An effective economical intradermal regimen of human diploid cell rabies vaccination for post-exposure treatment.

A closely-spaced multisite intradermal regimen of human diploid cell rabies vaccine (HDCV) was evaluated in 39 patients after low-risk exposure to rabies, in comparison to full-dose intramuscular HDCV and sheep brain-derived rabies (Semple) vaccine. The regimen consisted of four intradermal injections, 0.1 ml each of HDCV on days 0, 3 and 7, followed by two booster doses of only 0.1 ml each on days 28 and 91 administered intradermally. Although the total amount of HDCV used in this intradermal regimen was 1.4 ml or one-quarter of the conventional intramuscular regimen, a higher proportion of the recipients of this economical intradermal regimen, as compared to the full-dose intramuscular regimen, developed neutralizing antibodies above the hypothetical protective level of 0.5 iu/ml 7 days after starting immunization. Besides the earlier antibody response, the peak antibody level of the intradermal regimen was also satisfactorily high and not significantly different from that after the intramuscular regimen. Simultaneous administration of inosiplex, an antiviral and immunopotentiating agent, during the first 10 days of intradermal immunization resulted in an even higher antibody response for as long as 91 days. In contrast, but not unexpectedly, Semple vaccine evoked lower, more sluggish and inconsistent antibody responses. The side-effects of intradermal HDCV were mild, mainly local and self-remitting. We therefore recommend our intensive intradermal regimen of HDCV vaccination for safe, effective and economical use in post-exposure rabies immunization.     

Reduced levels of transforming growth factor-beta1, interleukin-12 and increased migration inhibitory factor are associated with severe malaria

In the present study, we investigated plasma levels of interleukin (IL)-12 and transforming growth factor (TGF-beta1) in malaria patients as these two cytokines regulate the balance between pro- and anti-inflammatory cytokines. We compared plasma IL-12 and TGF-beta1 levels in groups of malaria patients categorized as uncomplicated, severe, cerebral and placental malaria. Both TGF-beta1 and IL-12 levels were significantly reduced in peripheral plasma of adults with severe and cerebral malaria as well as in plasma of Tanzanian children with cerebral malaria (P<0.05). Similar results were observed with both placental and peripheral plasma of pregnant women who were infected with Plasmodium falciparum. IL-18, a cytokine known to be critical for the induction of IFN-gamma along with IL-1, was produced more in uncomplicated adult patients than in aparasitimic healthy controls (P<0.05). However, IL-18 response rate declined as the symptoms of the disease became more severe suggesting that the IL-18 response may be impaired with increased malaria severity. Together, the results of the three cytokines support the notion that imbalance between pro- and anti-inflammatory cytokines may contribute to the development of severe malaria infection. With malaria infection during pregnancy, we demonstrated that macrophage migration inhibitory factor (MIF) levels in infected placental plasma were significantly higher than those in the paired peripheral plasma (P<0.05). MIF, therefore, may play an important role in the local immune response to placental P. falciparum infection.     

Involvement of ATP synthase β subunit in chikungunya virus entry into insect cells

Chikungunya virus (CHIKV), the virus responsible for the disease chikungunya fever in humans, is transmitted by Aedes mosquitoes. While significant progress has been made in understanding the process by which CHIKV enters into mammalian cells, far less progress has been made in understanding the CHIKV entry process in insect cells. This study sought to identify mosquito-cell-expressed CHIKV-binding proteins through a combination of virus overlay protein binding assays (VOPBA) and mass spectroscopy. A 50-kDa CHIKV-binding protein was identified as the ATP synthase β subunit (ATPSβ). Co-immunoprecipitation studies confirmed the interaction, and colocalization analysis showed cell-surface and intracellular co-localization between CHIKV and ATPSβ. Both antibody inhibition and siRNA-mediated downregulation experiments targeted to ATPSβ showed a significant reduction in viral entry and virus production. These results suggest that ATPSβ is a CHIKV-binding protein capable of mediating the entry of CHIKV into insect cells.     

The molecular epidemiology of dengue virus serotype 4 in Bangkok, Thailand

Dengue represents a major public health problem in Thailand, with all four viral serotypes co-circulating. Dengue virus serotype 4 (DENV-4) is the least frequently sampled serotype, although one that is often associated with hemorrhagic fever during secondary infection. To determine the evolutionary forces shaping the genetic diversity of DENV-4, and particularly whether its changing prevalence could be attributed to instances of adaptive evolution in the viral genome, we undertook a large-scale molecular epidemiological analysis of DENV-4 in Bangkok, Thailand, using both E gene and complete coding region sequences. This analysis revealed extensive genetic diversity within a single locality at a single time, including the discovery of a new and divergent genotype of DENV-4, as well as a pattern of continual lineage turnover. We also recorded the highest average rate of evolutionary change for this serotype, at 1.072 x 10(-3) nucleotide substitutions per site, per year. However, despite this abundant genetic variation, there was no evidence for adaptive evolution in any gene, codon, or lineage of DENV-4, with the highest rate of nonsynonymous substitution observed in NS2A. Consequently, the rapid turnover of DENV-4 lineages through time is most likely the consequence of a high rate of deleterious mutation in the viral genome coupled to seasonal fluctuations in the size of the vector population.     

