Antibodies that block virus attachment to Vero cells are a major component of the human neutralizing antibody response against dengue virus type 2.

Epidemiological data strongly implicate a role for the host humoral immune response in both protection against and exacerbation of dengue virus-caused disease. In an effort to characterize elements of the normal human immune response against dengue virus we have addressed the issue of antibody-mediated neutralization of dengue virus. We show here the ability of both mouse monoclonal antibody 3H5 and human anti-dengue neutralizing sera to block binding of dengue-2 virus to monkey kidney (Vero) cells. Since Vero cells possess virus receptors but not Fc receptors we conclude that the major effect of host neutralizing antibodies is to block virus attachment to Vero cell dengue virus receptors. Analysis of 61 patient antisera yielded good correlation (Pearson's coefficient = 0.90; P < 0.001) between neutralizing activity and ability to block virus-cell attachment suggesting that antibody-mediated neutralization of dengue virus occurs primarily extracellularly and less by a postattachment mechanism as has been described for certain other viruses.

Comparison of the protective efficacy on mekongi schistosomiasis in mice induced by antigens derived from cercariae, schistosomulae and adult worms.

Protective efficacy of the extracts of cercariae, schistosomulae and adult worms of S. mekongi was studied in mice receiving immunizations with these extracts emulsified with Freund's complete adjuvant initially and incomplete adjuvant subsequently, and compared with mice receiving physiological saline with or without adjuvants as controls. After challenge with cercariae, the animals were sacrificed and the larvae or adult worms harvested by lung recovery and perfusion techniques on day 5 and weeks 6-8, respectively. Worm reduction rates were significantly higher in mice receiving extracts of schistosomula (59%) and adult worms (51%) than in those receiving the cercarial extracts (31%). Similar findings were obtained with the perfusion technique showing worm reduction rates of 57%, 53% and 30% in mice receiving extracts of schistosomulae, adult worms and cercariae, respectively. ELISA antibody titers were correspondingly increased in mice receiving extracts of schistosomulae and adult worms, but not in those receiving cercariae. This apparent association may be inadequate to suggest that the increase in ELISA titer be used as an indicator for resistance in mekongi schistosomiasis.

Applications of polymerase chain reaction for identification of dengue viruses isolated from patient sera.

A simple and sensitive procedure of reverse transcriptase polymerase chain reaction (RT-PCR) was developed previously such that all 4 serotypes of dengue viruses could be detected and their serotypes identified simultaneously in a single-step procedure. In this study we compared the RT-PCR with a conventional immunoperoxidase (PAP) staining method for the identification of dengue viruses currently isolated from patient sera. Sixty-six sera taken from dengue hemorrhagic fever (DHF) patients were subjected to virus isolation by inoculating onto C6/36 cell cultures. Screening for the presence of dengue viruses in culture fluids was done after 7 days of incubation by PAP staining using hyperimmune rabbit anti-dengue virus antibody as the primary reagent. Dengue viruses in positive cultures were further identified for their serotypes by PAP using type-specific monoclonal antibodies (MAb) and by RT-PCR. Thirty-two out of the 66 serum specimens tested (48.5%) were positive for dengue viruses. Of these, 5 were type 1 (DEN-1), 25 were type 2 (DEN-2) and 2 contained both DEN-1 and DEN-2. All cultures that were positive by PAP method were also positive by RT-PCR and vice versa. Thus, the results obtained by RT-PCR were in good agreement with those by PAP. It is important to point out that while all 5 DEN-1 isolates reacted readily with the MAb 1F1, only 2 of them could be identified by the MAb 15F3. Our data suggest that antigenic variation among DEN-1 isolates occur frequently and this should be taken into consideration in the selection of appropriate type-specific MAb for serotyping of dengue viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Hantaanvirus among urban rats from a slum area in Bangkok.

