Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents.

Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.

Aedes albolateralis, a potential vector of nocturnally subperiodic Wuchereria bancrofti and dengue type 2 virus.

The susceptibility of Aedes albolateralis to nocturnally subperiodic Wuchereria bancrofti and dengue type 2 virus was investigated by using artificial membrane feeding and intrathoracic inoculation techniques, respectively. The results indicated that Ae. albolateralis was susceptible to nocturnally subperiodic W. bancrofti (susceptibility rate = 9.43%) and dengue type 2 virus (susceptibility rate = 100%), suggesting the potential vector of the two pathogens.

Differentiation of Anopheles minimus species complex by RAPD-PCR technique.

Amplification of random regions of genomic DNA using 10-base primers in the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to differentiate Anopheles minimus A and An. minimus C. Genomic DNA was extracted from individual mosquitoes of An. minimus A and An. minimus C and amplified in PCR reactions using single primers of arbitrary nucleotide sequence. Fifteen different commercially available primers (Operon oligonucleotides kit M from Operon Technologies, Inc.), six primers were selected on the basis of presence or absence of the bands of A and C. They gave 8 different amplified DNA fragments of these two species of An. minimus. The primers revealed only species A are OPM8 and OPM13 at the 0.8 and 2.15 and 2, while both species A and C were in OPM17 at 0.8 Kb revealed A, at the 0.55 and 1.5 Kb revealed C. OPM 12 gave a 0.5 Kb DNA fragment in A while 0.4 Kb in C. OPM15 showed C at 0.7 Kb. These findings indicated that An. minimus species A and C can be differentiated by RAPD-PCR technique.

Integration of control measures for malaria vectors in endemic areas of Thailand.

Various vector control measures were applied in different endemic areas in two provinces, Saraburi and Chanthaburi, with comparison among different control measures. Application of IGR (insect growth regurator, pyriproxyfen) was introduced at Wat Tam Pra Pothisat, Tab-Kwang District, Saraburi Province. Some integration measures were performed at villages 6 and 8, Patavee, Makham District, Chanthaburi Province. In Tab-Kwang District with low malaria endemicity at the study site predators were not able to be released due to rapid velocity of running water. IGR could effectively control malaria compared to the basin released predators. Another endemic areas villagers 6 and 8, Patavee, Makham, Chanthaburi Province was chosen. Highly endemic multidrug resistant malaria has been prevalent for many years in this area. Integration of Kanda's trapping system, application of IGR, use of both residual spraying and impregnated bed-net methods with etofenprox successfully interrupted malaria infection. The application of these methods as an integrated control system could be adjusted to environmental conditions. The results of this study suggest rapid effective vector control.

Anthropophilic and zoophilic phlebotomine sand flies (Diptera, Psychodidae) from Thailand.

As a result of extensive surveys of sand flies carried out during 1987-90 in Thailand, species including a man-biter, Phlebotomus hoepplii, and cattle-biters, P. argentipes and P. major major, were discovered in Kanchanaburi Province. The human-baited catches revealed a low density of P. hoepplii and the biting occurred during 2100-2300 hours. Phlebotomus hoepplii was also found feeding on buffalo and inhabiting Pha-Thai Cave, Lampang Province. Phlebotomus argentipes was also a common cave-dweller and therefore had a wider distribution than P. major major, which was only collected in Sara Buri and Kanchanaburi provinces. The 3 species inhabited forest environments close to foothills and on stream banks. Although there is yet no evidence that they are involved in leishmaniasis transmission, the bionomics of the species are of importance and should be studied further.

Recent advances in vector control in filariasis.

Three factors are involved when vector control is incorporated into an integrated control program for filariasis: (1) the vector species or species complex of each filarial worm; (2) type of control measures employed; (3) pesticide application. Recent advances in vector control in filariasis include the use of bed nets, environmental manipulation, insecticides and biological control. Three promising advances in vectrol control are the use of pathogenic bacteria such as Bacillus thuringiensis H-14 and B. sphaericus, juvenile hormone mimics or insect growth regulator (IGR) and predatory crustaceans.

Vectors of Japanese encephalitis virus (JEV): species complexes of the vectors.

