RESUMO
In 2013, the national surveillance case definition for West Nile virus (WNV) disease was revised to remove fever as a criterion for neuroinvasive disease and require at most subjective fever for non-neuroinvasive disease. The aims of this project were to determine how often afebrile WNV disease occurs and assess differences among patients with and without fever. We included cases with laboratory evidence of WNV disease reported from four states in 2014. We compared demographics, clinical symptoms and laboratory evidence for patients with and without fever and stratified the analysis by neuroinvasive and non-neuroinvasive presentations. Among 956 included patients, 39 (4%) had no fever; this proportion was similar among patients with and without neuroinvasive disease symptoms. For neuroinvasive and non-neuroinvasive patients, there were no differences in age, sex, or laboratory evidence between febrile and afebrile patients, but hospitalisations were more common among patients with fever (P < 0.01). The only significant difference in symptoms was for ataxia, which was more common in neuroinvasive patients without fever (P = 0.04). Only 5% of non-neuroinvasive patients did not meet the WNV case definition due to lack of fever. The evidence presented here supports the changes made to the national case definition in 2013.
Assuntos
Doenças Assintomáticas/epidemiologia , Febre/epidemiologia , Febre do Nilo Ocidental/diagnóstico , Febre do Nilo Ocidental/epidemiologia , Vírus do Nilo Ocidental/isolamento & purificação , California/epidemiologia , Técnicas de Laboratório Clínico/métodos , Feminino , Febre/diagnóstico , Humanos , Incidência , Louisiana/epidemiologia , Masculino , Massachusetts/epidemiologia , Minnesota/epidemiologia , Vigilância da População , Estudos Retrospectivos , Medição de Risco , Índice de Gravidade de DoençaRESUMO
AIMS: We report on a measles outbreak largely occurring in Minnesota's under-vaccinated Somali community in the spring of 2017. The outbreak was already into its third generation when the first two cases were confirmed, and rapid public health actions were needed. The aim of our response was to quickly end transmission and contain the outbreak. METHODS: The state public health department performed laboratory testing on suspect cases and activated an Incident Command staffed by subject matter experts that was operational within 2 h of case confirmation. Epidemiologic interviews identified exposures in settings where risk of transmission was high, that is, healthcare, childcare, and school settings. Vaccination status of exposed persons was assessed, and postexposure prophylaxis (PEP) was offered, if applicable. Exposed persons who did not receive PEP were excluded from childcare centers or schools for 21 days. An accelerated statewide measles, mumps, and rubella (MMR) recommendation was made for Somali Minnesota children and children in affected outbreak counties. Partnerships with the Somali Minnesota community were deepened, building off outreach work done with the community since 2008. RESULTS: Public health identified 75 measles cases from 30 March to 25 August 2017: 43% were female, 81% Somali Minnesotan, 91% unvaccinated, and 28% hospitalized. The median age of cases was 2 years (range: 3 months-57 years). Most transmission (78%) occurred in childcare centers and households. A secondary attack rate of 91% was calculated for unvaccinated household contacts. Over 51,000 doses of MMR were administered during the outbreak above expected baseline. At least 8490 individuals were exposed to measles; 155 individuals received PEP; and over 500 persons were excluded from childcare and school. State and key public health partners spent an estimated $2.3 million on response. CONCLUSION: This outbreak demonstrates the necessity of immediate, targeted disease control actions and strong public health, healthcare, and community partnerships to end a measles outbreak.
Assuntos
Controle de Doenças Transmissíveis/organização & administração , Sarampo/epidemiologia , Sarampo/prevenção & controle , Adolescente , Adulto , Criança , Pré-Escolar , Controle de Doenças Transmissíveis/economia , Surtos de Doenças , Feminino , Humanos , Programas de Imunização/organização & administração , Lactente , Masculino , Vacina contra Sarampo-Caxumba-Rubéola/administração & dosagem , Pessoa de Meia-Idade , Minnesota/epidemiologia , Profilaxia Pós-Exposição/organização & administração , Adulto JovemRESUMO
Human hepatocyte growth factor (hHGF) has recently been expressed as a recombinant polypeptide from Chinese hampster ovary cell transfectants. Using a primary rat hepatocyte bioassay, we have tested the biological activity of recombinant hHGF and compared it with native hHGF. Dose-response curves were almost identical, with half-maximal stimulation of DNA synthesis at 1-2 ng/ml (equivalent to approximately 10 pM). S-phase labeling index was similarly enhanced and numerous mitotic cells were observed. Recombinant and native hHGF also stimulated DNA synthesis and S-phase labeling index in primary adult human hepatocytes. Human cells were more responsive than rat hepatocytes, with recombinant hHGF slightly more potent than native hHGF (half-maximal stimulation 0.3 and 0.6 ng/ml, respectively). Since HGF levels rise in patients with fulminant hepatic failure and in animals after partial hepatectomy or administration of hepatotoxins, situations where liver regeneration occurs, HGF is suggested to play a key role in regulation of hepatic growth. The high potency of the factor on human hepatocytes reinforces its candidacy as a critical mitogen in human liver growth. The availability of a recombinant hHGF opens the way for in vivo experimental studies and to the possibility of using hHGF as a clinical therapeutic agent, either alone or in combination with other factors.
