RESUMO
Delayed awakening after general anaesthesia due to psychogenic coma is a phenomenon that rarely presents to the oral and maxillofacial surgeon. A case of psychogenic coma following general anaesthesia for dental extractions is presented here. It is recommended that patients at risk of conversion disorder should be counselled about the risks of psychogenic coma. Early diagnosis of this condition could lead to better patient management.
Assuntos
Anestesia Geral/efeitos adversos , Coma/induzido quimicamente , Coma/psicologia , Extração Dentária , Adulto , Feminino , Escala de Coma de Glasgow , Humanos , Fatores de RiscoRESUMO
Studies on the in vitro hepatitis C virus (HCV) infection are hampered by the lack of an appropriate system to culture permissive cells to be continuously infected with HCV. Trypsinization required for cell passage can lead to possible temporary loss of permissiveness for infection, whereas refreshment of the medium can result in loss of infectious particles necessary for perpetuation of the infection; it is therefore very difficult to maintain a continuous HCV infection in cell cultures. A new infection method was designed and evaluated in order to prevent these unfavourable circumstances. A cell line derived from the human hepatoblastoma cell line Hep G2 was grown in the extracapillary space of a haemodialysis cartridge, in the presence of a HCV-positive inoculum, while the culture medium was recirculated through the intracapillary space, supplying the cells with nutrients and oxygen. HCV RNA could continuously be detected in the cells up to 77 days of culture. Sequence analysis of the HCV hypervariable region 1 (HVR1) revealed that 56% and 75%, respectively, of the clones obtained from the cells at day 20 and 40 after start of the infection were different from the clones obtained from the original inoculum and that certain nucleotide positions in this region were more susceptible to mutations, leading to an alteration in amino acid sequence. As none of these sequences were present in the clones from the inoculum, it is suggested that new HCV quasispecies have emerged as a result of viral replication in the hepatocytes in vitro. This system seems a valuable tool for the in vitro evaluation of antiviral drugs.
Assuntos
Hepacivirus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Técnicas de Cultura de Células/métodos , Linhagem Celular , Meios de Cultura , Hepacivirus/química , Humanos , Dados de Sequência Molecular , RNA Viral/análise , Análise de Sequência de Proteína , Fatores de Tempo , Proteínas Virais/química , Replicação ViralRESUMO
HYPOTHESIS: A reproducible and potentially reversible model of acute liver failure in the pig is feasible based on transient ischemia of the liver. DESIGN: To determine the shortest period of liver ischemia sufficient to cause 100% mortality, ischemia of the liver was induced for different lengths of time, starting with 6 hours. If the pig survived, ischemia time was prolonged for 2 hours in the next animal. In the first group, the common bile duct was not tightened. In the second group, the common bile duct was tightened. SETTING: The Laboratory for Hepatopathophysiology, Catholic University, Leuven, Belgium. PARTICIPANTS: Female stress-negative Belgian Landrace pigs weighing 18 to 22 kg. INTERVENTIONS: During preparatory surgery, all ligaments around the liver and connective tissue around the liver hilum were transected and an end-to-side portacaval shunt was made. Vessel loops were placed around the branches of the hepatic artery and bile duct. Three days later, in fully awake pigs, the loops were tightened. MAIN OUTCOME MEASURES: Mortality. Development of acute liver failure was determined based on neurologic, biochemical, and pathological variables. RESULTS: When occluded for 10 hours, all pigs in group 2 (n = 5) [corrected] died between 12 and 17 hours after the induction of ischemia. All pigs developed typical acute liver failure. Tissue specimens showed 90% necrosis of the liver parenchyma. CONCLUSION: A highly reproducible and potentially reversible model of acute liver failure in the large animal has been established.
