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.

Acyclovir susceptibility of herpes simplex virus isolates at King Chulalongkorn Memorial Hospital, Bangkok.

OBJECTIVE: To determine the ACV susceptibility in Thai HSV clinical isolates. MATERIAL AND METHOD: One hundred thirty HSV isolates from the Virology Laboratory Unit, King Chulalongkorn Memorial Hospital, Bangkok Thailand had typing done by immunofluoresent assay using monoclonal antibody specific to either HSV-1 or HSV-2. Their sensitivity to ACV (IC50) was determined by plaque reduction assay. RESULTS: The IC50 of 77 HSV-1 isolates ranged from 0.07-0.97 microg/ml and that of 53 HSV-2 isolates was 0.13-1.66 microg/ml. The standard HSV-1 (KOS) and HSV-2 (Baylor 186) were included in each run. The mean + standard deviation (SD) of ACV IC50 among HSV-1 and HSV-2 isolates were 0.38 +/- 0.23 and 0.50 +/- 0.32 microg/ml while that of standard HSV-1 and HSV-2 were 0.45 +/- 0.13 and 0.57 +/- 0.04 microg/ml. Statistically significant difference between IC50 of HSV-1 and HSV-2 isolates was indicated (p = 0.02). CONCLUSION: No ACV(r) HSV has been detected and ACV susceptibility of HSV-2 has more resistance than that of HSV-1.

Prevalence of human cytomegalovirus (HCMV) gB genotypes in Thai patients.

Human cytomegalovirus (HCMV) infection can cause asymptomatic to symptomic diseases leading to morbidity and mortality especially in immunocompromized patients. One factor of the difference in clinical outcome is the distinction of HCMV strain. As HCMV glycoprotein (g)B plays an important role in viral entry and neutralizing antibody induction, HCMV gB genotypes were determined in 161 clinical specimens containing HCMV-DNA obtained from patients at King Chulalongkorn Memorial Hospital, Bangkok, Thailand during the year 2000 and 2004. Of the 113 (70%) samples that were able to be genotyped, mixed gB genotype was demonstrated in 35%, followed by gB1 (33%), gB3 (15%), gB2 (11%), and untyped (7%); gB4 was not detected. The distribution of HCMV gB genotypes between genders was not significantly different. Mixed gB genotype (35%) was found in HIV- infected patients.

LipL32, an outer membrane protein of Leptospira, as an antigen in a dipstick assay for diagnosis of leptospirosis.

Microscopic agglutination test (MAT), as well as other serological assays that aimed at detecting antibodies to Leptospira, supplements the leptospirosis diagnosis based on the clinical features. Nevertheless, false positive results have been occasionally reported when the crude antigen was used in those antibody-based tests due either to the presence of antibodies stimulated by other antigenically related pathogens in the patient's serum, or the antibodies in the serum may be stimulated by a previously unrecognized Leptospira infection, especially in the disease endemic areas. Thus, the more refined antigen should improve the serodiagnostic accuracy. Among Leptospira spp., LipL32, which is a pathogenic Leptospira outer membrane protein (OMP), expressed by the bacteria grown both in vitro and in vivo. In this study, recombinant LipL32 protein was tested by a dipstick method for its potential in serodiagnosis of leptospirosis. Preliminary results suggest that the recombinant LipL32 is a good diagnostic detection reagent for specific Leptospira IgG. Diagnostic sensitivity and specificity of the Lip32 dipstick assay, when compared to those of MAT, were 100% and 98.33%, respectively.

The prevalence of varicella-zoster virus infection in normal healthy individuals aged above 6 months.

The prevalence of varicella-zoster virus (VZV) infection was studied by determining the presence of IgG antibody to VZV (anti-VZV IgG) using ELISA method. Three hundred and fifty sera collected from Thai healthy individuals aged above 6 months (mean age +/- standard deviation = 14.9 +/- 11.4) were tested, the prevalence of VZV infection was 64.6% (225/350). All samples were randomly sampling from healthy children and blood donors who visited the hospital and clssified into 7 groups, 50 samples each, according to their age, i.e., group 1; 6 months-3 years, group 2; 4-6 years, group 3; 7-9 years, group 4; 10-14 years, group 5; 15-19 years, group 6; 20-24 years and group 7; above 25 years. The prevalence of VZV infection were 12%, 42%, 64%, 70%, 78%, 84% and 100% respectively. The mean amount of anti-VZV IgG among groups of positive VZV infection (225 samples) was 86.8 +/- 29.7 unit/ml. The mean amount of anti-VZV IgG was highest in 6 months-3 years age group (113.6 +/- 39.2 unit/ml). Significant difference of the mean amount of anti- VZV IgG was found between group 1, 3, 6 and other groups (p-value < 0.05). There was a correlation between history of varicella and the presence of anti-VZV IgG in the serum. 95.3% of individuals with positive history has already had the antibody. The important associated factors that might involve VZV infection were age, number of members in family and place of exposure to VZV infection. Other factors, such as sex and income did not show any association to VZV infection.

