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.

Prevalence of Listeria monocytogenes in Raw Meats Marketed in Bangkok and Characterization of the Isolates by Phenotypic and Molecular Methods.

Listeria monocytogenes causes listeriosis characterized by septicaemia, encephalitis, and abortion or stillbirth. Regular monitoring of its prevalence in food and characterization of its phenotypes and genotypes are necessary for disease surveillance and tracing the epidemic outbreaks. In this study, the prevalence of L. monocytogenes in raw meats marketed in Bangkok was 15.4%. The bacteria isolated from meat were serotyped and genotyped using enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC-PCR). Their virulence-associated genes, antimicrobial susceptibility, and ability to invade intestinal epithelial cells were studied. All 22 L. monocytogenes strains isolated from 104 raw meat samples carried virulence-associated genes, such as actA, flaA, hlyA, iap, inlA, inlB, and prfA. These were serotype 4b, suggesting their pathogenic and epidemic potential. These isolates could be classified into six ERIC-PCR groups: A-F. The majority (59.1%) of the isolates belonged to Group A, and three isolates were Group D which was closely related to the Group A. Two isolates each were Group C and E, and one isolate each was group B and F. Although the isolates belonged to the same serotype and genotype and were all equipped with the virulence-associated genes, they showed a different cell invasion capability and antibiotic susceptibility. All the isolates were susceptible to ampicillin, amikacin, chloramphenicol, gentamicin, imipenem, penicillin G, sulphamethoxazole-trimethoprim, and tetracycline. However, one isolate showed only intermediate susceptibility to tetracycline. The data provide the first molecular insight into the L. monocytogenes isolates in Thailand and elucidate a potential risk of people contracting listeriosis.

Human monoclonal ScFv that inhibits cellular entry and metalloprotease activity of tetanus neurotoxin.

Tetanus is a deadly disease of warm blooded animals and humans caused by an exotoxin called te-tanospasmin or tetanus neurotoxin (TeNT) produced by anaerobic bacterium named Clostridium tetani. TeNT is an A-B toxin; each molecule consists of a heavy chain (HC) containing cellular receptor binding domain and a light chain (LC) with zinc metalloprotease activity. TeNT produced in the infected tissue by the bacteria grown under anaerobic condition binds to ganglioside receptors of peripheral nerve, and endocytosed. The A subunit exits from the endosome and undergoes a retrograde transport via the nerve axon to the spinal cord. This highly toxic enzyme specifically cleaves one of the nerve cell SNARE proteins, i.e., synaptobrevin, resulting in inhibition of the release of neurotransmitters (glycine and GABA) from inhibitory interneuron causing spastic paralysis, the characteristic of tetanus. Current treatment mainstay of human tetanus is by passively administering anti-tetanus toxin produced from animals immunized with adjuvanted tetanus toxoid (TT). There are several obstacles in production and use of the animal derived therapeutic antibody especially the allergic reaction and serum sickness induced by the host immune response to the foreign protein. The animal antibody, mainly IgG, blocks nerve cell entry of the TeNT but does not neutralize the TeNT protease activity per se and cannot reverse the tetanus symptoms. In this study, fully human single chain antibody fragments (HuScFv) were produced from a human antibody phage display library. TT was used as antigen in a single round phage bio-panning to select phage clones that display TT bound-HuScFv from the library. HuScFv from 4 selected huscfv-phagemid transformed E. coli clones inhibited binding of the native TeNT to retinoic acid pulsed human neuroblastoma cells when used at the molecular TeNT:HuScFv ratio of 1:100. HuScFv from one of the 4 clones also inhibited the TeNT mediated cleavage of recombinant synaptobrevin. Further investigation is needed for identification of epitope specificity of these HuScFv and HuScFv effector mechanisms towards the TeNT. Cell penetrating version of the HuScFv that inhibited the TeNT zinc metalloprotease activity should be made. The HuScFv produced in this study either singly or in their suitable combination warrant developing further to a real use in humans as a surrogate of the animal antibody for treatment of tetanus.

