Quantification of Der f 1 in houses of patients allergic to house dust mite, Dermatophagoides farinae, using a locally produced detection reagents.

Background: House dust mite (HDM) allergen quantification in house dust samples before and after the allergen elimination is one means of convincing the target population about the health benefits of allergen removal from their environment. Objective: To produce local reagents for quantification of Der f 1 (major allergen of Dermatophagoides farinae) in dust samples from houses of HDM allergic Thai patients. Methods: Recombinant Der f 1 was used for immunization of a BALB/c mouse for hybridoma production. Polyclonal antibody (PAb) to whole body extract of D. farinae was prepared from an immunized rabbit. A sandwich ELISA (MAb-allergen-PAb) was used, in comparison with the commercialized reagents (Indoor Biotechnology, UK), to quantify Der f 1 in dust samples. Results: Two hybridoma clones, Df1-1 and Df1-2, were established. Their secreted MAbs (MAbDf1-1 and MAbDf1-2, respectively) bound to the homologous antigen as well as native Der f 1 and a crude extract of D. farinae. Epitopes of MAbDf1-1 and MAbDf1-2 were located at amino acid residues 206NSQHYGISNYCQ217 and 283DYW---NSWD-WGDSG298 of Der f 1. MAbDf1-1 had higher affinity to Der f 1 than the MAbDf1-2. A sandwich ELISA (MAbDf1-1-allergen-PAb) and commercialized reagents (MAb1-allergen-MAb2 sandwich ELISA) were used in comparison for quantification of Der f 1 in 42 dust samples collected from bedrooms and living rooms of 21 houses of the HDM allergic patients. All of the 42 dust samples measured by both ELISAs had the Der f 1 levels higher than 2 mg per gram of fine dust which is the HDM allergy sensitizing level. In addition, Der f 1 levels in 41 samples (except 1 sample from a living room) measured by the MAbDf1-1-PAb and MAb1-MAb2 sandwich ELISAs were higher than 10 mg per g of dust which is the morbidity level of HDM allergen. The local sandwich ELISA showed a high coefficient correlation (r = 0.91) in measuring known amounts of recombinant and native Der f 1. The results indicate that the reagents produced in the present study can be used for measuring the environmental levels of HDM Der f 1. The assay can also be used for standardization of the HDM extract for monitoring patient's allergenic status or for immunotherapeutic purpose.

In-vivo allergenic potency of Siriraj Mite Allergen Vaccine (SMAV) comparing with standardized vaccine in mite-sensitive patients.

Background: The prevalence of allergic diseases, particularly asthma and allergic rhinitis, has increased tremendously in Thailand and worldwide. House dust mite (HDM) is the major IgE sensitizer among allergic children and adults. We have developed local standardized mite allergen extracts, Siriraj Mite Allergen Vaccine (SMAV) from Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df) from our source materials which were highly purified (99%). Objective: To compare in-vivo allergenic potency of both SMAV Dp and Df with commercial standardized mite allergen vaccine by using skin prick testing in mite-sensitive individuals. Methods: This was a double-blind, randomized, self controlled study comparing SMAV and commercial standardized mite allergen vaccine (Dp and Df) by using skin prick testing in mite-sensitive adult volunteers, 18 – 60 years of age. Results: The study was performed in 54 adult volunteers (19 males, mean age 26.6 + 5.5 years old) who had positive skin test to commercial Dp and Df. Seventeen of them had no allergic disease. The most common allergic disease among the volunteers was allergic rhinitis (21/37). Mean wheal diameter of SMAV Dp and commercial Dp at the concentration of 10,000 and 5, 000 AU/ml were equivalent but at the concentration of 2,500 AU/ml was inequivalent. Mean wheal diameter of SMAV Dp was significantly larger than commercial Dp at concentration of 2,500 AU/ml (p < 0.05). Mean wheal diameter of SMAV Df and commercial Df at all 3 concentrations were equivalent. There was no systemic side effect in all subjects. Conclusion: The study demonstrated that in mite-sensitive adults, SPT using SMAV Dp (10,000 AU) and Df (10,000 AU) had equivalent allergenic potency to the commercial comparator without any systemic side effect.

