RESUMEN
Mediterranean spotted fever caused by Rickettsia conorii is a potentially lethal disease characterized by vascular inflammation affecting multiple organs. Studies of R. conorii so far have focused on activation of inflammatory cells and their release of inflammatory cytokines, but complement activation has not been investigated in R. conorii-infected patients. Here, we performed a comprehensive analysis of complement activation markers and the soluble cross-talking co-receptor CD14 (sCD14) in plasma from R. conorii-infected patients. The clinical data were supplemented with ex vivo experiments where the cytokine response was characterized in human whole blood stimulated with R. conorii. Complement activation markers at the level of C3 (C3bc, C3bBbP) and terminal pathway activation (sC5b-9), as well as sCD14, were markedly elevated (p <0.01 for all), and closely correlated (p <0.05 for all), in patients at admission compared with healthy matched controls. All tested markers were significantly reduced to baseline values at time of follow up. Rickettsia conorii incubated in human whole blood was shown to trigger complement activation accompanied by release of the inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, IL-8 and tumour necrosis factor. Whereas inhibition of either C3 or CD14 had only a minor effect on released cytokines, combined inhibition of C3 and CD14 resulted in significant reduction, virtually to baseline levels, of the four cytokines (p <0.05 for all). Our data show that complement is markedly activated upon R. conorii infection and complement activation is, together with CD14, responsible for a major part of the cytokine response induced by R. conorii in human whole blood.
Asunto(s)
Fiebre Botonosa/inmunología , Fiebre Botonosa/metabolismo , Activación de Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Citocinas/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Rickettsia conorii/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores , Fiebre Botonosa/microbiología , Estudios de Casos y Controles , Citocinas/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
We constructed Salmonella enterica serovar Typhimurium double-knockout mutants in which either the lipoprotein A (lppA) or the lipoprotein B (lppB) gene was deleted from an msbB-negative background strain by marker exchange mutagenesis. These mutants were highly attenuated when tested with in vitro and in vivo models of Salmonella pathogenesis.
Asunto(s)
Proteínas Bacterianas/genética , Lipoproteínas/genética , Infecciones por Salmonella/microbiología , Salmonella typhimurium/patogenicidad , Aciltransferasas/genética , Animales , Proteínas Bacterianas/inmunología , Eliminación de Gen , Sueros Inmunes/inmunología , Lipopolisacáridos/inmunología , Lipoproteína(a)/genética , Lipoproteína(a)/inmunología , Lipoproteínas/inmunología , Ratones , Mutación , Infecciones por Salmonella/inmunología , Salmonella typhimurium/genética , Salmonella typhimurium/inmunologíaRESUMEN
Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppA and lppB) located on the chromosome of Salmonella enterica serovar Typhimurium contributed to bacterial virulence. In this study, we characterized lppA and lppB single-knockout (SKO) mutants and compared them with an lpp double-knockout (DKO) mutant using in vitro and in vivo models. Compared to the lpp DKO mutant, which was nonmotile, the motility of the lpp SKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT) S. enterica serovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with the lpp SKO mutants compared to the cytotoxicity when cells were infected with the lpp DKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with the lppB SKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WT S. enterica serovar Typhimurium, than the level in host cells infected with the lppA SKO mutant. The lpp DKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with the lppB SKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-alpha and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in the lppA SKO mutant-infected mice than in animals challenged with the lpp DKO mutant. The increased cytokine levels observed with the lppB SKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with the lppA SKO and lpp DKO mutants. Moreover, the lppB SKO mutant-infected mice had increased susceptibility to death. Since the lpp DKO mutant retained intact LPS, we constructed an S. enterica serovar Typhimurium triple-knockout (TKO) mutant in which the lppA and lppB genes were deleted from an existing msbB mutant (msbB encodes an enzyme required for the acylation of lipid A). Compared to the lpp DKO and msbB SKO mutants, the lpp-msbB TKO mutant was unable to induce cytotoxicity and to produce cytokines and chemokines in vitro and in vivo. These studies provided the first evidence of the relative contributions of Lpp and lipid A acylation to Salmonella pathogenesis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Citocinas/metabolismo , Humanos , Lipoproteínas/genética , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Virulencia/genética , Virulencia/fisiologíaRESUMEN
By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.