Increase of genital HSV-1 and mixed HSV-1 and HSV-2 infection in Bangkok, Thailand.

From January 1998 to December 2004, 207 out of 1125 samples were HSV isolation positive and typed. Two groups of patients, Thai and foreigner, as well as site of infection, non-genital and genital area, were identified. The prevalence of non-genital HSV-1 infection from 27 samples of Thai patients was 81.84%. Out of 180 genital samples, 39.02% HSV-1 and 43.09% HSV-2 from 123 Thai patients and 36.84% HSV-1 and 49.12% HSV-2 of 57 foreigner patients were determined. Moreover mixed infection of HSV-1 and HSV-2 was found in both Thai and foreigner groups, 17.89% and 14.04%, respectively. The prevalence of genital HSV-1 infection in Thai patients chronologically increases from 1.6% to 56.91% from of 1985 to 2004. Increase of HSV-1 genital infection and mixed HSV-1 and HSV-2 infection in Thai people might probably be due to changing of sexual behavior in the AIDS era.

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.

Intratypic variations among Thai herpes simplex virus (HSV) isolates determined by restriction fragment length polymorphism (RFLP) analysis.

Whole genomic polymorphisms for 20 HSV-1 and 20 HSV-2 isolates from Thai patients were analyzed by means of Restriction Fragment Length Polymorphism (RFLP) analysis using 4 restriction endonucleases: BamHI, Kpnl, HindIII, and EcoRI. Variations in cleavage sites among the HSV-1 and HSV-2 isolates were compared to the cleavage patterns of standard HSV-1 strain KOS and HSV-2 strain Baylor 186. Although 70% of HSV-1 isolates with BamHI digestion, 50% with Kpnl, 75% with HindIII and 70% with EcoRI digestion were found to be similar to the standard HSV-1 (KOS) pattern, new BamHI restriction sites were detected in some HSV-1 isolates. For HSV-2 isolates, 85% had the same pattern as the standard HSV-2 (Baylor 186) after digestion with BamHI, HindIII, and EcoRI. No difference was observed with Kpnl digestion. When the patterns from the 4 enzymes were combined, HSV-1 isolates showed more divergence than the HSV-2 isolates. HSV-1 isolates found in both non-genital and genital lesions had more variety than the HSV-2 isolates. This suggests that intratypic variations in HSV-2 are fewer than in HSV-1.

Induction of apoptosis by herpes simplex virus in Jurkat cells is partly through caspase-3, -8 and -9 activation.

Herpes simplex virus (HSV), a large DNA containing virus, is endemic in all human populations investigated. After infection of mucocutaneuos surfaces, HSV establishes a latent infection in nerve cells. Various immune evasion mechanisms have been shown to be utilized by HSV including apoptosis induction in Tlymphocytes. However, the mechanisms of T cell infection and apoptosis by HSV are still unknown. The present study investigated the molecular mechanisms of apoptosis induction in T cells by HSV The Jurkat T cell line was used as a representative for T cells. Apoptosis detection by Annexin Vassay demonstrated that both HSV-1 and HSV-2 induced apoptosis in Jurkat cells and caspase-3, -8, and -9 inhibitors blocked apoptosis induced by HSV-1 and HSV-2. The data suggested that HSV-1 and HSV-2 induced apoptosis in T lymphocytes by caspase-dependent pathway. However, apoptosis may occur through other mechanism(s) since caspase inhibitors used in the present study could not completely inhibit apoptosis induced by HSV infection. In addition, the data demonstrated that the number of apoptotic cells induced by HSV-2 was significantly higher than byHSV-1 at 12 hour post-infection (h p.i.) (p = 0.003). Further studies in peripheral blood T cells and the proteins of viruses involved in apoptosis induction should be further performed in order to elucidate the molecular mechanisms of apoptosis induced by these viruses.

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.

Seroprevalence of anti-RSV IgG in Thai children aged 6 months to 5 years.

Respiratory syncytial virus (RSV) is the single most important cause of lower respiratory tract infection during infancy and early childhood worldwide including Thailand. The magnitude as well as the intensity of both infection and host response to the RSV infection determine the severity of disease. To determine the presence of anti-RSV IgG in children of different age groups, 124 serum samples were randomly collected from healthy children aged 6 months to 5 years. All of them were assayed for anti-RSV IgG using a commercial ELISA kit. The mean prevalence rate was found to be 68.91%. The prevalence increased with age: from 6 to 11 months, 11.76%; from 12 to 17 months, 41.67%; from 18 to 23 months, 60.87%; from 24 to 29 months, 88.24%; from 30 to 35 months, 78.57%; from 36 to 41 months, 94.44% and from 42 to 60 months, 100%. The amount of anti-RSV IgG among the groups was significantly different (p = 0.006). No sexual preponderance was found. RSV infection commonly occurred in children aged 12 to 29 months. All children older than 5 years had experienced a RSV infection.

Prevalence of parvovirus B19 infection in Thai young adults.