Comparison between siriraj mite allergen vaccine and Standardized Commercial mite vaccine by skin prick testing in normal Thai adults.

House dust mite is a major cause of allergic asthma and rhinitis in Thai population. Skin prick test (SPT) is a useful tool for the diagnosis of the IgE-mediated reactions. The imported commercial mite vaccine for SPT is available but it is relatively expensive. Aim of this studyis to compare Siriraj Mite Allergen Vaccine (SMAV) with standardized commercial mite allergen vaccine by skin prick testing in normal Thai adults. A double blind, self-controlled study between the SMAV and standardized commercial mite allergen vaccine was performed by SPT in 17 normal Thai adult males and non-pregnant or non-lactating females aged 18-60 years. The study showed that35.29 % of non atopic adults had positive SPT reaction to Dp and Df of both SMAV andstandardized commercial mite allergen vaccine. Mean wheal and flare diameters from SPT of Dp and Df of SMAV showed strong correlation with standardized commercial mite allergen vaccine (r = 0.768 and 0.897 in Dp and Df respectively, p <0.001). The intraclass correlation was also excellent (0.893 and 0.775 in Dp and Df respectively). There was no significant difference in wheal and flare diameter between SMAV andstandardized commercial mite allergen vaccine. No systemic or large local reaction was found in any of the study cases.

Allergic rhinitis and its impact on asthma update (ARIA 2008) - western and asian-pacific perspective.

The prevalence of allergic diseases such as allergic rhinitis (AR) and asthma is markedly increasing worldwide as societies adopt western life styles. Allergic sensitization is an important risk factor for asthma and AR, and asthma often co-exists with AR. An estimated 300 million people worldwide have asthma, about 50% of whom live in developing countries and about 400 million people suffer from AR. Yet, AR is often under-diagnosed and under-treated due to a lack of appreciation of the disease burden and its impact on quality of life, as well as its social impact at school and at the workplace. However, AR with or without asthma is a huge economic burden. Thus, there was clearly a need for a global evidence-based document which would highlight the interactions between the upper and lower airways including diagnosis, epidemiology, common risk factors, management and prevention. The Allergic Rhinitis and its Impact on Asthma (ARIA) document was first published in 2001 as a state-of-the-art guide-line for the specialist, the general practitioner and other health care professionals. Subsequent new evidence re-garding the pathomechanisms, new drugs and increased knowledge have resulted in the publication of the ARIA 2008 update. The present review summarizes the ARIA update with particular emphasis on the current status of AR and asthma in the Asia-Pacific region and discusses the Western and Asian perspective

Allergenicity of native/recombinant tropomyosin, per a 7, of American cockroach (CR), periplaneta Americana, among CR allergic Thais.

In this study, native tropomyosin (Per a 7) of American cockroach (CR), Periplaneta americana, caught in Thailand was purified. Also, gene sequence encoding full length tropomyosin of the CR was PCR ampli-fied by using degenerate primers designed from gene sequences coding for P. americana tropomyosin of the data-base (Per a 7.0101 and Per a 7.0102; accession no.Y14854 and AF106961, respectively). Amino acid sequence deduced from the nucleotide sequence encoding P. americana tropomyosin of this study (GenBank accession no. FJ976895) had 98.59% identity with the sequences of Per a 7.0101 and Per a 7.0102 and was 97.18% identical to the Bla g 7 sequence of German cockroach, Blatella germanica (accession no. AF260897). The native and recom-binant tropomyosins (~34 kDa) were used as antigens in sandwich ELISA for detecting specific IgE in serum sam-ples of 14 consented allergic patients who were positive by skin test to crude CR extract in comparison to 5 indi-viduals who were skin test negative. It was found that 8 (57%) and 6 (43%) of the CR allergic patients gave positive IgE binding results to the native and the recombinant proteins, respectively, while none of the non-allergic counter-parts was positive. Results of immunoblotting conformed to the ELISA results. Tropomyosin extracted from the P. americana caught in Thailand has potential as standard P. americana allergen in clinical monitoring of the allergic Thai patients.