Giardia intestinalis in Thailand: Identification of Genotypes.

This study was undertaken to determine the genetic diversities of Giardia intestinalis isolated in Thailand. G. intestinalis cysts were collected from stool samples of 61 subjects residing in Bangkok or in rural communities of Thailand with and without gastrointestinal symptoms. All the cyst samples gave positive tpi amplicons (100% sensitivity), either of the 148- or the 81-bp tpi segments. Cyst assemblage identification of the 148- and 81-bp tpi gene segments by polymerase chain reaction showed that 8% of the cysts were assemblage A, 41% assemblage A and B combined, and 51% assemblage B. The prevalence of assemblage A was significantly lower than that of assemblage B and the mixed types. Restriction fragment length polymorphism (RFLP) of the 384-bp ß-giardin gene segment revealed that 12% and 88% of the assemblage A cysts were AI and AII respectively. RFLP, based on the 432-bp gdh gene segment, showed 45.5% of the assemblage B cysts to be BIII and 54.5% to be BIV. The AI sub-assemblage was less prevalent than the others. All subjects with AI and 50% of the subjects with BIII sub-assemblage cysts were symptomatic; 80% of symptomatic Bangkok residents were adults/elderly while 85% of the rural cases were children.

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.

Seasonal levels of the major American cockroach allergen per a 9 (arginine kinase) in Bangkok and their rele-vance for disease severity.

Monitoring the levels of cockroach (CR) allergen in the environment has medical relevance as a clear dose response relationship between CR allergen exposure, sensitization and hospitalization has been reported. In this study, a cross-sectional survey of the levels of a major American cockroach (Periplaneta americana) allergen, i.e. Per a 9 (arginine kinase) in dust samples collected in various seasons throughout the year 2007 from 76 houses of CR allergic Thai patients in the Bangkok metropolitan area were determined. A monoclonal antibody-polyclonal antibody (MAb-PAb) based-sandwich ELISA was used. The MAb was specific to Per a 9 and the PAb was raised in a rabbit against the crude extract of P. americana. The detection limit of the assay was 122 pg of the allergen or 0.024 μg per gram of fine dust powder. The concentrations of Per a 9 were found to be highest during the winter months and lowest in summer. The levels of this CR allergen had a direct correlation with disease exac-erbation; i.e. the majority of the CR allergic patients had their most severe clinical manifestations during winter. Moreover, the CR allergen levels were found to be higher in wood based-houses than in concrete houses.

A murine model of allergy caused by American cockroach (CR), Periplaneta americana.

An animal model resembling the human immuno-pathological features of CR allergy is needed for CR allergy research, e.g., measuring allergenicity of novel allergens, testing immunotherapeutic efficacies of drugs and vaccines. In this study we develop a murine model of American CR, P. americana allergy. BALB/c mice, 6 weeks old, were individually intraperitoneally injected with three doses (days 0, 7 and 14) of alum adjuvanted-crude extract of P. americana. On days 21 and 23, they were given crude CR extract in PBS intranasally (10 microl) and aerosolically (10 ml) via an air-pressure nebulizer, respectively. Mice received alum alone and PBS instead of the CR extract served as non-allergenic controls. All mice were bled twenty four hours after the nebulization and sacrificed. Their serum samples, broncho-alveolar lavage fluids (BALF), and lung tissues were collected. BALF of all allergen-treated mice had marked cellular infiltration notably neutrophils, eosinophils and lymphocytes. The average total cell count in BALF of the allergenic mice was 1.9 x 10(5) cells/ml which out-numbered those of the non-allergenic controls (8 x 10(4) cells/ml). The eosinophil infiltration was pronounced in lungs of the allergen-treated mice. Specific serum IgE to the CR extract elevated in serum samples of all allergen treated mice and nil in the sera of the controls. None of the mice showed detectable level of IgG2a to the CR extract. RT-PCR revealed that all allergen-treated mice had marked increase of IL-13, IL-4 and TNF-alpha gene expressions, slight increase of IL-5 gene expression, and absence of detectable IFN-gamma gene expression in comparison to the non-allergenic controls. None of the allergen-treated mice and 50% of the non-allergenic controls had IL-12 gene expression as detected by RT- PCR. One allergen treated-mouse (25%) had subpar level of the IL-18 gene expression compared to the controls. Results of the quantitative real-time PCR conformed to those of the RT-PCR. A murine model of P. americana resembling human allergic manifestations was successfully developed.

