Hepatitis B virus upregulates host microRNAs that target apoptosis-regulatory genes in an in vitro cell model.

Chronic hepatitis B (CHB) infection increases the risk of developing severe liver disease including cirrhosis and hepatocellular carcinoma (HCC). As microRNAs may modulate host - virus interactions, we here investigated if hepatitis B virus (HBV) infection modulate microRNA expression using an in vitro HepG2 cell model system with inducible HBV replication. We found that HBV replication was associated with upregulation of miR-192-5p, miR-194-5p and miR-215-5p, of which miR-192-5p and miR-215-5p have identical seed sequences. Bioinformatics analyses revealed a significant enrichment of potential target genes involved in apoptosis signaling of all three microRNAs. In line with this, transfection with a mimic of miR-192-5p suppressed the protein level of pro-apoptotic BIM and reduced endoplasmic reticulum (ER) stress-induced apoptosis in HepG2 cells. In contrast, transfection with a mimic of miR-194-5p downregulated the anti-apoptotic proteins SODD and cFLIP, and sensitized HepG2 cells to both ER stress- and cytokine-induced apoptosis. In conclusion, our study suggests that HBV upregulates the expression of miR-192-5p and miR-194-5p in the host cell. These microRNAs target important apoptosis-regulatory proteins, and may thus contribute to the development of HBV-related liver disease.

Hepatitis B virus suppresses the secretion of insulin-like growth factor binding protein 1 to facilitate anti-apoptotic IGF-1 effects in HepG2 cells.

Hepatitis B virus (HBV) infection is a major global health burden as chronic hepatitis B (CHB) is associated with the development of liver diseases including hepatocellular carcinoma (HCC). To gain insight into the mechanisms causing HBV-related HCC, we investigated the effects of HBV replication on global host cell gene expression using human HepG2 liver cells. By microarray analysis, we identified 54 differentially expressed genes in HBV-replicating HepG2 cells. One of the differentially-expressed genes was insulin-like growth factor binding protein 1 (IGFBP1) which was downregulated in HBV-replicating cells. Consistent with the gene expression data, IGFBP1 was suppressed at both the cellular and secreted protein levels in the presence of HBV replication. Transient transfection experiments with an inducible plasmid encoding the HBV X protein (HBx) revealed that HBx alone was sufficient to modulate IGFBP1 expression. Small interference RNA (siRNA)-mediated loss of function studies revealed that knockdown of IGFBP1 reduced apoptosis induced by either thapsigargin (TG) or staurosporine (STS). Treatment of cells with recombinant insulin-like growth factor 1 (IGF-1) decreased both TG- or STS-induced apoptosis. Interestingly, addition of recombinant IGFBP1 reversed the anti-apoptotic effect of IGF-1 on TG-induced, but not STS-induced, apoptosis. In conclusion, our results suggest an anti-apoptotic autocrine function of HBV-mediated downregulation of IGFBP1 in HepG2 cells. Such an effect may contribute to the development of HBV-mediated HCC by increasing pro-survival and anti-apoptotic IGF-1 effects.

Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line.

BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes for the human HCC-derived cell line HepG2. RESULTS: A panel of 739 microRNAs was screened to identify the most stably expressed microRNAs, followed by a PubMed search identifying microRNAs previously used as reference genes. Sixteen candidate reference genes were validated by RT-qPCR. Reference gene stabilities were calculated first by standard deviations of ΔCt values and then by geNorm and NormFinder analyses, taking into account the amplification efficiency of each microRNA primer set. The optimal set of reference genes was verified by a target analysis using RT-qPCR on miR-215-5p. CONCLUSION: We identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model.

Circulating MicroRNAs in Plasma of Hepatitis B e Antigen Positive Children Reveal Liver-Specific Target Genes.