Asunto(s)
Aeromonas hydrophila/patogenicidad , Proteínas Bacterianas/genética , Enterotoxinas/genética , Regulación Bacteriana de la Expresión Génica , Aeromonas hydrophila/genética , Animales , Proteínas Bacterianas/fisiología , Secuencia de Bases , Clonación Molecular , Elementos Transponibles de ADN , Femenino , Prueba de Complementación Genética , Ratones , Datos de Secuencia Molecular , Mutación , Transcripción Genética , VirulenciaRESUMEN
Natural killer (NK) cell activity was significantly increased on days 2-6 of infection in the Rickettsia conorii-infected C3H/HeN mice and on day 2 in the Rickettsia typhi-infected C57BL/6 mice. Depletion of NK cell activity utilizing anti-NK1.1 monoclonal antibody enhanced the susceptibility of normally resistant C57BL/6 mice to infection with R. typhi, and depletion of NK cell activity with antibody to asialo GM1 enhanced the susceptibility of C3H/HeN mice to infection with R. conorii. Serum gamma interferon was increased in R. conorii-infected C3H/HeN mice compared with NK cell-depleted, infected mice during the early course of infection. Additionally, the NK cell activating cytokine IL-12 was elevated in the sera of infected mice during the time period representing enhanced NK cell activity compared with uninfected mice. Thus, it appears that NK cells contribute to the early anti-rickettsial immune response, likely via a mechanism involving gamma interferon.
Asunto(s)
Interferón gamma/biosíntesis , Células Asesinas Naturales/inmunología , Infecciones por Rickettsia/inmunología , Rickettsia conorii/inmunología , Rickettsia typhi/inmunología , Animales , Anticuerpos Monoclonales , Fiebre Botonosa/sangre , Fiebre Botonosa/inmunología , Embrión de Pollo , Chlorocebus aethiops , Modelos Animales de Enfermedad , Citometría de Flujo , Interferón gamma/sangre , Interferón gamma/inmunología , Interleucina-12/sangre , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Infecciones por Rickettsia/sangre , Bazo/inmunología , Tifus Endémico Transmitido por Pulgas/sangre , Tifus Endémico Transmitido por Pulgas/inmunología , Células VeroRESUMEN
Cytotoxic T-lymphocyte (CTL) activity developed against the major infected target cells of rickettsial infections, endothelial cells and macrophages. Spleen cells from mice immune to Rickettsia conorii exerted specific major histocompatibility complex (MHC) class I-matched CTL activity against R. conorii-infected SVEC-10 endothelial cells, with peak activity on day 10. Similarly, spleen cells from Rickettsia australis-immune mice exerted specific CTL activity against an R. australis-infected macrophage-like cell line. Gamma interferon (IFN-gamma) gene knockout mice were more than 100-fold more susceptible to R. australis infection than wild-type C57BL/6 mice. MHC class I gene knockout mice were the most susceptible, more than 50,000-fold more susceptible to a lethal outcome of R. australis infection than wild-type C57BL/6 mice. These results indicate that CTL activity was more critical to recovery from rickettsial infection than were the effects of IFN-gamma. The observation that perforin gene knockout mice were more than 100-fold more susceptible than wild-type C57BL/6 mice indicates that perforin-mediated activity accounts for a large component, but not all, of the CTL-mediated antirickettsial effect. CTL activity was expressed by immune CD8 T lymphocytes. Adoptive transfer of immune CD8 T lymphocytes from IFN-gamma gene knockout mice into R. australis-infected IFN-gamma gene knockout mice dramatically reduced the infectious rickettsial content in the organs, confirming that CD8 T lymphocytes provide immunity against rickettsiae besides that provided by the secretion of IFN-gamma. CTLs appear to be crucial to recovery from rickettsial infection.
