenged with LP-BM5.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida del Murino/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Células TH1/inmunología , Células Th2/inmunología , Animales , Citocinas/análisis , Femenino , Granuloma/patología , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Conejos , Esquistosomiasis mansoni/complicaciones , Esquistosomiasis mansoni/patología , Bazo/patología , Bazo/virologíaRESUMEN
OBJECTIVE: To analyse the relationship between CD8+ lymphocyte phenotype alterations and plasma HIV RNA levels in HIV-infected patients treated with the zidovudine-didanosine combination. METHODS: A total of 30 HIV-infected patients who had never received antiretroviral therapy and who were starting treatment with a combination of zidovudine and didanosine were prospectively studied. Multiparameter flow cytometric analysis of CD8+ lymphocytes and plasma HIV RNA determination were performed on day 0, day 15 and monthly from months 1 to 6. RESULTS: Patients were divided into three categories according to the time-course of plasma HIV RNA levels. In 14 patients, an early and sustained fall in plasma HIV RNA to below the detection limit (500 copies/ml) was observed; in 10 patients, the fall was transient; in six patients, plasma HIV RNA was always detectable (non-responders). The mean CD4+ lymphocyte gain was 120 x 10(6)/l at month 6 in sustained and transient responders, and 55 x 10(6)/l in non-responders. A significant fall in the proportion of CD8+ lymphocytes with an activated phenotype was observed only in the two groups of responders, and was higher in the sustained responders (CD38+HLA-DR+, -56.8%; CD38+CD45RO+, -54.0%; HLA-DR+CD45RO+, -48.4%; CD38+CD28-, -47.3%). CONCLUSION: A fall in the proportion of activated CD8+ lymphocytes is associated with the disappearance of HIV RNA from plasma during antiretroviral therapy. Undetectable plasma HIV RNA is not associated with a return to normal CD8+ lymphocyte activation status after 6 months of treatment, suggesting that viral replication persists in lymphoid tissues.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Didanosina/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1 , Activación de Linfocitos , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Zidovudina/uso terapéutico , Adulto , Recuento de Linfocito CD4 , Linfocitos T CD8-positivos/citología , Quimioterapia Combinada , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , Humanos , Inmunofenotipificación , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , ARN Viral/sangre , Carga ViralRESUMEN
We studied susceptibility to experimental systemic cryptococcosis in mice previously infected with the retroviral complex LP-BM5 (responsible for murine AIDS). LP-BM5 was inoculated to C57B1/6 mice by intravenous (i.v.) injection 8 weeks before an i.v. challenge with 4 x 10(3) CFU of Cryptococcus neoformans. Uninfected and singly infected mice were used as controls. LP-BM5 infection did not result in a significant increase in fungal burdens in the lungs or brains of co-infected animals compared to mice infected with C. neoformans alone. However, mortality was enhanced in the co-infected animals. The kinetics of splenocyte subsets differed in co-infected mice and LP-BM5-infected mice; the increase in CD4+, CD8+ and Ly5+ cells was only moderate in the former. Cytokine production by concanavalin A (Con A)-stimulated splenocytes from co-infected mice showed a marked decrease in the Th1 response (IFN-gamma, IL-2) and an increase in the Th2 response (IL-4, IL-10). Furthermore, cryptococcosis altered the course of MAIDS, inhibiting splenomegaly. This effect was not related to a decrease in ecotropic virus titres in the spleen or to improved in vitro responsiveness of spleen cells to Con A. The marked decrease in IFN-gamma production in co-infected animals could partly explain the inhibition of LP-BM5-induced splenomegaly. This model of murine retroviral infection does not seem to be suitable for studying cryptococcosis in immunosuppressed animals, but remains valuable for investigating in vivo interactions between two pathogens.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA , Criptococosis/etiología , Cryptococcus neoformans , Síndrome de Inmunodeficiencia Adquirida del Murino/complicaciones , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Criptococosis/inmunología , Citocinas/inmunología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Ratones , Ratones Endogámicos C57BL , Síndrome de Inmunodeficiencia Adquirida del Murino/inmunologíaRESUMEN
Infection and tumors provoke substantial changes accompanied with the disbalance of many neuroendocrine factors which in their summarizing effects influence the life span of animals. Our previous results showed enhanced mortality after one injection of morphine in association with Friend leukaemia virus infection. The aim of this study was to examine the effects of some other opioids (pethidine and pentazocine) and an acetylcholine esterase inhibitor neostigmine on the survival of animals under two conditions: (1) Friend leukaemia virus infection which mostly depressed immune functions, and (2) Toxoplasma gondii infection which in general enhanced the immune status. In contrast to our previous observation with morphine, the mortality induced by single doses of pethidine (150 mg/kg) or pentazocine (50-75 mg/kg) was unchanged during the Friend leukaemia virus infection. A single injection of neostigmine (0.42 or 0.56 mg/kg) was significantly more lethal in DBA-2 mice infected with Friend leukaemia virus. Neostigmine in doses of 0.33 and 0.4 mg/kg caused death in 46 % and 57 %, respectively, of animals infected with Toxoplasma gondii which was significantly higher in comparison with only 8 % and 12.5 % in control groups. Pethidine (150 mg/kg) killed 70 % of Toxoplasma gondii infected animals and even 90 % of non-infected mice. Thus, the Friend leukaemia virus and Toxoplasma gondii infections increased toxicity only of some drugs which may, at least partly, be associated with altered immune status during infection and involvement of the cholinergic system.