The accuracy of limited CT in the evaluation of chronic rhinosinusitis and for endoscopic sinus surgery: a multicenter study.

Objective: To compare the accuracy of Limited CT with the Full CT as the standard evaluation for inflammatory disease of PNS and the identification of anatomical variations. Methods: From Full CT of PNS, Limited CT were retrieved. Computerized tomographic scans were performed for the preoperative planning of endoscopic sinus surgery (ESS) in 3 tertiary care university hospitals in Thailand. The two types of examinations were reviewed independently and in random order by two experienced radiologists. Using Full CT as the standard, the accuracy of Limited CT were evaluated for 1) the radiologic staging of rhinosinusitis (Lund-Mckay scoring system) and 2) the anatomic variations which are an important landmark for surgical operations in rhinosinusitis. Results: Totally 132 patients were included. Two hundred and sixty four half-faces were reviewed. Lund-McKay radiographic sinus staging system showed 97-99% specificity except for the ostiomeatal complex region. Regarding anatomic variation, Limited CT was able to yield accurate results for the frontal cell type II-IV, Haller cell, Agger nasi cell, paradoxical middle turbinate, concha bullosa and the protrusion of the optic nerve. Conclusion: Limited CT can be used as a surgical roadmap for the cases with the anterior group of sinus involvement. It may be utilized for surgical planning of chronic CRS involving the anterior group of sinuses. For the posterior group of sinuses, it may not yield enough accuracy and the standard Full CT should be requested to prevent erroneous estimation.

Native troponin-T of the American cockroach (CR), Periplaneta americana, binds to IgE in sera of CR allergic Thais.

The American cockroach, Periplaneta americana, is the predominant cockroach (CR) species in Thailand and a major source of indoor allergens second only to the house dust mite. The incidence of CR allergy among allergic Thai patients is increasing but basic information on the allergenic components is scarce. In this study a recombinant troponin-T was produced by using cDNA prepared from RNA of the P. americana as a template and PCR primers designed from the P. americana troponin-T sequence deposited in the GenBank database. The recombinant protein (Mr approximately 50) did not bind to IgE in the sera of 18 skin prick test positive CR allergic patients. Rabbit polyclonal antiserum (PAb) against the recombinant troponin-T was produced and used in preparing an affinity column for the purification of native troponin-T from the crude P. americana extract (Mr approximately 47). IgE-immunoblotting revealed that the native protein bound to IgE in 3 of the 18 (16.7%) patients. Our results imply that native P. americana troponin-T, but not its recombinant counterpart, is a minor allergen among the CR allergic Thais.

A comparative study of patients' preferences and sensory perceptions of three forms of inhalers among Thai asthma and COPD patients.

In 9 study centers, 419 patients with asthma or COPD were randomized to receive two forms of salbutamol metered-dose-inhalers (MDIs), i.e. CFC-driven MDI, non-CFC (HFA) MDI and one salbutamol dry powder inhaler (DPI), in a multi-center, comparative, cross-over and randomized study, performed to facilitate the formulation of a strategic plan to phase out CFC MDIs. After having received all three forms of test products, the patients completed an evaluation questionnaire indicating their preferences, likelihood of treatment compliance on each product and the easiest one to use. Statistical analysis showed that the CFC MDI was significantly less irritating (p < 0.014) but lower in its overall appeal (p < 0.0001). The "most preferred form to be prescribed" was DPI at 47.5% followed by non-CFC at 32.5% and CFC MDI at 20.1%. Concerning the ease of use among the three forms of test products, 59.9% of the patients indicated "no difference". Adverse events were mild and occurred in only 8.2%. In conclusion, patients' preference and sensory perception among the three forms of inhalers were comparable except that the CFC MDI was significantly less irritating but lower in its overall appeal. DPI was the most preferred and easiest form to use but also the most expensive. Taking public health into consideration, a non-CFC MDI with a similar market price to the CFC MDI would be the obvious choice in a strategic plan to phase out CFC MDIs with the least difficulty to the consumers.