Assuntos
Isquemia/patologia , Isquemia/fisiopatologia , Falência Hepática Aguda/patologia , Falência Hepática Aguda/fisiopatologia , Fígado/irrigação sanguínea , Animais , Modelos Animais de Doenças , Feminino , Fígado/patologia , Sensibilidade e Especificidade , Análise de Sobrevida , SuínosAssuntos
Carcinoma Hepatocelular/epidemiologia , Hepatite C/epidemiologia , Neoplasias Hepáticas/epidemiologia , Bélgica/epidemiologia , Carcinoma Hepatocelular/diagnóstico , Comorbidade , Feminino , Hepatite C/diagnóstico , Hepatite C/prevenção & controle , Humanos , Incidência , Neoplasias Hepáticas/diagnóstico , Masculino , Medição de RiscoAssuntos
Colo/irrigação sanguínea , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Neoplasias Hepáticas/secundário , Animais , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/mortalidade , Humanos , Neoplasias Hepáticas/mortalidade , Invasividade Neoplásica/patologia , Neovascularização Patológica/diagnóstico , Neovascularização Patológica/fisiopatologia , Prognóstico , Análise de SobrevidaRESUMO
Previously, we have determined that human annexin V (hAV), a Ca2+-dependent phospholipid-binding protein, and not rat AV, binds specifically to small hepatitis B surface antigen (SHBsAg), and that transfection of a rat hepatoma cell line with a construct containing the hAV gene led to hAV expression and conferred susceptibility to hepatitis B virus (HBV) infection. In this work, we have examined the effect of administration of hAV on in vitro binding of SHBsAg to human and to rat hepatocytes and on in vitro HBV infection. The results showed that hAV could bind to human as well as to rat hepatocytes. Because of this property, excess hAV was unable to prevent HBV infection in primary cultures of human hepatocytes. On the other hand, it enabled rat hepatocytes to specifically bind SHBsAg and conferred susceptibility to HBV infection. After infection of primary cultures of rat hepatocytes in the presence of hAV, HBV mRNA, covalently closed circular (ccc) DNA, replicative intermediates, hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and secreted HBV DNA were detected. After infection in the absence of hAV, no markers of HBV replication were detected. Hence, from the present study we conclude that hAV is involved in facilitating HBV entry, leading to successful HBV infection in primary cultures of rat hepatocytes, while it is not effective in preventing HBV infection in primary cultures of human hepatocytes.
Assuntos
Anexina A5/farmacologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Fígado/metabolismo , Animais , Células Cultivadas , Criopreservação , Suscetibilidade a Doenças , Hepatite B/metabolismo , Hepatite B/prevenção & controle , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/virologia , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
PURPOSE: Exfoliated or soiled free malignant cells have serious consequences in patients undergoing gastrointestinal cancer surgery. The present study evaluates the toxicity and efficacy of cytotoxic agents in the prevention of cell seeding and tumor growth in the peritoneal cavity in an experimental model. METHODS: Mtln3 adenocarcinoma cell viability was tested in vitro using the trypan blue exclusion test after incubation with povidone-iodine or chlorhexidine. In vivo, Fischer rats were inoculated with 10(5) or 10(6) cells followed by peritoneal lavage with physiological saline, chlorhexidine 0.02 percent, povidone-iodine low molecular weight 1 percent or povidone-iodine high molecular weight 1 and 2 percent in different quantities and incubation times. RESULTS: Chlorhexidine 0.02 percent and povidone-iodine low molecular weight 1 percent or high molecular weight 2 percent, killed over 98 percent of 10(5) or 10(6) tumor cells in vitro. Povidone-iodine low molecular weight 1 percent and high molecular weight 2 percent were toxic and lethal when 5 ml were applied in the peritoneal cavity three times for five minutes. Chlorhexidine 0.02 percent applied after inoculation of 10(5) or 10(6) cells, reduced the tumor development only to 70 and 80 percent. Application of 5 ml povidone-iodine 1 percent low molecular weight or high molecular weight, three times for one and five minutes, after inoculation of 10(6) cells did not change the tumor take. However, inhibition of Mtln3 cells to form metastases was observed. When povidone-iodine low molecular weight 1 percent was used three times for one minute after 10(5) tumor cells were "soiled", no toxicity was observed and the tumor take was reduced to 30 percent (P < 0.05). CONCLUSIONS: Povidone-iodine toxicity proved to be a major issue in vivo. However, povidone-iodine low molecular weight 1 percent was safe when used for short periods and very effective when a limited number of tumor cells was inoculated. The use of cytotoxic agents to prevent recurrent disease caused by tumor cell seeding in patients seems to make sense only when the "inoculum size" of exfoliated or soiled cancer cells is limited.
