Flow cytometric detection of intracellular cytokines in peripheral blood of HIV-1 infected Thai children.

The objective of this study was to determine changes in Th1/Th2 cytokine production at the cellular level which occur during the progression of HIV-1 subtype E infection in Thai children born to HIV-1 subtype E infected mothers. Mitogen stimulated whole blood cultures from 12 uninfected and 27 HIV-1 subtype E infected Thai children were stained intracellularly with fluorescein labelled monoclonal antibodies against Interleukin (IL)-2 and IFN-gamma (Th1 cytokines) and IL-4 (Th2 cytokine). Additionally, co-staining of CD4+ and CD8+ T cells was performed. Results were analyzed by two and three color flow cytometry. The percentage of IFN-gamma expressing cells in CD4+ T cells was increased in HIV-1 subtype E infected Thai children with mild and moderate immunosuppression (Immunological categories 1 + 2, Centers for Diseases Control and Prevention (CDC) staging system, 1994). The percentages of IFN-gamma expression was continuously enhanced accompanied by remaining preserved in the proportion of IL-2 producing T cells in HIV-1 subtype E infected Thai children with severe immunosuppression (Immunological category 3, CDC staging system, 1994). The percentages of IFN-gamma expression was continuously augmented whereas the proportion of IL-2 producing T cells remained unchanged in HIV-1 subtype E infected Thai Children with severe immunosuppression (immunological category 3, CDC staging system, 1994). The percentage of Th2 cytokine producing cells within the CD4+ ad CD8+ T cells increased in HIV-1 subtype E infected individuals and showed a significant difference in HIV-1 subtype E infected Thai children with AIDS compared with uninfected infants. These results suggest that in vertically acquired HIV-1 infection with severe immunosuppression, the percentages of IL-2 producing CD4+ T cell was consistent but the percentages of IL-4 and IFN-gamma producing cell were increased. Similar results were found for CD8+ T cells in which IL-4 producing cells were increased in conjunction with a remaining in the number of IL-2 producing cells in HIV-1 subtype E infected Thai children. Thus, changes in the Th1 and Th2 cytokine pattern during HIV-1 infection may contribute to the prognosis of HIV disease in children.

TNF-alpha induces caspase 3 (CPP 32) dependent apoptosis in human cholangiocarcinoma cell line.

Cholangiocarcinoma (CCA), a malignant tumor derived from bile duct epithelium, occurs with a higher incidence in tropical countires especially in some areas of Southeast Asian countries such as Thailand. This tumor is relatively resistant to chemotherapy. In this study, molecular mechanism of killing of this tumor by TNF-alpha was investigated. Human cholangiocarcinoma cell line (HuCCA-1) was developed and used as a model for treatment. Activation of HuCCA-1 with TNF-alpha in the present of actinomycin D (1 microg/ml) caused death of the tumor cells. Western blotting analysis of the cells extracted demonstrated the cleavage of poly (ADP-ribose) polymerase (PARP) within 6-8 hours following TNF-alpha treatment indicating apoptotic death. The cleavage of PARP was inhibited when the cell line was pretreated with peptide inhibitor, Ac-DEVD-CHO, suggesting that apoptosis induced by TNF-alpha of this cell line involves activation of caspase II subfamily. The procaspase 3 (proCPP-32), one of the caspase group II subfamily was degraded after the HuCCA- I cell line was treated with TNF-alpha. Furthermore, Gelsolin, an 83 kDa protein which is identified as caspase 3 substrate, was cleaved to 43 kDa fragments after the cells were treated with TNF-alpha. These results indicate that apoptosis of human cholangiocarcinoma cell line as induced by TNF-alpha treatment is mediated through caspase 3.

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.

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.

Cell-mediated immune response following intracutaneous immunisation with human diploid cell rabies vaccine.

Specific cell-mediated immune response (CMIR) against rabies antigens was studied in recipients of two regimens of human diploid cell rabies vaccine (HDCV) using the antigen-stimulated lymphocyte transformation test (LTT) as a measure of CMIR. Reconstituted HDCV could be conveniently used as the in vitro stimulating antigen and the response was antigen-dependent. Conventional intramuscular immunization with full-dose HDCV resulted in positive LTT as early as 14 days after starting immunisation, and peaked on day 28. Intracutaneous immunisation with 0.1 ml of HDCV at four sites on days 0, 3 and 7 was a more efficient means of inducing specific lymphocyte response. Specific CMIR was evident as early as seven days and became maximal on day 14. In addition to the more rapid induction of specific CMIR, our intracutaneous regimen also resulted in a brisker and higher antibody response than the intramuscular regimen. The peak antibody level of the intracutaneous regimen was reached on day 14 whereas that of the intramuscular regimen was reached on day 28 and the geometric mean antibody titre on day 14 of the intracutaneous route was significantly higher than that of the intramuscular regimen. We therefore conclude that our closely spaced intracutaneous immunisation with HDCV was effective both in the induction of specific antibodies and the cell-mediated immune response.