An antigen detection assay for diagnosing filariasis

In this study we examined the diagnostic potential of monoclonal antibodies (MAb) reactive to antigens of adult Brugia malayi, their microfilariae and antigen of Dirofilaria immitis. The MAb of clone 17E10, which were of IgM isotype, reacted to the inner cuticles and internal content of both male and female worms and also to the sheath and internal content of microfilariae in utero. However, these MAb did not react to the sheath of blood circulating microfilariae. The MAb 17E10 produced a smear pattern between 37 to > 200 kDa in the Western blot analysis against a SDS-PAGE separated extract of B. malayi. The epitopes were non-protein in nature as indicated by their resistance to proteinase-K treatment. The MAb 17E10 were applied in a sandwich ELISA to detect filarial antigen in the buffy coat and plasma of patients. We tested patients with different clinical manifestations of brugian filariasis, i.e. microfilaremia (M), lymphangitis (L) and elephantiasis (E), as well as non-symptomatic inhabitants of a filariasis endemic area (NE), and compared them to samples from non-symptomatic inhabitants of disease non-endemic areas (NNE). It was found that 22 of 31 (70.9%) of M, 7 of 13 (53.8%) of L, 2 of 14 (14.2%) of E, 10 of 100 (10.0%) of NE and none (0%) of the NNE were positive for antigenaemia. The assay was also positive in 14 of 15 (93.3%) blood samples from B. malayi microfilaremic cats and in 7 of 7 (100%) blood samples of Dirofilaria immitis microfilaremic dogs. The so-developed test has a high potential for routine diagnosis of active filariasis, for epidemiological studies in both humans and reservoir animals and for monitoring treatment efficacy.     

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     

Augmented sympathetic vasoconstriction in exercising forearms of postmenopausal women is reversed by oestrogen therapy

Sympathetic vasoconstriction is normally attenuated in exercising muscles of young men and women. Recent evidence indicates that such modulation, termed functional sympatholysis, may be impaired in older men. Whether a similar impairment occurs in older women, and what role oestrogen deficiency might play in this impairment, are not known. Based on the strong positive correlation between circulating oestrogen levels and functional sympatholysis previously reported in female rats, we hypothesized that sympatholysis would be impaired in oestrogen-deficient postmenopausal women, and that this impairment would be reversed by oestrogen replacement. To test these hypotheses, we measured vasoconstrictor responses in the forearms of pre- and postmenopausal women using near infrared spectroscopy to detect decreases in muscle oxygenation in response to reflex activation of sympathetic nerves evoked by lower body negative pressure (LBNP). In eight premenopausal women, LBNP decreased muscle oxygenation by 20 ± 1% in resting forearm, but only by 3 ± 2% in exercising forearm  ( P < 0.05)  . In contrast, in eight postmenopausal women, LBNP decreased muscle oxygenation by 15 ± 3% in resting forearm, and by 12 ± 4% in exercising forearm  ( P > 0.05)  . After 1 month of transdermal oestradiol replacement in these women, the normal effect of exercise to blunt sympathetic vasoconstriction was restored (rest, −19 ± 3%; exercise, −2 ± 3%;   P < 0.05  ). These data indicate that functional sympatholysis is impaired in oestrogen-deficient postmenopausal women. The effect of short-term unopposed oestrogen replacement to correct this impairment implicates a role for oestrogen in the sympathetic neural control of muscle haemodynamics during exercise.     

Short-term effects of an intensive lifestyle modification program on lipid peroxidation and antioxidant systems in patients with coronary artery disease

The purpose of this study was to compare the short-term effects of an intensive lifestyle modification (ILM) program on lipid peroxidation and antioxidant systems in patients with coronary artery disease (CAD). Twenty-two patients in the control group continued to receive their conventional treatment with lipid-lowering drugs, whereas 22 patients in the experimental group were assigned to intensive lifestyle modification (ILM) without taking any lipid-lowering agent. The ILM program comprised dietary advice on low-fat diets, high antioxidants and high fiber intakes, yoga exercise, stress management and smoking cessation. After 4 months of intervention, patients in the experimental group revealed a statistically significant increase in plasma total antioxidants, plasma vitamin E and erythrocyte glutathione (GSH) compared to patients in the control group. There was no significant change in plasma malondialdehyde (MDA), a circulating product of lipid peroxidation, in either group. We concluded that the ILM program increased circulating antioxidants and reduced oxidative stress in patients with CAD.     