Assuntos
DNA/biossíntese , Substâncias de Crescimento/farmacologia , Fígado/efeitos dos fármacos , Adulto , Animais , Relação Dose-Resposta a Droga , Substâncias de Crescimento/análise , Fator de Crescimento de Hepatócito , Humanos , Fígado/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Proteínas Recombinantes/farmacologiaRESUMO
In previous studies, intrahepatic human biliary epithelial cells (BEC) were isolated in high purity. However, these cells demonstrated only limited growth responses. Here we report that human BEC proliferate in response to human hepatocyte growth factor (hHGF), retain BEC-specific phenotype, and can be serially passaged. BEC showed dose-dependent growth in response to 0.01-100 ng/ml hHGF. The maximum S-phase labeling index reached 40% with half-maximal stimulation at 1 ng/ml. The response of cells from normal and primary biliary cirrhotic liver to hHGF was similar. Cultures were immunostained with specific antibodies and then processed for [3H]thymidine autoradiography. Proliferating cells expressed BEC-specific markers (HEA125 and CK-19), but were negative for desmin and factor VIII-related antigen. Occasional vimentin-positive cells were observed, but these were nonproliferative. In conclusion, cells responding to hHGF were clearly BEC in origin. The observation that HGF is mitogenic for BEC as well as hepatocytes has important implications. First, greater yields of intrahepatic BEC are available for subsequent studies of the pathogenesis and etiology of diseases of the biliary epithelium. Secondly, some means of regulating the cellular response to HGF in vivo must operate, in that HGF levels rise early after partial hepatectomy and yet BEC proliferate 24 h later than hepatocytes.
Assuntos
Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Animais , Ductos Biliares Intra-Hepáticos/química , Ductos Biliares Intra-Hepáticos/citologia , Células CHO , Divisão Celular , Células Cultivadas , Cricetinae , Epitélio/efeitos dos fármacos , Humanos , Vimentina/análiseRESUMO
Mammalian cells take up exogenous DNA very inefficiently. However, in the absence of viral vectors, DNA can be transfected into cells by co-precipitation with calcium phosphate and usually also with carrier DNA or by lipofection or electroporation. Such DNA can be expressed efficiently by cells. Alternatively, direct injection can also result in uptake and expression of transgenes. Without carefully designed means to target DNA specifically to integrate into the host genome, the vast majority of internalised DNA remains extra-chromosomal and is degraded. The likely fate of DNA which in low levels may contaminate vaccines derived from mammalian cell lines, will also be destruction. There is a theoretical risk of DNA integration events with random sequences of donor-derived DNA but the probability of that leading to serious adverse effects to the host is extremely small.
Assuntos
DNA/metabolismo , Administração Oral , Animais , Transporte Biológico , Linhagem Celular , Células Cultivadas , Desoxirribonucleases/metabolismo , Endocitose , Endossomos/fisiologia , Humanos , Mamíferos , Vacinas de DNA/administração & dosagemRESUMO
A hypernephroma removed from a male patient who had lost 30 kg in weight in the 2 months preceding surgery was established in immunosuppressed CBA/Lac mice as a nonmetastasizing transplantable xenograft. The xenografted tumors, although comprising less than 5% of the total body weight of the mice, produced considerable weight loss (greater than 25%). A slight reduction in food intake of tumor-bearing mice was noted, but some animals bearing mouse or human tumors not inducing cachexia had equally low food intake without accompanying weight losses. No obvious defects in gastrointestinal histology or absorption were observed. The precise mechanism(s) producing the severe cachexia remains to be established.