A surveillance study of bacteriologic profile in rhinosinusitis.

Objective: To review the types of bacteria found in rhinosinusitis and the prevalence of a beta-lactamase producing organism in a tertiary care hospital during the year 2004 and compared with our previous reports. Methods: Charts of patients who underwent endoscopic sinus surgery or maxillary antral puncture or endoscopic-guided culture at the Rhinology & Allergy Division, Department of Otolaryngology and the Department of Microbiology, Siriraj Hospital from January 2004 to December 2004 were reviewed. Information regarding the patient’s age, site of specimens, culture and sensitivity results were obtained. Results: There were 162 specimens and 29 bacterial species isolated. There were 50.4% positive aerobic cultures, gram-negative bacteria were more common than gram-positive bacteria (68.5% vs 31.5%). Common aerobes were Pseudomonas aeruginosa (16.2%), non-fermentative gram negative rod: NF-GNR (10.8%), Coagulase- negative Staphylococcus aureus :CNS (9.9%) and Klebsiella pneumoniae (9.9%).The most common anaerobes were Peptostreptococcus sp., Bacteroides fragilis and Fusobacterium sp. Conclusion: Contrary to our previous studies, gram negative organisms play a more important role than gram positive organisms. The causative pathogens of rhinosinusitis should be studied continuously because rapid progress in the development of new antimicrobial agents has a significant impact on their bacteriologic profile.

Availability and consumption status of CFC and non-CFC inhalers for asthma and chronic obstructive pulmonary diseases in Thailand.

In response to the Montreal Protocol and the calls for global early-bird CFC phase-out before 2010, the demand and supply status of both CFC and non-CFC inhalers for prevention and treatment of asthma and COPD in Thailand were evaluated to determine how soon the country would be able to discontinue CFC MDIs with least impacts to both consumers and importers. Availability and supply of the inhalers were collected from registration and importation database of the Thai FDA. Demand and product cost were obtained from the local importers and from IMS, Thailand. Available inhaled products comprise of 39% CFC MDIs, 28% DPIs, 20% solutions for nebulizers and 13% HFA MDls, respectively. All 31 brands of portable hand-held inhalers, comprising 16 CFC MDIs, 6 HFA MDIs and 9 DPIs, are imported, only solutions for nebulization are locally manufactured. Salbutamol is mostly prescribed MDI, its consumption is over 50% of all. The transition to non-CFC alternatives (HFA MDIs and DPIs) has become evidence since 2000. After being informed about the demand and supply of the inhalers, in 2005, Thai FDA has announced its CFC phase-out policy and encouraged importation of HFA alternatives by facilitating the registration and approval process. When the most prescribing CFC MDls, salbutamol, is completely replaced with non-CFC form in 2006, Thailand would be able to reduce considerable amount of CFCs into our atmosphere.

Development of a health-related quality of life questionnaire for Thai patients with rhinoconjunctivitis.

