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Biomedical subjects

B W Lee

Publications and source records attributed to B W Lee.

At least 19 recordsLinked to original sources
PubMed2006

Endotoxin levels in rural Thai and urban Singaporean homes.

BACKGROUND: Exposure to dust endotoxin and allergens in early childhood may influence the development of allergic diseases. AIMS: This study aimed to evaluate dust endotoxin and dust mite allergens in urban Singapore and rural Thai homes of young children and study potential environmental influences. METHODS: Mattress dust endotoxin and Der p 1, Der f 1, group 2 (Der f 2 and Der p 2) and Blo t 5 allergen levels were quantified in 101 infant mattress dust samples, 51 from urban Singapore and 50 from rural Thailand. Comprehensive questionnaires on the home environment and cleaning practices were completed. RESULTS: Endotoxin levels in rural Thailand were significantly higher than in urban Singapore (geometric mean 26,334.12 +/- 4.60 and 18,377.85 +/- 2.52 endotoxin units/g, respectively; p = 0.032). In contrast, higher levels of Der f 1 (p = 0.02), group 2 (p < 0.01) and Blo t 5 (p < 0.01) allergens were found in Singapore homes compared with rural Thai homes. Multiple logistic regression analysis showed that the use of detergents (p = 0.001) and disinfectants (p = 0.024) to clean floors and mattress protectors (p = 0.021) were independently associated with lower endotoxin levels. CONCLUSION: Endotoxin levels are higher in rural compared with urban homes in South East Asia. The reverse was true for dust mite allergen levels. Certain identifiable home environmental conditions and practices accounted for the differences in endotoxin levels.

Air Pollution, Indoor↗
PubMed2006

Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis.

Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events.

Apoptosis↗
PubMed2006

Distinct pattern of commensal gut microbiota in toddlers with eczema.

BACKGROUND: Recent studies have demonstrated differences in the composition of gut microbiota in infants with and without allergic diseases, particularly eczema. METHODS: A case-control study involving 21 toddlers (age 3.0 +/- 0.5 years) with and 28 age-matched toddlers without eczema was conducted. Four groups of aerobic gut microbiota were identified and quantitated in stool samples grown on selective media. Three groups of anaerobes were enumerated by fluorescent in situ hybridization followed by quantitative flow cytometry. We also performed molecular typing of lactic-acid-producing bacteria (LAB) and enterococcal isolates to facilitate detailed analysis at species level by bacterial 16S rDNA sequencing. RESULTS: Toddlers with eczema harbored significantly lower counts of Bifidobacterium [(median 0.14 (25th and 75th percentile: 0.04 and 0.47) vs. 0.71% (0.16, 1.79) of cells acquired, p = 0.003)] and Clostridium [(0.28 (0.09, 0.78) vs. 0.83% (0.35, 1.82) of cells acquired, p = 0.012)] but significantly higher counts of total LAB [7.3 (6.1, 8.5) vs. 5.7 (4.4, 7.3) log CFU/g, p = 0.006] in particular enterococci [6.3 (4.8, 7.4) vs. 5.0 (3.4, 6.4) log CFU/g, p = 0.018]. There was no significant correlation between eczema severity score and bifidobacterial counts. CONCLUSION: The results further confirm previous reports that the gut microecosystem differs between children with and without eczema and extend them beyond infancy.

Bacteroides↗
PubMed2006

Characterization of glutathione S-transferase from dust mite, Der p 8 and its immunoglobulin E cross-reactivity with cockroach glutathione S-transferase.