Assuntos
Caquexia/etiologia , Neoplasias Experimentais/complicações , Adenocarcinoma/complicações , Animais , Água Corporal/análise , Peso Corporal , Modelos Animais de Doenças , Ingestão de Energia , Feminino , Humanos , Neoplasias Renais/complicações , Camundongos , Camundongos Endogâmicos CBA , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Tamanho do Órgão , Transplante HeterólogoRESUMO
The regulation of DNA replication by growth hormone and the production of somatomedin C/insulin-like growth factor I (SM-C/IGF-I) and insulin by fetal rat islets in culture has been studied. Islets were cultured for 3 days in medium containing 2.7 or 16.7 mM glucose, various concentrations of fetal calf serum (FCS), and 100-1000 ng/ml human growth hormone (GH). DNA replication was determined by incorporation of [3H]thymidine into islet DNA; SM-C/IGF-I and insulin secreted into the medium were measured by specific radioimmunoassays. Glucose caused a twofold stimulation of islet DNA replication in medium containing greater than or equal to 1% FCS but failed to stimulate DNA replication at lower serum concentrations. In the presence of 16.7 mM glucose, GH (100-1000 ng/ml) stimulated DNA replication at all serum concentrations. In medium containing 2.7 mM glucose, GH was stimulatory only in the presence of 1% FCS. Somatomedin C/IGF-I release into the culture medium could be detected in all experimental groups. Glucose alone did not affect SM-C/IGF-I release, and in serum concentrations less than 0.1% FCS, GH also failed to increase the release of the peptide. In medium containing 1% FCS and 16.7 mM glucose, 100-1000 ng/ml GH caused a 50-100% increase in SM-C/IGF-I release into the medium. Addition of 100 ng/ml exogenous SM-C/IGF-I to medium containing 16.7 mM glucose and 0.1-1.0% FCS caused a twofold stimulation of the islet DNA replication. This effect could be abolished by the addition of an antibody to SM-C/IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Replicação do DNA/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Fator de Crescimento Insulin-Like I/biossíntese , Ilhotas Pancreáticas/efeitos dos fármacos , Somatomedinas/biossíntese , Animais , Células Cultivadas , Feminino , Feto/metabolismo , Insulina/metabolismo , Secreção de Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Patients with glucocorticoid excess develop central obesity, yet in simple obesity, circulating glucocorticoid levels are normal. We have suggested that the increased activity and expression of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) generating active cortisol from cortisone within adipose tissue may be crucial in the pathogenesis of obesity. In this study primary cultures of human hepatocytes and adipose stromal cells (ASC) were used as in vitro models to investigate the tissue-specific regulation of 11betaHSD1 expression and activity. Treatment with tumor necrosis factor-alpha (TNFalpha) caused a dose-dependent increase in 11betaHSD1 activity in primary cultures of both sc [1743.1 +/- 1015.4% (TNFalpha, 10 ng/ml); P < 0.05 vs. control (100%)] and omental [375.8 +/- 57.0% (TNFalpha, 10 ng/ml); P < 0.01 vs. control (100%)] ASC, but had no effect on activity in human hepatocytes [90.2 +/- 2.8% (TNFalpha, 10 ng/ml); P = NS vs. control (100%)]. Insulin-like growth factor I (IGF-I) caused a dose-dependent inhibition of 11betaHSD1 activity in sc [49.7 +/- 15.0% (IGF-I, 100 ng/ml]; P < 0.05 vs. control (100%)] and omental [71.6 +/- 7.5 (IGF-I, 100 ng/ml); P < 0.01 vs. control (100%)] stromal cells, but not in human hepatocytes [101.8 +/- 15.7% (IGF-I, 100 ng/ml); P = NS vs. control (100%)]. Leptin treatment did not alter 11betaHSD1 activity in human hepatocytes, but increased activity in omental ASC [135.8 +/- 14.1% (leptin, 100 ng/ml); P = 0.08 vs. control (100%)]. Treatment with interleukin-1beta induced 11betaHSD1 activity and expression in sc and omental ASC in a time- and dose-dependent manner. 15-Deoxy-12,14-PGJ2, the putative endogenous ligand of the orphan nuclear receptor peroxisome proliferator-gamma, significantly increased 11betaHSD1 activity in omental cells [179.7 +/- 29.6% (1 microM); P < 0.05 vs. control (100%)] and sc [185.3 +/- 12.6% (1 microM); P < 0.01 vs. control (100%)] ASC, and it is possible that expression of this ligand may ensure continued cortisol generation to permit adipocyte differentiation. Protease inhibitors used in the treatment of human immunodeficiency virus infection are known to cause a lipodystrophic syndrome and central obesity, but saquinavir, indinavir, and neflinavir caused a dose-dependent inhibition of 11betaHSD1 activity in primary cultures of human omental ASC. 11betaHSD1 expression is increased in human adipose tissue by TNFalpha, interleukin-1beta, leptin, and orphan nuclear receptor peroxisome proliferator-gamma agonists, but is inhibited by IGF-I. This autocrine and/or paracrine regulation is tissue specific and explains recent clinical data and animal studies evaluating cortisol metabolism in obesity. Tissue-specific 11betaHSD1 regulation offers the potential for selective enzyme inhibition within adipose tissue as a novel therapy for visceral obesity.
