Viral gastroenteritis among children of 0-5 years in Nigeria: Characterization of the first Nigerian aichivirus, recombinant noroviruses and detection of a zoonotic astrovirus.

BACKGROUND: Viruses are the leading cause of acute gastroenteritis in children worldwide. Understanding of the occurrence and genetic diversity of these viruses can help to prevent infections. OBJECTIVES: The present study describes the presence, genetic diversity and possible recombination of five enteric viruses in children with gastroenteritis in Southwestern Nigeria. STUDY DESIGN: From August 2012 to December 2013, stool samples and sociodemographic data of 103 diarrheic children <5 years were collected to detect and characterize rotavirus A, norovirus, human astrovirus, aichivirus and sapovirus using PCR techniques followed by sequencing and phylogenetic analyses. RESULTS: At least one virus was identified in 58.3% (60/103) of the stool samples. Rotavirus, norovirus and astrovirus were detected in 39.8% (41/103), 10.7% (11/103), and 6.8% (7/103) respectively. Notably, aichivirus was detected for the first time in Nigeria (1/103; 0.97%). Sapovirus was not detected in the study. Co-infections with rotavirus were observed in eight samples either with norovirus or astrovirus or aichivirus. Phylogenetic analyses of different genome regions of norovirus positive samples provided indication for recombinant norovirus strains. A novel astrovirus strain closely related to canine astrovirus was identified and further characterized for the first time. CONCLUSIONS: Viruses are the common cause of acute gastroenteritis in Nigerian infants with rotavirus as most frequently detected pathogen. New norovirus recombinants and a not yet detected zoonotic astrovirus were circulating in Southwestern Nigeria, providing new information about emerging and unusual strains of viruses causing diarrhea.

A large proportion of people who inject drugs are susceptible to hepatitis B: Results from a bio-behavioural study in eight German cities.

BACKGROUND: People who inject drugs (PWID) are at high risk of hepatitis B virus (HBV) infection by sharing needles and drug use paraphernalia. In Germany, no routine surveillance of HBV prevalence and vaccination coverage among PWID exists. METHODS: Socio-demographic and behavioural data were collected between 2011 and 2014 through face-to-face interviews, during a bio-behavioural survey of PWID recruited in eight German cities. Dried blood spots (DBS) prepared with capillary blood were tested for HBV markers. Factors associated with past/current HBV infection and vaccination status were analysed by univariable and multivariable analysis using logistic regression. The validity of self-reported HBV infection and vaccination status was analysed by comparison to the laboratory results. RESULTS: Among 2077 participants, the prevalence of current HBV infection was 1.1%, of past HBV infection was 24%, and of vaccine-induced HBV antibodies was 32%. No detectable HBV antibodies were found in 43%. HBV infection status was significantly associated with study city, age, years of injecting, use of stimulants, migration status, and homelessness; HBV vaccination status was significantly associated with study city, age, and level of education. Correct infection status was reported by 71% and correct vaccination status by 45%. CONCLUSIONS: HBV seroprevalence among PWID was about five times higher than in the general population in Germany, confirming PWID as an important risk group. Targeted information campaigns on HBV and HBV prevention for PWID and professionals in contact with PWID need to be intensified. Routinely offered HBV vaccination during imprisonment and opioid substitution therapy would likely improve vaccination rates among PWID.

Steep rise in norovirus cases and emergence of a new recombinant strain GII.P16-GII.2, Germany, 2016.

Since early November 2016, the number of laboratory-confirmed norovirus infections reported in Germany has been increasing steeply. Here, we report the detection and genetic characterisation of an emerging norovirus recombinant, GII.P16-GII.2. This strain was frequently identified as the cause of sporadic cases as well as outbreaks in nine federal states of Germany. Our findings suggest that the emergence of GII.P16-GII.2 contributed to rising case numbers of norovirus gastroenteritis in Germany.

Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

Identification of a natural intergenotypic recombinant hepatitis delta virus genotype 1 and 2 in Vietnamese HBsAg-positive patients.

