Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA

Nucleic acid sequence-based amplification (NASBA) is a technique that allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A NASBA technique has been developed that allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from inoculated chick embryos. The amplified viral RNA is detected by electrochemiluminescence. The NASBA technique described below is rapid and specific for the identification of influenza A subtype H5 viruses of the Eurasian lineage. More importantly, it can be used to distinguish highly pathogenic and low pathogenic strains of the H5 subtype.     

Persistent infection of SARS coronavirus in colonic cells in vitro

Severe acute respiratory syndrome coronavirus (SARS-CoV) can produce gastrointestinal symptoms. The intestinal tract is the only extrapulmonary site where viable viruses have been detected. This study examined seven established human intestinal cell lines, DLD-1, HCT-116, HT-29, LoVo, LS-180, SW-480 and SW-620, for their permissiveness to SARS-CoV infection. The results showed that only LoVo cells were permissive to SARS-CoV infection as evident by positive findings from indirect immunofluorescence staining for intracellular viral antigens, in situ hybridization for intracellular viral RNA, and electron microscopy for intracellular viral particles. In contrast to Vero cells, SARS-CoV did not produce cytopathic effects on LoVo cells. However, LoVo cells were found to be highly permissive for productive infection with a high viral titre (>3 x 10(7) viral copies/ml) produced in culture supernatant following a few days of incubation. SARS-CoV established a stable persistent chronic infection that could be maintained after multiple passages. Being a cell line of human origin, LoVo cells could be a useful in vitro model for studying the biology and persistent infection of SARS-CoV. Our results on the expression of angiotensin-converting enzyme 2 (ACE2), a recently identified cellular receptor for SARS-CoV, in these cell lines indicated that it might not be the sole determinant for cells to be susceptible to SARS-CoV infection.     

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.     

Downregulation and abnormal expression of E-cadherin and beta-catenin in nasopharyngeal carcinoma: close association with advanced disease stage and lymph node metastasis

Nasopharyngeal carcinoma (NPC) is predominantly of the undifferentiated histological subtype. Histological differentiation is of limited prognostic significance in NPC. Recent studies have suggested that downregulation of the cadherin-catenin cell adhesion complex may play a crucial role in the initial stage of cancer invasion and metastasis and is associated with poor prognosis in human cancers. Expression of E-cadherin has not been reported previously in NPC, and its prognostic value in NPC is unknown. The purpose of this study was to examine the expression pattern of E-cadherin and its associated partner, beta-catenin, in NPC and their possible applications as prognostic markers to predict the clinical outcome of NPC. Expression of the E-cadherin and beta-catenin was examined by immunohistochemical methods in 74 cases of primary NPC and 17 of their corresponding lymph node metastases. Normal nasopharyngeal epithelium showed strong and homogeneous immunocytochemical staining of E-cadherin and beta-catenin at the cell membranes and intercellular junctions. In contrast, primary NPC showed variable and heterogeneous staining patterns of E-cadherin and beta-catenin. Loss of membranous E-cadherin expression was significantly associated with advanced stages of diseases (P<.001). Eighty percent to ninety percent of NPC in stages IV and V (Ho's staging), respectively, showed a reduced (<35%) membranous staining of E-cadherin compared with normal nasopharyngeal epithelium. Expression of beta-catenin also was downregulated in advanced NPC. Ninety percent to one hundred percent of NPC in stages IV and V (Ho's staging) expressed a reduction (<35%) of imnmunocytochemical staining of beta-catenin. The expression pattern of beta-catenin staining was strongly associated with the expression of E-cadherin (P<.001). Unlike E-cadherin, nuclear staining of beta-catenin expression was observed in some of the primary NPC and lymph node metastasis. Reduced expression of E-cadherin and beta-catenin expression was associated with a shorter survival of NPC patients (P<.001). In advanced NPC patients (stages IV and V), a significant difference in survival was observed in tumors with higher or lower levels of E-cadherin expression (P=.0224, log-rank test). These observations suggests that expression of E-cadherin and beta-catenin may have prognostic values in NPC patients.     