BACKGROUND: Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems. OBJECTIVE: To assess the allergenicity of native and recombinant mite glutathione S-transferase (GST) (Der p 8) and study the IgE cross-reactivity between Der p 8 and cockroach GST. METHODS: Der p 8 cDNA encoding a new isoform was isolated and expressed in yeast. Native Der p 8 was affinity purified from mite extract. IgE reactivity to native and recombinant Der p 8 was assessed by ELISA using sera from allergic subjects from Taiwan, Singapore and Malaysia. IgE cross-reactivity between Der p 8 and cockroach GST was examined by IgE inhibition assays. RESULTS: Our Der p 8 cDNA encoded a basic isoform (pI=8.5) containing six polymorphic residues located at positions 46, 106, 149, 160, 167 and 184. At least 8 isoforms of native Der p 8 were detected by two-dimensionalgel and immunoblot analyses. Sera from Taiwanese asthmatics showed 96% and 84% IgE reactivity to native Der p 8 and recombinant Der p 8, respectively. Native Der p 8 showed 75% and 65% IgE reactivity with sera from Malaysia and Singapore, respectively. CONCLUSIONS: A high frequency of sensitization to mite GST among allergic subjects was observed but the titres of IgE reactivity were low. The IgE cross-reactivity between mite and cockroach GST suggests that GST is a panallergen.

Adolescent↗
PubMed2006

A rotavirus vaccine for infants: the Asian experience.

INTRODUCTION: Severe rotavirus gastroenteritis in children causes significant morbidity worldwide and substantial deaths in developing countries. Hence, a live attenuated vaccine Rotarix was developed with human strain RIX4414 of G1P1A P[8] specificity. RIX4414 trials in infants have begun in developed and developing countries worldwide. An overview of RIX4414 in developed and developing countries and prospects with this vaccine in Asia are presented. METHODS: Completed RIX4414 trials have been reviewed. RESULTS: Two oral doses of RIX4414 were well tolerated with a reactogenicity profile similar to placebo. RIX4414 was also highly immunogenic, e.g., in a dose-ranging study conducted in Singapore, 98.8% to 100% of infants had a vaccine take after 2 doses. RIX4414 did not affect the immune response of simultaneously administered routine infant vaccines. RIX4414 significantly reduced severe rotavirus gastroenteritis in settings where multiple serotypes including the emerging G9 type co-circulated. CONCLUSION: These encouraging results warrant further evaluation of the vaccine worldwide and especially in developing countries with the highest need. Therefore, evaluation of the Rotarix vaccine is continuing in large phase III trials in Asia and worldwide.

Asia↗
PubMed2005

Characterization of a serine protease-mediated cell death program activated in human leukemia cells.

Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis.

Apoptosis↗
PubMed2005

Quantification of Blo t 5 in mite and dust extracts by two-site ELISA.

BACKGROUND: Blomia tropicalis is an important mite species in the tropical and sub-tropical regions of the world. Blo t 5 is the major allergen with up to 70% sensitization rates in B. tropicalis allergic populations. METHODS: Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 5 gene with in vivo electroporation. Blo t 5 monoclonal antibodies were generated using methylcellulose-based hybridoma kit. Monoclonal antibody (mAb) 4A7 was characterized by two-dimensional electrophoresis immunoblotting. A specific quantitative two-site enzyme-linked immunosorbent assay (ELISA) was developed with mAb 4A7 and guinea pigs Blo t 5 polyclonal antibody as capture and detection antibodies, respectively. This system was tested with Blo t 5 in crude extracts and dust samples. RESULTS: A high-affinity mAb 4A7 recognizing several isoforms of Blo t 5 has been generated. Monoclonal antibody 4A7 is useful for immunoblotting and two-site ELISA. The two-site ELISA developed has a high sensitivity, with a detection limit of 10 pg/ml. The assay is species-specific and recognized the same epitopes on both native and recombinant Blo t 5. The assay developed is able to detect Blo t 5 in commercial diagnostic and therapeutic B. tropicalis extract. Blo t 5 quantification in dust samples showed that Blo t 5 is present in a high quantity in Singapore dust. CONCLUSIONS: A highly sensitive and specific two-site ELISA has been developed. The assay system developed is useful for the quantification of Blo t 5 in mite and environmental dust extracts.

Allergens↗
PubMed2005

Childhood injuries in Singapore: a community nationwide study.

