PURPOSE: HPV (human papillomavirus) are known as the major causative agent for development of cervical cancer. High-risk HPVs, especially HPV-16 /18 DNA, are often found to be integrated into the human genome in high grade CINs as well as cervial cancer. Investigation of the relationship between the genomic states of HPV genes and their antibody response against the HPV-16 Ll/L2 virus-like particles (VLPs) and the in vitro translated E6 and E7 proteins may help to explain the mechanism of HPV-related cervical carcinogenesis and host immune responses. MATERIALS AND METHODS: Cervical cancer tissues obtained from 41 patients with cervical cancer were studied by PCR, Southern blot hybridization and the antibody response against HPV-16 Ll/L2 VLPs and HPV-16 E6, E7 proteins of serum were tested by ELISA and radioimmunoprecipitation assay (RIPA), respectively. RESULTS: Integrated forms of the HPV-16 DNA were found in 23 of the 38 patients (60.5%). The HPV-16 positive cervial cancer patients had a significantly higher prevalence (39.5%; 15/38) of antibodies to HPV-16 Ll/L2 VLPs than 8.7% (2/28) of the the control group (p<0.05). Antibodies to HPV-16 Ll/L2 VLPs were more detectable in 60% (9/15) of the cervical cancer patients with episomal forms of HPV-16 DNA than those who having only integrated HPV-16 (26.1%; 6/23) (p<0.05). Antibodies to E6 and E7 proteins were positive in 36.8% (14/38) and 50% (19/38) of the patients with HPV-16 positive cervical cancer. And those were siginificantly higher than the positivities for the control group (8.3% and 2.8%), (p<0.05). The difference between seroreactivities to E6 and E7 proteins in the patients with episomal forms of HPV-16 DNA (pure episomal and mixed forms) and those with integrated froms of HPV-16 DNA was not significant (P>0.05). CONCLUSION: Integrated forms of HPV-16 DNA were prevalent in most patients with cervical cancer. Antibodies to HPV-16 Ll/L2 VLPs, in vitro translated HPV-16 E6 and E7 proteins appeared in the significantly larger proportions of the HPV-associated cervical cancer patients than in the controls. Antibodies to HPV-16 Ll/L2 VLPs were more detectable in the cervical cancer patients with episomal form of HPV-16 DNA than those who having only integrated forms of HPV-16. Antibody responses to HPV-16 E6 and E7 proteins were not influenced by the different viral states. More numbers of studies would be necessary to determine the relationship between the genomic states of HPV and the immune responses to their proteins by the such genomic and serologic parameters.
Antibodies ; Antibody Formation ; Blotting, Southern ; Carcinogenesis ; DNA ; Enzyme-Linked Immunosorbent Assay ; Genome, Human ; Human papillomavirus 16 ; Humans ; Polymerase Chain Reaction ; Prevalence ; Radioimmunoprecipitation Assay ; Uterine Cervical Neoplasms*

BACKGROUND: Infection can activate the immune system and may trigger the production of autoantibodies. It has been reported that malaria infection triggers the production of various autoantibodies. Therefore, we investigated the pattern and significance of antinuclear antibodies (ANA) found in patients with malaria infection. METHODS: Our study group included 36 patients who were diagnosed with malaria infection at Mokdong Hospital from July 1998 to July 2001. We performed antinuclear antibody test using indirect immunofluorescence method (Quantafluor, Sanofi Diagnostics Pasteur Inc., USA), extractable nuclear antigen test (ENA) using double immunodiffusion method (Nova Gel, Inova Diagnostics Inc., USA), anti-double stranded DNA Ab test (anti-ds DNA Ab) using Farr assay (DPC anti-DNA, Diagnostic products Corporation, USA), and anti-single stranded DNA Ab test (anti-ssDNA Ab) using enzyme immunoassay method (QUANTA, Lite ssDNA, Inova Diagnostics Inc., USA). RESULTS: Among the 36 patients, 32 patients (88.9%) showed ANA positivity and 27 patients (75.0%) showed cytoskeleton or speckled pattern of ANA. Anti-ssDNA Ab was found in 3 of 20 patients; however, anti-dsDNA Ab and ENA were not found in all patients. Patients who had ANA showed higher levels of IgG, IgM and IgA, compared with those patients who did not have ANA. Follow up (11-37 month) of the 13 patients with ANA positivity revealed no symptoms associated with autoimmune disorder. CONCLUSIONS: Malaria infection may develop ANA, especially cytoskeleton or speckled pattern. The follow up of patients with ANA positivity showed no symptoms associated with any autoimmune disorder, but further evaluation would be necessary to reveal the relationship between malaria infection and development of autoimmune disorder.
Antibodies, Antinuclear* ; Autoantibodies ; Cytoskeleton ; DNA ; Fluorescent Antibody Technique, Indirect ; Follow-Up Studies ; Humans ; Immune System ; Immunodiffusion ; Immunoenzyme Techniques ; Immunoglobulin A ; Immunoglobulin G ; Immunoglobulin M ; Malaria* ; Radioimmunoprecipitation Assay

BACKGROUNDThe acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML.

METHODSQualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PCR), and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids (including mutant plasmids) of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence of EYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay.

RESULTSEYA4 gene was hypermethylated in AML1-ETO+ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA4 gene by binding at AML1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AML1-ETO+ cell lines. We also found EYA4 transfection increased apoptosis of Kasumi-1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively.

CONCLUSIONSOur study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML1-ETO+ t (8;21) AML.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; genetics ; physiology ; Chromatin Immunoprecipitation ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; DNA Methylation ; genetics ; Epigenesis, Genetic ; genetics ; Gene Silencing ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; RUNX1 Translocation Partner 1 Protein ; Radioimmunoprecipitation Assay ; Trans-Activators ; genetics ; metabolism