Background and Aim. Hepatitis B e antigen positive (HBeAg-positive) children are at high risk of severe complications such as hepatocellular carcinoma and cirrhosis. Liver damage is caused by the host immune response to infected hepatocytes, and we hypothesise that specific microRNAs play a role in this complex interaction between virus and host. The study aimed to identify microRNAs with aberrant plasma expressions in HBeAg-positive children and with liver-specific target genes. Methods. By revisiting our previous screen of microRNA plasma levels in HBeAg-positive and HBeAg-negative children with chronic hepatitis B (CHB) and in healthy controls, candidate microRNAs with aberrant plasma expressions in HBeAg-positive children were identified. MicroRNAs targeting liver-specific genes were selected based on bioinformatics analysis and validated by qRT-PCR using plasma samples from 34 HBeAg-positive, 26 HBeAg-negative, and 60 healthy control children. Results. Thirteen microRNAs showed aberrant plasma expressions in HBeAg-positive children and targeted liver-specific genes. In particular, three microRNAs were upregulated and one was downregulated in HBeAg-positive children compared to HBeAg-negative and healthy control children, which showed equal levels. Conclusion. The identified microRNAs might impact the progression of CHB in children. Functional studies are warranted, however, to elucidate the microRNAs' role in the immunopathogenesis of childhood CHB.

Hepatitis B surface antigen quantity positively correlates with plasma levels of microRNAs differentially expressed in immunological phases of chronic hepatitis B in children.

BACKGROUND AND AIM: Children with chronic hepatitis B (CHB) are at high risk of progressive liver disease. It is suggested that a newly-identified panel of 16 microRNAs is important in the pathogenesis of CHB in children. Subviral hepatitis B surface antigen (HBsAg) particles are produced in large excess over infectious virions. Interestingly, circulating HBsAg particles have been shown to carry microRNAs. A thorough characterisation of the identified microRNAs and HBsAg over time in plasma from children with CHB may provide useful information about the natural course of childhood CHB. PATIENTS AND METHODS: A cohort of 42 children with CHB was followed over time. Three to five blood samples were obtained from each child at minimum intervals of half a year; in total 180 blood samples. Plasma levels of the 16 microRNAs previously identified were analysed by quantitative real-time polymerase-chain-reaction. Plasma HBsAg was quantified using ARCHITECT® HBsAg assay. RESULTS: The presence of 14/16 plasma microRNAs in children with CHB was confirmed. All 14 microRNAs were significantly differentially expressed in different immunological phases of the disease. MicroRNA plasma levels were highest in immune-tolerant children, lower in immune-active children, and reached the lowest values in immune-inactive children, p<0.001. Plasma levels of four microRNAs decreased significantly over time in immune-tolerant and immune-active children whereas the microRNA plasma levels were stable in immune-inactive children, p<0.004. HBsAg quantity was positively correlated with plasma levels of 11/14 microRNAs, p<0.004. CONCLUSION: This is the first study to characterise plasma microRNAs and HBsAg over time in children with CHB. Our data suggest that plasma levels of selected microRNAs and HBsAg are inversely correlated with immunological control of CHB in children. Further studies are, however, needed to advance the understanding of microRNAs and HBsAg in the pathogenesis of CHB in children.

Differential plasma microRNA profiles in HBeAg positive and HBeAg negative children with chronic hepatitis B.