Asunto(s)
Analgésicos Opioides/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Leucemia Experimental/inmunología , Leucemia Experimental/mortalidad , Meperidina/toxicidad , Neostigmina/toxicidad , Pentazocina/toxicidad , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/mortalidad , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/mortalidad , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/mortalidad , Acetilcolina/fisiología , Acetilcolinesterasa/fisiología , Animales , Virus de la Leucemia Murina de Friend , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Monitorización InmunológicaRESUMEN
The causative agent of murine AIDS (MAIDS) is the defective murine leukemia virus BM5d, that requires the replication-competent ecotropic MuLV (BM5e) helper virus. We developed a competitive quantitative PCR method including specific internal standards to quantify the expression of BM5d in the spleen of infected mice and to characterize BM5d expression kinetics following experimental infection. Specimen RNA was reverse-transcribed and co-amplified with a competitive template containing a gag sequence specific for BM5d that can be discriminated from that corresponding to wild-type cDNA by the presence of a unique restriction site, Bg/II. PCR products were quantified by means of densitometric analysis after ethidium bromide staining of gels. To standardise the RNA extraction and reverse transcription steps, the amount of defective-virus mRNA was compared to a constant copy number of murine beta actin mRNA. LP-BM5 production was measured in the spleen of infected mice. Defective gag mRNA production was compared to that of the ecotropic virus. The mRNA level of the defective virus and the titre of replicative virus increased with the duration of infection, and the amount of defective virus mRNA correlated with the titre of replicating virus.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus de la Leucemia Murina/genética , Síndrome de Inmunodeficiencia Adquirida del Murino/genética , Síndrome de Inmunodeficiencia Adquirida del Murino/virología , Reacción en Cadena de la Polimerasa/métodos , Actinas/genética , Animales , Células Cultivadas , Clonación Molecular , ADN Complementario/genética , Virus Defectuosos/genética , Electroforesis en Gel de Poliacrilamida , Femenino , Productos del Gen gag/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Plásmidos , ARN Mensajero/análisis , ARN Viral/genética , Bazo/virología , Replicación Viral/genéticaRESUMEN
To examine the role of nitric oxide (NO) in murine AIDS (MAIDS) pathogenesis, we determined NO production and inducible NOS (iNOS) mRNA expression in the macrophages of LP-BM5-infected mice, together with the in vivo effects of L-NAME, a competitive inhibitor of NO synthase. LP-BM5 infection induced neither spontaneous nitrite production nor iNOS mRNA expression. No differences in IFN gamma + LPS-induced nitrite production or iNOS mRNA expression were observed in macrophages, from non-infected or infected mice. Spleen weight, ecotropic MuLV replication, the blood lymphocyte phenotype and proliferative response of splenocytes were not modified by L-NAME. LP-BM5 infection did not increase macrophage NO production and NO production did not appear to protect against LP-BM5-induced immunodeficiency.