The prevalence of antibody to human parvovirus B19 in 128 Thai healthy young adults was measured. Antibodies of the immunoglobulin G (IgG) class were investigated in serum samples of 51 males and 77 females aged 18-24 years (mean 19.83; SD 1.07) by a commercial enzyme-linked immunosorbent assay (ELISA) using high specific recombinant parvovirus B 19 antigen. Only 14 out of 128 (10.94%) sera were found positive, including 6 males and 8 females. No sex preponderance was observed. The amount of antibody calculated as antibody index was not statistically significant difference between genders.

Rectal prolapse associated with cytomegalovirus pseudomembranous colitis in a child infected by human immunodeficiency virus.

We report a newly recognized presentation of cytomegalovirus (CMV) enterocolitis in a 4-year-old girl with newly diagnosed HIV disease who presented with rectal prolapse. Gross findings showed multiple whitish punctate lesions. An endoscopic examination revealed multiple shallow ulcers and pseudomembranes along the colon. A biopsy from colonic tissues demonstrated CMV-like inclusion bodies. A direct immunofluorescence assay using specific CMV monoclonal antibody was positive for CMV-infected cells in specimens from the rectal smear.

Seroprevalence of cytomegalovirus infection in children born to HIV-1 infected women.

Cytomegalovirus (CMV) is a frequent opportunistic infectious agent in children infected with human immunodeficiency virus type 1 (HIV-1). It has been implicated as a factor in the progression of HIV-1 disease. The aim of the present study was to evaluate the prevalence of CMV infection in Thai children born to HIV-1 infected women. The prevalence of CMV infection was 13, 89 and 84% in HIV-infected children and 9, 61 and 75% in HIV uninfected at age ranges of 0-12, 13-36 and 37-79 months, respectively. The prevalence of CMV infection was significantly different between HIV infected children (89%) and HIV uninfected (61%) at the age of 13-36 months (p < 0.05). The presence of CMV IgM in some children of age < 1 year suggested that CMV infection could occur early in life. Early co-infection may be important as they remain a risk factor for reactivation of latent CMV infection throughout the course of the HIV diseases. Clinical monitoring and appropriate work up may be of benefit in the early diagnosis and treatment of CMV disease.

Detection and differentiation of human herpesviruses 1-5 by consensus primer PCR and RFLP.

Eight human viruses of the Herpesviridae family represent a significant public health problem world-wide. Detection and typing of five of the human herpesviruses (HSV-1, HSV-2, VZV, EBV, and CMV) was performed by applying a consensus primer polymerase chain reaction (PCR). The amplified PCR products from the five human herpesviruses were typed based on their restriction enzyme digestion polymorphism with Hinf I and Alu I. Fifteen clinically suspected specimens from herpesvirus-infected patients were also evaluated. A fragment of the DNA polymerase gene from each of the five human herpesviruses was successfully amplified by the set of consensus primers. Their amplicons obtained by PCR from the template DNAs were subjected to restriction endonuclease digestion and human herpesviruses 1-5 could be clearly differentiated and typed. This method can be used to detect and differentiate between the five human herpesviruses in clinical specimens. This study demonstrates the value of testing for five human herpesviruses by consensus PCR and restricted fragment length polymorphism (RFLP). These procedures are simple and straightforward techniques for the investigation of clinical specimens.

Replication of herpes simplex virus in T lymphocytes.

HSV is known to cause infection at various parts in the human body such as skin, mouth, eyes, genital area, and brain. In this study, the authors showed the possibility of HSV replication in Jurkat, a human leukemic T lymphocytes. Although the yield production was very low when compared to the other 2 epithelial cells, Vero and HEp-2 cells, the yield production could enhance after PHA activation. Delayed viral protein expression was observed in Jurkat cells. This might be the reason for low production. However, the exactly mechanism is unknown. Replication of viruses have been examined in a number of cell systems and the duration of successive steps in the replication cycle depends upon the types of cells, the virus strain, and the multiplicity of infection.

Survey of human papillomavirus infection in cervical intraepithelial neoplasia in Thai women.

HPV infection is known to be associated with cervical cancer development. Precancerous lesions named cervical intraepithelial neoplasia (CIN) are divided into 3 grades, i.e., CIN-1, CIN-2, and CIN-3. Here, HPV infection determined by PCR and dot hybridization was observed in these 3 different grades of formalin-fixed paraffin embedded tissues. The HPV infection was demonstrated in 33.3 per cent of CIN-1, 36.8 per cent of CIN-2 and 75 per cent of CIN-3. Using type specific probes for HPV-6, 11, 16, 18 and 33, HPV-16 was the most prevalent type (44.44%) followed by HPV-18 (16.05%) in CIN-3. Only one HPV-18 was identified in CIN-1 while CIN-2 contained one HPV-6 and one HPV-18. Mixed infection was found in CIN-3 (12.35%). All of them had HPV-16. The cervicitis cases with normal histopathology were included as control. Only 2.7 per cent of HPV infection was shown. The relative risk of HPV infection was high in CIN-3 (OR = 107.25, 95% CI = 50.29-228.73). Our data confirm the association between high-risk HPV types and development of CIN.