Assuntos
Tecido Adiposo/enzimologia , Citocinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hidroxiesteroide Desidrogenases/genética , Isoenzimas/genética , 11-beta-Hidroxiesteroide Desidrogenases , Tecido Adiposo/citologia , Células Cultivadas , Humanos , Indinavir/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Interleucina-1/farmacologia , Leptina/farmacologia , Especificidade de Órgãos , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Células Estromais/enzimologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The specific binding of human placental lactogen (hPL) and human GH (hGH) to particulate cell membranes from human fetal liver and skeletal muscle at 12-19 weeks gestation was examined. Fetal liver and muscle specifically bound [125I]hPL. This binding was inhibited by increasing concentrations of unlabeled hPL (half-maximal concentrations, 2.2 and 3.4 nmol/L, respectively). Scatchard analysis of the hepatic membrane binding revealed curvilinear plots with higher (Kd, 2.2 nmol/L) and lower (Kd, 24 nmol/L) affinity sites, while binding to muscle involved a single receptor class of Kd 5.6 nmol/L. The binding capacities for the two hepatic sites correlated positively with fetal body weight. [125I]hGH specifically bound to liver, but not muscle, with higher (Kd, 1.6 nmol/L) and lower (Kd, 8.6 nmol/L) affinity sites. [125I]PRL bound to hepatic membranes, but was preferentially displaced by hPL or hGH. Between 4 and 500 micrograms/L (mean, 82 micrograms/L, 3.8 nmol/L) hPL were present in fetal plasma. The findings identify distinct hPL receptors in human fetal liver and skeletal muscle and a hepatic hGH receptor in midgestation.
Assuntos
Desenvolvimento Embrionário e Fetal , Feto/metabolismo , Lactogênio Placentário/sangue , Receptores de Peptídeos , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/metabolismo , Sangue Fetal , Hormônio do Crescimento/metabolismo , Humanos , Membranas/metabolismo , Microssomos/metabolismo , Microssomos Hepáticos/metabolismo , Músculos/metabolismo , Concentração Osmolar , Lactogênio Placentário/metabolismo , Prolactina/metabolismoRESUMO
The two major insulin-like growth factor-binding protein (IGFBP) species in the human circulation, IGFBP-1 and IGFBP-3, are synthesized in large amounts by liver. To determine which hepatic cell populations in human liver were responsible for the synthesis and release of these IGFBPs, we 1) performed in situ hybridization with specific complementary RNA (RNA) probes for human IGFBP-1 or-3 or performed immunohistochemical analysis to reveal the sites of messenger RNA (mRNA) presence and peptide translation, respectively, in sections of normal liver derived from organ donors; and 2) examined the release of IGFBP species by Western ligand and immunoblots of medium conditioned by isolated cultures of hepatocytes, lipocytes, and Kupffer cells. In situ hybridization showed that IGFBP-1 mRNA was distributed widely among the parenchymal cell population, which also showed immunohistochemical staining for IGFBP-1 peptide. Conversely, IGFBP-3 mRNA and immunoreactive peptide were mainly localized to Kupffer cells, which were positively identified by immunoreactivity with antiserum against the glycoprotein marker, CD68. Isolated hepatocytes released two species of IGFBP of 28 and 30-32 kilodaltons, which were recognized immunologically as IGFBP-1. Isolated Kupffer cells released only a 43- to 46-kilodalton IGFBP immunologically recognized as IGFBP-3. Lipocyte cultures released no detectable IGFBP species. The results suggest that IGFBP-1 and IGFBP-3 are derived from separate cell populations in human liver.
Assuntos
Proteínas de Transporte/biossíntese , Fígado/metabolismo , Adolescente , Adulto , Western Blotting , Proteínas de Transporte/genética , Células Cultivadas , Meios de Cultivo Condicionados , Humanos , Imuno-Histoquímica , Hibridização In Situ , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Células de Kupffer/metabolismo , Fígado/citologia , Masculino , Pessoa de Meia-Idade , Sondas RNA , RNA Mensageiro/análiseRESUMO
We compared the abilities of human placental lactogen (hPL), somatomedin-C/insulin-like growth factor I (SM-C/IGF-I), multiplication-stimulating activity (MSA), and insulin to induce a rapid anabolic event, the uptake of the nonmetabolizable amino acid [3H]alpha-aminoisobutyric acid ([3H] AIB) or the more long term action of increasing [3H]thymidine incorporation, as a measure of DNA synthesis, in isolated human fetal fibroblasts and myoblasts. Myoblasts were derived from skeletal muscle and fibroblasts from skin explants removed from human fetuses delivered between 12 and 19 weeks gestation after prostaglandin-induced abortion. Each of the four peptides caused a dose-dependent increase in [3H]AIB uptake by both fibroblasts and myoblasts, with mean half-maximal concentrations (ED50) ranging from 0.9-1.9 nM. The concentration of each peptide required to stimulate [3H]thymidine uptake was significantly greater, with the exception of insulin, which was inactive. For myoblast cultures, the mean ED50 values were: hPL, 7.9 nM; SM-C/IGF-I, 2.0 nM; and MSA, 2.2 nM. For fibroblast cultures, the mean ED50 values were: hPL, 2.3 nM; SM-C/IGF-I, 3.3 nM; and MSA, 4.3 nM. Insulin did not stimulate [3H]thymidine incorporation into either cell type at concentrations up to 6.9 nM. Incubation in the presence of monoclonal antibody against SM-C/IGF-I abolished the ability of SM-C/IGF-I to stimulate either [3H]thymidine or [3H]AIB uptake into fetal fibroblasts. The antibody substantially inhibited the incorporation of [3H]thymidine by these cells in response to hPL, but was less effective in blocking hPL-stimulated [3H]AIB uptake. It did not inhibit the uptake of either radioisotope in response to MSA or [3H]AIB uptake in response to insulin. The actions of SM-C/IGF-I and hPL on thymidine incorporation were additive at submaximal concentrations, but not so at maximal individual concentrations. Their actions on AIB uptake were additive at both submaximal and maximal concentrations. The results suggest that hPL as well as the SMs may contribute to the growth stimulus in human fetal connective tissues. Since incubation with SM-C/IGF-I antibody reduced the mitogenic response of fetal cells to hPL, the actions on DNA synthesis may be partially mediated by local release of SM. However, the similar ED50 values with which these peptides stimulated [3H]AIB uptake during a short incubation, and their additive effects at maximal individual concentrations, suggest that hPL may also have direct actions.