Asunto(s)
Macrófagos/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Murino/metabolismo , Óxido Nítrico/metabolismo , Animales , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Virus de la Leucemia Murina/fisiología , Activación de Linfocitos/efectos de los fármacos , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Endogámicos C57BL , Síndrome de Inmunodeficiencia Adquirida del Murino/inmunología , Síndrome de Inmunodeficiencia Adquirida del Murino/virología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/metabolismo , Nitritos/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/virología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Replicación Viral/efectos de los fármacosRESUMEN
The murine leukemia virus LP-BM5 has been used to reproduce the model of murine AIDS in order to evaluate the course of infection with the MO-1 strain of Mycobacterium avium complex (MAC). LP-BM5 was inoculated in C57BL/6 mice by intravenous (i.v.) injection either 8 weeks before an i.v. challenge with 10(3) or 10(6) CFU of MAC (coinfection 1) or 10 days after an i.v. challenge with 10(3) CFU of MAC (coinfection 2). During coinfection 2 experiments, the phenotypic alterations in blood lymphocyte subsets were analyzed. During coinfection 1, LP-BM5 infection tended to decrease the mycobacterial growth, with the difference reaching statistical significance for the lower inoculum (10(3) CFU of MAC) (P<0.001). During coinfection 2, LP-BM5 did not exacerbate MAC infection except in the spleen, at day 90 after LP-BM5 challenge (P<0.001). LP-BM5 infection and the LP-BM5-MAC coinfection increased the numbers of activated CD4+ lymphocytes (CD4+ Ly6AE+) (P<0.001), activated CD8+ lymphocytes (CD8+ Ly6AE+) (P<0.001), and activated B lymphocytes (Ly5+ Ly6AE+) (P<0.001). This activation of T lymphocytes could explain the lack of exacerbation of MAC infection and even the trend to a lower level of MAC infection. Thus, this model of retroviral infection of mice does not seem to be a reliable model of immunodepression for the study of MAC infection and its treatments.
Asunto(s)
Virus de la Leucemia Murina , Mycobacterium avium , Infecciones por Retroviridae/inmunología , Tuberculosis/inmunología , Infecciones Tumorales por Virus/inmunología , Animales , Femenino , Subgrupos Linfocitarios , Ratones , Ratones Endogámicos C57BL , Tamaño de los ÓrganosRESUMEN
The relationship between CD8 lymphocyte phenotypic alterations and virological parameters was studied in 47 asymptomatic subjects with human immunodeficiency virus type 1 (HIV-1) infection and CD4 T cell counts above 400/microliters. CD8 subsets were examined by means of three-color flow cytometry, using an extensive panel of monoclonal antibody combinations. Virological parameters were measured by both end-point dilution culture of peripheral blood mononuclear cells (PBMCs) and plasma and branched-DNA (bDNA) signal amplification of plasma HIV RNA. Whereas HIV-infected patients had a near-normal CD4 cell count (mean, 782 cells/microliter), several subsets of activated CD8 cells were markedly expanded relative to values in 23 HIV-seronegative controls. The PBMC cultures were positive in 38 cases and plasma HIV RNA was detected in 31. The percentage of CD4 cells correlated negatively with both cellular viremia and plasma HIV RNA levels. Conversely, a positive correlation was observed between viral load and the percentage of CD8 cells. Among CD8 lymphocytes, the CD38+CD8 and HLA-DR+CD8 subsets correlated best with viral load. Three-color analysis showed that the subpopulations involved in this relationship were CD38+HLA-DR+, CD38+CD28-, HLA-DR+CD28+, HLA-DR+CD57-, CD38+CD57-, CD38+CD45RO+, and HLA-DR+CD45RO+. Our data provide the first evidence that viral load correlates with subsets of activated CD8 lymphocytes in asymptomatic HIV-infected subjects who have near-normal numbers of CD4 lymphocytes.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , ARN Viral/sangre , Adulto , Antígenos CD/sangre , Antígenos CD/clasificación , Recuento de Linfocito CD4 , Linfocitos T CD8-positivos/clasificación , Células Cultivadas , Femenino , Infecciones por VIH/sangre , VIH-1/inmunología , VIH-1/aislamiento & purificación , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/virología , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Subgrupos de Linfocitos T/clasificación , ViremiaRESUMEN
Nitric oxide (NO) exerts microbicidal effects on a broad spectrum of pathogens, including viruses, but its antiretrovirus properties have not yet been described. The purpose of this study was to determine whether NO inhibits murine Friend leukemia virus (FV) replication in vitro and to what extent NO may play a role in defenses against FV infection in mice. Three NO-generating compounds were studied: 3-morpholino-sydononimine (SIN-1), sodium nitroprusside (SNP), and S-nitroso-N-acetylpenicillamine (SNAP). The effects of these three compounds were compared with those of their controls (SIN-1C, potassium ferricyanide, and N-acetylpenicillamine, respectively), which do not generate NO and with that of sodium nitrite (NaNO2). SIN-1, SNP, and SNAP inhibited FV replication in dunni cells in a concentration-dependent manner. In contrast, no significant inhibitory effect was observed with the three controls or NaNO2. Furthermore, the addition of superoxide dismutase did not alter the inhibitory effect of SIN-1, which is also known to generate superoxide anions. No dunni cell toxicity was observed in the range of concentrations tested. We also assessed the effect of NO produced by activated macrophages on FV replication. Macrophages activated by gamma interferon and lipopolysaccharide inhibited FV replication in a concentration-dependent manner. This inhibition was due in part to NO production, since it was reversed by NG-monomethyl L-arginine, a competitive inhibitor of NO synthase. In vivo administration of NG-nitro-L-arginine methyl ester, a competitive inhibitor of NO synthase, significantly increased the viral load in spleen cells of FV-infected mice. These results suggested that NO may play a role in defenses against the murine Friend leukemia retrovirus.