The vectors of JEV are Cx. tritaeniorhynchus, Cx. vishnui, Cx. pseudovishnui, Cx. gelidus, Cx. fuscocephala, Cx. quinquefasciatus, Cx. pipiens pallens, Cx. bitaeniorhynchus, Cx. annulirostris, Aedes togoi, Ae. japonicus, Ae. vexans nipponii, Anopheles annularis and An. vagus. Cx. tritaeniorhynchus is in the tritaeniorhynchus complex, breeds in rice fields, ground pools in vast areas. Two types of mating behavior, eurygamy and moderate stenogamy were detected. In the case of the eurygamy type, the mosquitoes were from Southern Thailand and hilly areas near Kanchanaburi, Thailand. Female mosquitoes are usually dark in color, the cibarial armature has rod teeth and the posterior end of the cibarial armature is bowl shaped with a typical rim. The rim of the bowl is everted. The moderate stenogamy type were mosquitoes from the plain areas such as Bangkok, Ayutthaya, Suphan Buri and Saraburi. The posterior end of the cibarial armature is bowl shaped with a stout rim. The larvae were characteristic in their siphon index, antennal index, hair O of prothoracic segment, and comb scale number and arrangement. Cx. tritaeniorhynchus summorosus from Japan, Los Banos and Luzon, Philippines, differed from Cx. tritaeniorhynchus in that on the lateral plate of the phallosome tritaeniorhynchus teeth are somewhat weakly developed and only gently curved whereas in tritaeniorhynchus summorosus they are strongly developed, considerably longer, and sharply recurved. The siphons of larvae are short, the sides parallel and the apex truncate in tritaeniorhynchus whereas in tritaeniorhynchus summorosus they are long and slender. Cx. tritaeniorhynchus var. siamensis is possibly present. Colonies have been maintained in the Department of Medical Entomology for 31 generations. The characteristics are in hair O (short, less than 20 branches, and without secondary branching and the larval siphon (short and broad where the others are long). Cx. vishnui and Cx. pseudovishnui are in the vishnui complex. Cx. quinquefasciatus and Cx. pipiens pallens are in the Cx. pipiens complex comprising: (1) Cx. pipiens; (2) Cx. quinquefasciatus Say; (3) Cx. molestus Forskal; (4) Cx. pipiens pallens; (5) Cx. australicus; (6) Cx. globocoxitus. Anopheles annularis is a species complex evidenced by two types of polytene chromosomes.

A brief survey of phlebotomine sandflies in Thailand.

Sandflies were found in widely scattered localities in Thailand in varied numbers. Daytime resting places for the adults included caves, termite hills, abandoned houses, ancient stone sanctuaries, air-raid shelters, tree hollows, loose bark of dead standing trees and rock crevices. Of fifteen species, P. major major, P. teshi, S. anodontis, S. gemmea, S. hodgsoni hodgsoni, S. perturbans, S. punjabensis and N. vietnamensis were here recorded for the first time in Thailand. P. argentipes and P. major major are interesting in view of their potential as disease vectors.

Alteration in Aedes togoi susceptibility to Brugia pahangi microfilariae induced by Aedes albopictus thoracic homogenate.

Refractoriness to Brugia pahangi microfilarial infection could be induced in the normally susceptible Aedes togoi mosquitoes by intrathoracic injection with crude thoracic homogenate of the refractory Aedes albopictus mosquitoes. The crude thoracic homogenate contained three Sephadex G-200 protein profiles of which the first profile showed strongest inhibition to the parasite development.

Population genetic studies on the Anopheles minimus complex in Thailand.

The population genetic studies on the Anopheles minimus complex revealed that An. minimus is a species complex evident by IK value were less than 0.0534 and 0.4163 for the morphotaxonomy and isozyme studies. There are two sibling species in the An. minimus species complex. The first is the typical minimus and its variations described by Harrison. Esterase-2 alleles 100 and 102 are predominant with a few other alleles of 96 and 104. It is exophagic and zoophilic. It is widely distributed in all provinces where minimus breeds. The second of the two sibling species is An. minimus species C, with 2 pale spots on the humeral dark band and Est-2 allele 98. It is found most frequently in Pu Toei District of Kanchanaburi Province, Thailand. An. minimus probably species B showing M1 + 2 wing vein entirely dark is found in China.

Investigation of malaria sporozoites by immunoradiometric assay.