Hepatitis D virus (HDV) infection is acquired as a co- /superinfection of Hepatitis B virus (HBV) and can modulate the pathophysiology of chronic hepatitis B and related liver diseases including hepatocellular carcinoma. Among the eight distinct HDV genotypes reported, relatively few studies have attempted to investigate the prevalence of HDV mixed genotypes and RNA recombination of HDV. With a recorded prevalence of 10-20% HBV infection in Vietnam, this study investigated the HDV variability, HDV genotypes and HDV recombination among twenty-one HDV isolates in Vietnamese HBsAg-positive patients. HDV subgenomic and full-length genome sequences were obtained using newly established HDV-specific RT-PCR techniques. The nucleotide homology was observed from 74.6% to 99.4% among the investigated full-length genome of the HDV isolates. We observed HDV genotype 1 and HDV genotype 2 in the investigated Vietnamese patients. Although no HDV genotype mixtures were observed, we report here a newly identified recombinant of HDV genotypes (HDV 1 and HDV 2). The identified recombinant HDV isolate C03 revealed sequence homology to both HDV genotype 1 (nt1 to nt907) and HDV genotype 2 (nt908 to nt1675; HDAg coding region) with a breakpoint at nt908. Our findings demonstrate the prevalence of intergenotypic recombination between HDV genotypes 1 and 2 in a Vietnamese HBsAg-positive patient. Extended investigation on the distribution and prevalence of HDV, HDV mixed genotypes and recombinant HDV genotypes in a larger Vietnamese population offers vital insights into understanding of the micro-epidemiology of HDV and subsequent pathophysiology in chronic HBV- /HDV-related liver diseases.

Treatment of severe, nonfulminant acute hepatitis B with lamivudine vs placebo: a prospective randomized double-blinded multicentre trial.

Acute hepatitis B virus (aHBV) infection can lead to fulminant liver failure, which likely is prevented by early lamivudine therapy. Even nonfulminant but severe acute hepatitis B can lead to significant morbidity and impaired quality of life. Therefore, lamivudine was evaluated in patients with severe aHBV in a placebo-controlled trial. Patients with severe aHBV infection (ALT >10× ULN, bilirubin >85 µm, prothrombin time >50%) were prospectively treated with lamivudine 100 mg/day or with placebo within 8 days after the diagnosis. The primary end point was time to bilirubin <34.2 µm. Secondary end points were time to clear HBsAg and HBV-DNA, development of anti-HBs and normalization of ALT. Eighteen cases were randomized to lamivudine, 17 to placebo. 94% of patients were hospitalized. No individual progressed to hepatic failure; all but one patient achieved the primary end point. Due to smaller than expected patient numbers, all study end points did not become statistically significant between treatment arms. Median time end points [in days] were bilirubin <34.2 µm (26.5 vs 32), ALT normalization (35 vs 48) and HBsAg clearance (48 vs 67) referring to earlier recovery under lamivudine, in contrast to loss of HBV-DNA (62 vs 54) and development of anti-HBs (119 vs 109). In all but two patients (one in every group), HBsAg clearance was reached in the study. Adverse events occurred more frequently during lamivudine therapy, but did not reach statistical significance. Lamivudine may ameliorate severe aHBV infection, but limited patient numbers prevented definite conclusions.

Hepatitis B virus-induced hepatocellular carcinoma: functional roles of MICA variants.