The objective of this study was to develop a disease-specific questionnaire for patients with rhinoconjunctivitis. All patients were recruited at the Out-Patient Clinic at Siriraj Hospital. Related topics were gathered from several sources, and a list of 63 items was produced. In phase I, the first version of the questionnaire was completed by 363 patients. Forty-eight items were identified by clinical impact analysis during the item removal process, two more questions were then added, giving a total of 50. Two hundred and forty-three patients completed the second version questionnaire in phase II. The average time taken to complete the questionnaire was 6.38 minutes. The item removal process in phase II was achieved by a multi-step process. There were 36 items in the third version questionnaire which consisted of six dimensions and two independent items as follows: symptoms (17 items), physical functioning (3 items), role limitations (3 items), sleep (3 items), social functioning (3 items), emotions (5 items), general health (1 item), and absenteeism (1 item). The scores of each item ranged from 1 to 5; a lower score indicating a better quality of life. Data from the selected 36 items was extracted to test the validity and reliability of the final version. The floor and ceiling effects of the scores for each dimension were low. Multitrait multi-item analysis was conducted to examine construct validity. The scaling success of convergent and divergent validity was 100% and 94%, respectively. Internal consistency determined by Cronbach's alpha coefficient, was satisfactory (0.79-0.87). The study indicates that the questionnaire is suitable for use in clinical settings. While the test results are encouraging, further work needs to be done on the test-retest reliability and on responsiveness.

The levels of cockroach allergen in relation to cockroach species and allergic diseases in Thai patients.

Recently, cockroaches have been established as the second most Important allergen, producing allergic diseases, especially in low socioeconomic populations. In Thailand, about 44-61% of atopic patients were positive to cockroach extract by a skin-prick test. This study examined cockroach allergen levels in relation to cockroach species and allergic diseases in the houses of cockroach-sensitive patients. Sixty households of allergic patients in the Bangkok metropolitan area were surveyed using open- and closed-ended questionnaires. Cockroaches were collected using commercial cockroach traps, while dust samples were obtained from the bedrooms, kitchens and living rooms of the houses using a vacuum cleaner. The cockroaches were counted and their species Identified. The levels of cockroach allergens were determined by specific monoclonal antibodies using a monoclonal antibody-polyclonal antibody based sandwich ELISA kit. Six cockroach species were Identified: Periplaneta americana (American cockroach, 72.15%), Supella longlpalpa (2.75%, found in only one house), Periplaneta brunnea (0.78%), Periplaneta australaslae (0.78%), Neostylopyga rhombifolla (0.78%), Blattella germanica (German cockroach, 0.39%) and nymphs (22.35%). Allergens of the predominant species, P. americana, were detectable in all homes studied, with the highest levels in the kitchen areas. The range of allergen levels in house dust varied from 0.40-162.00 microg per g of dust. The median and mean allergen levels in kitchen dust were 59.16 microg and 62.80 microg per g of dust, respectively, while the median allergen level in bedroom dust was only 15.90 microg per g of dust. The German cockroach allergen (Bla g 2) was undetectable in any of the houses. IN CONCLUSION: P. americana was the most common cockroach and may be the species causing allergic diseases, especially asthma, in Thailand, which differs from the USA and Europe

An open-label, prospective study of an oral polyvalent bacterial lysate (Luivac) in the treatment of recurrent respiratory tract infections in Thai patients.

An open-label, non-comparative study was performed in the Department of Otolaryngology, Siriraj Hospital, Bangkok, Thailand, to assess the safety, tolerability, acceptability and efficacy of an oral polyvalent bacterial lysate (Luivac) in the treatment of recurrent respiratory tract infections (RTIs) in Thai patients. Thirty-three patients were included in this study, 18 males and 15 females, with a mean age of 34.0 +/- 14.7 years. The mean number of RTIs during the 12-month period preceding the study was 9.5 per patient. During the study each patient received one tablet of Luivac daily for 28 days followed by a treatment-free period of 28 days. This was followed with another 28 days on Luivac, after which there was a 28-day treatment-free follow-up period. This study lasted 4 months with five scheduled patient visits (V1-V5). Laboratory studies were done at baseline (V1) and after treatment (V4), which included complete blood count and serum immunoglobulins (IgA, IgE, IgG and IgM). The incidence of all adverse events was 15.2% and no case was related to the studied drug. There were no clinical relevant changes in laboratory parameters after treatment. The reduction rate of RTIs per month at the end of the study period was 63.5% when compared to the average RTIs rate per month during the 12 months preceding the study. A comparison of the first study period (V1-V3) and the second study period (V3-V5) showed a reduction in duration of RTIs (23.1%), in the clinical infection score (17.5%), in the number of antibiotics used (2.1%), in the number of symptomatic treatments (3.5%), and in the number of days absent from school or work (50.0%). Overall tolerability and acceptability were assessed as very good and good in 96.8% of the patients. This study suggests that oral polyvalent bacterial lysate (Luivac) was safe and also showed a tendency to be effective in preventing RTIs in Thai patients with or without risk factors for recurrent RTIs. Other clinical advantages were reduction in the severity and duration of infection as well as in reduction of the cost of treatment and the number of days absent from school or work.