Assuntos
Adenocarcinoma/patologia , Adenocarcinoma/terapia , Clorexidina/administração & dosagem , Recidiva Local de Neoplasia/prevenção & controle , Inoculação de Neoplasia , Povidona-Iodo/administração & dosagem , Neoplasias Retais/terapia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Recidiva Local de Neoplasia/patologia , Neoplasias Experimentais , Lavagem Peritoneal/métodos , Ratos , Ratos Endogâmicos F344 , Neoplasias Retais/patologia , Sensibilidade e Especificidade , Resultado do TratamentoRESUMO
Mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid (MPA), is currently used as an immunosuppressive agent in kidney transplant recipients. After oral administration, MMF is hydrolysed to MPA, the active compound, which is a potent inhibitor of inosine monophosphate dehydrogenase (IMP-DH). Inhibition of this enzyme results in a depletion of the intracellular GTP and dGTP pools. MPA has been shown to inhibit the replication of a number of viruses, including arena viruses (Junin and Tacaribe), yellow fever virus, reovirus-1, parainfluenza-3 virus, Coxsackie B4 virus, Epstein-Barr virus and human immunodeficiency virus. To examine whether MPA also has an inhibitory effect on HBV replication, experiments were performed using cultures of primary human hepatocytes and HBV-transfected, HepG2 2.2.15 cells. After in vitro infection with HBV in human hepatocytes, HBV covalently-closed-circular (ccc) DNA and HBV mRNAs were detectable in the cells during the 10 days following infection. HBV DNA and hepatitis B surface antigen (HBsAg) were also secreted into the culture medium. In the presence of 10 microg ml-1 MPA (the therapeutic serum level of MPA as an immunosuppressive agent) in culture medium, HBV ccc DNA and HBV mRNAs became undetectable 5 days after treatment was started. The secretion of HBV DNA and HBsAg into the medium was also markedly reduced. No cytotoxic effect of the drug was noted during the experiments. The effect of MPA on HBV replication was abolished by the presence of guanosine (50 microg ml-1). In HepG2 2.2.15 cells (which contain an integrated tandem dimer of the HBV genome), MPA treatment had no significant inhibitory effect on the secretion of HBV DNA and HBsAg into the culture medium. HBV ccc DNA and HBV mRNAs in HepG2 2.2.15 cells were also not affected. The observed effect of MPA on HBV replication in primary human hepatocyte cultures may involve only episomal replication and may have clinical implications, especially before integration of HBV DNA into the host genome.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Fígado/virologia , Ácido Micofenólico/farmacologia , Células Cultivadas , DNA Viral/metabolismo , Inibidores Enzimáticos/farmacologia , Guanosina/farmacologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Humanos , Imunossupressores/farmacologia , Fígado/citologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacosRESUMO
Previously, we have shown that small hepatitis B surface antigen (SHBsAg) binds specifically to human annexin V (hAV) and that hAV plays a key role in the initial steps of hepatitis B virus (HBV) infection. We have also demonstrated the spontaneous development of anti-idiotypic antibodies (antibodies to HBsAg Ab2) in rabbits immunized with hAV. As Ab2 is able to inhibit the binding of hAV to SHBsAg, Ab2 might contain epitope(s) mimicking a region of hAV for binding to SHBsAg. Identification of this epitope will therefore reveal a SHBsAg sequence involved in hAV binding. Using a panel of synthetic peptides covering the region of SHBsAg located on the outer surface of the virus, binding studies showed that the region incorporating amino acids (aa) 125-131 of SHBsAg is important for binding to Ab2 and consequently also for binding to hAV. Further experiments revealed that not only this region, but also the region incorporating aa 158-169, is involved in the binding of SHBsAg to hAV. As these regions are located in the structural vicinity according to the topological model of HBsAg proposed by Chen et al., our findings suggest that these regions are parts of a conformational epitope of SHBsAg for binding to hAV. Because of the crucial role of hAV in HBV infection, further studies on the HBsAg epitopes for hAV binding may lead to the development of a new generation of vaccines or molecules for prevention and for treatment of patients with chronic hepatitis B.