Hepatitis B virus infection is a high-risk factor for hepatocellular carcinoma. The human major histocompatibility complex class I chain-related gene A (MICA) is a ligand of the NKG2D receptor that modulates the NK and T-cell-mediated immune responses and is associated with several diseases. This study determined the effects of MICA polymorphisms during HBV infection and HBV-induced HCC. We conducted a case-controlled study in a Vietnamese cohort and genotyped ten functional MICA polymorphisms including the microsatellite motif in 552 clinically classified hepatitis B virus patients and 418 healthy controls. The serum soluble MICA levels (sMICA) were correlated with MICA variants and liver enzyme levels. We demonstrated a significant contribution of MICA rs2596542G/A promoter variant and nonsynonymous substitutions MICA-129Met/Val, MICA-251Gln/Arg, MICA-175Gly/Ser, triplet repeat polymorphism and respective haplotypes with HBV-induced HCC and HBV persistence. The circulating sMICA levels in HBV patient groups were elevated significantly compared with healthy controls. A significant contribution of studied MICA variants to sMICA levels was also observed. The liver enzymes alanine amino transferase (ALT), aspartate transaminase (AST), total bilirubin and direct bilirubin were positively correlated with sMICA levels suggesting sMICA as a biomarker for liver injury. We conclude that MICA polymorphisms play a crucial role in modulating innate immune responses, tumour surveillance and regulate disease susceptibility during HBV infection.

[Molecular mechanisms and consequences of cardiac viral infections]. / Molekulare Mechanismen und Konsequenzen kardialer Virusinfektionen.

Molecular biological methods have confirmed the pathogenetic role of enteroviruses, primarily coxsackieviruses of group B (CVB), in the induction and maintenance of inflammatory cardiomyopathy. More recently, adenoviruses, various herpes viruses, and increasingly parvovirus B19 (B19) have been identified as potential cardiotropic agents. While cardiac myocytes are target cells for enterovirus and adenovirus infections with virus-induced cytolysis, B19-associated inflammatory cardiomyopathy is characterized by infection of intracardiac endothelial cells of small arterioles and veins, which may be associated with endothelial dysfunction, impairment of myocardial microcirculation, penetration of inflammatory cells, and secondary myocyte necrosis. Recent observations showed that B19 is involved in intracellular calcium regulation by the viral phospholipase. B19-induced caspase activation can lead to proinflammatory/proapoptotic processes through dysregulation of STAT signaling. These cellular interactions may contribute to mechanisms by which B19 establishes persistent infection in endothelial cells and play a critical role in viral pathogenesis of inflammatory cardiomyopathy.

Simplified detection of microsatellite instability in colorectal cancer without the need for corresponding germline DNA analysis.

A panel of five quasimonomorphic mononucleotide repeats that dispenses with the need to analyse corresponding germline DNA was proposed by Suraweera et al for the detection of high-frequency microsatellite instability (MSI) in colorectal cancer. Using this panel, a simplified and a more sensitive (compared with the original) algorithm (p<0.05) was developed to define the instability of each repeat by assessing the morphological shape of its plot and not its absolute length. 103 cases of colorectal tumours were investigated and the results compared with those obtained by the analysis of five consensus microsatellites (Bethesda reference panel). By the proposed method, a higher specificity, but no loss of sensitivity, was found. Thus, the use of the five mononucleotide repeats in combination with the modified assessment technique simplifies the assessment of MSI, while retaining the sensitivity of the Bethesda panel for the detection of high-frequency MSI.

Parvovirus B19: a new emerging pathogenic agent of inflammatory cardiomyopathy.

The human parvovirus B19 (PVB19), an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild to more severe outcomes such as hydrops fetalis, is the only currently known human pathogenic parvovirus. Recently, PVB19 has been identified as a causative agent of pediatric and adult inflammatory cardiac diseases. The first hints for a possible etiopathogenetic role of the PVB19 infection and the development of cardiac dysfunction were demonstrated by molecular biology methods such as in situ hybridization (ISH) and polymerase chain reaction (PCR). In this regard, PVB19-associated inflammatory cardiomyopathy is characterized by infection of endothelial cells of small intracardiac arterioles and venules, which may be associated with endothelial dysfunction, impairment of myocardial microcirculation, and penetration of inflammatory cells in the myocardium.

Human parvovirus B19: a new emerging pathogen of inflammatory cardiomyopathy.