Asunto(s)
Antivirales/farmacología , Virus de la Leucemia Murina de Friend/fisiología , Molsidomina/análogos & derivados , Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Penicilamina/análogos & derivados , Replicación Viral/efectos de los fármacos , Animales , Arginina/análogos & derivados , Arginina/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Fibroblastos , Virus de la Leucemia Murina de Friend/efectos de los fármacos , Cinética , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/fisiología , Macrófagos Peritoneales/virología , Masculino , Ratones , Ratones Endogámicos DBA , Molsidomina/farmacología , NG-Nitroarginina Metil Éster , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina , Penicilamina/farmacología , S-Nitroso-N-Acetilpenicilamina , Nitrito de Sodio/farmacologíaRESUMEN
The role of T helper 1 (Th1) and T helper 2 (Th2) responses in the murine acquired immunodeficiency syndrome (MAIDS) is unclear. It has been suggested that differential activation of T cell subsets, particularly a shift to Th2 cytokine production, may be associated with disease progression. To clarify the regulation of the cytokine network in the course of MAIDS, we examined the kinetics of cytokine production by isolated splenocytes. C57/BL6 mice were infected with the LP-BM5 mixture. The spleen cell proliferative response, together with IL-2, IFN-gamma, IL-10 and IL-4 production by unstimulated and ConA or anti-CD3 MoAb-stimulated spleen cells, were determined at various times after inoculation (weeks 1, 3, 6 and 9). Spleen cells isolated from murine leukemia virus complex (LP-BM5) infected mice spontaneously produced significant amounts of IL-2 and IFN-gamma one and three weeks post-infection, compared to uninfected controls. The capacity of isolated T cells to produce the Th1 cytokines IL-2 and IFN-gamma in response to stimulation with ConA and anti-CD3 MoAb decreased after 3 weeks of infection. The fall in IL-2 production ran parallel to the fall in the T cell proliferative response to ConA. IL-10 production in response to ConA and anti-CD3 MoAb increased after three weeks post-inoculation, and followed the reverse kinetic pattern to IFN-gamma and IL-2. In contrast, no significant spontaneous IL-4 production and no increase in IL-4 production in response to ConA or anti-CD3 MoAb occurred during the course of MAIDS, relative to uninfected controls. These results suggest that LP-BM5 infection leads to a fall in Th1 cytokine production rather than a clear switch to Th2 cytokine production.
Asunto(s)
Citocinas/biosíntesis , Síndrome de Inmunodeficiencia Adquirida del Murino/patología , Bazo/metabolismo , Células TH1/metabolismo , Células Th2/metabolismo , Animales , Células Cultivadas , Concanavalina A/farmacología , Citocinas/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-10/biosíntesis , Interleucina-10/genética , Interleucina-2/biosíntesis , Interleucina-2/genética , Interleucina-4/biosíntesis , Interleucina-4/genética , Cinética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Mitógenos/farmacología , Muromonab-CD3/farmacología , Bazo/patologíaRESUMEN
We have developed a sensitive high-performance liquid chromatographic assay for the determination of the zidovudine metabolite 3'-amino-3'-deoxythimidine (AMT) using fluorescence and sensitivity in the picomolar range. Plasma was diluted with 0.05 M sodium phosphate buffer pH 7.2 and subsequently prepared for analysis using solid-phase extraction. AMT was derivatized with 9-fluorenyl methylchloroformate and chromatographed using a reversed-phase system. The mobile phase consisted of acetonitrile-0.01 M potassium phosphate buffer (pH 7) (32.68, v/v). The fluorescence of the column effluent was monitored at 262 nm (excitation) and 306 nm (emission). Good resolution of AMT from endogenous plasma components was obtained. Within- and between-day variability was less than 10%. The limit of quantitation was 0.9 microgram/l. The assay was successfully applied to the determination of AMT in human plasma and in plasma of mice treated with zidovudine.