Assuntos
Anexina A5/metabolismo , Mapeamento de Epitopos , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/imunologia , Sequência de Aminoácidos , Animais , Anexina A5/imunologia , Anticorpos Anti-Idiotípicos/sangue , Anticorpos Anti-Idiotípicos/metabolismo , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/imunologia , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Coelhos , Proteínas RecombinantesRESUMO
The use of digoxigenin-labelled probes was studied for quantitation of HBV-DNA during antiviral drug evaluation. Digoxigenin (dig)-labelled probes were generated either via incorporation of dig-dUTP in a polymerase chain reaction (PCR) or a random priming reaction. Using the PCR-labelled probe (delineating a 523 bp fragment in the core gene of the HBV) as little as 1 pg of immobilized HBV-DNA could be detected following an 8 h exposure of the hybridized membrane. A close correlation (r = 0.95) was found between the amount of HBV-DNA (range 2.5-200 pg) and the signal generated by the probe hybridized to its target DNA. By using a probe that was labelled with digoxigenin via random priming, the minimal quantity of immobilized HBV plasmid DNA that could be detected following an 8 h exposure was 4 pg, whereas a 32P-labelled probe, generated in parallel by random priming, allowed the detection of 16 pg of HBV plasmid DNA following a 4-day exposure. The PCR-generated digoxigenin-labelled probe proved to be useful for antiviral drug evaluation, i.e. to detect HBV-DNA in total cellular DNA from HBV-positive hepatoma cells (HepG2.2.15) that had either been treated with reference antiviral agents or left untreated. The 50% effective concentrations (EC50) that were calculated for inhibition of HBV-DNA production by lamivudine (3TC), penciclovir (PCV), lobucavir (LBV), adefovir (PMEA) and tenofovir (PMPA) were comparable to those reported in the literature. The use of digoxigenin-labelled probes thus appears to be a simple, convenient, rapid, reliable and non-radioactive method for use for anti-HBV screening. In addition, and in contrast to 32P-labelled probes, digoxigenin-labelled probes can be stored for >1 year without loss of specific activity, which makes these probes particularly attractive for large-scale antiviral drug evaluation purposes.
Assuntos
Antivirais/farmacologia , Sondas de DNA/metabolismo , DNA Viral/análise , Digoxigenina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Vírus da Hepatite B/genética , Sondas de DNA/efeitos dos fármacos , Nucleotídeos de Desoxiuracil/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
Previously, we have found that human liver annexin V (hA-V; in earlier reports referred as Endonexin II) is a specific hepatitis B surface antigen (HBsAg) binding protein. In this study, we demonstrate that transfection of rat hepatoma FTO 2B cells, a cell line that is not infectable by hepatitis B virus (HBV) and does not express hA-V, with a construct containing the hA-V gene, resulted in hA-V expressing cells susceptible to HBV infection. After in vitro infection, transfected FTO cells (assigned as FTO 9.1 cells) expressing hA-V in cultures were shown to contain HBV-precore/core, X mRNAs, and covalently closed circular (ccc) DNA as detected by polymerase chain reaction (PCR). The presence of HBV ccc and replicative intermediate DNA was also demonstrated by Southern blot hybridization assay. HBV DNA secreted in the culture medium was also evident as determined by quantitative branched DNA (bDNA) assay. HBsAg and hepatitis B core antigen (HBcAg) could also be detected by an immunocytochemical method in 10% to 15% of the cells at day 3 and day 5 after infection. Infectivity of in vitro-propagated HBV was demonstrated by infection of the naive FTO 9.1 cells with the culture supernatant from HBV-carrier cultures. In contrast to primary cultures of human hepatocytes and FTO 9.1 cells, primary rat and mouse hepatocytes, as well as rat hepatoma cell lines that do not express hA-V, are not susceptible to HBV infection. These findings suggest that hA-V plays a key role in the initial step of HBV infection and that the species-specific susceptibility to HBV infection and replication in hepatocytes is associated with the expression of hA-V.
