Recombinant VP9-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Banna virus (genus Seadornavirus)

Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans. Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9.The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector. VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i). VP9 is a major protein in virus-infected cells and is a capsid protein (ii). among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid.The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past.The specificity of this ELISA was >98%. In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells.The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV. This test should be a useful tool for epidemiological survey of BAV.     

Impaired generation of bone marrow CD34-derived dendritic cells with low peripheral blood subsets in patients with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective haematopoiesis and blood cytopenias. The present study investigated the potential of bone marrow CD34(+) progenitors in MDS patients to proliferate and differentiate into dendritic cells (DCs) in a cytokine-supplemented liquid culture system and analysed the status of blood DC subsets in these patients. CD34(+) progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34(+) cell was significantly lower compared with controls (median value 0.2 vs. 4, P = 0.003). In patients, the survival and proliferation of CD34(+) cells in culture was not correlated to the degree of apoptosis. Phenotypically and functionally CD34(+)-derived DCs were similar in MDS patients and normal subjects. The percentage of both circulating DC subsets in patients was extremely diminished compared with controls (myeloid DC: 0.10 +/- 0.10% vs. 0.35 +/- 0.13%, P < 0.001; plasmacytoid DC: 0.11 +/- 0.10% vs. 0.37 +/- 0.14%, P < 0.001). In cases with the 5q deletion both CD34-derived DCs and blood DCs harboured the cytogenetic abnormality. Our results indicate that, in MDS, the production of DCs is affected by the neoplastic process resulting in ineffective 'dendritopoiesis' with low blood DC precursor numbers. This quantitative DC defect probably contributes to the poor immune response against infectious agents and to the escape of the malignant clone from immune recognition with disease progression towards acute leukaemia.     

CHEK2 gene alterations independently increase the risk of death from breast cancer in Bulgarian patients

Checkpoint kinase 2 (CHEK2) is a?DNA damage-activated protein kinase implicated in cell cycle checkpoint control. The significance of CHEK2 alterations for breast cancer incidence and clinical behavior is not clear. In this study we determined the mutational spectrum and the level of promoter hypermethylation of CHEK2 gene in a?group of 145 Bulgarian patients with breast cancer. A?special emphasis was put on the clinical impact of CHEK2 alterations for breast cancerogenesis.PCR-SSCP-sequencing analysis of the entire coding sequence of CHEK2 gene was performed to estimate the mutational profile of tumor samples. Methylation-sensitive SSCP was applied to determine the methylation status in CpG clusters implicated in CHEK2 silencing. Clinical significance of CHEK2 alterations was evaluated using standard statistical methods.Mutations in CHEK2 were identified in 9.65 % of the patients. Two novel missense substitutions Thr476Met (C >T) and Ala507Gly (C>G), and a?novel silent variant Glu79Glu (A>G) were registered. However, hypermethylation was not found in any of the studied cases. Comparison with clinical characteristics showed that CHEK2 positive women have predominantly lobular type of breast carcinoma (р=0.04) and PR+ status (p=0.092). CHEK2 mutations correlated significantly with ATM+ status (p=0.046). All patients with the Glu79Glu variant were progesterone receptor positive (p=0.004). A?decrease in overall survival (p = 0.6301) and a?threefold increased independent risk of death (HR = 3.295, 95%CI 0.850-12.778, p?= 0.085) in CHEK2+patients was found.Our data indicate the significance of CHEK2 gene alterations in contrast to promoter hypermethylation in breast cancerogenesis. Specificity of CHEK2 mutational profile for the Bulgarian population was found. Though CHEK2 mutational status correlated with more favorable clinical characteristics, including positive progesterone receptor and lobular histological type, it independently increased the risk of death in these patients. Keywords: breast cancer, CHEK2, mutation, hypermethylation, clinico-pathological characteristics, overall survival.     