Assuntos
Aminoácidos/metabolismo , DNA/biossíntese , Feto/metabolismo , Fator de Crescimento Insulin-Like II/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Insulina/farmacologia , Lactogênio Placentário/farmacologia , Somatomedinas/farmacologia , Ácidos Aminoisobutíricos/metabolismo , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Músculos/embriologia , Músculos/metabolismo , Timidina/metabolismoRESUMO
As the liver represents a major target organ for thyroid hormone action, we compared the expression of thyroid hormone receptor (TR) alpha and beta variants in normal human liver and liver affected by primary biliary cirrhosis, sclerosing cholangitis, cryptogenic cirrhosis, and alcoholic cirrhosis (n = 6 in each group). Western blot analysis using specific polyclonal antibodies to alpha 1 or beta 1 TRs or to the related non-T3-binding c-erbA alpha 2 variant revealed abundant expression of TRs in normal and diseased liver, with no difference in size or abundance of TR proteins. Immunocytochemistry likewise revealed abundant nuclear expression of TR proteins in normal and diseased liver, with similar patterns and intensity of staining. Despite abundant TR protein expression, Northern blot hybridization of polyadenylated ribonucleic acid (RNA; 10 micrograms) to TR complementary DNAs revealed only a weak signal for c-erbA alpha 2 messenger RNA (mRNA). Comparison of the level of expression of the thyroid hormone-regulated mRNAs encoding T4-binding globulin, sex hormone-binding globulin, cortisol-binding globulin, and transthyretin in normal and diseased tissue revealed no significant difference, suggesting that hepatocellular expression of these mRNAs is maintained in chronic liver disease despite a marked reduction in circulating T3 concentrations.
Assuntos
Hepatopatias/metabolismo , Fígado/química , Receptores dos Hormônios Tireóideos/análise , Animais , Western Blotting , Doença Crônica , Humanos , Imuno-Histoquímica , Camundongos , RNA Mensageiro/análise , Ratos , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Hormônios Tireóideos/sangue , Tireotropina/sangueRESUMO
To assess DNA damage caused by lipid peroxidation due to copper and iron storage disorders in the human liver, the formation of the etheno adducts 1,N6-ethenodeoxyadenosine (epsilon dA) and 3,N4-ethenodeoxycytine (epsilon dC) was measured in liver DNA from normal subjects and from patients with Wilson's disease (WD) and primary hemochromatosis. The mean epsilon dA and epsilon dC levels per 10(9) parent nucleotides in normal liver were 19.3 +/- 4.9 and 27.5 +/- 10.0, respectively. The mean epsilon dA and epsilon dC levels per 10(9) parent nucleotides in WD were 61.03 +/- 7.95 and 91.50 +/- 36.02, and in primary hemochromatosis, they were 46.62 +/- 32.83 and 64.32 +/- 11.55, respectively, two to three times higher than those in the normal liver. The etheno adduct levels were highly correlated with the copper content of the liver in the normal and WD samples. This study demonstrates for the first time the formation of promutagenic etheno adducts in humans in association with copper and iron storage-induced lipid peroxidation. Thus, the etheno adducts are implicated as initiating DNA damage in copper/iron-induced carcinogenesis in humans and should also be explored as biomarkers in disease progression and prevention trials.