Assuntos
Anexina A5/genética , Suscetibilidade a Doenças , Hepatite B , Neoplasias Hepáticas Experimentais/metabolismo , Fígado/química , Transfecção , Animais , DNA Circular/análise , DNA Viral/análise , Hepatite B/metabolismo , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/análise , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/análise , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Humanos , RNA Mensageiro/análise , RNA Viral/análise , Ratos , Ratos Wistar , Transativadores/genética , Células Tumorais Cultivadas , Proteínas Virais Reguladoras e Acessórias , Replicação ViralRESUMO
Studies on the pathobiology of chronic (long-term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an in vitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier-attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12,000-14,000) membrane (dialysis) bags for a duration of 2 months. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86- and 3.28-fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long-term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.
Assuntos
Vírus da Hepatite B/fisiologia , Hepatite B Crônica/fisiopatologia , Fígado/citologia , Replicação Viral , Southern Blotting , Cápsulas , Células Cultivadas , Células Imobilizadas , Meios de Cultura , DNA Circular/análise , DNA Viral/análise , Antígenos do Núcleo do Vírus da Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/biossíntese , Humanos , Imuno-Histoquímica , Fígado/metabolismo , RNA Mensageiro/análise , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virologia/métodosRESUMO
In a previous study, we have determined the prevalence of serum HGV-RNA in patients with congenital clotting disorders. Twenty-six (15%) of 175 patients investigated were serum HGV-RNA positive. In addition, HGV-RNA was detectable in peripheral blood mononuclear cells (PBMC) in ten percent of the cases, three of these patients were serum HGV-RNA negative. In the present study, we have determined the prevalence of anti-HGV-E2 antibodies in the same patient population. Anti-HGV-E2 as determined by ELISA was detected in 45 patients (25.7%). Forty of these patients were serum HGV-RNA negative. Ninety-two percent of the 26 HGV viremic patients and all but one patient (44 patients) with detectable anti-HGV-E2 had coinfection with the hepatitis C virus (HCV). Of these coinfected patients, 62.5% of HGV viremic patients and 53% of anti-HGV-E2 positive patients showed elevated serum ALT levels. Anti-HGV-E2 seroconversion is thus not associated with HCV infection. Two patients who were solely infected with HGV had normal serum ALT levels. In a retrospective longitudinal study, we have observed in 15 patients that serum HGV-RNA persisted during one to 19 years of follow-up, while anti-HGV-E2 was repeatedly negative. Five additional patients who were anti-HGV-E2 positive with concomitant detectable HGV-RNA (4 patients in serum and 1 patient in PBMC) became HGV-RNA negative during follow-up, ranging from 1 to 8 years after the first detection of anti-HGV-E2 antibodies. Two patients had lost anti-HGV-E2 antibodies 3 to 6 years after the seroconversion without the re-appearance of serum HGV-RNA. From these findings, it is clear that the prevalence rate of HGV infection in patients with clotting disorders as determined by PCR assay for HGV-RNA and anti-HGV-E2 by ELISA is actually higher than the prevalence of HGV viremia. Although HGV viremia may persist for longer than 19 years, most of the patients infected with HGV may clear the viremia spontaneously. The clearance of viremia is usually associated with seroconversion to anti-HGV-E2. In addition, anti-HGV-E2 may be lost during years of follow-up without the reappearance of the HGV-RNA. Although HGV infection does not seem to influence the fate of HCV infection and does not induce increased levels of serum ALT, the clinical significance of long-term infection remains to be established.