Asunto(s)
Fiebre Botonosa/inmunología , Infecciones por Rickettsia/inmunología , Linfocitos T Citotóxicos , Traslado Adoptivo , Animales , Apoptosis , Fiebre Botonosa/terapia , Endotelio Vascular/microbiología , Genes MHC Clase I , Inmunidad Innata , Interferón gamma/genética , Macrófagos/microbiología , Glicoproteínas de Membrana , Ratones , Ratones Noqueados , Perforina , Proteínas Citotóxicas Formadoras de Poros , Infecciones por Rickettsia/terapiaRESUMEN
Human monocytotropic ehrlichiosis caused by Ehrlichia chaffeensis is a life-threatening, tick-borne, emerging infectious disease for which no satisfactory animal model has been developed. Strain HF565, an ehrlichial organism closely related to E. chaffeensis isolated from Ixodes ovatus ticks in Japan, causes fatal infection of mice. C57BL/6 mice became ill on day 7 after inoculation and died on day 9. The liver revealed confluent necrosis, ballooning cell injury, apoptosis, poorly formed granulomas, Kupffer cell hyperplasia, erythrophagocytosis, and microvesicular fatty metamorphosis. The other significant histological findings consisted of marked expansion of the marginal zone and infiltration of the red pulp of the spleen by macrophages, interstitial pneumonitis, and increased numbers of immature myeloid cells and areas of necrosis in the bone marrow. Ehrlichiae were detected by immunohistology and electron microscopy in the liver, lungs, and spleen. The main target cells were macrophages, including Kupffer cells, hepatocytes, and endothelial cells. Apoptosis was detected in Kupffer cells, hepatocytes, and macrophages in the lungs and spleen. This tropism for macrophages and the pathological lesions closely resemble those of human monocytotropic ehrlichiosis for which it is a promising model for investigation of immunity and pathogenesis.
Asunto(s)
Ehrlichia , Ehrlichiosis/patología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Apoptosis , Médula Ósea/microbiología , Médula Ósea/patología , Modelos Animales de Enfermedad , Ehrlichia/inmunología , Ehrlichia chaffeensis/inmunología , Ehrlichiosis/metabolismo , Ehrlichiosis/microbiología , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Recuento de Leucocitos , Hígado/microbiología , Hígado/patología , Hígado/ultraestructura , Pulmón/microbiología , Pulmón/patología , Pulmón/ultraestructura , Ratones , Ratones Endogámicos C57BL , Recuento de Plaquetas , Bazo/microbiología , Bazo/patología , Bazo/ultraestructura , Distribución TisularRESUMEN
Rapid diagnosis of Mycobacterium avium complex (MAC) bacteremia is important for management of patients with the acquired immunodeficiency syndrome who have disseminated MAC. The purpose of this study was to determine the reliability of the MycoAKT latex agglutination test for direct detection of MAC in positive mycobacterial blood cultures. First, colonies of isolates of previously identified mycobacteria, including 35 MAC, were tested. Of the 55 isolates evaluated, 33 were identified as MAC by the latex test, including 31 of the known MAC and 2 M. chelonae (sensitivity, 88.6%; specificity, 90.0%). Second, broth from 20 ESP II and 20 BACTEC 12B bottles seeded with isolates of MAC were tested. Aliquots from 19 (95%) ESP II cultures and 16 (80%) 12B cultures were positive by the latex test. In phase 3, broth from 115 signal-positive ESP II blood cultures were tested by latex agglutination. Forty-three subcultures from these bottles grew mycobacteria (41 MAC and 2 Mycobacterium tuberculosis complex); the remainder grew no organisms. Broth from 40 of the blood cultures (39 that grew MAC and 1 from which no organisms were recovered) were latex positive; thus, the sensitivity, specificity, and positive and negative predictive values of the latex test for direct identification of MAC in ESP II blood cultures were 95.1, 98.6, 97.5, and 97.3%, respectively. The mean time to detection of MAC was 14.6 days (range, 6-34 days) with the direct latex test, compared with 18.3 days (range, 9-36 days) with subculture and probe (p < 0.05).