INTRODUCTION: Childhood injuries are the leading cause of death for children between 5 to 14 years of age in Singapore. However, there is no information or data on childhood injuries in the community in Singapore. There was a need to conduct a nationwide study on childhood injuries in Singapore in order to estimate the prevalence rate, types of injuries, and to identify the hazards in the homes of the children. METHODS: A cross-sectional nationwide study with a two-stage stratified random sampling was conducted to obtain a representative sample of the Singapore population. Families that had children younger than 15 years of age were selected for the study. Parents and caregivers were interviewed at their homes. RESULTS: There were 2322 children in the study, of which 452 had one or more injuries in the past one year, giving an overall prevalence rate of 19.5 percent. Of the children who had injuries, the home was the commonest place where injuries occurred (45 percent) and falls were the leading cause of injuries (77 percent). There were many hazards identified in the homes studied and it was found that the more hazards present in the homes, the more likely it is for the child to get injuries. CONCLUSION: There is a need for educational and interventional programmes to help Singaporeans understand the need to make the home environment safe for children and to prevent home injuries.

Accidents↗
PubMed2005

Knowledge, attitude and practices of childhood injuries and their prevention by primary caregivers in Singapore.

INTRODUCTION: Childhood injuries are the leading cause of death for children between 5 to 14 years of age in Singapore. There have been no studies done on primary caregivers' knowledge, attitude and practices on childhood injuries and its prevention in Singapore. Thus there is a need to conduct this type of study in order to develop appropriate educational programmes. METHODS: A cross-sectional nationwide study with a two-stage stratified random sampling was conducted to obtain a representative sample of the Singapore population. Families that had children younger than 15 years of age were selected for the study. Parents and caregivers were interviewed at their homes. RESULTS: The primary caregivers had good knowledge of road safety but poor knowledge on home safety and first aid. The higher the education of the mother, the more likely it is that she would possess the correct knowledge and practice on childhood injuries and its prevention. The media plays an important role on information on child safety of the caregivers. Only 38 percent said they obtained information from doctors and other health personnel. CONCLUSION: There is a need to educate parents and caregivers on home safety and first aid. Doctors and nurses should take a more active role in giving advice on child safety.

Accident Prevention↗
PubMed2004

High-level expression of a codon optimized recombinant dust mite allergen, Blo t 5, in Chinese hamster ovary cells.

Blo t 5 is a major allergen from house dust mite Blomia tropicalis. Purification of native Blo t 5 (nBlo t 5) from whole dust mite extract is tedious and gave low yield. In this study, we demonstrated that codon optimization facilitated high-level expression of Blo t 5 in Chinese hamster ovary (CHO)-K1 cells and thus allows production of sufficient recombinant cBlo t 5 for specific immunotherapy. A codon optimized Blo t 5 gene was synthesized by PCR and the codon optimized or wild-type Blo t 5 gene in pcDNA3.0 was transfected into CHO-K1 cells and stably selected with Geneticin (G418). Western-immunoblot analysis of spent culture media detected a positive band at 14kDa for the codon optimized but not wild-type gene transfectants. In addition, a stable CHO-K1 clone produced up to 13 mg/L of the cBlo t 5 protein having a high correlation of human IgE reactivities and allergenicity to the native Blo t 5, thus indicating proper conformation of this recombinant form.

Allergens↗
PubMed2004

DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis.