Assuntos
Adutos de DNA/metabolismo , Hemocromatose/genética , Degeneração Hepatolenticular/genética , Peroxidação de Lipídeos/genética , Fígado/metabolismo , Erros Inatos do Metabolismo dos Metais/genética , Adolescente , Adulto , Idoso , Criança , Cobre/metabolismo , DNA/química , DNA/genética , Adutos de DNA/genética , Desoxiadenosinas/análise , Feminino , Hemocromatose/metabolismo , Degeneração Hepatolenticular/metabolismo , Humanos , Recém-Nascido , Ferro/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
The levels of malic-enzyme mRNA and activity were determined in primary cultures of adult rat hepatocytes maintained on either rat-tail collagen or a laminin-rich substratum. Cells plated on laminin-rich gels exhibited substantially improved patterns of albumin and malic-enzyme expression when compared with cells maintained on rat-tail collagen. Moreover, hepatocytes plated on the laminin-rich matrix displayed marked malic-enzyme inducibility in response to tri-iodothyronine and dichloroacetate, especially in the presence of insulin. However, Northern blot analysis revealed that the ratio of the amounts of the two major malic-enzyme mRNA species (2.0 and 3.1 kb) was reversed when compared with that found in the liver in vivo, the altered levels of these two species being closer to those found in non-hepatic tissues. These findings indicate that, although the hormonal responsiveness of isolated hepatocytes maintained on laminin-rich gels is markedly improved, and approaches the degree of induction demonstrated in the liver in vivo, the mechanisms of control differ, indicating a loss of liver-specific expression.
Assuntos
Hormônios/fisiologia , Fígado/enzimologia , Malato Desidrogenase/biossíntese , Animais , Células Cultivadas , Colágeno , Ácido Dicloroacético/farmacologia , Indução Enzimática , Regulação Enzimológica da Expressão Gênica , Insulina/fisiologia , Fígado/citologia , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Tri-Iodotironina/fisiologiaRESUMO
Insulin-like growth factors (IGFs) are expressed by, and are biologically active on, human fetal cells. The mitogenic actions of IGF-I are modulated by the 21-41 kDa class of IGF-binding proteins (IGF-BPs). Using a rabbit anti-human IGF-BP antibody raised against a highly pure 26 kDa IGF-BP derived from amniotic fluid, we have compared the cellular location of IGF-BP and IGF peptides in tissue sections from prostaglandin-induced human abortuses of 14-16 weeks of gestation. The monoclonal and polyclonal antibodies used were raised against human IGF-I, but did not distinguish between IGF-I and IGF-II. Positive staining for IGF-BP was seen in every tissue except brain, spleen and thyroid. With the exception of skin, the cellular distribution of IGF-BP was similar to that of IGF peptides. Strong immunostaining was found in hepatocytes, hepatic erythropoietic cells, pulmonary epithelium, the tubular epithelium of kidney, intestinal epithelia, the fetal adrenal cortex and cardiac and skeletal muscle fibres. In skin, IGF-BP was located throughout the dermis and in the germinal layer of the epidermis. IGF peptide in skin was restricted to the deeper dermal layers. In the tibial epiphyseal growth plate both IGF-BP and IGF peptide were located in chondrocytes throughout the proliferation and hypertrophic zones. The similarity in distribution of IGF-BP and IGF peptides in fetal tissues suggests that the latter may exist predominantly complexed to IGF-BP in or on the surfaces of cells in vivo. The distribution of IGF-BP may define the sites of biological action of IGF peptides.
Assuntos
Proteínas Fetais/metabolismo , Feto/análise , Receptores de Superfície Celular/análise , Somatomedinas/análise , Córtex Suprarrenal/análise , Córtex Suprarrenal/embriologia , Sistema Digestório/análise , Sistema Digestório/embriologia , Coração Fetal/análise , Idade Gestacional , Humanos , Imuno-Histoquímica , Rim/análise , Rim/embriologia , Pulmão/análise , Pulmão/embriologia , Sistema Musculoesquelético/análise , Sistema Musculoesquelético/embriologia , Peptídeos/metabolismo , Receptores de Somatomedina , Pele/análise , Pele/embriologia , Somatomedinas/metabolismo , Timo/análise , Timo/embriologiaRESUMO
We have investigated the ability of glucose, human GH and human placental lactogen (hPL) to alter the content and release of somatomedin C/insulin-like growth factor I (SM-C/IGF-I), and the biosynthesis, content and release of insulin from cultured human fetal pancreas. Fetal pancreatic explants obtained from glands following prostaglandin-induced abortion between 12 and 21 weeks of gestation were maintained in free-floating culture for 3-5 days before the experiments. The explants were then cultured for 3 days in medium containing either 2.7 or 16.7 mmol glucose/l with or without GH (4.5 or 45.5 nmol/l) or hPL (4.6 or 46.5 nmol/l). Serum-free medium from the final 24 h of culture was collected and SM-C/IGF-I and insulin were measured radioimmunologically in both conditioned medium and tissue explants extracted with acid ethanol. Insulin biosynthesis, determined by immunoprecipitation of [3H]leucine incorporated into insulin, was not significantly altered by any experimental variable. Incubation in the presence of 16.7 mmol glucose/l caused an increase of insulin release from explants, but had no consistent action on insulin content, compared with medium containing 2.7 mmol glucose/l. The pancreatic content and release of SM-C/IGF-I were independent of these glucose concentrations. Neither GH nor hPL altered insulin or SM-C/IGF-I content or release in the presence of the lower glucose concentration. At the higher glucose concentration, 45.5 nmol GH/l did not alter insulin release but caused a significant increase in SM-C/IGF-I content.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hormônio do Crescimento/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Pâncreas/embriologia , Lactogênio Placentário/farmacologia , Somatomedinas/metabolismo , Técnicas de Cultura , Glucose/farmacologia , Humanos , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismoRESUMO
Insulin-like growth factors (IGFs) are bound to specific binding proteins in extracellular fluids in vivo and when released by cells in vitro. One class of binding protein (IGF-BP), a peptide of 26 kDa purified from amniotic fluid, has been shown to modulate IGF bioactivity on isolated human fibroblasts. We have determined the factors that control release of IGF-BP from monolayers of human fetal fibroblasts using a radioimmunoassay, and have compared this with the effects of these factors on the release of IGF-I and -II. Separation of cell-conditioned cultured medium on SDS-PAGE, and subsequent immunoblotting with antibody against IGF-BP showed that fibroblasts released a single species of immunoreactive protein of an estimated molecular weight of 30 kDa. This was not the predominant binding protein released by cells since major bands of approximately 42 kDa and 39 kDa were visualized following separation by SDS-PAGE and ligand blotting with 125I-labelled IGF-I. The 30 kDa IGF-BP was released in parallel with radioimmunoassayable IGF-I and -II over 48 h. However, a significant inverse correlation was found between the release of IGF-BP, IGF-I, IGF-II and cell density. The exposure of fibroblasts to 1.3 nmol/l or greater of IGF-I or -II caused a significant release of IGF-BP. Maximum release was seen in sparse cultures with little or no release from confluent cultures. IGF-I and -II were approximately equipotent with a fourfold increase in IGF-BP release at 19.7 nmol/l. Insulin caused a release of IGF-BP and IGF-I and -II from fibroblasts at supraphysiological concentrations (16.7 nmol/l) which again was maximal on sparse cell cultures. Increases in IGF-BP, IGF-I and -II release were also found in the presence of human placental lactogen (23.3 nmol/l), but human GH, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor were without effect. The results show that human fetal fibroblasts released an IGF-BP immunologically similar to that seen in amniotic fluid together with IGF-I and -II, that IGF-BP release was enhanced by exogenous addition of IGF peptides, and that the release of all three peptides was a property of sparsely plated, rapidly growing cells. These findings strengthen the hypothesis that the cellular expression of IGF-binding proteins may represent an important level of control in IGF physiology.
Assuntos
Proteínas de Transporte/metabolismo , Fator de Crescimento Insulin-Like II/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Somatomedinas/farmacologia , Contagem de Células , Células Cultivadas , Feto , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a InsulinaRESUMO
Two isozymes of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) are responsible for the interconversion of the active glucocorticoid, cortisol in man, (corticosterone in the rodent), to the inactive 11-keto metabolite, cortisone (11-dehydrocorticosterone). We have examined the regulation of type 1 11 beta-HSD (11 beta-HSD1) using primary cultures of rat and human hepatocytes, both of which express only 11 beta-HSD1. Only 11 oxo-reductase activity could be demonstrated in cultured hepatocytes (apparent Km for cortisone 382 +/- 43 nM in human hepatocytes, apparent Km for 11-dehydrocorticosterone 14.6 +/- 1.5 microM in rat hepatocytes). There exists a marked discrepancy between 11 beta-HSD oxo-reductase activity and 11 beta-HSD1 mRNA levels in cultured human hepatocytes and human liver. Thus oxo-reductase specific activity is much higher in the cultured hepatocytes (7.2 +/- 0.01 nmoles cortisol/mg/h vs 0.89 +/- 0.06 for whole liver homogenates) whilst the converse is true for steady state 11 beta-HSD1 mRNA levels (0.78 +/- 0.02 vs 1.94 +/- 0.07 in whole liver, 11 beta-HSD1/18S expressed as arbitrary units). Carbenoxolone has a significant inhibitory effect on 11 oxo-reductase activity in both rat and human hepatocytes. However, there is clear species-specific regulation of 11 oxo-reductase activity by thyroid hormone (tri-iodothyronine (T3)), which increases 11 oxo-reductase activity in rat hepatocytes but has no effect on activity in human hepatocytes, and progesterone which inhibits activity in human hepatocytes, but has no effect on activity in rat hepatocytes. Neither T3 nor progesterone altered 11 beta-HSD1 mRNA levels. A series of growth factors (hepatocyte growth factor, epidermal growth factor, basic fibroblast growth factor, transforming growth factor beta 1) were without effect on 11 oxo-reductase activity in cultured rat hepatocytes. In contrast to homogenates of human liver, cultured hepatocytes express only 11 beta-HSD oxo-reductase activity. This is inhibited by carbenoxolone and shows species-specific regulation by T3 and progesterone. Growth factors do not appear to regulate activity or expression of 11 beta-HSD1. The discrepant enzyme activity data and 11 beta-HSD1 mRNA expression in hepatocytes and whole liver could reflect unstable 11 beta-HSD1 oxo-reductase activity or, alternatively, an additional 11 beta-HSD oxo-reductase isoform in cultured hepatocytes.