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.

The potential usefulness of the modified Kato thick smear technique in the detection of intestinal sarcocystosis during field surveys.

A total of 479 stool specimens were collected from rural communities of Ubon Ratchathani Province, Thailand and examined by two techniques: the modified Kato thick smear and the direct smear. The prevalence of Opisthorchis viverrini (14.8%), hookworm (10.2%), Sarcocystis spp (4.6%), Taenia spp (2.9%), Strongyloides stercoralis (2.1%), Giardia lamblia (1.2%), Echinostoma spp (0.6%), Ascaris lumbricoides (0.4%), Entamoeba histolytica (0.2%), Chilomastix mesnili (0.2%) and Endolimax nana (0.2%) were determined. The morphology of the Sarcocystis spp sporocysts examined by both procedures looked similar and was found to be easily recognizable. Among these specimens, 22 cases (4.6%) were positive for Sarcocystis infection detected by the modified Kato technique, whereas only one case (0.2%) was detected by both techniques. These differences were found to be statistically significant (p < 0.05), indicating that the modified Kato technique was decidedly more sensitive than the direct smear procedure in identifying Sarcocystis infection. An epidemiological survey was conducted in Khon Kaen Province involving 1124 stool samples using the modified Kato technique. The greatest frequency was Opisthorchis viverrini at 32.0% while the second highest was Sarcocystis spp at 8.0%. The prevalences of hookworm, Echinostoma spp, Taenia spp, Trichuris trichiura and Enterobius vermicularis were 2.7, 2.1, 1.0, 0.2 and 0.2%, respectively. Other than opisthorchiasis, northeastern Thailand may be an endemic area for sarcocystosis. This is the first report of the applicability and potential usefulness of the Kato thick smear technique for the diagnosis of Sarcocystis infection in a field survey.

Ganoderma lucidum: a cause of pseudoparasitosis.

We report a pseudoparasitosis case due to Ganoderma lucidum, (lingzhi or reishi mushroom); we believe this to be a first reported case in Thailand. A 49-year-old male patient with non-Hodgkins lymphoma presented with chronic watery diarrhea. He had a history of consumption of powdered lingzhi extract as a dietary supplement and herbal medicine. Stool examination demonstrated many spores of G. lucidum, which must be differentiated from intestinal helminth ova and coccidia. After discontinuation of mushroom spores ingestion, the diarrheal symptoms improved and fecal examination subsequently showed no Ganoderma spores. Many artifacts in the stool may be confused with parasites. Differentiation of parasites from artifacts depends on characterization of the size, shape, structure, and reactivity with common stains.

Riboflavin-deficient and Trichinella spiralis-induced stresses on plasma corticosterone associated with spermatogenesis in male Wistar rats.

The objective of this study was to investigate the effects of riboflavin-deficient and Trichinella spiralis-induced stresses on corticosterone associated with spermatogenesis in male Wistar rats. Rats were allocated into 4 groups: Group 1: control; group 2: riboflavin-deficient diet; group 3: T. spiralis infection; group 4: riboflavin deficient diet with T. spiralis infection. This experiment lasted for 12 weeks. Plasma corticosterone was significantly enhanced when exposed to acute riboflavin deficiency and/or T. spiralis infection stress. When the rats were chronically subjected to such stresses, T. spiralis per se had prolonged effects, in a marked increase in corticosterone. T. spiralis per se tended to impact on such sperm characteristics as sperm motility, sperm count and daily sperm production, even defected seminiferous tubules. It was proposed that the Trichinella spiralis-induced stress probably had adverse effects on the level of adrenocortical-testicular axis whenever their habitats on muscle fibers were evident. However, riboflavin-deficient-induced stress had little implication in the adrenocortical-testicular axis.