A multicentre randomized controlled trial of oral misoprostol and i.m. syntometrine in the management of the third stage of labour

Postpartum haemorrhage accounts for nearly 28% of maternal mortality in developing countries. Syntometrine is an effective and commonly used oxytocic in preventing postpartum haemorrhage, but it requires a controlled storage environment and i.m. administration. Misoprostol is an orally active uterotonic agent. A total of 2058 patients having a singleton pregnancy, low risk for postpartum haemorrhage and vaginal delivery were randomized to receive either 1 ml syntometrine or 600 microgram misoprostol for the management of the third stage of labour. There were no significant differences between the two groups in the mean blood loss, the incidence of postpartum haemorrhage and the fall in haemoglobin concentration. The need for additional oxytocic injection was significantly higher in the misoprostol group [relative risk (RR) 1.62, 95% confidence interval (CI) 1.34-1.96], but that of manual removal of placenta was reduced (RR 0.29, 95% CI 0.09-0.87). Shivering and transient pyrexia were more common in the misoprostol group. Oral misoprostol might be used in the management of the third stage, especially in situations where the use of syntometrine is contraindicated and facilities for storage and parenteral administration of oxytocics are limited.     

Three distinct loci on human chromosome 21 contribute to interferon-α/β responsiveness

The species specificity of interferons (IFNs) depends on restricted recognition of these ligands by multisubunit cell surface receptors. Expression of the human receptor subunit IFNAR in mouse cells conferred sensitivity only to one subtype of human IFN, IFN-B. Other genes on human chromosome 21 were required for responses to other subtypes of type I IFN. In contrast, IFNAR expression in hamster cells did not confer sensitivity to any human IFN tested, including IFN-B. Using human-hamster somatic cell hybrids, we mapped theIfnabr gene, encoding a ligand-binding subunit of the IFN-/ (type I) receptor, to human chromosome 21.Ifnabr colocalized withIfnar to the distal region of q22.1. The presence of a chromosomal fragment encoding IFNABR and IFNAR was also not sufficient to confer sensitivity to human IFN. In contrast, hybrids carrying in addition the region 21q22.2 showed a full response to human IFN-B, suggesting that a gene located in this region encodes a third factor required for type I IFN receptor activity.     

Functional studies of yeast actin mutants corresponding to human cardiomyopathy mutations

The molecular mechanisms by which different mutations in actin lead to distinct cardiomyopathies are unknown. Here, actin mutants corresponding to α-cardiac actin mutations causing hypertrophic cardiomyopathy [(HCM) P164A and A331P] and dilated cardiomyopathy [(DCM) R312H and E361G] were expressed in yeast and purified for in vitro functional studies. While P164A appeared unaltered compared to wild-type (WT) actin, A331P function was impaired. A331P showed reduced stability in circular dichroism melting experiments; its monomer unfolding transition was 10°C lower compared to WT actin. Additionally, in vitro filament formation was hampered, and yeast cell cultures were temperature sensitive, implying perturbations in actin–actin interactions. Filament instability of the A331P mutant actin could lead to actomyosin dysfunction observed in HCM. Yeast strains harboring the R312H mutation did not grow well in culture, suggesting that cell viability is compromised. The E361G substitution is located at an α-actinin binding region where the actin filament is anchored. The mutant actin, though unaltered in the in vitro motility and standard actomyosin functions, had a threefold reduction in α-actinin binding. This could result in impairment of force-transduction in muscle fibers, and a DCM phenotype. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of quantitative PCR and branched-chain DNA assay for detection of hepatitis B virus DNA in sera from hepatocellular carcinoma and liver transplant patients

This study evaluated the applicability of quantitative PCR (Q-PCR) and branched-chain DNA assays for detection of hepatitis B virus (HBV) DNA in sera. For 42 samples, the detection rates were 81 and 41%, respectively, with a correlation coefficient of 0.633. The Q-PCR is useful for early monitoring of HBV load in high-risk patients.     

Recurrent and novel mutations of GCDH gene in Chinese glutaric acidemia type I families

Glutaric acidemia type I is caused by mutations of the glutaryl-CoA dehydrogenase (GCDH) gene resulting in loss of GCDH enzyme activity. Patients present with progressive dystonia and lesions in basal ganglia. Dietary treatment, when instituted from the early neonatal period, markedly reduces dystonia and morbidity. Early diagnosis and prenatal diagnosis will be facilitated by knowledge of locally prevalent GCDH mutations. Several common GCDH mutations have been found in different ethnic groups. GCDH mutations were studied in 5 Chinese glutaric acidemia type I families. We detected two novel recurrent mutations (A219T and IVS10-2A>C) which were found in two unrelated families. An asymptomatic carrier of IVS10-2A>C was also found on screening of 120 individuals. Other mutations were identified, including two other novel (R386G & IVS3+1G>A) and two known mutations (G178R & R355H). Fibroblasts from patients carrying the novel mutations were confirmed to be deficient for GCDH activity. This is the first report of GCDH mutations describing recurrent mutations in Chinese patients. The carrier rate of IVS10-2A>C may be particularly high in Chinese.