BACKGROUND AND AIM: Children chronically infected with hepatitis B virus (HBV) are at high risk of progressive liver disease. However, no treatment is available that is consistently effective in curing chronic hepatitis B (CHB) in children. Improved understanding of the natural course of disease is warranted. Identification of specific microRNA (miRNA) profiles in children chronically infected with HBV may provide insight into the pathogenesis of CHB and lead to advances in the management of children with CHB. PATIENTS AND METHODS: MiRNA PCR panels were employed to screen plasma levels of 739 miRNAs in pooled samples from HBeAg positive, HBeAg negative, and healthy children. The three groups' plasma miRNA profiles were compared, and aberrantly expressed miRNAs were identified. The identified miRNAs were then validated. Individual RT-qPCRs were performed on plasma from 34 HBeAg positive, 26 HBeAg negative, and 60 healthy children. RESULTS: A panel of 16 plasma miRNAs were identified as aberrantly expressed in HBeAg positive and HBeAg negative children (p<0.001). Levels of all of the miRNAs were upregulated in HBeAg positive children compared with in HBeAg negative children. A positive correlation was furthermore found between plasma levels of the identified miRNAs and HBV DNA (p<0.001). CONCLUSION: We are the first to investigate the plasma miRNA profile of children chronically infected with HBV. Our data indicates the existence of a relationship between abundance of circulating miRNAs and immunological stages in the natural course of disease. Certain miRNAs may contribute to the establishment and maintenance of CHB in children. Further studies are warranted to advance understanding of miRNAs in the pathogenesis of CHB, hopefully leading to the identification of future therapeutic targets.

Safety of a 2-dose regimen of a combined measles, mumps, rubella and varicella live vaccine manufactured with recombinant human albumin.

BACKGROUND: ProQuad, a vaccine containing antigens from M-M-RVAXPRO (measles, mumps and rubella vaccine) and VARIVAX (varicella vaccine), is indicated for simultaneous vaccination against measles, mumps, rubella and varicella (MMRV) in individuals from 12 months of age. To eliminate blood-derived products of human origin from the manufacturing process of the MMRV vaccine, recombinant human albumin was selected as a replacement for human serum albumin. METHODS: This open-label, multicenter clinical trial (clinicaltrials.gov identifier NCT00560755) was designed to describe the safety profile of a 2-dose schedule of the MMRV vaccine at a 1-month interval in healthy children aged 12-22 months. RESULTS: In total, 3388 children received at least 1 dose of the MMRV vaccine. Overall, 3376 (99.65%) children were included in the post-dose 1 safety analysis and 3342 (98.64%) in the post-dose 2 safety analysis. After doses 1 and 2, the frequencies of children experiencing solicited injection-site reactions (post-dose 1: erythema 14.31%; swelling 5.57% and pain 10.31%; post-dose 2: erythema 30.46%; swelling 13.23% and pain 11.49%), rashes of interest (post-dose 1: 11.4%; post-dose 2: 2.78%), vaccine-related nonserious systemic adverse events (post-dose 1: 34.86%; post-dose 2: 13.4%) and temperature ≥39.4 °C (post-dose 1: 25.24%; post-dose 2: 12.06%) were consistent with those observed in previous studies of the MMRV vaccine manufactured with human serum albumin. Neither serious allergic-type adverse events nor anaphylactic reactions were reported. CONCLUSION: The results confirm the good safety profiles of MMRV and of measles, mumps and rubella vaccines manufactured with recombinant human albumin.

Pattern recognition receptor responses in children with chronic hepatitis B virus infection.

BACKGROUND: Several studies have demonstrated that hepatitis B virus (HBV) affects the expression and function of Toll like receptors (TLRs), but data on TLR function in HBV infection are mainly from adult patients. The natural history of chronic hepatitis B (CHB) infection is distinctly different in children, since 90% of children become chronic carriers compared to 5% of adults when infected with HBV. OBJECTIVES: We wanted to study the function of TLRs and cytosolic DNA receptors in children with CHB infection compared to healthy children. STUDY DESIGN: PBMCs from 19 children with CHB and 19 healthy children were stimulated with ligands for TLR 2, 3, 4, 7 and 9 for 24 h. For activation of cytosolic DNA receptors, cells were transfected with a double-stranded DNA using Lipofectamine 2000. Supernatants were analyzed for levels of IFN-α, TNF-α, IL-6, CXCL10 and CCL3 by Luminex. RESULTS: Stimulation with ligands for TLR2, TLR3 and TLR9 induced IL-6, CCL3 and CXCL10 to a significantly higher level in children with CHB compared to healthy children. CHB patients displayed significantly lower IFN-α production compared to healthy children after stimulation with ligands for TLR2, TLR3 and TLR4. Stimulation of intracellular DNA sensors with synthetic double-stranded DNA elicited significantly higher induction of the inflammatory cytokines and chemokines IL-6, TNF-α and CCL3 in the CHB patients as compared to the healthy children. CONCLUSIONS: Our results indicate a TLR-mediated inflammatory response in children with CHB infection. Furthermore, our study is the first to show that the responses of intracellular DNA receptors are affected in CHB.