Assuntos
Transtornos da Coagulação Sanguínea/complicações , Flaviviridae/imunologia , Anticorpos Anti-Hepatite/sangue , Hepatite Viral Humana/sangue , RNA Viral/sangue , Proteínas do Envelope Viral/imunologia , Viremia/sangue , Adolescente , Adulto , Idoso , Alanina Transaminase/sangue , Biomarcadores , Transtornos da Coagulação Sanguínea/congênito , Criança , Pré-Escolar , Comorbidade , Infecção Hospitalar/sangue , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Flaviviridae/isolamento & purificação , Seguimentos , Hepatite B/epidemiologia , Hepatite C/epidemiologia , Hepatite Viral Humana/epidemiologia , Hepatite Viral Humana/transmissão , Hepatite Viral Humana/virologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Prevalência , Remissão Espontânea , Diálise Renal/efeitos adversos , Estudos Retrospectivos , Estudos Soroepidemiológicos , Viremia/epidemiologia , Viremia/virologiaRESUMO
A recently discovered non-A-E hepatitis virus has been designated as hepatitis G virus (HGV) and identified as a new member of the Flaviviridae family. Infection by this virus is thought to be associated with blood-borne hepatitis and usually in the presence of hepatitis C or hepatitis B virus (HBV) infection. In this study, the presence of HGV-RNA in serum or plasma and the prevalence of antibodies against an HGV envelope protein (E2) were investigated in patients undergoing chronic hemodialysis using a sensitive reverse-transcriptase polymerase chain reaction and an enzyme-linked immunosorbent assay, respectively. HGV-RNA was detected in 19 of 112 patients investigated (17%) and anti-E2 antibodies were detected in 15 of 106 patients studied (14.2%). With the exception of two patients, the appearance of anti-E2 is associated with the clearance of serum HGV-RNA. The total prevalence of current (HGV-RNA positivity) and/or past (anti-E2 positivity) HGV infection in this patient population is thus 28.6% (32 of 112 patients were positive for serum HGV-RNA and/or anti-E2 antibodies). In apparently healthy blood donors, serum HGV-RNA was detected in four of 358 individuals (1.12%) and anti-E2 was not detected in 50 individuals investigated. From the 19 patients with serum HGV-RNA positivity, nine were coinfected with other hepatitis viruses (seven with HBV; one with HBV, hepatitis C virus [HCV], and hepatitis D virus; and one with HBV and cytomegalovirus). Thirteen of 15 patients with anti-E2 positivity (10 were positive for only anti-E2 and three were also positive for anti-HBc) had no detectable HGV-RNA. In two patients, both HGV-RNA and anti-E2 antibodies were concomitantly present (both patients were coinfected with HCV or HBV). Of the HGV-infected patients, only three who were coinfected with HBV showed elevated serum alanine aminotransferase levels. The serum HCV-RNA and/or anti-HCV were detected in five (4.5%) of 112 patients. From these findings, we conclude that there is a high prevalence of HGV infection (28.6%) compared with HCV (4.5%) in patients undergoing hemodialysis in our hospital. However, approximately 50% of patients had spontaneously lost the viremia and developed anti-HGV-E2 antibodies. We confirm that HGV infection alone is not associated with elevated serum transaminases, and the appearance of anti-HGV-E2 is usually accompanied with clearance of serum HGV-RNA. In contrast to the results of our previous study, the majority of patients infected with HGV are not coinfected with HCV, indicating that HGV is capable of independent transmission. It is likely that there is a preferential HGV acquisition in the hemodialysis unit. The clinical significance of long-term infection with HGV remains to be established.
Assuntos
Flaviviridae , Hepatite C/transmissão , Hepatite Viral Humana/transmissão , Diálise Renal/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Flaviviridae/isolamento & purificação , Anticorpos Anti-Hepatite/análise , Hepatite C/diagnóstico , Hepatite Viral Humana/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Viral/análise , Proteínas do Envelope Viral/imunologiaRESUMO
Hepatitis B virus (HBV) infection is still a major public health problem worldwide. Although much information about the molecular biology of HBV has been gained in the last decades, little is known about the mechanism of attachment and penetration of the HBV particle into human hepatocytes. The HBV envelope proteins are important for the interaction between the HBV particle and the hepatocyte plasma membrane. Although initially it was suggested that the preS2 domain could act, via polymerized human serum albumin, as an attachment site to human hepatocytes, in recent years other observations showed that the preS1 domain is probably the most important attachment site to human hepatocytes. However, controversial findings on cellular proteins for binding to the preS1 domain has been described, namely the IgA-, the IL6-, the asialoglycoprotein receptor and GAPD. Although the preS1 attachment site may be important, apo H has been shown to bind specifically to small HBsAg. Recently, we have identified human liver Annexin V as a specific small HBsAg-binding protein. In a preliminary report, the direct involvement of human Annexin V in the initial step of HBV infection has been demonstrated. A rat hepatoma cell line, which does not express human Annexin V and which is not infectable by HBV, gained the ability to become infected by HBV after transfection with human Annexin V. This result may facilitate the progress of HBV receptor research and elucidate the molecular mechanism of the initial step of HBV infection.
