Characterization of a monoclonal antibody against the 3D polymerase of enterovirus 71 and its use for the detection of human enterovirus A infection

Over the last decade, frequent epidemic outbreaks of hand, foot and mouth disease have been observed in the Asia-Pacific region. Hand, foot and mouth disease is caused by different viruses from the enterovirus family, mainly coxsackievirus A16 and enterovirus 71 (EV71) from the human enterovirus A family. Severe disease and neurological complications are associated more often with EV71 infection, and can lead occasionally to fatal brain stem encephalitis in young children. The rapid progression and high mortality of severe hand, foot and mouth disease makes the direct detection of antigens early in infection essential. The best method for virus detection is the use of specific monoclonal antibodies. The generation and characterization of a monoclonal antibody specific for the 3D polymerase of human enterovirus A and the development of a virus detection dot blot assay are described. A recombinant 3CD protein from EV71 C4 strain was used as an immunogen to generate monoclonal antibodies (MAbs). Screening of hybridoma cells led to the isolation of monoclonal antibody 4B12 of the immunoglobulin IgG1 isotype. MAb 4B12 recognizes the linear epitope DFEQALFS close to the active site of the 3D polymerase, corresponding to amino acid positions 53-60 of 3D and 1784-1791 of enterovirus 71 polyprotein. The presence of 3D polymerase and its precursor 3CD proteinase in purified virus particles was confirmed. MAb 4B12 was used successfully to detect all enterovirus 71 subgenotypes in a denaturing dot blot assay with a sensitivity of 10 pg of 3D protein and 10(4) tissue culture infective dose of virus particles.     

IRF4 deficiency abrogates lupus nephritis despite enhancing systemic cytokine production

The IFN-regulatory factors IRF1, IRF3, IRF5, and IRF7 modulate processes involved in the pathogenesis of systemic lupus and lupus nephritis, but the contribution of IRF4, which has multiple roles in innate and adaptive immunity, is unknown. To determine a putative pathogenic role of IRF4 in lupus, we crossed Irf4-deficient mice with autoimmune C57BL/6-(Fas)lpr mice. IRF4 deficiency associated with increased activation of antigen-presenting cells in C57BL/6-(Fas)lpr mice, resulting in a massive increase in plasma levels of TNF and IL-12p40, suggesting that IRF4 suppresses cytokine release in these mice. Nevertheless, IRF4 deficiency completely protected these mice from glomerulonephritis and lung disease. The mice were hypogammaglobulinemic and lacked antinuclear and anti-dsDNA autoantibodies, revealing the requirement of IRF4 for the maturation of plasma cells. As a consequence, Irf4-deficient C57BL/6-(Fas)lpr mice neither developed immune complex disease nor glomerular activation of complement. In addition, lack of IRF4 impaired the maturation of Th17 effector T cells and reduced plasma levels of IL-17 and IL-21, which are cytokines known to contribute to autoimmune tissue injury. In summary, IRF4 deficiency enhances systemic inflammation and the activation of antigen-presenting cells but also prevents the maturation of plasma cells and effector T cells. Because these adaptive immune effectors are essential for the evolution of lupus nephritis, we conclude that IRF4 promotes the development of lupus nephritis despite suppressing antigen-presenting cells.     

Profile of men＇s health in Malaysia： problems and ：hallenges

Men's health concerns have evolved from the traditional andrology and male sexual health to a more holistic approach that encompasses male psychological, social and physical health. The poor state of health in men compared to their female counterparts is well documented. A review of the epidemiological data from Malaysia noted a similar trend in which men die at higher rates in under 1 and above 15 years old groups and most disease categories compared to women. In Malaysia, the main causes of death in men are non-communicable diseases and injuries. Risk factors, such as risk-taking behaviour, smoking and hypertension, are prevalent and amenable to early interventions. Erectile dysfunction, premature ejaculation and prostate disorders are also prevalent. However, many of these morbidities go unreported and are not diagnosed early; therefore, opportunities for early intervention are missed. This reflects poor health knowledge and inadequate health-care utilisation among Malaysian men. Their health-seeking behaviour has been shown to be strongly influenced by family members and friends. However, more research is needed to identify men's unmet health-care needs and to develop optimal strategies for addressing them. Because the Malaysian population is aging and there is an increase in sedentary lifestyles, optimizing men's health will remain a challenge unless effective measures are implemented. The existing male-unfriendly health-care system and the negative influence of masculinity on men's health behaviour must be addressed. A national men's health policy based on a male-friendly approach to health-care delivery is urgently needed to provide a framework for addressing these challenges.     

New considerations on the management of osteoporosis in Central and Eastern Europe (CEE): summary of the ��3rd Summit on Osteoporosis��CEE��, November 2009, Budapest, Hungary

Introduction

In November 2009, the ??3rd Summit on Osteoporosis??Central and Eastern Europe (CEE)?? was held in Budapest, Hungary. The conference aimed to tackle issues regarding osteoporosis management in CEE identified during the second CEE summit in 2008 and to agree on approaches that allow most efficient and cost-effective diagnosis and therapy of osteoporosis in CEE countries in the future.

Discussion

The following topics were covered: past year experience from FRAX? implementation into local diagnostic algorithms; causes of secondary osteoporosis as a FRAX? risk factor; bone turnover markers to estimate bone loss, fracture risk, or monitor therapies; role of quantitative ultrasound in osteoporosis management; compliance and economical aspects of osteoporosis; and osteoporosis and genetics. Consensus and recommendations developed on these topics are summarised in the present progress report.