Prospective study of the burden of rotavirus gastroenteritis in Danish children and their families.

This was the first study to characterize the total burden of rotavirus gastroenteritis (RVGE) at both hospital and general physician (GP) clinics in Denmark, and also the first to confirm rotavirus (RV) as the leading cause of acute gastroenteritis (GE) among children <5 years in GP clinics nationwide. Several aspects of RVGE were reported, including the impact of RVGE on family life by changes in HRQoL and by the number of days absent from day care. RV was detected in 225 (63.6%) children, and the median number of days absent from day care was 5 days. In 43.0% of the families, at least one family member, a total of 170 individuals, experienced symptoms of acute GE. Reduced health-related quality of life was observed both among children and parents. Our data suggested that RVGE indirectly as well as directly is a major public health burden in Denmark and comparable with data from other European countries.

Evidence of Bordetella pertussis infection in vaccinated 1-year-old Danish children.

We measured IgA and IgG antibodies to pertussis toxin (PT) and filamentous hemagglutinin (FHA) in sera from 203 1-year-old children who had received one to three doses of a monocomponent PT toxoid vaccine. Ten children (5%) had IgA antibody to PT indicating recent infection; seven of these children had received three doses of vaccine. PT IgA responders did not have significantly longer coughing episodes than PT IgA non-responders. Since an IgA antibody response occurs in only approximately 50% of infected children, the actual infection rate in our cohort is estimated to approximately 10%. The apparent high Bordetella pertussis infection rate in Danish infants suggests that the monocomponent PT toxoid vaccine used in Denmark has limited efficacy against B. pertussis infection. A prospective immunization study comparing a multi-component vaccine with the present monocomponent PT toxoid vaccine should be undertaken.

Hepatitis B virus DNA in saliva from children with chronic hepatitis B infection: implications for saliva as a potential mode of horizontal transmission.

To explore the mechanism of horizontal transmission of hepatitis B virus (HBV) among children, we investigated the quantitative relationship between HBV in saliva and blood from 46 children with chronic hepatitis B. We found high levels of HBV DNA in saliva of HBeAg (+) children, suggesting saliva as a vehicle for horizontal transmission of HBV among children.

Immunogenicity of a 2-dose priming and booster vaccination with the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine.

BACKGROUND: The immunogenicity of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D-conjugate vaccine (PHiD-CV) was determined following a simplified 2-dose priming and the more commonly employed 3-dose priming both followed by a booster dose. METHODS: A total of 351 healthy subjects were primed with PHiD-CV at either 3 and 5 or 3, 4 and 5 months of age followed in all subjects by a booster dose at 11 to 12 months of age. Serotype-specific pneumococcal responses were measured by 22F-inhibition ELISA and opsonophagocytic assays 1 month following primary and booster vaccinations. RESULTS: Depending on the serotype, the percentages of subjects reaching the ELISA antibody threshold of 0.2 microg/mL were 92.8% to 98.0% following 2 primary doses and 96.1% to 100% following 3 primary doses except for serotype 6B (55.7% and 63.1%, respectively) and serotype 23F (69.3% and 77.6%, respectively). Opsonophagocytic activity (OPA) could be measured in 74.4% to 100% and 88.9% to 100% of the subjects after the 2-dose or 3-dose priming, respectively, except for serotype 1 (60.8% and 62.9%, respectively). In both groups, robust increases in ELISA antibodies and OPA titers were observed for all serotypes after the booster dose. Higher postprimary and postbooster ELISA antibody levels and OPA titers were observed for most serotypes following the 3+1 schedule. CONCLUSION: PHiD-CV was immunogenic in both schedules, but further effectiveness data are needed to fully understand the public health benefit to be expected from these schedules in terms of prevention against invasive and mucosal infections.