Assuntos
Vírus da Hepatite B/patogenicidade , Hepatite B/virologia , Fígado/virologia , Animais , Genoma Viral , Vírus da Hepatite B/genética , Vírus da Hepatite B/ultraestrutura , Humanos , Fígado/citologia , Especificidade de Órgãos , Ratos , Especificidade da Espécie , Transcrição GênicaRESUMO
We perfused livers from fed rats with a balanced salt solution containing 1 mmol/L glucose. Under these conditions a low steady rate of glycogenolysis was observed (approximately 1.7 micromol glucose equivalents/g/min; 20% of the maximal glycogenolytic activity). Nitric oxide (NO) transiently stimulated hepatic glucose production. A maximal response (on average doubling basal glucose output) was observed with 34 micromol/L NO. The same concentration of nitrite (NO2-) was ineffective. Half-maximal effects were seen at 8 to 10 micromol/L NO, irrespective of the flow direction (portocaval or retrograde). This glycogenolytic response to NO corresponded to a partial activation of phosphorylase. The NO effect was not additive to maximal stimulation of glycogenolysis (7.7 +/- 0.2 micromol hexose equivalents/g/min; n = 4) by 100 micromol/L dibutyryl cyclic adenosine monophosphate (Bt2cAMP). The requirement for activation of phosphorylase was also evidenced by the ineffectiveness of NO in phosphorylase-kinase-deficient livers of gsd/gsd rats. The NO effect was blocked by co-administration of cyclooxygenase inhibitors (50 micromol/L ibuprofen, 50 micromol/L indomethacin, or 2 mmol/L aspirin), suggesting a mediatory role of prostanoids from nonparenchymal cells. This conclusion was confirmed by the fact that NO did not activate phosphorylase in isolated hepatocytes. Moreover, NO was no longer glycogenolytic in livers perfused with Ca2+-free medium, in agreement with the known mediatory role of Ca2+ in prostanoid-mediated responses. Surprisingly, in Ca2+-free medium NO inhibited the basal glucose production. This coincided with an increased elution of cyclic guanosine monophosphate (cGMP). Inhibition of glycogenolysis by NO under these conditions was blocked by 1 mmol/L theophylline, suggestive for involvement of cGMP-stimulated cAMP phosphodiesterase. However, we could not confirm that an increase in cGMP resulted in a drop in cAMP. In conclusion, NO recruits opposing mechanisms with respect to modulation of basal hepatic glycogenolysis. In the presence of Ca2+, activation of phosphorylase with stimulation of glycogenolysis dominates. Cyclooxygenase inhibitors abolish this effect. Activation by NO of the cyclooxygenase in nonparenchymal cells is a distinct possibility. In the absence of Ca2+, inhibition of basal glycogenolysis becomes observable. It remains to be established whether this results from cGMP-mediated stimulation of hydrolysis of cAMP.
Assuntos
Glicogênio Hepático/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Óxido Nítrico/farmacologia , Animais , Bucladesina/farmacologia , Cálcio/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Glucose/metabolismo , Técnicas In Vitro , Masculino , Óxido Nítrico/administração & dosagem , Perfusão , Fosforilases/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos WistarRESUMO
Octreotide has been proposed for the treatment of variceal bleeding. The effects on portal pressure, however, have been variable in published studies. As bleeding is more directly related to pressure in the varices, this study investigated the effect on variceal pressure of octreotide and terlipressin, a vasoactive drug with a well established effect. Variceal pressure was measured during four to eight minutes by a continuous non-invasive endoscopic registration method. Thirty patients in whom a stable variceal pressure recording had been obtained during at least one minute, were randomised to receive either 2 mg terlipressin, 50 micrograms octreotide or an identical volume of saline, as a single intravenous injection given over 60 seconds. For the final analysis three patients had to be excluded because of lack of a satisfactory recording. There were no significant clinical differences between the three groups of patients. Placebo administration did not induce significant changes, but a mean decrease in variceal pressure of -27% was noted with terlipressin, starting from two minutes onwards. Variceal pressure changes after injection of octreotide were variable and the mean change in pressure did not reach statistical significance. Seven of 10 patients showed a temporary increase in variceal pressure. In conclusion, terlipressin induces a significant and progressive decrease in variceal pressure but inconsistent variations of variceal pressure changes were seen after octreotide administration. This is probably related to its effect on central venous pressure. This study also shows that continuous variceal pressure recording with the non-invasive endoscopic registration technique detects in an accurate way the effect of vasoactive drugs on variceal pressure, because placebo injection did not produce significant changes.
