Transcriptional repression of serum amyloid A1 by AP -2 contributes to its liver -specific expression

Studies on the transcriptional regulation of serum amyloid A1 (SAA1) gene, a liver specific acute-phase gene, identified a regulatory element in its promoter that functioned to repress (SAA1) gene transcription in nonliver cells. This silencer element interacts with a nuclear protein that is detectable in HeLa cells, fibroblasts and placental tissues but not in liver or liver-derived cells. As the expression pattern of this repressor is consistent with its potential regulatory role in repressing SAA1 expression, and that many other liver gene promoters also contain this repressor binding site, we sought to investigate whether this repressor may have a broader functional role in repressing liver genes. ^ We have utilized protein purification, cell culture, transient and stable gene transfection, and molecular biology approaches to identify this protein and investigate its possible function in the regulation of (SAA1) and other liver genes. Analyses of amino acid sequence of the purified nuclear protein, and western blot and gel shift studies identified the repressor as transcription factor AP-2 or AP-2-like protein. Using transient transfection of DNA into cultured cells, we demonstrate that AP-2 can indeed function as a repressor to inhibit transcription of SAA1 gene promoter. This conclusion is supported by the following experimental results: (1) overexpression of AP-2 in hepatoma cells inhibits conditioned medium (CM)-induced expression of SAA1 promoter; (2) binding of AP-2 to the SAA1 promoter is required for AP-2 repression function; (3) one mechanism by which AP-2 inhibits SAA1 may be by antagonizing the activation function of the strong transactivator NFκB; (4) mutation of AP-2 binding sites results in derepression of SAM promoter in HeLa cells; and (5) inhibition of endogenous AP-2 activity by a dominant-negative mutant abolishes AP-2's inhibitory effect on SAM promoter in HeLa cells. In addition to the SAM promoter, AP-2 also can bind to the promoter regions of six other liver genes tested, suggesting that it may have a broad functional role in restricting the expression of many liver genes in nonliver cells. Consistent with this notion, ectopic expression of AP-2 also represses CM-mediated activation of human third component of complement 3 promoter. Finally, in AP-2-expressing stable hepatoma cell lines, AP-2 inhibits not only the expression of endogenous SAA, but also the expression of several other endogenous liver genes including albumin, α-fetoprotein. ^ Our findings that AP-2 has the ability to repress the expression of liver genes in nonliver cells opens a new avenue of investigation of negative regulation of gene transcription, and should improve our understanding of tissue-specific expression of liver genes. In summary, our data provide evidence suggesting a novel role of AP-2 as a repressor, inhibiting the expression of liver genes in nonliver cells. Thus, the tissue-specific expression of AP-2 may constitute an important mechanism contributing to the liver-specific expression of liver genes. ^

Imagen Corporal y Calidad de vida de mayores incluidos en un programa de Rehabilitación Cardiaca

La mayor parte delas personas que conviven con algún tipo de enfermedad, tienden a adoptar un mayor número de hábitos saludables, pudiendo crear nuevas maneras de ver la vida y a sí mismos. El objetivo de este estudio fue verificar la relación entre índices de calidad de vida y percepción de la imagen corporal de los pacientes incluidos en un programa de rehabilitación cardiovascular en Florianópolis-Brasil. La muestra estuvo compuesta por 24 sujetos varones con una edad de 62 ± 1,3 años, portadores de Enfermedad Arterial Coronaria. Para evaluar la calidad de vida, se utilizó el cuestionario Minnesota Living With Heart Failure Questionnaire (MLHFQ) y para identificar el grado de descontento de la muestra con la imagen corporal, se aplicó el cuestionario de Stunkard y Sorensen (1993). El análisis de las pruebas fue hecho a través de un programa de estadísticas utilizando para tal fin, el software SPSS 11.0. El grado de asociación entre variables fue estudiado a partir del test de Kendall. Se verificó que cuanto mayor es el IMC y la silueta actual, mayor el grado de insatisfacción con la imagen corporal. Los síntomas emocionales también parecen estar correlacionados significativamente con un deseo de obtener una menor silueta corporal y con indicadores de menor calidad de vida (r= 0,474 y r= 0,735; p mayor 0,05). Los síntomas físicostambién se encuentran correlacionados de manera significativa con los síntomas emocionales. Estos resultados sugieren que las variables referentes a la calidad de vida poseen un peso significativo en la imagen corporal y la satisfacción con ésta, parece correlacionar con una menor cantidad de problemas emocionales y en un mejor afrontamiento de la enfermedad. Los programas de rehabilitación cardiovascular que implementan actividad física en los hábitos diarios se muestran adecuados como herramienta para la mejora de dichas dolencias en esta fase post aguda