The human parvovirus B19 (PVB19), an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild to more severe outcomes such as hydrops fetalis, is the only known human pathogenic parvovirus so far. Although enteroviruses have long been considered the most common cause of inflammatory cardiomyopathy, PVB19 is emerging as a important candidate. Recent studies have indicated an association of PVB19 with paediatric and adult inflammatory cardiac disease. However, whether or not PVB19 has an impact on inflammatory cardiomyopathy in adult patients is still unclear. The first hints for a possible aetiopathogenetic role of the PVB19-infection and the development of cardiac dysfunction were demonstrated by molecular biology utilizing in situ hybridization (ISH) and polymerase chain reaction (PCR). According to available evidence, PVB19-associated inflammatory cardiomyopathy is characterized by infection of endothelial cells of small intracardiac arterioles and venules, which may be associated with endothelial dysfunction, impairment of myocardial microcirculation, and penetration of inflammatory cells into the myocardium.

High prevalence of cardiac parvovirus B19 infection in patients with isolated left ventricular diastolic dysfunction.

BACKGROUND: The etiology of left ventricular (LV) isolated diastolic dysfunction often remains unclear. In the present study, we report a strong association between parvovirus B19 (PVB19) genomes and isolated LV diastolic dysfunction. METHODS AND RESULTS: In 70 patients (mean+/-SD age, 43+/-11 years) admitted with exertional dyspnea and/or reduced exercise tolerance despite preserved LV systolic contractility (ejection fraction=68%), isolated diastolic dysfunction was clinically suspected. Patients with classic risk factors for diastolic dysfunction such as hypertension, coronary heart disease, diabetes mellitus, or pulmonary disease had been excluded. Diastolic function was assessed by echocardiography and LV and RV catheterization. Endomyocardial biopsies (EMBs) were analyzed for the presence of storage or infiltrative diseases or myocarditis, including molecular screening for cardiotropic virus genomes. In a substudy of 24 patients who reported atypical angina, coronary endothelial function was additionally investigated with a coronary Doppler flow-wire technique. In 37 of 70 patients (53%), isolated diastolic dysfunction was confirmed as the cause of their clinical symptoms. No evidence for cardiac storage or infiltrative diseases was found in these cases, but in 35 of 37 of these patients (95%), cardiotropic virus genomes were detected in EMBs (P<0.001). PVB19 was the most frequent pathogen in 31 of 37 patients (84%). In a subgroup of 10 patients with diastolic dysfunction and coexisting endothelial dysfunction, all 10 (100%) were PVB19 positive. CONCLUSIONS: PVB19 genomes were predominant in patients with unexplained, isolated diastolic dysfunction. A strong association with the incidence of endothelial dysfunction was obvious, consistent with the hypothesis that PVB19-induced endothelial dysfunction may be a possible pathomechanism underlying diastolic dysfunction.

Low frequency of mutations in the X gene, core promoter and precore region of hepatitis B virus infected Vietnamese.

Numerous mutations in the hepatitis B virus (HBV) genome have been described, but in most cases their role in the pathogenesis of HBV infection is still unclear. Therefore, we analysed specific mutations in HBV-infected Vietnamese patients and assessed their potential relationship with their clinical outcome. A total of 153 HBV-infected Vietnamese patients with well-characterised clinical profiles were enrolled. None of the study participants had a history of alcohol or drug use and none received any antiviral or immunosuppressive therapy before or during the course of this study. The HBx- and core promoter regions were analysed by sequencing. The majority of isolates corresponded to genotype A. The presence of hepatitis B e antigen (HBeAg) was associated with significantly higher viral loads in the chronic HBV-infection group (P = 0.026). Double mutations in the core promoter (1762/1764) were more frequent in those with cancer than in noncancer patients (P < 0.01). Mutations at nucleotide (nt) 1766/1773 were found at low prevalence but with no obvious association to clinical presentation. Cytosine at nt 1858 was predominant but the stop codon mutation in the precore region was not detected. In the study, 4/48 hepatocellular carcinoma (HCC) patients revealed truncated HBx, whilst the serine to alanine mutation (codon 31) of HBx was more prevalent in cancer patients than in asymptomatic HBV carriers (P < 0.01). Thus, the low frequency of mutations indicates the relation of the absence of antiviral pressure in this population. The exclusively found prevalence of certain mutations detected in those with HBV-related carcinoma nevertheless indicates a degree of association with disease progression.