A comparative study of the major allergenic components in house dust extracts between siriraj and commercially prepared extracts.

The quantity and concentration of major allergens in mite allergenic extracts are crucial for skin testing, which is the recommended standard method for the diagnosis of house dust mite allergic disease. The purposes of this study were 1) To compare the constituents of major mite allergens in 3 types of mite extracts, i.e., extracts from mite reared in-house (Dermatophagoides pteronyssinus and Dermatophagoides farinae), extracts from commercial mite products and commercial ready-made mite extracts. These in-house extracts were prepared either with or without the preservative, glycerine. The concentrations of the major constituents of group 1 and 2 allergens of the extracts were determined by a two-site monoclonal based ELISA. 2 ) Biological assays were also carried out to determine the relative potency of the extracts in 120 allergic patients by skin prick test. It was found that the mean concentrations of Der p1, Der f1 and group 2 mite allergens in extracts from mites reared in-house were 102, 195 and 94 ตg/ml, respectively, compared to 169, 238 and 91 ตg/ml, respectively in commercial mite extracts. The commercial with product had the lowest concentrations of mite allergens (1 ตg/ml). Comparison of mite extracts with and without glycerine preservative showed no significant difference in concentrations of major allergens. Reduction of allergens concentration from 10,000 to 1,000 PNU/ml also reduced the concentration of mite allergen proportionately. Siriraj mite extracts were stable for at least 1 year at 4 oC without any significant change in composition or concentration. In conclusion, mite reared in-house can be used as raw material for preparation of mite allergenic vaccine.

An open-label, non-comparative phase IIIb study of oral gatifloxacin in the treatment of acute, uncomplicated bacterial sinusitis in Thai patients.

