Significantly higher values were obtained for the DF and DHF groups (mean values equivalent to maximum detectable absorbance of 1 1.80; p<0.001) (Table 1). Table 1 Reactivity of patients sera to total MSP in ELISA saliva could be concentrated by TCA precipitation and fractionated by non-denaturing PAGE, reproducibly resolving seven major protein bands with molecular sizes ranging from 14 kDa to 68 kDa. DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans. Keywords: dengue, dengue haemorrhagic fever, mosquitoes. After an incubation period FK866 of 3 to 14 days, a person bitten by an infected mosquito can experience acute fever accompanied by a variety of nonspecific signs and symptoms. In the last 50 years, the incidence of DEN disease has increased and hyperendemic transmission of DENV has been established 1, 2, resulting in a dramatic global increase in the most severe form of the disease, DHF, which was first described in Southeast Asia 3. A major risk factor for developing DHF is re-infection with a virus serotype different from the first infection 4. Molecular epidemiologic evidence has shown that there also is an increased risk of developing DHF when the infection is caused by a virulent DENV genotype 5, 6, 7. Inoculation of MSP at the time of infection has been associated with enhanced pathogenesis of arboviruses 8, 9, 10, 11. Co-inoculation of salivary proteins or salivary gland extracts and arboviruses facilitates the dissemination of the pathogen due to modulation of the host immune response 12, 13, 14, 15. Other recognised functions of MSP that might potentiate pathogen transmission include vasodilation, inhibition of platelet activation, and suppression of inflammation 16, 17; for example, sequestration of inflammatory mediators such as biogenic amines and leukotrienes are important functions of the abundant D7 protein in saliva18, 19. Exposure to MSP has been shown to elicit specific IgE and IgG1 antibodies 20. In experimental models, disease severity was reduced in mice pre-exposed to sandfly saliva before infection, and to mosquito saliva prior to infection 21, 22, 23. In contrast, a report on the effect of pre-exposure to MSP on West Nile virus infection indicated that it enhances mortality in a mouse model 24. In this retrospective study using well-characterized serum specimens from children FK866 who were hospitalized in Bangkok, Thailand, we examined the relationship between the human immune response to MSP and DEN disease severity. MATERIALS AND METHODS Patients A total of 101 paired acute and convalescent coded serum specimens were obtained upon admission and 2C6 days later, respectively, from Thai patients who had been admitted to FK866 a Bangkok childrens hospital. These paired de-personalized serum specimens had been subjected to laboratory diagnosis, a service provided by the Armed Forces Research Institute of Medical Sciences (AFRIMS) to the Bangkok community. All sera were collected in compliance with Thai and U.S. regulations on human use research. Serologic diagnosis Rabbit Polyclonal to PKR1 of acute DENV infection was made and Japanese encephalitis virus infection was excluded by IgM and IgG capture ELISA for both viruses. Dengue infection status (i.e., acute primary, acute secondary, or no DENV infection) was assigned to all patients based on established AFRIMS criteria for IgM and IgG capture ELISA results 25, 26. Reverse transcription-nested polymerase chain reaction (RT-PCR) was used to determine DENV serotype using primers described by Lanciotti et al. 27. Severity of infection was assigned using World Health Organization (WHO) criteria 28. Results reported in this study were limited to acute serum samples collected at the time of hospital admission from 96 patients whose subsequent laboratory diagnosis indicated they had DENV2 secondary infections or did not have DENV infections, determined after the patient identification code was broken, and whose sera reacted with salivary proteins in immunoblots. Of the 50 DHF patients, 46% were male; age range 3C28 yr, mean 8.5 yr; median 10 yr. Of the 28 DF individuals, 36% were male; age range 3C17 yr, mean 9 yr, median 9 yr. Of the 18 non-DENV-infected (NI) individuals, 50% were male; age range 2C10 yr, mean 5.7 yr, median 6 yr. All sera were collected between July and December 2001, inclusive of the peak transmission time of year. Mosquito saliva collection (Rex-D strain, Puerto FK866 Rico) were reared at 28C, 80% relative moisture, and a 12 h light:12 h dark photocycle in the Arthropod-borne and Infectious Diseases Laboratory (AIDL), Colorado State University or college. After eclosion, 250 female mosquitoes were placed in containers and sugars and water were supplied salivary glands were dissected into lysis buffer comprising 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5mM EDTA, 1% sodium deoxycholate, 1% Triton-X-100, 0.1% SDS and 1 mM PMSF. The salivary glands were mechanically disrupted and proteins were concentrated by TCA precipitation and.