[Parvovirus B19 myocarditis in a young man with previous non-bacterial meningitis]. / Parvovirus B19-Myokarditis eines jungen Erwachsenen nach abakterieller Meningitis.

HISTORY AND CLINICAL FINDINGS: A 22-year old man presented with fatigue, dyspnea NYHA III and presyncopes that had persisted since a non-bacterial meningitis 3 months before. INVESTIGATIONS: Transthoracic echocardiography revealed a dilated left ventricle with an ejection fraction (EF) reduced to 35-40 % due to global hypokinesia. No pericardial effusion was seen; ECG and lung function test were normal. Serological, immunological and microbiological tests as well as nested PCR analysis of blood leucocytes for detection of cardiotropic pathogens were inconclusive. In endomyocardial biopsies retrieved from the left ventricular posterolateral wall, a chronic macro-phage-rich myocarditis was shown by histopathology and, in addition, Parvovirus B19 was identified as specific pathogen by use of nested PCR analysis. TREATMENT AND COURSE: At physical rest and with ACE inhibitor therapy (2.5 mg ramipril/day), heart failure decreased steadily. Follow-up echocardiography 1 month later revealed a left ventricle that was only slightly dilated with an EF of 50 %. 3 months later, the patient was markedly more load-bearing; the EF amounted to 55-60 %. CONCLUSIONS: Parvovirus B19 should be regarded as potential pathogen in case of suspected myocarditis in adulthood. Whether the previous non-bacterial meningitis was also attributable to this specific pathogen, remains open. Of note, however, the present case report by demonstrating a localized myocardial Parvovirus B19 infection without detectable systemic infection underscores the importance of molecular tests for diagnostic accuracy in manifest organ failure.

Cross-resistance testing of antihepadnaviral compounds using novel recombinant baculoviruses which encode drug-resistant strains of hepatitis B virus.

Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in the selection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistant variants are clearly warranted but have been difficult to do because of the lack of convenient and reliable in vitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replication that relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBV genome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently been used to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation. The further development of this novel system and its application to determine the cross-resistance profiles of drug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses which encoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examine the effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir), and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526M mutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutations M550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir is active against each of these mutants. These findings are supported by the limited amount of clinical data currently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecular characterization of clinically significant HBV strains.

Structural organization of the hepatitis B virus minichromosome.

The replicative intermediate of hepatitis B virus (HBV), the covalently closed, circular DNA, is organized into minichromosomes in the nucleus of the infected cell by histone and non-histone proteins. In this study we investigated the architecture of the HBV minichromosome in more detail. In contrast to cellular chromatin the nucleosomal spacing of the HBV minichromosome has been shown to be unusually reduced by approximately 10 %. A potential candidate responsible for an alteration in the chromatin structure of the HBV minichromosome is the HBV core protein. The HBV core protein has been implicated in the nuclear targeting process of the viral genome. The association of the HBV core protein with nuclear HBV replicative intermediates could strengthen this role. Our findings, confirmed by in vivo and in vitro experiments indicate that HBV core protein is a component of the HBV minichromosome, binds preferentially to HBV double-stranded DNA, and its binding results in a reduction of the nucleosomal spacing of the HBV nucleoprotein complexes by 10 %. From this model of the HBV minichromosome we propose that the HBV core protein may have an impact on the nuclear targeting of the HBV genome and be involved in viral transcription by regulating the nucleosomal arrangement of the HBV regulatory elements, probably in a positive manner.

The enhancer I core region contributes to the replication level of hepatitis B virus in vivo and in vitro.