Assuntos
Hidroxiesteroide Desidrogenases/metabolismo , Isoenzimas/metabolismo , Fígado/enzimologia , 11-beta-Hidroxiesteroide Desidrogenases , Adolescente , Adulto , Animais , Northern Blotting , Carbenoxolona/farmacologia , Células Cultivadas , Criança , Corticosterona/análogos & derivados , Corticosterona/metabolismo , Cortisona/metabolismo , Humanos , Hidroxiesteroide Desidrogenases/genética , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Progesterona/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Especificidade da Espécie , Tri-Iodotironina/farmacologiaRESUMO
The presence of insulin-like growth factors (IGFs) in blood is regulated by their association with specific IGF-binding proteins (IGFBPs). In turn, the level of IGFBPs in the blood is likely to depend on a dynamic equilibrium between peptide production and clearance to extravascular tissues or organ-specific degradation. Since circulating IGFBPs may largely derive from liver we have employed partial hepatectomy in the rat to study the clearance rate of endogenous IGFBPs from blood once a major site of production is removed. Adult male rats were partially hepatectomized and serum and the remaining liver removed between 30 min and 7 days after surgery. Ligand blot analysis revealed two major species of IGFBP, of 28-30 kDa and 40-44 kDa in sera from control rats or sham-operated rats respectively. The larger species corresponded in size to rat IGFBP-3, but the smaller form was not recognized by antisera against rat IGFBP-1, bovine IGFBP-2 or human IGFBP-5 following Western immunoblot. Following hepatectomy, the levels of both IGFBP forms in the serum declined within 30 min and were barely detectable after 3 h or 6 h. They began to increase again in serum 24 h following surgery. The reduction in IGFBPs following hepatectomy was not primarily due to degradation by specific proteases in serum. Circulating levels of insulin were increased fivefold 3 h after hepatectomy but subsequently returned to control values. The rise in insulin was accompanied by a significant (P < 0.05) reduction in circulating IGF-I after 3 h which persisted at 24 h. Glucose levels in serum showed a transient but non-significant reduction between 90 min and 6 h after hepatectomy. Total RNA was extracted from remnant liver and subjected to Northern blot hybridization with 32P-labelled cDNAs encoding rat IGFBP-1, -2 or -3. Messenger RNA encoding IGFBP-1 was barely detectable in liver from control or sham-operated animals, but increased within 30 min of partial hepatectomy and peaked at 3 h. It subsequently declined and was again barely detectable after 24 h. No expression of IGFBP-2 or -3 mRNAs was found by Northern blot analysis in the liver of control animals or following partial hepatectomy. These results suggest that both IGF-I and IGFBPs in rat serum decreased rapidly following partial hepatectomy, and that this was due largely to the rapid clearance of the peptide and its binding proteins once the major source of production was removed.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Proteínas de Transporte/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/metabolismo , Somatomedinas/metabolismo , Animais , Glicemia/metabolismo , Northern Blotting , Western Blotting , Hepatectomia , Insulina/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Masculino , Radioimunoensaio , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Hepatocyte growth factor (HGF) was first described as a hepatotrophic factor in partially hepatectomized rat plasma in the early 1980's and was purified from plasma of a patient with fulminant hepatic failure and from rat platelets in 1986-1987. Recent progress has revealed that HGF is the same protein as scatter factor and tumor cytotoxic factor, and is now known to be a broad-spectrum growth factor which stimulates cell growth not only of hepatocytes but also of many other types of epithelial and endothelial cells. In this review, however, we concentrate on the role of HGF, mainly human HGF, on liver regeneration after injury. In humans, plasma levels of hHGF increase to greater than 10 ng/ml during severe liver disease such as fulminant hepatic failure and decrease rapidly to normal levels when the patients recover from the disease. In less severe liver damage such as occurs in acute hepatitis, levels of hHGF in plasma increase to 0.5-1 ng/ml which is approaching the half maximal concentration for the stimulation of DNA synthesis in human hepatocytes in culture. Thus, HGF is believed to be involved in control of liver regeneration. Although the cell type(s) which produces HGF during liver disease is not yet identified, it is possible that circulating leukocytes or splenocytes are responsible. Synthesis of HGF is though to be regulated by a putative inducer(s) derived from damaged liver tissue. A control mechanism for HGF production during liver disease is proposed.