Cytotoxic function of gamma delta (ϒ/δ) T cells against pamidronate- treated cervical cancer cells.

The cytotoxic function of polyclonal expanded ϒ/δ T cells against pamidronate-treated cervical cancer cells in vitro and in vivo were determined. The ϒ/δ T cells were isolated and purified from PBMCs by using miniMACS and were later treated with 10 μM pamidronate. The expansion of ϒ/δ T cells was 15 times more than the non-stimulated cells. Among the expanded ϒ/δ T cells, 47% were Vϒ9/Vδ2 T cells with a purity of 87%. Analyzing the cytotoxic function of ϒ/δ T cells against 3 cervical cancer cells in vitro by LDH cytotoxicity test revealed that the killing efficacy increased if the cervical cancer cells (HeLa, SiHa and CaSki) were pretreated with pamidronate. The presence of CD107 on ϒ/δ T cells indicated the degranulation of perforin and granzyme pathway is one of the mechanisms used by the ϒ/δ T cells to kill cancer cells. The killing ability of ϒ/δ T cells against cancer cells in vivo was preliminary assessed by using mouse baring HeLa cells. The results demonstrated that ϒ/δ T cells induce apoptosis in tumor cells. Our study supports the usefulness of ϒ/δ T cells in future development of immunotherapy for cervical cancer.

Establishment of real-time polymerase chain reaction-based assay for quantitation of Epstein-Barr virus DNA in healthy donors and in patients with EBV associated lymphoma.

Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma and lymphoma in immunocompromised patients. Quantitative monitoring of EBV DNA in these patients has recently become essential for management of the disease. In the present study the authors developed a rapid and reliable real-time PCR to quantify the EBV DNA in peripheral blood mononuclear cell (PBMC) using hybridization probe technique. The real-time primers and probes in this real-time PCR system were designed based on EBNA-1 sequence. The newly-established real-time PCR demonstrated its high sensitivity (as few as 10 copies of EBV could be detected) and specificity. The intra- and inter-assay variations of the assay were shown to be within a 0.5-log10-difference range. A total of 2 EBV-seronegative, 14 EBV-seropositive healthy donors and 4 patients with PCNSL were enrolled into the study. Our results revealed the median of EBV-DNA in lymphoma patients (7886 copies/10(6) PBMC or 15,150 copies /microg DNA) was higher than that of healthy donors (<10 copies/l0(6) PBMC or <10 copies/microg DNA) with statistic significance (P < 0.01). Assessment of this assay in larger number of donors and patients will provide clinical cut-off values which are essential for monitoring and diagnosis of EBV-associated diseases.

Establishment of cytotoxic T lymphocytes specific for autologous Epstein-Barr virus in HIV-infected patients: the feasibility study of EBV-specific immunotherapy for patients with EBV-associated lymphoma.

Cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV) have previously been successfully used in immunotherapy of Posttransplant lymphoproliferative disease (PTLD) and Hodgkin's disease. A similar strategy has never been employed in HIV/AIDS patients who also have high risk of developing EBV-associated lymphoma. A total of 5 HIV-infected patients were enrolled to evaluate their EBV-specific T cell responses by Interferon-gamma (IFNgamma) ELISpot assays. Most patients had detectable T cell responses, mainly directed at Epstein-Barr nuclear antigen (EBNA-3). The authors wanted to see whether it was possible to augment magnitude and spectrum of the EBV responses by stimulating patient PBMC with cells presenting autologous EBV antigens. The authors successfully established spontaneously EBV-transformed lymphoblastoid cell lines (EBVh-BCL) and used them for generation of EBV-specific CTL (EBV-CTL). The EBVh-CTL lines established in the present study were not only highly cytotoxic against the autologous virus but also able to secrete IFNgamma detected by ELISpot. The authors are now in the process of generating these lines in a large number and in a clinical grade for adoptive immunotherapy.

Prevalence of dengue virus in Aedes mosquitoes during dry season by semi-nested reverse transcriptase-polymerase chain reaction (semi-nested RT-PCR).

Dengue hemorrhagic fever remains a major health concern in Thailand. Much effort has focused on the prevention and control of the disease. Detection of dengue virus infection rate in mosquitoes would evaluate dengue control programs and predict the epidemics of dengue hemorrhagic fever. To determine dengue virus infection rate in mosquitoes by Semi-nested RT-PCR. A total of 400 mosquitoes were collected from Rom Kao Community representing a crowded community and another 9 non-crowded communities in Bangkok. Mosquitoes were then divided into 40 pools, each contained 10 mosquitoes. A total of 391 Aedes aegypti and 9 Aedes albopictus were screened for dengue virus. The mosquito infection rate in the Rom Klao community was 5% of the mosquito pool equal to that found in non-crowded communities. Both groups were found to have dengue virus serotype 3. The present study suggests a circulation of dengue virus serotype 3 in both crowded and non-crowed communities, the infection rates of which are indifferent during the dry season.