A total of 106 rodents sera from slum Wat Phai Ton and slum Klong Toey were examined by immunofluorescent antibody assay during May to August 1990. The positive sera were further tested by plaque reduction neutralization test with the prototype hantaanvirus and the rat-associated hantaan like virus. Isolation attempts were also performed from their tissues. Antibody-positive rats were found in both slum areas, 32.7% in slum Wat Phai Ton and 5.6% in slum Klong Toey. Rattus norvegicus was the major species found positive. Positive plaque reduction neutralization results indicated that the infecting virus was antigenically similar to the strain of rat-associated hantaanvirus. The presence of low titer antibodies (IFA titer 32 to 128) may be an obstacle to isolation of associated virus using tissue culture.

Investigation on immunity induced by Schistosoma spindale against S. mekongi in experimental mice.

An investigation on immunity induced by Schistosoma spindale cercariae (cattle and swamp buffalo schistosome) against S. mekongi (human schistosome) was conducted in Swiss albino mice. The studies comprised the development patterns of homologous immunity of S. spindale and heterologous immunity induced by S. spindale against S. mekongi. The development pattern of homologous immunity was studied in mice with an immunization of 100 S. spindale cercariae. At one week intervals, between 2 to 16 weeks after immunization, they were each challenged with 500 S. spindale cercariae. Significant homologous immunity, as judged by lung recovery assay five days after challenge, occurred from week 5 to week 16 with week 8 giving the highest homologous immunity (68.1% of schistosomular reduction). Using the above information mice, with an eight-week immunization period of 100 S. spindale cercariae, were tested for resistance to heterologous S. mekongi infection. The criteria used to evaluate their immune status was schistosomular lung recovery, daily egg output, worm recovery and tissue egg count. The results showed that mice immunized with S. spindale cercariae could develop heterologous immunity against S. mekongi infection. Manifestation of immunity was demonstrated by significant reduction in mean schistosomular recovery (31.4%), in mean daily egg output per female worm (16.7%), in mean worm recovery (64.2%) and in mean egg deposition in the liver tissue and intestines per female worm (37.05%).

Lung-migration patterns of Schistosoma mekongi and S. spindale in mouse.

Two groups of laboratory-bred Swiss albino mice were used to study the lung-migration patterns of Schistosoma mekongi and S. spindale. The first group was individually infected with 100 S. mekongi cercariae by hair-looping application on shaved abdomen. The latter group was individually exposed to 500 S. spindale cercariae by tail immersion. Each group of these infected mice was then divided into subgroups. The number of schistosomulae was determined using a lung recovery assay starting from the second day after infection and continuing for 15 consecutive days. The results revealed a sharp peak of both S. mekongi and S. spindale on the fifth day post cercarial infection.

Endotoxin and dengue haemorrhagic fever.

Limulus amoebocyte lysate test (LALT) was used to detect endotoxin-like substances in the plasma of 57 patients with dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS), four patients with dengue fever and 20 control patients with other diseases. The LALT positivity rates in DHF/DSS and dengue fever patients were 43.9 and 25 per cent respectively, whereas all control patients were negative (p less than 0.0025). LALT positivity was highest on 5th and 6th days of admission with positive rates of 46 and 50 per cent respectively whereas the positive rates in those admitted on fourth and seventh days of admission were 29 and 33 per cent respectively. A follow-up in LALT positive patients showed a decline in the positive rates after admission. LALT positivity was observed in 48.8 per cent of DHF/DSS patients with shock and in 26.6 per cent of patients without shock.

Factors contributing to the development of cerebral malaria. II. Endotoxin.

Limulus amoebocyte lysate test (LALT) was used to detect endotoxin-like substances in the plasma of 15 patients with cerebral malaria, 28 patients with uncomplicated falciparum malaria and 30 healthy controls. On admission, 67% of cerebral malaria patients were positive, whereas only 21.4% of uncomplicated malaria patients and none of controls were positive. Among uncomplicated malaria cases, four of eight patients with parasitaemia over 90,000/mm3 were LALT positive whereas only two of 20 patients with parasitaemia of less than 90,000/mm3 were positive. A follow-up study in cerebral malaria patients showed some variation in LALT positivity rate from day to day (85.7% on day 1, 53.3% on day 3 and all negative on discharge from hospital). LALT positivity bore no relationship to gram negative bacteraemia. Leucocytosis and elevated serum enzymes were more frequently found in LALT-positive patients. Our results suggest that endotoxin (LALT positivity) of the plasma of malaria patients is derived from either the parasites themselves or from the gut. It relates to parasitaemia, leucocytosis and elevated serum enzymes, but not to the clinical syndrome of cerebral malaria.