Imagen Corporal y Calidad de vida de mayores incluidos en un programa de Rehabilitación Cardiaca

La mayor parte delas personas que conviven con algún tipo de enfermedad, tienden a adoptar un mayor número de hábitos saludables, pudiendo crear nuevas maneras de ver la vida y a sí mismos. El objetivo de este estudio fue verificar la relación entre índices de calidad de vida y percepción de la imagen corporal de los pacientes incluidos en un programa de rehabilitación cardiovascular en Florianópolis-Brasil. La muestra estuvo compuesta por 24 sujetos varones con una edad de 62 ± 1,3 años, portadores de Enfermedad Arterial Coronaria. Para evaluar la calidad de vida, se utilizó el cuestionario Minnesota Living With Heart Failure Questionnaire (MLHFQ) y para identificar el grado de descontento de la muestra con la imagen corporal, se aplicó el cuestionario de Stunkard y Sorensen (1993). El análisis de las pruebas fue hecho a través de un programa de estadísticas utilizando para tal fin, el software SPSS 11.0. El grado de asociación entre variables fue estudiado a partir del test de Kendall. Se verificó que cuanto mayor es el IMC y la silueta actual, mayor el grado de insatisfacción con la imagen corporal. Los síntomas emocionales también parecen estar correlacionados significativamente con un deseo de obtener una menor silueta corporal y con indicadores de menor calidad de vida (r= 0,474 y r= 0,735; p mayor 0,05). Los síntomas físicostambién se encuentran correlacionados de manera significativa con los síntomas emocionales. Estos resultados sugieren que las variables referentes a la calidad de vida poseen un peso significativo en la imagen corporal y la satisfacción con ésta, parece correlacionar con una menor cantidad de problemas emocionales y en un mejor afrontamiento de la enfermedad. Los programas de rehabilitación cardiovascular que implementan actividad física en los hábitos diarios se muestran adecuados como herramienta para la mejora de dichas dolencias en esta fase post aguda

Imagen Corporal y Calidad de vida de mayores incluidos en un programa de Rehabilitación Cardiaca

La mayor parte delas personas que conviven con algún tipo de enfermedad, tienden a adoptar un mayor número de hábitos saludables, pudiendo crear nuevas maneras de ver la vida y a sí mismos. El objetivo de este estudio fue verificar la relación entre índices de calidad de vida y percepción de la imagen corporal de los pacientes incluidos en un programa de rehabilitación cardiovascular en Florianópolis-Brasil. La muestra estuvo compuesta por 24 sujetos varones con una edad de 62 ± 1,3 años, portadores de Enfermedad Arterial Coronaria. Para evaluar la calidad de vida, se utilizó el cuestionario Minnesota Living With Heart Failure Questionnaire (MLHFQ) y para identificar el grado de descontento de la muestra con la imagen corporal, se aplicó el cuestionario de Stunkard y Sorensen (1993). El análisis de las pruebas fue hecho a través de un programa de estadísticas utilizando para tal fin, el software SPSS 11.0. El grado de asociación entre variables fue estudiado a partir del test de Kendall. Se verificó que cuanto mayor es el IMC y la silueta actual, mayor el grado de insatisfacción con la imagen corporal. Los síntomas emocionales también parecen estar correlacionados significativamente con un deseo de obtener una menor silueta corporal y con indicadores de menor calidad de vida (r= 0,474 y r= 0,735; p mayor 0,05). Los síntomas físicostambién se encuentran correlacionados de manera significativa con los síntomas emocionales. Estos resultados sugieren que las variables referentes a la calidad de vida poseen un peso significativo en la imagen corporal y la satisfacción con ésta, parece correlacionar con una menor cantidad de problemas emocionales y en un mejor afrontamiento de la enfermedad. Los programas de rehabilitación cardiovascular que implementan actividad física en los hábitos diarios se muestran adecuados como herramienta para la mejora de dichas dolencias en esta fase post aguda