Conclusion

Lectures on up-to-date data of topical interest, the distinct regional provenances of the participants, a special focus on practical aspects, intense mutual exchange of individual experiences, strong interest in cross-border cooperations, as well as the readiness to learn from each other considerably contributed to the establishment of these recommendations. The ??4th Summit on Osteoporosis??CEE?? held in Prague, Czech Republic, in December 2010 will reveal whether these recommendations prove of value when implemented in the clinical routine or whether further improvements are still required.     

The protective role of bone morphogenetic protein-8 in the glucocorticoid-induced apoptosis on bone cells

One of the side effects associated with glucocorticoid therapy is glucocorticoid-induced bone loss. Glucocorticoids partly detain bone formation via the inhibition of osteoblastic function, however, the exact mechanism of this inhibition remains elusive. In this study, we examined the effect of dexamethasone, an active glucocorticoid analogue, on cell viability and expression of bone remodelling-related genes in primary mouse calvarial and cloned MC3T3-E1 osteoblasts. Using sensitive biochemical assays, we demonstrated the apoptotic effect of dexamethasone on osteoblastic cells. Then, utilizing Taqman probe-based quantitative RT-PCR technology, gene expression profiles of 111 bone metabolism-related genes were determined. As a result of dexamethasone treatment we have detected significant apoptotic cell death, and six genes, including Smad3, type-2 collagen α-1, type-9 collagen α-1, matrix metalloproteinase-2, bone morphogenetic protein-4 and bone morphogenetic protein-8 showed (BMP-8) significant changes in their expression on a time- and concentration-dependent manner. BMP-8, (a novel player in bone-metabolism) exhibited a two orders of magnitude elevation in its mRNA level and highly elevated protein concentration by Western blot in response to dexamethasone treatment. The knockdown of BMP-8 by RNA interference significantly increased dexamethasone-induced cell death, confirming a protective role for BMP-8 in the glucocorticoid-induced apoptosis of osteoblasts. Our results suggest that BMP-8 might be an essential player in bone metabolism, especially in response to glucocorticoids.     

Mortality of therapeutic fish Garra rufa caused by Aeromonas sobria

Objective

To investigate a case of mass mortality of Garra rufa (G. rufa) from a fish hatchery farm in Slovakia.

Methods

Causative bacterial agent was swabbing out of affected fish skin area and subsequently identified using commercial test system. Antibiotic susceptibility was determined by the disk diffusion method.

Results

Infected G. rufa was characterized by abnormal swimming behaviour, bleeding of skin lesions and local haemorrhages. Despite of using recommended aquatic antibiotic treatment no improvement was achieved and Aeromonas sobria (A. sobria) was identified as a causative agent of fish mortality. Due to massive fish mortality, antibiotic susceptibility of pure isolated culture of A. sobria was evaluated employing eight antibiotics against human infections. A. sobria was resistant only against one antibiotic, namely ampicilin.

Conclusions

These results indicate that A. sobria can act as a primary pathogen of G. rufa and may be a potential risk factor for immunodeficient or immunoincompetent patients during the ichthyotherapy.     

Living and working with the people of 'the bush': A foundation for rural and remote clinical placements in undergraduate medical education

Background: The Australian Government's policies and programmes to redress the medical workforce shortage in rural and remote areas focus on recruitment of rural students and provision of rural clinical placements. The University of Notre Dame's Rural and Remote Health Placement Programme (RRHPP) uses an additional approach to address this issue. Aim: This article describes the RRHPP undertaken by all medical students in the first 2-years of their course and examines the educational worth of this approach. Method: Data were obtained from curricular documents, publications about the RRHPP and evaluation questionnaires administered to students and supervisors. Results: The RRHPP provides students with opportunities to develop a patient- and community-centred perspective on the health issues of rural and remote populations by having them live and work with people in these areas prior to clinical placements. It is based on sound educational principles and underpinned by participation of rural/remote communities as experts and equal teaching partners. The RRHPP is valued and perceived by a majority of students and placement hosts as a useful strategy to develop medical students' understanding of the rural/remote community context and its impact on health. Conclusion: This community participatory approach benefits medical students and rural/remote communities.     

Isolation and characterization of Acanthamoeba spp. from air-conditioners in Kuala Lumpur, Malaysia

During a study on the quality of the indoor environment, Acanthamoeba spp. were detected in 20 out of 87 dust samples collected from air-conditioners installed in a four-story campus building located in Kuala Lumpur, Malaysia. Twenty-one cloned Acanthamoeba isolates designated as IMU1 to IMU21 were established from the positive primary cultures. Five species were identified from the 16 isolates according to the morphological criteria of Pussard and Pons; i.e. A. castellanii, A. culbertsoni, A. griffini, A. hatchetti and A. polyphaga. Species identities for the remaining five isolates (IMU4, IMU5, IMU15, IMU20 and IMU21), however, could not be determined morphologically. At genotypic characterization, these isolates were placed into T3 (IMU14); T5 (IMU16 and IMU17) and T4 (all the remaining isolates). To predict the potential pathogenicity of these Acanthamoeba isolates, thermo- and osmotolerance tests were employed; many isolates were predicted as potential human pathogens based on the outcome of these tests. This is the first time potentially pathogenic Acanthamoeba have been isolated from air-conditioners in Malaysia.     

Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA

Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families.     

Statin therapy in lupus-mediated atherogenesis: two birds with one stone?

The atherosclerotic process is accelerated in patients with systemic lupus erythematosus (SLE). In addition to a robust lipid-lowering effect, various immunomodulatory functions have been ascribed to statins. By virtue of the latter they may be able to reduce atherosclerotic vascular disease in SLE by inhibiting immune activation within the arterial wall and by attenuating lupus activity. The effects of statins on SLE as well as on lupus-mediated atherogenesis in vivo are discussed in this viewpoint.