This was an open-label, non-comparative phase IIIb study of oral gatifloxacin in the treatment of acute, uncomplicated bacterial sinusitis in Thai patients. The study was conducted at three otolaryngologic centers in Thailand, i.e. Siriraj Hospital, Srinakarind Hospital, and Bhumipol Hospital, during the period of November 1999 through October 2000. Thirty patients with symptoms and signs of acute maxillary sinusitis, which were confirmed by abnormal radiological findings, were included. Documentation of infection was made by pre- and post-therapy culture and susceptibility testing. All patients received gatifloxacin 400 mg tablet orally once daily for 10 days. Symptoms and signs were evaluated on four occasions, i.e. before treatment started (day 0, V1), day 3-5 (V2), the third (V3) on day 11-14 (V3), and day 21-28 (V4). Primary outcome measures were the changes in the percentage of patients who had general clinical symptoms (i.e. fever, malaise, chill, sore throat, headache, cough, and halitosis), and also the clinical symptoms and signs of sinusitis (i.e. sinus pain, sinus tenderness, purulent nasal discharge, facial pressure, nasal congestion, postnasal drip, and anosmia). The clinical responses were classified as cure, improvement, relapse or failure at the end of treatment (V3) and at follow up (V4). Treatment success was defined as cure or improvement. The safety of gatifloxacin was assessed from vital signs, general physical examination, hemato-biochemical parameters, and adverse events reported. There were 13 males and 17 females, with a mean age of 34.7 + 12.2 years (range 21-70). The mean duration of symptoms was 2.4 + 8.8 weeks. At V1,23 patients (76.7%) had headache, 19 patients (63.3%) had cough, and 14 patients (46.7%) had halitosis. At V3 and V4 all these symptoms had resolved, except in 1 patients who still had cough. The percentages of patients who had symptoms and sign of sinusitis at V1 were as follows : purulent nasal discharge 90%, nasal congestion 86.7%, postnasal drip 86.7%, sinus pain 80%, facial pain 66.7%, and sinus tenderness 63.6%. After 3-5 days of treatment (V2), the percentages of patients who had symptoms and signs of sinusitis decreased significantly for every symptom and sign. The same was true at V3 and V4. The total percent improvement in radiological findings was 83.4% (41.7% resolution, 41.7% improvement). Pretreatment cultures were positive in 23 out of 30 patients (76.7%). The most common pathogens were Hemophilus influenzae (18.4%), Streptococcus pneumoniae (9.4%), other Streptococcus species (9.4%), Klebsiella pneumoniae (6.3%) and coagulase negative Staphylococcus (6.3%). The anaerobes found were Peptostreptococcus prevotii (9.4%), Fusobacterium nucleatum (9.4%), Bacteroides species (6.1%) and Prevotella species (3.1%). Bacteriological responses at V3 were 30.4% eradication, 65.2% presumed eradication, and 4.4% persistence. Clinical responses at V4 were 88.9% cured, 7.4% relapsed, and 3.7% failed. Adverse events were found in 4 patients (13.3%) which were transient and resolved spontaneously. Adverse events found were nausea, vomiting, dry mouth, constipation, agitation and tremor. This study showed the efficacy and safety of a 10-day course of oral gatifloxacin 400 mg once daily in the treatment of acute uncomplicated bacterial sinusitis. 88.9% of patients experienced a cure, and bacteriological eradication was achieved in 95.6%. The excellent profile of gatifloxacin makes it suitable for the treatment of community acquired acute bacterial sinusitis. The once daily regimen enhances patient compliance.

Cockroach allergen detection and cockroach allergens of allergic Thai patients.

Hybridomas secreting monoclonal antibodies (MAb) specific to American cockroach (Periplaneta americana) were produced through a fusion of immune splenocytes of a BALB/c mouse immunized with crude cockroach (CR) extract and mouse myeloma cells. Two hybridomas namely 38G6 and 3C2 were established. These specific hybridomas secreted IgG1 monoclonal immunoglobulins with antigenic specificities to CR protein components of over 207 to 72 kDa and 45 to 40 kDa, respectively. The monoclonal antibodies were applied to select their specific epitopes out of the crude CR extract using affinity chromatography. A Prausnitz-Kustner test revealed that these epitopes were allergens which caused wheals and flares of the skin of a guinea-pig previously sensitized with a pool of serum samples from CR allergic patients. The monoclonal antibodies were also used in a capture ELISA to detect specific IgE in serum samples of allergic Thai patients. It was found that 72% and 76% of the patients had IgE antibodies to the epitopes of MAb 38G6 and MAb 3C2, respectively, indicating that the two epitopes are major CR allergens among the CR allergic Thai patients. An antibody-sandwich ELISA was developed for quantitative detection of CR allergens using the two monoclonal antibodies as a capture reagent and rabbit polyclonal antibodies to crude CR extract as a detection reagent. The assay could detect allergenic epitopes contained in as little as 122 pg of crude cockroach extract, and has high potential for direct measurement of the marker allergens in extracts of environmental samples.

Recombinant American cockroach component, Per a 1, reactive to IgE of allergic Thai patients.