Hypotension and inflammatory cytokine gene expression triggered by factor Xa–nitric oxide signaling

The signaling pathway initiated by factor Xa on vascular endothelial cells was investigated. Factor Xa stimulated a 5- to 10-fold increased release of nitric oxide (NO) in a dose-dependent reaction (0.1–2.5 μg/ml) unaffected by the thrombin inhibitor hirudin but abolished by active site inhibitors, tick anticoagulant peptide, or Glu-Gly-Arg-chloromethyl ketone. In contrast, the homologous clotting protease factor IXa or another endothelial cell ligand, fibrinogen, was ineffective. A factor Xa inter-epidermal growth factor synthetic peptide L83FTRKL88(G) blocking ligand binding to effector cell protease receptor-1 inhibited NO release by factor Xa in a dose-dependent manner, whereas a control scrambled peptide KFTGRLL was ineffective. Catalytically active factor Xa induced hypotension in rats and vasorelaxation in the isolated rat mesentery, which was blocked by the NO synthase inhibitor l-NG-nitroarginine methyl ester (l-NAME) but not by d-NAME. Factor Xa/NO signaling also produced a dose-dependent endothelial cell release of interleukin 6 (range 0.55–3.1 ng/ml) in a reaction inhibited by l-NAME and by the inter-epidermal growth factor peptide Leu83–Leu88 but unaffected by hirudin. Maximal induction of interleukin 6 mRNA required a brief, 30-min stimulation with factor Xa, unaffected by subsequent addition of tissue factor pathway inhibitor. These data suggest that factor Xa-induced NO release modulates endothelial cell-dependent vasorelaxation and cytokine gene expression. This pathway requiring factor Xa binding to effector cell protease receptor-1 and a secondary step of ligand-dependent proteolysis may preserve an anti-thrombotic phenotype of endothelium but also trigger acute phase responses during activation of coagulation in vivo.

Recruitment of IRAK to the interleukin 1 receptor complex requires interleukin 1 receptor accessory protein

The proinflammatory cytokine interleukin 1 (IL-1) activates the transcription of many genes encoding acute phase and proinflammatory proteins, a function mediated primarily by the transcription factor NF-κB. An early IL-1 signaling event is the recruitment of the Ser/Thr kinase IRAK to the type I IL-1 receptor (IL-1RI). Here we describe the function of a previously identified IL-1 receptor subunit designated IL-1 receptor accessory protein (IL-1RAcP). IL-1 treatment of cells induces the formation of a complex containing both IL-1RI and IL-1RAcP. IRAK is recruited to this complex through its association with IL-1RAcP. Overexpression of an IL-1RAcP mutant lacking its intracellular domain, the IRAK-binding domain, prevented the recruitment of IRAK to the receptor complex and blocked IL-1-induced NF-κB activation.

Fetuin (α2-HS-glycoprotein) opsonizes cationic macrophagedeactivating molecules

Macrophages become activated by bacterial endotoxin (lipopolysaccharide) and other stimuli to release proinflammatory cytokines and NO. To prevent release of toxic or potentially lethal quantities of these factors, the state of macrophage activation is counter-regulated by anti-inflammatory mediators (e.g., glucocorticoid hormones, interleukin 10, and transforming growth factor type β). Fetuin, a negative acute-phase protein, recently was implicated as an anti-inflammatory mediator, because it is required for macrophage deactivation by spermine. In the present studies, we found that fetuin is necessary for macrophages to respond to CNI-1493, a tetravalent guanylhydrazone inhibitor of p38 mitogen-activated protein kinase phosphorylation. Fetuin dose-dependently increases macrophage uptake of CNI-1493, which can be specifically inhibited by anti-human fetuin antibodies. Anti-human fetuin antibodies render primary human peripheral blood mononuclear cells insensitive to deactivation by CNI-1493. Thus, macrophages use fetuin as an opsonin for cationic-deactivating molecules, both endogenous (e.g., spermine) and pharmacologic (e.g., CNI-1493). This role of fetuin as an opsonic participant in macrophage-deactivating mechanisms has implications for understanding and manipulating the innate immune response.