Genetic control of immune responses to Schistosoma japonicum.

The mouse IgE antibody response to S. japonicum antigen (Sj) was found to be under control of a gene(s) linked to the major histocompatibility complex. In some strains but not all among low responders, however, T cell responsiveness to Sj could be demonstrated by the induction of carrier effect as well as by proliferation response. Resistance to reinfection with a large dose of S. japonicum cercariae was demonstrated in most strains examined, except C57BL/6, irrespective of the immune responsiveness. Further studies will be needed to elucidate whether genetically regulated immune responses may affect susceptibility to or pathogenesis of schistosomiasis japonica in the mouse.

Comparison of recovery of schistosomula of Schistosoma japonicum from lungs of mice and rats.

Various strains of mice and rats were exposed to small doses of Schistosoma japonicum cercariae. At intervals, they were challenged with 200 to 230 cercariae per mouse and 500 to 520 cercariae per rat. Age-matched control animals received only the challenge infection from the same pool of cercariae. Recoveries of schistosomula from experimental and control groups were different on day 3 through day 5, with peak recovery on day 3 or 4 in four strains of mice examined (DBA/2, C57BL/6, C3H/He, and SWM) as well as in Fischer rats, indicating rapid migration of S. japonicum to the lung compared with that of S. mansoni. Significant reductions of recovery of schistosomula from lungs were demonstrated in DBA/2, C3H/He, SWM, and B10.S mice at week 3, but not in BALB/c, CBA, and C57BL/6 mice. Significant reductions were also demonstrated in Donryu, Wistar, Sprague-Dawley and Fischer rats at week 6. The degree of reduction depended on cercarial doses used for the initial exposure. Studies in DBA/2 mice revealed that the degree of reduction intensified biphasically, being greatest at week 5 and at week 12. The lowest recovery was demonstrated at week 6 in Fischer rats, when recovery of schistosomula tentatively increased in DBA/2 mice. A good correlation was found between recovery of schistosomula from lungs and recovery of adults by portal perfusion when DBA/2 and C57BL/6 mice, and Fischer rats were used for comparison. Thus, the present results provide basic information on the lung recovery of schistosomula of S. japonicum in suitable and unsuitable hosts.

Mouse IgE response to an allergen from Entamoeba histolytica.

Attempts were made to demonstrate the presence of allergenic components in an extract of axenically cultured Entamoeba histolytica (HK 9). Various strains of mice were immunized i.p. with a low dose of the extract mixed with Al(OH)3 and boosted with the same mixture 4 wk later. A high titer of IgE antibody response was demonstrated in BALB/c, DBA/2 (H-2d), CBA (H-2k), and SWM strains but not in C3H/He (H-2k) strain. The extract was fractionated by gel filtration with Sephadex G-200 and then by DEAE-cellulose chromatography with a gradient buffer with progressive increase in molarity. Fractions having the strongest activity to stimulate the IgE antibody response in the mouse were subjected to SDS polyacrylamide gel electrophoresis. From the results of gel filtration and polyacrylamide gel electrophoresis, the molecular weight on the partially purified allergen was estimated to be 25,000 to 27,000 daltons.

Tachypleus lysate test for endotoxin in patients with Gram negative bacterial infections.

Amoebocyte lysate from the horseshoe crabs (Tachypleus gigas) which abounds in the Gulf of Thailand was used to detect endotoxin in patients with Gram-negative bacteremia, in patients with Gram-positive bacterial infections as well as in the control. The Tachypleus lysate test (TLT) was positive in 94.4% of 36 patients with Gram-negative bacteremia before initiation of antibiotic therapy. Only 4% of 50 healthy individuals were positive and all 7 patients with Gram-positive bacterial infections were negative. The threshold sensitivity of TLT was 0.625 micrograms endotoxin per ml of the plasma. In comparison with the commercial Limulus lysate test (LLT), the TLT was slightly more sensitive in exhibiting higher grade of reaction, eventhough the threshold sensitivity was the same.