Twelve similar recombinant Per a 1 clones were produced from an American cockroach (CR) cDNA library. The nucleotide sequence of a representative cline, i.e. clone A6, contained 579 base pairs (bp) and a 372 bp open reading frame (2-373) encoding 124 amino acids. A stop codon was found at position 374-376 followed by a 3' end untranslated region with an AATAAA polyadenylation signal and a poly (A) tail. The estimated molecular mass of the 24 amino acid residue protein was 13.8 kDa, with a predicted isoelectric point value of 4.74. Cysteine or N-linked glycosylation was not found. The deduced amino acid sequence of the A6 revealed 84.68-95.97% identity to other previously reported Per a 1 clones and 65.87-69.60% homology to the previously reported Bla g 1 clones. However, while previously reported Per a 1 clones showed homology to ANG12, a precursor protein in the midgut of the female Anopheles gambiae secreted after the blood meal, the A6 DNA sequence was found to have homology (37.1%) to DNA of G2, a putative protein in the midgut of Aedes aegypti (AY 050565). The deduced amino acid sequence of A6 contained a mitochondrial energy transfer protein signature, phosphorylation sites for the cAMP-and cGMP-dependent protein kinase C and casein kinase II. Hydrophobic and hydrophilic characteristics of the A6 deduced peptide indicated that it was a transmembrane protein. This is the first report that Per a 1 is a transmembrane protein. The deduced amino acid sequence of the A6, which contained the sequence LIRSLFGLP, differed in one amino acid from two previously reported epitopes, i.e. LIRALFGL and IRSWFGLP, of Per a 1.0104 which bound 80% and 100%, respectively, to IgE of the allergic patients tested. The A6 DNA sequence was deposited in the GenBank (Accession number AY 259514) and has been designated Per a 1.0105. The A6 expressed protein bound to monoclonal antibodies (MAb 3C2) specific to American cockroach and also bound to IgE of all (100%) of the 20 allergic Thai patients.

Clinical and histopathological classification of nasal polyps in Thais.

One hundred and forty five patients with nasal polyps (NP) who underwent a first operation at the Department of Otolaryngology, Faculty of Medicine Siriraj Hospital during the 2-year period January 1998 to December 1999, were studied to determine the incidence of clinical and histolopathologic types of NP in Thai patients. The clinical types of NP were classified into 5 groups, using Stammberger's criteria i.e. isolated polyps (IP); antrochoanal polyps (ACP); NP and chronic rhinosinusitis (NPCRS) associated with non-eosinophilic infiltration; NPCRS associated with eosinophilic infiltration or NP with asthma (NPA); and NP with specific diseases (NPSD). The histopathologic types were classified into 4 types, using Hellquist's criteria i.e. Type I, edematous, eosinophilic (allergic) polyps; Type II, chronic inflammatory (fibroinflammatory) polyps; Type III, polyps with hyperplasia of seromucinous glands; and Type IV, polyps with stromal atypia. There were 86 males (59.3%) and 59 females (40.7%), with a male : female ratio = 1.5 : 1. The mean age was 36.1 + 16.5 years, ranging from 9 to 74 years. The mean duration of symptoms was 63.6 + 75.2 months, ranging from 1-420 months. The clinical classification study showed that 17 patients (11.7%) had IP, 13 patients (8.9%) had ACP, 105 patients (72.4%) had NPCRS, 5 patients (3.5%) had NPA, and 5 patients (3.5%) had NPSD (one case of Katargener's syndrome, bronchiectasis, aspirin intolerance, immotile cilia syndrome, and AIDS respectively). The histopathologic study showed that 17 specimens (11.7%) were Type I, 118 specimens (81.4%) were Type II, 9 specimens (6.2%) were Type III, and 1 specimen (0.7%) was Type IV. In the group of patients with NPCRS (105 patients), the most common histopathologic type was type II (86 patients, 81.9%). Type I and Type III were found in 12 patients (11.4%) and 7 patients (6.7%) respectively. The incidence of eosinophilic polyps in the groups of NPCRS was only 18.1% (Type I + Type III). This finding is different from that of NP in the western countries, in which the incidence of eosinophilic polyps is 80-90%. The pathogenetic mechanism underlying this difference is still not known. Is this difference due to racial or genetic factors, or geographic differences? The answers to these question are to be studied further.