BACKGROUND: Blo t 11 is a high molecular weight allergen from Blomia tropicalis with significant immunoglobulin (Ig)E binding frequency. Native and recombinant Blo t 11 are susceptible to degradation and the isolation and expression of the allergen is problematic thus obtaining sufficient amounts of purified Blo t 11 for antibody production is limiting. DNA-based immunization is an attractive alternative strategy that bypasses antigen purification for antibody production. OBJECTIVES: To use a DNA-based immunization protocol for the production and characterization of Blo t 11 monoclonal antibodies (mAbs). METHODS: The 2625 bp cDNA coding for Blo t 11 was cloned into a mammalian expression vector and immunized intramuscularly with electroporation into mice. Monoclonal antibodies to Blo t 11 were generated using a methylcellulose-based hybridoma cloning kit. These mAbs were utilized for native Blo t 11 isolation and the development of sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Six mAbs recognizing the native and recombinant Blo t 11 were generated and characterized. Native Blo t 11 was affinity purified from Bt extract and its identity was confirmed by matrix assisted laser desorption/ionization - time of flight mass spectrometry. The native Blo t 11 showed IgE reactivity with 67% of mite allergic sera. A two-site ELISA developed showed a detection limit of 100 pg/ml of Blo t 11. CONCLUSION: A DNA-based immunization protocol was successfully used to generate Blo t 11 mAbs with a spectrum of distinct epitopes located throughout the whole molecule, and they are useful for immunoaffinity purification and immunoassays.

Allergens↗
PubMed2004

Acoustic rhinometry in nasal allergen challenge study: which dimensional measures are meaningful?

BACKGROUND: Acoustic rhinometry (AR) is commonly used as a quantitative assessment of nasal response to nasal allergen challenge (NAC). However, sources of error and physical limitations of various AR area-distance measurements have not been adequately evaluated. OBJECTIVE: To investigate the clinical value of AR measurements, and the relationship between subjective sensation and objective AR measurements in the NAC study. METHODS: Nasal challenge using increasing concentrations of crude Blomia tropicalis (Bt) extracts (0.6, 6, and 60 microg/mL) was performed in 15 adult patients (eight males and seven females) with ongoing persistent allergic rhinitis. Subjective symptom scores of nasal obstruction were recorded together with the objective AR measurements of the minimum cross-sectional area (MCA), distance to MCA and cross-sectional area (CSA) at 3.3, 4.0 and 6.4 cm from the nostril, during the 7 h after the last challenge. RESULTS: The dose-response increase in nasal obstruction score was significantly (P<0.001 for all) associated with decreases in mean MCA (r=0.75), mean CSA3.3 (r=0.54), mean CSA4.0 (r=0.53) and mean CSA6.4 (r=0.20). The mean MCA (+/-SD) for each subjective symptom score 0, 1, 2 and 3 was found to be 0.73 (+/-0.22) cm2, 0.63 (+/-0.29) cm2, 0.33 (+/-0.17) cm2 and 0.21 (+/-0.14) cm2, respectively. When the MCA (left and right separately) reached an area <0.2 cm2, measurements of CSA3.3 and CSA4.0 were significantly reduced by 60-70%. CONCLUSION: This study demonstrates that AR is a useful and objective investigational tool, which correlates well with the sensation of nasal obstruction. MCA, CSA3.3 and CSA4.0 are more reliable measurements than CSA6.4 due to physical limitations. It is important to note that when the MCA is smaller than 0.2 cm2, a common condition in the early-phase reaction, area-distance measurements beyond this point can be misinterpreted and should be considered with caution.

Adult↗
PubMed2004

Immunoglobulin E reactivity of native Blo t 5, a major allergen of Blomia tropicalis.

BACKGROUND: Blo t 5 is a major allergen of Blomia tropicalis and its complementary DNA (cDNA) has been expressed in both prokaryotic and eukaryotic expression systems. Although the recombinant Blo t 5 has been well characterized, relatively less is known about its native counterparts and the allergenicity comparison of the native and recombinant Blo t 5 allergens has not been reported. OBJECTIVE: The aims of this study are to characterize the native counterpart of Blo t 5, and compare the allergenicity of native and recombinant Blo t 5 by in vivo and in vitro assays. METHODS: Native Blo t 5 were purified by immuno-affinity chromatography and characterized by proteomic means. The allergenicity of the allergen was evaluated by skin prick tests, human IgE ELISA, ELISA inhibition and histamine release assays. RESULTS: Native Blo t 5 consists of at least five distinct isoforms, ranging from pI 3 to 5.5. Allergenicity assessment of recombinant and native Blo t 5 based on skin reaction, IgE-binding capacity and histamine release in allergic individuals indicated that there was a good correlation between both forms of Blo t 5 in general. However, data from IgE ELISA inhibition assay revealed the presence of additional unique IgE epitopes in native Blo t 5. CONCLUSIONS: At least five distinct isoforms of Blo t 5 have been identified. Comparative assessment of native and recombinant Blo t 5 revealed that the allergenicity of these two forms was similar but not completely identical suggesting that the various isoforms of native Blo t 5 may exhibit additional unique IgE epitopes.