Components of pathogenic Leptospira spp. with potentials for diagnosis of human leptospirosis

Existing serological methods for diagnosis of leptospirosis are still unsatisfactorily due mainly to their low accuracy. In this study, serum samples of 18 clinically diagnosed-, IgM dipstick positive-, MAT positive-leptospirosis patients (group 1) were analyzed by IgG Western blotting against SDS-PAGE separated-whole cell homogenates of pathogenic and non-pathogenic Leptospira spp. belonging to 20 serovars of 15 serogroups. The samples of group 1 were collected from the patients at days 3 to 10 after the fever onset (fist samples). Second and third samples could be obtained from 4 patients. Sera of the 22 patients with other febrile illnesses (group 2) and 22 healthy counterparts (group 3) were used as patient- and normal- controls, respectively. Irrespective of the serovar or serogroup of the pathogenic Leptospira spp. used as antigen in the Western blotting, all of the 18 sera of patients with leptospirosis (group 1) gave characteristic diffuse antigen-antibody reactive bands located at approximately 35-38 and 22-26 kDa; and thus 100% diagnostic sensitivity of the Western blot assay. Some serum samples of the leptospirosis patients also reacted to components located at 80-100, approximately 70, 60, 54, and 48 kDa. More bands or the early recognized bands with increased intensity were observed when tested the second and third samples. The characteristic bands were not seen when homogenates of L. biflexa, serogroup Semaranga, serovar Patoc (saprophytic) and L. biflexa, serogroup Andamana, serovar Andamana (non-pathogenic but can infect host) were used in the assay. Sera of groups 2 and 3 did not react to the components at the seven locations implying 100% diagnostic specificity of the IgG Western blot assay. While awaiting validation with more patients' samples, the IgG Western Blot analysis aiming at the detection of the characteristic antigen-antibody reactive bands described in this study has high potential for early, rapid, simple and accurate diagnosis of human leptospirosis.     

Histogenesis and disappearance of the teeth of the Mekong giant catfish, Pangasianodon gigas (Teleostei)

Juveniles of the Mekong giant catfish, Pangasianodon gigas (Teleostei), have 3 sorts of tooth-upper and lower jaw teeth, palatal teeth, and pharyngeal teeth--but adults are toothless. To investigate the histogenesis and disappearance of the teeth, we made serial sections of the mouth and teeth of juvenile fish at 10 developmental stages (from ca. 8.5 to ca. 30 cm in total length) and examined them under scanning electron microscope and light microscope. Observations of teeth and surrounding tissues in the serial sections revealed the process of tooth resorption by active odontoclast-like cells. Numbers of jaw and palatal teeth decreased with age. When the fish reached ca. 14 cm in total length, the numbers of functional upper jaw teeth and successional tooth germs decreased rapidly, and the developmental rate of successional tooth germs slowed. When the fish reached ca. 24 cm, no teeth existed in the upper jaw. It is clear that tooth disappearance results from the shedding of functional teeth and the lack of replacement tooth germs.     

Preferences for treatment of diarrhoea and dysentery in Kaengkhoi district, Saraburi province, Thailand

To estimate the proportion of cases missed in a passive surveillance study of diarrhoea and dysentery at health centres and hospitals in Kaengkhoi district, Saraburi province, Thailand, a community-based cluster survey of treatment-seeking behaviours was conducted during 21-23 June 2002. Interviews were conducted at 224 households among a study population of 78,744. The respondents reported where they sought care for diarrhoea and dysentery in children aged less than five years and adults aged over 15 years. Health centres or hospitals were the first treatment choice for 78% of children with dysentery (95% confidence interval [CI] 63-94%), 64% of children with diarrhoea (95% CI 54-74%), 61% of adults with dysentery (95% CI 40-82%), and 35% of adults with diarrhoea (95% CI 17-54%). A high degree of heterogeneity in responses resulted in a relatively large design effect (D=3.9) and poor intra-cluster correlation (rho=0.3). The community survey suggests that passive surveillance estimates of disease incidence will need to be interpreted with caution, since this method will miss nearly a quarter of dysentery cases in children and nearly two-thirds of diarrhoea cases in adults.     

CpG DNA, liposome and refined antigen oral cholera vaccine

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.