Chronic hepatitis B virus (HBV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Long-term interaction of the immune system with the virus results in the selection of escape mutants and viral persistence. In this work we characterize mutations in the enhancer I region isolated prior to liver transplantation from the HBV genomes of 10 patients with chronic HBV infection. The HBV-genomes were sequenced, and the enhancer I region was cloned into luciferase reporter constructs to determine the transcriptional activity. Functional studies were performed by transfecting HBV replication-competent plasmids into hepatoma cells. Analyses of the replication fitness of the mutant strains were conducted by biochemical analysis. In all HBV genomes the enhancer I region was mutated. Most of these mutations resulted in decreased transcriptional activity. The strongest effects were detectable in strains with mutations in the hepatocyte nuclear factor 3 and 4 (HNF3 and HNF4) binding sites of the enhancer I core domain. Replication-competent HBV constructs containing these mutations demonstrated up to 10-fold-reduced levels of virus replication. Before liver transplantation, when the mutant strains were detected in the patients' sera, low HBV DNA levels were found. After transplantation and reinfection with a wild-type virus, the levels of replication were up to 240-fold higher. Our results show that mutations in the enhancer I region of HBV have a major impact on HBV replication. These mutations may also determine the switch from high to low levels of viral replication which is frequently observed during chronic HBV infection.

NF-kappaB determines between apoptosis and proliferation in hepatocytes during liver regeneration.

Tumor necrosis factor (TNF)-alpha is a potent inducer of apoptotic cell death in various tissues, whereas the transcription factor nuclear factor (NF)-kappaB is essential to protect against TNF-alpha-induced apoptosis. Human hepatoma cell lines were used to investigate the effectiveness and specificity of the fungal metabolite gliotoxin in inhibiting TNF-alpha-induced NF-kappaB activation in transformed cells. Gliotoxin-TNF-alpha cotreatment induced massive apoptosis in these otherwise TNF-alpha-resistant cell lines. With the use of the mouse partial hepatectomy model, we were also able to demonstrate in vivo the capacity of gliotoxin to act as inhibitor of NF-kappaB activation. Bromodeoxyuridine staining of liver sections showed that the lack of NF-kappaB activation correlated with 80% reduction of DNA synthesis 48 h after hepatectomy compared with untreated controls. Additionally, animals treated with gliotoxin showed nuclear condensation and DNA laddering of hepatocytes indicative of apoptosis 24 h after hepatectomy. In summary, our results demonstrate that NF-kappaB is essential in defining the fate of liver cells in response to TNF-alpha in vivo and furthermore implicate gliotoxin as a potential new response modifier for TNF-alpha-based therapy.

Variation of human mucin gene expression in gastric cancer cell lines and gastric mucous cell primary cultures.

Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.

The pre-S region determines the intracellular localization and appearance of hepatitis B virus.

The functional role of the hepatitis B virus (HBV) pre-S region for assembly and appearance of the virus is not completely understood. In this study, 3 natural-occurring mutants were investigated. Three mutants of the pre-S region-a point mutation in the CCAAT box (MUT1), a 6-bp deletion (MUT2) 3' of the CCAAT box, and a 153-bp deletion (MUT3) in the preS2 domain-were cloned alone or in combinations in replication-competent HBV plasmids and transfected in hepatoma cells. The impact on HBV assembly and appearance was studied by Northern Blot, primer extension analysis, immunofluorescence studies, enzyme-linked immunosorbent assay, and electron microscopy. An inversed ratio of pre-S/S mRNA transcripts compared with wild-type (wt) HBV was found when either MUT1 or -2 were included into the plasmid. Intracellular localization with both mutants showed retention of large S-protein in the endoplasmic reticulum and nuclear accumulation of core protein. The extracellular amount of S-protein was reduced with MUT1 and -2 or combinations in which 1 of the mutants was included. However, the extracellular appearance of viral products was comparable with wtHBV. In contrast, MUT3 showed major changes. Virion-like particles had a fried-egg, and filaments a screw-like appearance. The S-promoter mutations MUT1 and MUT2 correlated with viral retention. MUT3 leads to malformed viral particles. Therefore, different regions in the pre-S domain are essential to determine the intracellular localization and extracellular appearance of HBV, and might contribute to the prognosis of chronic HBV infection.