Invasive pneumococcal disease in Danish children, 1996-2007, prior to the introduction of heptavalent pneumococcal conjugate vaccine.

AIM: The aim of this study was to document the epidemiology, microbiology and outcome of invasive pneumococcal disease (IPD) among children <16 years with quality surveillance data, just prior to the introduction of the heptavalent pneumococcal conjugate vaccine (PCV7) into the Danish routine immunization programme October 2007. METHODS: Clinical and microbiological records on cases of IPD in children <16 years admitted to Hvidovre Hospital, Denmark 1996-2007, were retrospectively reviewed. RESULTS: We identified 106 cases of IPD. The annual incidence of IPD was 11 per 100 000 in children <16 years, but considerably higher, 62 per 100 000, in children <2 years. Additionally, of the children with pneumococcal meningitis 86% were <2 years. We observed no fatalities. A total of 10% developed sequelae, but of the patients with pneumococcal meningitis 27% developed sequelae. Nine patients had known risk factors. The Streptococcus pneumoniae serotype was available for 81 cases. Seventy-five percent of the IPD cases in children aged <2 years were caused by one of the serotypes contained within PCV7, compared to only 24% in children >/=2 years. CONCLUSION: Our data indicate that an estimated 75% of all IPD cases among children <2 years are caused by PCV7 serotypes and might therefore be prevented by PCV7 vaccination.

[A study of the validity of urinary tract infection diagnosis in children younger than two years of age at Hvidovre Hospital]. / Undersøgelse af validiteten af diagnosen urinvejsinfektion hos børn under to år på Hvidovre Hospital.

INTRODUCTION: The aim of this study is to validate the diagnosis of urinary tract infection (UTI) concerning false-positive diagnoses in children younger than two years of age at Hvidovre Hospital. MATERIALS AND METHODS: The material consists of 89 children (50 girls and 39 boys) diagnosed with acute pyelonephritis from September 2002 until October 2004. Two patients dropped out as they were diagnosed in other countries. The patient records were investigated to identify the children who fulfilled the UTI criteria used in the department: 1) Two mid-stream urine samples with bacterial growth of = 10.000 cfu (colony forming units)/cc, 2) growth of = 100 cfu/cc in urine obtained by a suprapubic puncture of the bladder or 3) a mid-stream urine sample with bacterial growth of = 10.000 cfu/cc and a clinical picture of UTI together with elevated inflammatory laboratory parameters. RESULTS: 70 out of 87 patients (80%, 95% confidence limits 70-88%) had a valid UTI diagnosis. CONCLUSION: We consider a diagnostic validity of 80% as satisfactory although our aim is to increase the validity to 90%.

Clinical and epidemiologic characteristics of human bocavirus in Danish infants: results from a prospective birth cohort study.

BACKGROUND: Human bocavirus (HBoV) is a recently discovered parvovirus that has been detected in respiratory samples from children with acute respiratory tract infection (ARTI) and in feces from children with gastroenteritis. However, its role as a causative agent of respiratory disease is not determined. METHODS: We investigated the presence of HBoV by real-time polymerase-chain reaction of nasal swab specimens obtained from 228 healthy children followed in the community from birth to 1 year of age for a 2-year period from 2004 to 2006. Nasal swabs and symptom diaries were collected at monthly home visits. RESULTS: HBoV was detected in 57 (8.2%) of 697 nasal swab specimens from children with ARTI, in 1 (2.3%) of 44 swabs from children with diarrhea, and in 13 (8.6%) of 152 swabs from asymptomatic children. HBoV was present mainly during the winter months. An additional respiratory virus was identified in 27 (47.4%) HBoV-positive samples. Thirty-four (68%) of 50 children with ARTI shed HBoV for less than 1 month, 13 (26%) for 2 months, 2 (4%) for 3 months, and 1 (2%) for 4 months. Seven asymptomatic children shed HBoV for less than 1 month, 2 children for 2 months, and 1 asymptomatic child had 5 HBoV-positive nasal swabs detected for 6 consecutive months. HBoV infection was associated with maternal smoking, being born in the winter, and predisposition to asthma. CONCLUSIONS: Asymptomatic carriage of HBoV is common in infants <1 year of age, and an HBoV-positive test result does not imply that HBoV is the cause of the illness.