Characterization of serum amyloid A protein mRNA expression and secondary amyloidosis in the domestic duck

Secondary amyloidosis is a common disease of water fowl and is characterized by the deposition of extracellular fibrils of amyloid A (AA) protein in the liver and certain other organs. Neither the normal role of serum amyloid A (SAA), a major acute phase response protein, nor the causes of secondary amyloidosis are well understood. To investigate a possible genetic contribution to disease susceptibility, we cloned and sequenced SAA cDNA derived from livers of domestic ducks. This revealed that the three C-terminal amino acids of SAA are removed during conversion to insoluble AA fibrils. Analysis of SAA cDNA sequences from several animals identified a distinct genetic dimorphism that may be relevant to susceptibility to secondary amyloid disease. The duck genome contained a single copy of the SAA gene that was expressed in liver and lung tissue of ducklings, even in the absence of induction of acute phase response. Genetic analysis of heterozygotes indicated that only one SAA allele is expressed in livers of adult birds. Immunofluorescence staining of livers from adult ducks displaying early symptoms of amyloidosis revealed what appear to be amyloid deposits within hepatocytes that are expressing unusually high amounts of SAA protein. This observation suggests that intracellular deposition of AA may represent an early event during development of secondary amyloidosis in older birds.

Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein

The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

Multiple modes of cellular activation and virus transmission in HIV infection: A role for chronically and latently infected cells in sustaining viral replication

CD4+ T cell activation, required for virus replication in these cells, occurs in local microenvironmental domains in transient bursts. Thus, although most HIV originates from short-lived virus-producing cells, it is unlikely that chronic infection is generally sustained in rapid continuous cycles of productive infection as has been proposed. Such continuity of productive infection cycles would depend on efficient long-range transmission of HIV from one set of domains to another, in turn requiring the maintenance of sufficiently high concentrations of cell-free virus across lymphoid tissues at all times. By contrast, long-lived cellular sources of HIV maintain the capacity to infect newly activated cells at close range despite the temporal and spatial discontinuities of activation events. Such proximal activation and transmission (PAT) involving chronically and latently infected cells may be responsible for sustained infection, particularly when viral loads are low. Once CD4 cells are productively infected through PAT, they can infect other activated cells in their immediate vicinity. Such events propagate locally but generally do not spread systemically, unlike in the acute phase of the infection, because of the early establishment of protective anergy. Importantly, antiretroviral drug treatment is likely to differentially impact long-range transmission and PAT.

Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer binding protein δ

CCAAT/enhancer binding protein δ (C/EBPδ) is a transcriptional regulator implicated in the hepatic acute phase response and in adipogenic and myeloid cell differentiation. We found that C/EBPδ is widely expressed in the peripheral and central nervous systems, including neurons of the hippocampal formation, indicating a role in neural functions. To examine the role of C/EBPδ in vivo, we generated mice with a targeted deletion of the C/EBPδ gene. This mutation does not interfere with normal embryonic and postnatal development. Performance in a battery of behavioral tests indicates that basic neurological functions are normal. Furthermore, performance in a Morris water maze task suggests that C/EBPδ mutant mice have normal spatial learning. However, in the contextual and auditory-cue-conditioned fear task, C/EBPδ null mice displayed significantly more conditioned freezing to the test context than did wild-type controls, but equivalent conditioning to the auditory cue. These data demonstrate a selectively enhanced contextual fear response in mice carrying a targeted genomic mutation and implicate C/EBPδ in the regulation of a specific type of learning and memory.

Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation

Liver-specific and nonliver-specific methionine adenosyltransferases (MATs) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S-adenosylmethionine (AdoMet), the principal biological methyl donor. Mature liver expresses MAT1A, whereas MAT2A is expressed in extrahepatic tissues and is induced during liver growth and dedifferentiation. To examine the influence of MAT1A on hepatic growth, we studied the effects of a targeted disruption of the murine MAT1A gene. MAT1A mRNA and protein levels were absent in homozygous knockout mice. At 3 months, plasma methionine level increased 776% in knockouts. Hepatic AdoMet and glutathione levels were reduced by 74 and 40%, respectively, whereas S-adenosylhomocysteine, methylthioadenosine, and global DNA methylation were unchanged. The body weight of 3-month-old knockout mice was unchanged from wild-type littermates, but the liver weight was increased 40%. The Affymetrix genechip system and Northern and Western blot analyses were used to analyze differential expression of genes. The expression of many acute phase-response and inflammatory markers, including orosomucoid, amyloid, metallothionein, Fas antigen, and growth-related genes, including early growth response 1 and proliferating cell nuclear antigen, is increased in the knockout animal. At 3 months, knockout mice are more susceptible to choline-deficient diet-induced fatty liver. At 8 months, knockout mice developed spontaneous macrovesicular steatosis and predominantly periportal mononuclear cell infiltration. Thus, absence of MAT1A resulted in a liver that is more susceptible to injury, expresses markers of an acute phase response, and displays increased proliferation.

In vitro activation of human herpesviruses 6 and 7 from latency.

Human herpesviruses 6 and 7 (HHV-6 and HHV-7) are prevalent lymphotropic viruses that infect more than 80% of children at infancy or during early childhood. Infection ranges from asymptomatic to severe disease. HHV-6B causes exanthem subitum. The virus can be recovered from peripheral blood mononuclear cells during the acute phase of exanthem subitum, but the host remains latently infected throughout life. In immunocompromised patients undergoing kidney, liver, or bone marrow transplantation latent HHV-6B is reactivated, at times causing severe or fatal disease. Here, we describe the establishment of an in vitro system for reactivation of HHV-6B and HHV-7 from latency. HHV-7 is reactivated from latently infected peripheral blood mononuclear cells by T-cell activation. HHV-6B could not be reactivated under similar conditions; however, the latent HHV-6B could be recovered after the cells were infected with HHV-7. Once reactivated, the HHV-6B genomes became prominent and the HHV-7 disappeared. We conclude that HHV-7 can provide a transacting function(s) mediating HHV-6 reactivating from latency. Understanding the activation process is critical for the development of treatments to control the activation of latent viruses so as to avoid these sometimes life threatening infections in transplant recipients.

Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.

Human V delta 2+ gamma delta T-cell tolerance to foreign antigens of Toxoplasma gondii.

Little is known about the mechanisms involved in human gammadelta T-cell tolerance to self or to foreign antigens. Patients with congenital toxoplasmosis offer a unique opportunity to examine Vdelta2+ gammadelta T-cell tolerance. Analysis of gammadelta T cells in patients with congenital toxoplasmosis revealed evidence for anergy of these cells with or without clonal Vdelta2+ gammadelta T-cell expansion in the acute phase of the Toxoplasma infection. T cells in general were unresponsive and did not proliferate upon exposure to mitogens or to Toxoplasma lysate antigens or in response to live Toxoplasma-infected cells when the congenitally infected infants were 1 month of age, and they exhibited selective anergy to Toxoplasma lysate antigens and live Toxoplasma-infected cells when the infants were aged 5 months. During the chronic phase of congenital toxoplasmosis in the patients who were more than I year of age, the repertoires of the gammadelta T-cell receptors were found to be within normal ranges. In addition, in the chronic phase, the gammadelta T cells proliferated and secreted gamma-interferon in response to exposure to live Toxoplasmia-infected cells. By contrast, alphabeta T cells remained anergic. Vdelta2+ gammadelta T cells have been considered to undergo extrathymic maturation and thus to be subject to development of peripheral tolerance. Our findings indicate that Vdelta2+ gammadelta T-cell tolerance was lost in these infected infants earlier than alphabeta T-cell tolerance. These findings suggest that gammadelta T cells play a role in protection against Toxoplasma gondii in the chronic phase when congenitally infected children are more than 1 year of age, especially in those in whom alphabeta T cells continue to exhibit deficits in specific immune responses to Toxoplasma antigens.