Asunto(s)
Bacteriemia/diagnóstico , Pruebas de Fijación de Látex , Infección por Mycobacterium avium-intracellulare/diagnóstico , Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Estudios de Evaluación como Asunto , Humanos , Complejo Mycobacterium avium/aislamiento & purificación , Infección por Mycobacterium avium-intracellulare/microbiología , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The Texas Department of Criminal Justice (TDCJ) houses many subjects with acquired immunodeficiency syndrome (AIDS) who receive medical care in a comprehensive AIDS treatment center. In this case-control autopsy survey, we compared pathological outcomes of TDCJ inmates treated at the center (n = 155) with nonincarcerated patients who died during the same period (n = 155). Using multiple regression analysis and a proportional hazards model, survival time in the prisoners was equivalent to that in the controls. With few exceptions, the prevalences of opportunistic viral, fungal, protozoal, and bacterial infections contributing to mortality were equivalent between groups. Mycobacterium tuberculosis was isolated more frequently in the inmates, and M avium intracellulare was isolated less frequently (P < .0001). The inmates had a higher prevalence of bacterial infection of the central nervous system (CNS) (9.1% v 1.4%; P < .006); half of all CNS bacterial infections were caused by M tuberculosis. Inmates had significantly lower prevalences of vacuolar myelopathy (P < .006) and severe wasting disease (P < .0009). We conclude that survival of prison inmates with AIDS treated in a comprehensive AIDS treatment center was equivalent to that of nonincarcerated subjects with AIDS. Prevalences of certain complications of AIDS differed in the inmates, showing that the prison environment influenced some of the underlying causes of AIDS morbidity and mortality.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/patología , Prisioneros , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Infecciones Oportunistas Relacionadas con el SIDA/mortalidad , Infecciones Oportunistas Relacionadas con el SIDA/patología , Síndrome de Inmunodeficiencia Adquirida/microbiología , Adulto , Ambiente , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/aislamiento & purificación , Análisis de Regresión , Tasa de Supervivencia , TexasRESUMEN
Fragmentation of genomic DNA, a major biochemical feature of programmed cell death (apoptosis), is easily detected when apoptosis is prevalent. In brain tissue apoptotic cells are usually scarce and detection requires more sensitive techniques. We describe a highly sensitive method to quantify apoptosis in frozen human brain tissue using flow cytometry. Nuclei from homogenized brain specimens were isolated to purity using discontinuous isopyknic centrifugation through 2.2 M sucrose. DNA strand breaks in apoptotic nuclei were conjugated with biotinylated-dUTP using terminal deoxynucleotidyl transferase (TdT) and tagged with streptavidin-conjugated FITC (TUNEL). Negative controls excluding the TdT step, and positive controls using DNAase pretreatment to create 3'-OH strand breaks were run in parallel. The proportion of nuclei with TdT-dependent labeling in adult brain specimens was < 0.01% in 6 out of 7 specimens. In 3 fetal brains it averaged 0.86 +/- 0.11%. Apoptotic cells were readily detected in 2 malignant glial neoplasms and in a patient with HIV encephalitis. Comparable frequencies of stained nuclei were present in adjacent specimens embedded in paraffin and labeled in situ. By screening millions of nuclei cytometry detected very rare apoptotic events, producing quantitative results using banked frozen brains. The method has potential applications to studies of human brain development, neurodegenerative diseases, and brain tumors.
Asunto(s)
Apoptosis , Encéfalo/citología , Citometría de Flujo , Bancos de Tejidos , Adulto , Anciano , Encéfalo/ultraestructura , Núcleo Celular/ultraestructura , Centrifugación Isopicnica , Femenino , Feto , Fluoresceína-5-Isotiocianato , Congelación , Técnicas Genéticas , Humanos , Lactante , Masculino , Microscopía Fluorescente , Persona de Mediana EdadRESUMEN
Massive steatosis has recently been described among a few human immunodeficiency virus-seropositive patients who were receiving antiretroviral therapy. Although clinical and light-microscopic pathological findings were carefully described, no ultrastructural studies of the liver were performed in these cases. We report the light-microscopic and ultrastructural findings at autopsy of a 35-year-old woman with AIDS who developed severe lactic acidosis and hepatic failure. The patient had been receiving standard doses of zidovudine for 5 months when she was hospitalized because of the rapid onset of abdominal pain, nausea, and vomiting. The most significant findings at autopsy were massive hepatomegaly and steatosis. Ultrastructural examination of the liver and skeletal muscle showed slightly enlarged mitochondria in the liver but no mitochondrial changes in the skeletal muscle. The pathogenesis of mitochondrial toxicity associated with antiviral therapies is briefly discussed.