Acute respiratory symptoms and general illness during the first year of life: a population-based birth cohort study.

Respiratory symptoms are common in infancy. Most illnesses occurring among children are dealt with by parents and do not require medical attention. Nevertheless, few studies have prospectively and on a community-basis assessed the amount of respiratory symptoms and general illness in normal infants. In this population-based birth cohort study, 228 healthy infants from Copenhagen, Denmark were followed from birth to 1 year of age during 2004-2006. Symptoms were registered using daily diaries and monthly home visits. Interviews were performed at inclusion and every second month. Risk factor analysis was carried out by multiple logistic regression analysis. On average, children had general symptoms for 3.5 months during their first year of life, nasal discharge being most frequent followed by cough. Frequency of all symptoms increased steeply after 6 months of age. Each child had on average 6.3 episodes (median: 5.1, inter-quartile range (IQR): 3.3-7.8) of acute respiratory tract illness (ARTI) (nasal discharge and > or = 1 of the following symptoms: cough, fever, wheezing, tachypnea, malaise, or lost appetite) and 5.6 episodes (median: 4.3, IQR: 2.1-7.3) of simple rhinitis per 365 days at risk. Determinants for respiratory symptoms were increasing age, winter season, household size, size of residence, day-care attendance, and having siblings aged 1-3 years attending a day nursery. In conclusion, the present study provides detailed data on the occurrence of disease symptoms during the first year of life in a general population cohort and emphasizes the impact of increasing age, seasonality, and living conditions on the occurrence of ARTI.

A community study of clinical traits and risk factors for human metapneumovirus and respiratory syncytial virus infection during the first year of life.

Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are important respiratory pathogens with similar symptomatology. The aim of this prospective birth cohort study was to identify risk factors for an hMPV or RSV infection during the first year of life in unselected healthy children. We followed 217 children from birth to 1 year of age. Nasal swabs and symptom diaries were collected monthly. Anti-hMPV and anti-RSV IgG antibodies by age 1 year were detected by ELISA, and nasal swabs were analysed for hMPV and RSV by RT-PCR. Logistic regression was used for risk factor analysis. Anti-hMPV IgG was found in 38 children (17.5%), and anti-RSV IgG in 172 children (79%). Risk factors for being anti-hMPV IgG-positive were: (1) being born in the spring (OR = 2.36; 95% CI:1.06-5.27), and (2) having older siblings (OR = 3.82; 95% CI:1.75-8.34). Risk factors for being anti-RSV IgG-positive were: (1) gestational age <38 weeks (OR = 3.39; 95% CI:1.42-8.05), (2) increasing paternal age (OR = 1.85 per 5 yrs; 95% CI:1.28-2.68), and (3) wall-to-wall carpeting (OR = 3.15; 95% CI:1.29-7.68). Being born in the spring was associated with decreased odds of being anti-RSV IgG-positive (OR = 0.27, 95% CI:0.09-0.85). Risk factors for RSV hospitalisation (n = 11) were: (1) older siblings (OR = 4.49; 95% CI: 1.08-18.73) and (2) smoking in the household (OR = 5.06; 95% CI: 1.36-18.76). Exclusive breastfeeding for the first 14 days of life protected against hospitalisation (OR = 0.21; 95% CI:0.06-0.79). In conclusion, this study identifies risk factors for mild and asymptomatic hMPV infections in infancy.