Assuntos
Varizes Esofágicas e Gástricas/fisiopatologia , Fármacos Gastrointestinais/uso terapêutico , Hemorragia Gastrointestinal/fisiopatologia , Lipressina/análogos & derivados , Octreotida/uso terapêutico , Vasoconstritores/uso terapêutico , Adulto , Idoso , Determinação da Pressão Arterial/métodos , Endoscopia Gastrointestinal/métodos , Varizes Esofágicas e Gástricas/tratamento farmacológico , Feminino , Hemorragia Gastrointestinal/tratamento farmacológico , Humanos , Lipressina/uso terapêutico , Masculino , Pessoa de Meia-Idade , TerlipressinaRESUMO
The expression and cellular distribution of desmin, alpha-smooth muscle actin (A-SMA) and cytokeratin no. 8 (CK-8) and no. 18 (CK-18) in normal adult, neonatal and fetal rat liver were examined immunohistochemically on cryostat sections. At days 14 and 15 of gestation, nonhematopoietic cells in embryonic liver were strongly desmin-positive, and some of the cells, mainly located in the periphery, were also stained with anti-A-SMA. Desmin immunoreactivity gradually decreased from day 16 of gestation. A close association of desmin-positive cell processes with hematopoietic cells was observed during fetal and early neonatal development. From day 16 of gestation the pre-hepatocytes became desmin-negative, remained CK-8 and CK-18 positive. Desmin-expressing cells were numerous in the liver from the embryonic period to the neonatal age. However, their absolute number per unit area, as well as their number relative to hepatocytes, decreased with age. We suggest that desmin-positive cells in embryonic liver may act as stromal cells in the hepatic hematopoietic microenvironment and support hepatocyte development.
Assuntos
Actinas/análise , Desmina/análise , Fígado/química , Animais , Desenvolvimento Embrionário e Fetal/fisiologia , Células-Tronco Hematopoéticas/química , Imuno-Histoquímica , Queratinas/análise , Fígado/anatomia & histologia , Fígado/crescimento & desenvolvimento , Ratos , Ratos EndogâmicosRESUMO
Paediatric orthotopic liver transplant recipients may develop chronic hepatitis after surgery. To investigate the role of hepatitis C virus in this pathology a cohort of 249 paediatric orthotopic liver transplant recipients was studied. Sixteen children (6.4%) were found to have chronic hepatitis C virus hepatitis after orthotopic liver transplantation. All but one of them had serum transaminase values which were persistently raised two to eight times the upper limit of normal. Thirteen were positive for both serology and serum hepatitis C virus RNA. Serum hepatitis C virus RNA detection occurred five to 33 months before hepatitis C virus antibodies. Liver tissue hepatitis C virus RNA and hepatitis C virus core antigen were detected in five. In one patient, tissue hepatitis C virus core antigen was detected when other tests for hepatitis C were negative. Two patients had positive human cytomegalovirus serum antibodies and RNA before transplantation. Although serum hepatitis C virus RNA was not detected after transplantation, serum enzyme immunosorbent assay and tissue core antigen were still detectable in both patients. In another child, serum hepatitis C virus RNA was positive and hepatitis C virus core antigen was found on a liver biopsy specimen but antihepatitis C virus antibodies were negative as well as liver hepatitis C virus RNA. No patient developed severe liver disease or cirrhosis during a follow up of up to 72 months. It is concluded that hepatitis C virus is a significant cause of morbidity after paediatric orthotopic liver transplantation. Diagnosis cannot rely on serological testing only. The patients remained stable on follow up, but longer prospective histological studies remain necessary to establish prognosis.