Adolescent↗
PubMed2004

Production of monoclonal antibodies for immunoaffinity purification and quantitation of Blo t 1 allergen in mite and dust extracts.

BACKGROUND: Blo t 1 is a cysteine protease-like allergen from Blomia tropicalis. Recombinant Blo t 1 binds up to 90% of IgE from allergic patients and shows limited cross-reactivity to Der p 1. The generation of monoclonal antibodies (mAbs) against Blo t 1 is important for the detection, isolation and characterization of the native form of the allergen. METHODS: Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 1 gene with in vivo electroporation and boosted intraperitoneally with recombinant Blo t 1. mAbs against Blo t 1 were generated using a methylcellulose-based hybridoma cloning kit. The native Blo t 1 was isolated by mAb affinity purification and its allergenicity was determined by ELISA. A two-site ELISA for Blo t 1 was developed using the mAbs generated. RESULTS: A DNA-based immunization protocol induced high titre Blo t 1-specific antibodies in mice. Six stable hybridoma clones secreting mAbs recognizing the native and recombinant Blo t 1 were generated. The native Blo t 1 was affinity-purified from a B. tropicalis extract and its allergenicity was determined at 63% using a panel of Singaporean and Malaysian mite allergic patients' sera. A two-site ELISA was developed, which showed a detection limit of 10 ng/mL of Blot t 1. CONCLUSION: Six Blo t 1 mAbs were successfully generated by DNA immunization. These mAbs are useful for nBlo t 1 immunoaffinity isolation and quantitative immunoassays for Blo t 1 in mite and environmental dust extracts.

Allergens↗
PubMed2004

The prevalence of asthma and allergies in Singapore; data from two ISAAC surveys seven years apart.

BACKGROUND AND AIMS: Over the past few decades, the prevalence of asthma has been increasing in the industrialised world. Despite the suggestion of a similar increase in Singapore, the 12 month prevalence of wheeze among schoolchildren in 1994 was 2.5-fold less than that reported in western populations. It was hypothesised that with increasing affluence in Singapore, the asthma prevalence would further increase and approach Western figures. A second ISAAC survey was carried out seven years later to evaluate this hypothesis. METHODS: The cross-sectional data from two ISAAC questionnaire based surveys conducted in 1994 (n = 6238) and in 2001 (n = 9363) on two groups of schoolchildren aged 6-7 and 12-15 years were compared. The instruments used were identical and the procedures standardised in both surveys. RESULTS: Comparing data from both studies, the change in the prevalence of current wheeze occurred in opposing directions in both age groups--decreasing in the 6-7 year age group (16.6% to 10.2%) but increasing to a small extent in the 12-15 year age group (9.9% to 11.9%). The 12 month prevalence of rhinitis did not change; there was an increase in the current eczema symptoms in both age groups. CONCLUSION: The prevalence of current wheeze, a surrogate measure of asthma prevalence, has decreased significantly in the 6-7 year age group. Eczema was the only allergic disease that showed a modest increase in prevalence in both age groups.

Adolescent↗
PubMed2004

Mandatory temperature monitoring in schools during SARS.

During the SARS outbreak, temperature monitoring was mandatory for all Singapore schoolchildren. None of the Singapore children with SARS were detected through school temperature screening. However, temperature monitoring procedures have a powerful psychological effect of reassuring parents and the public that schools are safe during a SARS outbreak.

Adolescent↗