Contact sensitivity to hair dyes can be detected by the consumer open test

To avoid allergic reactions to hair dyes, an open test ("skin sensitivity test" or "dab test") is recommended 48 hours before the hair colouring procedure. We have investigated in a multicenter study, under standardized conditions and medical supervision, the validity of this test as a practical method to detect allergy to paraphenylenediamine (PPD)-containing hair dyes in 30 PPD patch test-positive and 30 PPD patch test-negative subjects. A defined volume of a marketed hair colouring product containing 1.8% PPD was applied in an open patch test to the retroauricular area. The grading method comprised the dermatologist's clinical evaluation and objective numerical scores. Allergic reactions were elicited in all PPD-positive subjects with a maximal intensity on Day 2; no allergic reactions were elicited in PPD-negative subjects. Increased severity of reactions by the dermatologist's clinical evaluation was correlated strongly with increasing numerical scores. The "skin sensitivity test" can be considered as an effective method to detect type IV hair dye allergy and as such, as an important factor in its secondary prevention.     

Rubella vaccination programme in Malaysia: analysis of a seroprevalence study in an antenatal clinic

In many developed countries, the incidence of rubella and congenital rubella syndrome (CRS) is considered to be negligible due to the availability of an effective vaccine. However, in Malaysia, several CRS cases are seen every year. This casts doubt on the effectiveness of the rubella vaccination programme. Very few seroprevalence studies were done over the years, making it difficult to discuss the effectiveness of the vaccination programme. The objective of this study is to determine the prevalence of rubella immunity among pregnant women attending antenatal clinics in a local teaching hospital. The hospital database on rubella immunity was assessed retrospectively from August 2001 to June 2002. A cross-sectional study of interviewed method as well as determination of rubella immunity by laboratory tests were carried out in July 2002. A total of 414 women were included, of whom 134 women were interviewed. The rubella immunity status was 92.3%. Based on this figure, rubella vaccination programme in Malaysia is a success despite the presence of CRS cases. Malaysia must ensure rubella vaccine coverage among target groups is high in order to minimise CRS cases.     

Clinical application of a fiber-reinforced post system

The tooth structure of an endodontically treated tooth was restored with a core on a post, both prefabricated with fiber-reinforced polymer-ceramic material (Sculpture/FibreKor) and subsequently with a metal-free crown. The tooth restoration is described in detail. It includes root canal preparation, polyvinyl siloxane impression taking, laboratory fabrication of the core on the post, adhesive cementation of the post-and-core system in the root canal, fabrication and cementation of the restorations, adjustment, and finishing. The success of this metal-free system is based on its increased flexural and tensile strength. Its application is safe, effective, and reliable. The high-quality aesthetics of the restorations is due to the translucency of the post-and-core material.     

Respiratory viruses detected in hospitalised paediatric patients with respiratory infections

Over 200 strains of respiratory viruses cause a variety of human infections ranging from common cold to life-threatening pneumonia. Respiratory viruses implicated in this study are respiratory syncytial viruses (RSV), adenovirus, influenza viruses and parainfluenza viruses. The objective of this study is to determine the epidemiology of respiratory viruses in paediatric patients with lower respiratory tract infection. The methods used were direct antigen detection method, shell vial culture method and conventional tube culture method. The samples included in this study are paediatric patients seen in Universiti Kebangsaan Malaysia Hospital, Kuala Lumpur with suspected acute viral respiratory infection, presenting with acute laryngotracheobronchitis (croup), bronchiolitis and pneumonia. Nasopharyngeal aspirates were collected and processed almost immediately. A total of 222 specimens were received during February 1999 to January 2000 showing a dual peak pattern in the months of April and December. The mean age of the patients was 13 months. Pneumonia (77.9%) was the most common clinical diagnosis in children with lower respiratory tract infection. This was followed by bronchiolitis (19.4%) and croup (27%). Viral aetiologies were confirmed in 23.4% of the patients. The most common respiratory virus isolated or detected was RSV, followed by parainfluenza viruses, influenza viruses and adenovirus.