Two-site immunoradiometric assay (IRMA) (Zavala et al., 1982) using monoclonal antibodies to P. falciparum and P. vivax was applied to detect sporozoites in laboratory-maintained An. dirus and also mosquitoes collected from endemic areas of malaria in Thailand. Study in P. falciparum infected mosquitoes revealed that the circumsporozoite (CS) antigen was first found in the abdominal portion on day 10 post-infection, while it could be observed in the salivary glands from day 15 onwards. The head-thorax portion of wild-caught mosquitoes were investigated by IRMA compared with the dissection technique. The results showed that none of the mosquitoes collected from Phrae was positive for malaria. The mosquitoes collected from Chantaburi showed 4 out of 1243 An. dirus that were positive for P. falciparum by IRMA, with sporozoites ranging from 207 to 3875. Among 3123 An. minimus collected from Kanchanaburi, 3 were positive by IRMA, 2 for P. falciparum and one P. vivax with sporozoites found in head-thorax portion were 1880, 2380 and 1026 respectively. Not a single sporozoite was found in the mosquitoes collected from these areas by the dissection technique. However 7 out of 1219 An. minimus from Kanchanaburi were found to possess undeveloped oocysts in the stomach wall. It is evident that the IRMA is efficient, convenient and suitable for the investigation of sporozoites in this region. The application of this technique in further epidemiological study of malaria is in progress.

Morphological differentiation in Anopheles maculatus of Thailand accompanied with genetical divergence assessed by hybridization.

Anopheles maculatus of Thailand were distinguished morphologically into densely and sparsely scaled types and cross-mating experiments between these types revealed a substantial amount of genetic divergence. Progeny of wild females of An. maculatus collected throughout Thailand from north to south were examined morphologically with respect to adult tergal pale scales. Northern progeny broods could be separated into densely scaled (willmori) and sparsely scaled (maculatus) types, or W and M types, respectively. Using these two types of An. maculatus from Chiang Rai and Nakhon Phanom, cross-mating experiments were conducted, which resulted in sterile F1 male hybrids in one direction of cross and very low survival rate of F1 male hybrids in the reciprocal cross. However, southern broods could not be separated clearly because of intergradation of scaling. These results indicate that Thai An. maculatus involves at least 2 forms differentiated morphologically, as well as by incomplete reproductive isolation.

Scanning electron microscopic study of nocturnally subperiodic Brugia malayi (Filarioidea: Onchocercidae), Narathiwat, Southern Thailand.

Scanning Electron Microscopic (SEM) observations were made on the adult females and males of nocturnally subperiodic Brugia malayi (Narathiwat, Southern Thailand) from 8-month-old intra-peritoneally infected jirds (Meriones unguiculatus). Descriptions of the morphological surfaces of anterior end, vulva, body cuticle, anus, posterior end of females and anterior end, body cuticle, cloaca, caudal papillae, spicules, sheath, posterior end of males were demonstrated. The comparison among these and other filarial parasites were also investigated.

Laboratory bionomics of Tripteroides aranoides.

Tripteroides aranoides was colonized in the laboratory. Total duration of the immature stages was ca. 3 weeks at 28 degrees C, L:D = 15.5:8.5 with an ample food supply. Retardation of 4th instar development was observed in larvae fed on insufficient food. Females were autogenous for the first clutch of eggs and required a blood meal for maturation of the second clutch. Mating was initiated in flight and copulation occurred on the cage wall. Gravid females hovered in small oblique loops above water in bamboo cups, whereupon a white egg appeared at the abdominal tip, which was propelled by the swing of the abdomen towards water surface. The females propelled eggs in the same manner into small apertures (11 x 4 mm) bored in bamboo.

In vitro studies on the sensitivities of Plasmodium falciparum in Thailand to pyrimethamine and proguanil.

The susceptibility to pyrimethamine and proguanil of a strain of Plasmodium falciparum from Thailand was assessed by using blood from the same patient cultured by two different techniques. Irrespective of the technique used, the strain of P. falciparum appeared more sensitive to proguanil than to pyrimethamine.

A small scale field trial with Temephos (Abate) for the control of four species of Mansonia (Mansonioides) (Diptera: Culicidae) in Thailand.

A 60-week control experiment with Temephos (Abate) against a largely isolated population of Mansonia annulata, Ma. annulifera, Ma. indiana and Ma. uniformis was carried out in a pool of 1.2 ha area and average depth of over 2 m in southern Thailand. Floating paper tea bags containing 24 g of 1% Abate sand granules were applied in two rounds at a density of 1 bag 4 m-2. Each round consisted of five applications at intervals of 13 days. During the application rounds mosquito larvae disappeared completely from the pool. This apparent effect of Abate was confirmed by bioassays made with pool water and larvae of Culex quinquefasciatus. Inactivation of Abate was linear during one week, with a slope of 5.8% per day. Adult densities dropped during the application rounds and, over a period of 48 weeks, were 78-85% less than during the corresponding time of the preceding reference year. The annual pattern of adult abundance that normally shows distinct seasonal peaks was strongly altered, although meterological data were comparable to the reference year.