Methylenetetrahydrofolate reductase (MTHFR) polymorphism (C677T) in relation to homocysteine concentration in overweight and obese Thais.

We analyzed the association between MTHFR (C677T) gene polymorphism with serum concentrations of homocysteine, folate, and vitamin B12 in 37 male and 112 female overweight/ obese Thai volunteers (BMI > or = 25.00 kg/m2), and compared them with 23 male and 90 female control subjects (BMI = 18.5-24.99 kg/m2). Statistically significant higher levels of serum homocysteine were found in the overweight/obese subjects than the control subjects (p < 0.05). Serum folic acid levels in the overweight/obese subjects were significantly lower than the control subjects (p < 0.05). When the data were grouped according to homocysteine concentration and MTHFR gene polymorphism, there were significantly higher homocysteine concentrations in the overweight/obese subjects than the control subjects in wild type gene polymorphism (CC) in the hyperhomocysteine group (homocysteine >10.0 mmol/l) (p < 0.05), but in genotype polymorphism (CC, CT, TT) there were lower folic acid and vitamin B12 concentrations in the overweight/obese subjects than in the control subjects. In the hyperhomocysteine groups, there was no significant difference in the frequencies of MTHFR (C677T) gene polymorphism between the overweight/obese subjects and the control subjects. Folic acid and gene polymorphism were found to be significantly related to the overweight/ obese and control groups in logistic regression analysis (p < 0.05). The results support the supposition that folic acid is more important than vitamin B12.

Relationship between soluble leptin receptor, leptin, lipid profiles and anthropometric parameters in overweight and obese Thai subjects.

Median, range and 95% confidence interval (CI) for median of age, anthropometric variables, soluble leptin receptor, serum leptin and lipid profile levels of 48 overweight (Body mass index (BMI) = 25.00-29.99 kg/m2) and obese (BMI > or = 30. 00 kg/m2) Thai males and 166 overweight and obese Thai females, compared with 26 males and 81 females in a control group (BMI = 18.50-24.99 kg/m2), were determined The study subjects were persons who turned up regularly for physical check-ups at the Out-patient Department, General Practice Section, Ratchawithi Hospital, Bangkok, aged between 18-60 years. Serum leptin, triglyceride and low density lipoprotein cholesterol/high density lipoprotein cholesterol ratios (LDL-C/ HDL-C ratio) were significantly higher in the overweight and obese males and females. Soluble leptin receptor and HDL-C were significantly lower in the overweight and obese males and females. Cholesterol and LDL-C were significantly higher in the overweight and obese females, but there was no significant difference in the overweight and obese males when compared with the control males. Low soluble leptin receptor levels were found in 38.1% (8/21) of the overweight and obese males, while 31.5% (29/92) were found in the overweight and obese females. Elevated leptin levels were found in 66.7% (32/48) and 89.8% (149/166) of the overweight and obese males and females, respectively. Both low soluble leptin receptor levels and elevated leptin levels were found in 9.5% (2/21) and 29.4% (27/92) of the overweight and obese males and females, respectively. A significant positive correlation was found between soluble leptin receptor and cholesterol, and between weight, BMI, waist, hip and HDL-C, with leptin. Serum soluble leptin receptor levels were significantly negatively correlated with leptin and BMI. The results can elucidate the causes and consequences of obesity, and are expected to aid the provision of care for overweight and obese Thai people.