Partial construction of apoptotic pathway in PBMC obtained from active SLE patients and the significance of plasma TNF-α on this pathway

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder that affects various organs and systems. Increased apoptosis, together with defects in the uptake of apoptotic bodies, are thought to have a pathogenic role in SLE. By detection of chromatin condensation, 30% of apoptosis was detected in peripheral blood mononuclear cells (PBMC) from Thai patients with active SLE. Therefore, understanding of the molecular processes in PBMC apoptosis may allow us to gain insight into pathophysiology of SLE. Thus, genes involved in the apoptosis of PBMC from these patients were investigated ex vivo by cDNA array analysis. Seventeen apoptosis-related genes were stimulated in active SLE, more than twofold higher than in inactive SLE. These genes are classified into six groups, namely death receptors, death ligands, caspases, bcl-family, and neutral proteases and genes involved in endoplasmic reticulum stress-mediated apoptosis, such as caspase-4 and GADD153. Among those stimulated genes, tumor necrosis factor (TNF) and the TNF-receptor family were drastically up-regulated 60- and 19-fold higher than in healthy controls, respectively. Moreover, the degree of apoptosis correlated with the level of TNF-α in plasma, suggesting that the TNF family plays a role in the induction of apoptosis in SLE. To verify this hypothesis, PBMC from healthy individuals were treated with plasma from active SLE patients in the presence or absence of etanercept, a TNF inhibitor. In the presence of etanercept, active SLE plasma reduced the level of apoptosis to 26.43%. In conclusion, massive apoptotic death of PBMC occurred during the active stage of SLE. The molecular pathway of SLE-PBMC apoptosis was mediated at least via TNF/TNFR signaling pathway, which was confirmed by functional test of TNF-α in SLE patients’ plasma.     

Anti-HIV positivity in Thailand: the usefulness of another ELISA test kit and Western blot as confirmatory tests

Sera from 47 individuals repeatedly reactive in one screening ELISA system (designated as ELISA-A) for antibodies against human immunodeficiency virus (HIV) were evaluated by a second ELISA system (designated as ELISA-B) as well as by the Western blot technique. Both ELISA systems and the Western blot were positive in all of the 14 patients with clinical diagnoses of AIDS and AIDS-related persistent generalized lymphadenopathy (PGL). Of the 7 asymptomatic gays whose sera were repeatedly reactive in ELISA-A, 5 were also reactive in ELISA-B and these were the ones with positive Western blot tests. Eight and 17 ELISA-A reactive individuals were uncovered during a survey of 2,699 female prostitutes and 15,210 potential workers for Saudi Arabia respectively. All of these 25 individuals were ELISA-B and Western blot negative, an indication of false-positive reactivity with ELISA-A. Our studies indicate that the prevalence of HIV infection among the general Thai population is still low, and that the specificity of two ELISA test kits for anti-HIV may differ considerably. We concluded that evaluation of test kits should include studies in tropical countries where ecological conditions, climate and background endemic disease patterns are different than in the countries producing the diagnostic systems. Such studies are needed to identify the most sensitive and specific kits for worldwide application. We did discover that concordant positivity of two different ELISA test kits served as a reliable and inexpensive confirmatory test for anti-HIV.     

Antibody to peptides of human myelin basic protein in post-rabies vaccine endophalomyelitis sera

Development of neurologic complications after Semple rabies vaccine is closely linked to deveopment of antibody to myelin basic protein (MBP). The portions of MBP against which the antibodies are directed were analyzed by enzyme immunoassays in sera and cerebrospinal fluid from 27 patients with vaccine complications. Most of the antibody was directed to regions of MBP peptides 45–89 ,and 90–170. There was no apparent correlation between antibody specificity for MBP peptides 1–44, 45–89 and 90–170 and the type of post-vaccinal neurologic comlication. We conclude that the immunoglobulin repertoire in human B lymphocytes for responding to human MBP favors the portion of the MBP molecule containing residues 42–170.     

Humoral and cell-mediated immune responses to various economical regimens of purified Vero cell rabies vaccine

Purified Vero cell rabies vaccine (PVRV) is a new effective but inexpensive tissue culture rabies vaccine for human use. We investigated if the cost of immunization with PVRV could be further reduced by intradermal immunization. Fifty-eight subjects with low-risk exposure to rabies were randomized into 4 groups to receive full-dose (0.5 ml) intramuscular injection of PVRV on days 0, 3, 7, 14 and 28 or 4, 2 or 1 intradermal injections of PVRV (0.1 ml) on days 0, 3, and 7, followed by another intradermal injection on day 28. Neutralizing antibodies and specific cell-mediated response (CMIR) were sequentially followed up to day 36. The antibody levels in the intradermal groups increased with the number of injection sites and the levels achieved by the 2-site i.d. regimen were not significantly different from those obtained by the full-dose i.m. even though only 1/3 of the amount of PVRV was used. Specific CMIR occurred 1 week sooner in the 2 and 4-site i.d. regimens than the full-dose i.m. We therefore recommended that our 2-site i.d. regimen of PVRV should be further tested with a view to substituting it for the more expensive full-dose i.m. regimen in order to further reduce the cost of rabies prophylaxis particularly in the developing countries.