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

Blastocystis hominis infection in irritable bowel syndrome patients.

Irritable bowel syndrome (IBS) is a functional bowel disorder in which abdominal pain is associated with a defect or a change in bowel habits. Subtle inflammation, especially after infectious enteritis, has been sometimes suspected as one mechanism of pathogenesis. This research was performed (1) to evaluate the prevalence of parasitic infections and (2) the possible association of IBS and parasitic infections. Fifty-nine IBS patients were recruited using symptom-based criteria (Rome Criteria II) with an absence of intestinal parasitic infection by direct smear method. Stool samples of individual patients were examined using 7 methods, ie examination for stool occult blood, simple saline smear method, formalin-ether technique, culture for Blastocystis hominis, modified trichrome stain, modified Ziehl-Neelsen method, and trichrome stain for parasitic and bacterial infections. Of the 59 patients, stool samples of 13 patients (22.1%) were positive for parasites. These were B. hominis (13.6%), Strongyloides stercoralis larvae (1.7%), Giardia lamblia cysts (1.7%), and non-pathogenic protozoa, ie Endolimax nana cysts (5.1%). The prevalence rate of parasitic infections in the control group (20%) was not statistically different from the patients. There was no statistical difference between B. hominis infection in IBS patients and control was found in this study (p = 0.87). In the IBS group, B. hominis infection predominated (13.6%), while other parasitic infections were found in 8.5%. The culture method for B. hominis is more sensitive than the direct (simple) stool smear method, which is the routine diagnostic method in most laboratories. These results were also found in control group.

Antioxidant enzyme levels in the erythrocytes of riboflavin-deficient and Trichinella spiralis-infected rats.

The erythrocyte antioxidant enzyme levels of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of riboflavin-deficient and Trichinella spiralis-infected rats were investigated. The rats were deprived of riboflavin at the 8th week of the experiment. At that time, the erythrocyte glutathione reductase activity coefficient (EGR AC), as an indicator of riboflavin status, was > or = 1.30 in rats fed a riboflavin-deficient diet and T. spiralis-infected rats fed a riboflavin-deficient diet showed no biochemical sign of riboflavin deficiency. At the 12th week of the experiment, the levels of catalase, SOD and GSH-Px were significantly lower in the riboflavin-deficient, T. spiralis-infected, and combined riboflavin-deficient and T. spiralis-infected, rats, compared to the control group. This may have been due to an increase in free oxygen radicals caused by riboflavin deficiency and parasitic infection.

Serum copper, zinc, ceruloplasmin and superoxide dismutase in Thai overweight and obese.

The serum copper, selenium, ceruloplasmin, superoxide dismutase (SOD) (specific activities of antioxidant enzymes), anthropometric measurements, including waist/hip ratio 51 male and 190 female overweight subjects (body mass index (BMI) > or = 25.0 kg/m2) compared with a 26 male and 83 female control group (BMI = 18.5-24.9 kg/m2) Thai volunteers who attended the Out-patient Department, General Practice Section, Rajvithi Hospital, Bangkok, for a physical check-up from March to October, 1998, were investigated. There was no age difference between the overweight group and the controls. All of the anthropometric variables, except the height of the overweight group, were significantly higher than those of the normal subjects. The medians of weight and waist/hip ratio of overweight and obese males were significantly higher than those of overweight and obese females. Serum ceruloplasmin, copper were statistically significantly higher in overweight subjects than in the controls. However, serum zinc and superoxide dismutase activity in the overweight group were found to be lower than in the control group. Higher serum ceruloplasmin, copper, zinc and superoxide dismutase activity were shown in the female overweight group than in the male overweight group. Ceruloplasmin was found to correlate positively with copper concentration but negatively related with superoxide dismutase enzyme activity. A negative correlation was found between serum copper and zinc concentrations in both sexes of the overweight and obese subjects. Low SOD activity found in the overweight and obese subjects might be caused by low zinc intake.

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.