T Cells Modified with CD70 as an Alternative Cellular Vaccine for Antitumor Immunity / Journal of the Korean Cancer Association, 대한암학회지

Purpose@#Successful tumor eradication primarily depends on generation and maintenance of a large population of tumor-reactive CD8 T cells. Dendritic cells (DCs) are well-known potent antigen-presenting cells and have applied to clinics as potent antitumor therapeutic agents. However, high cost and difficulty in obtaining sufficient amounts for clinical use are the crucial drawbacks of DC-based vaccines. Here, we aimed to develop T cell–based vaccine capable of eliciting potent antitumor therapeutic effects by providing effective costimulatory signals. @*Materials and Methods@#Antigenic peptide-loaded T cells transfected with retrovirus encoding costimulatory ligands CD70, CD80, OX40L, or 4-1BBL were assessed for antigen-specific CD8 T-cell responses and evaluated antitumor effects along with immunization of a mixture of synthetic peptides, poly-IC and anti-CD40 antibodies (TriVax). @*Results@#T cells expressing CD70 (CD70-T) exhibited similar level of stimulatory functionality and therapeutic efficacy as DCs. Moreover, CD70-T prime followed by TriVax booster heterologous vaccination elicited therapeutic antitumor effect against B16 melanoma where mediated by CD8 T cells but not CD4 T cells or natural killer cells. The combination with programmed death-ligand 1 blockade led to potent therapeutic efficacy which exhibited increased tumor-infiltrating CD8 T cells. CD70-T pulsed with multi-antigenic peptide generated multiple antigen-specific polyvalent CD8 T cells that were capable of inhibiting tumor growth effectively. Moreover, CD70-T vaccination resulted in higher expansion and migration of adoptively transferred T cells into tumor sites and elicits enhanced therapeutic effects with peptide-based booster immu-nization. @*Conclusion@#These results imply that T cells endowed with CD70 enable the design of effective vaccination strategies against solid cancer, which may overcome current limitations of DC-based vaccines.

Unrelated hematopoietic stem cell registry and the role of the Hematopoietic Stem Cell Bank

BACKGROUND: The hematopoietic stem cell bank has been actively recruiting registrants since 1994. This study systematically reviews its operations and outcomes over the last 20 years. METHODS: Retrospective data on a total of 47,711 registrants were reviewed. Relevant data were processed using PASW Statistics for Windows, version 18.0. RESULTS: As of 2013, the Korean Network for Organ Sharing database contained 265,307 registrants. Of these, 49,037 (18%) registrants committed to hematopoietic cell donation from 1994 to 2013. Fifty-seven percent of the registrants were men, and 43% were women. The reasons for opting out of the registry included refusal to donate (70%), family refusal (28%), and others (2%). The donation willingness of registrants was significantly higher than those who refused to receive a mail to confirm their continued enrollment (χ2=6.103, P=0.013). The bank successfully coordinated a total of 512 donors among newly matched donors from 1995 to 2013, of which the bone marrow and peripheral blood stem cell accounted for 40.8% and 59.2% of the total donations, respectively. CONCLUSION: Our recruitment activities focus on promoting voluntary registration and the importance of updating personal contact information. We expect that these data may be useful for diverse studies and demonstrate the positive impacts on the donation program.

The Past, Present, and Future of Adoptive T Cell Therapy

Although adoptive T cell therapy (ACT) has become a promising immunotherapeutic regime for cancer treatment, its effectiveness has been hindered by several inherent shortcomings regarding safety and efficacy. During the past few decades, several strategies for enhancing the efficacy of ACT have been developed and introduced in clinic. This review will summarize not only the past approaches but also the latest strategies which have been shown to enhance the anticancer activity of ACT.

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

BACKGROUND: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. METHODS: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-gamma ELISPOT assay, cytotoxicity assay and tetramer staining. RESULTS: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. CONCLUSION: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

HLA and Disease Associations in Koreans

The human leukocyte antigen (HLA), the major histocompatibility complex (MHC) in humans has been known to reside on chromosome 6 and encodes cell-surface antigen-presenting proteins and many other proteins related to immune system function. The HLA is highly polymorphic and the most genetically variable coding loci in humans. In addition to a critical role in transplantation medicine, HLA and disease associations have been widely studied across the populations world-wide and are found to be important in prediction of disease susceptibility, resistance and of evolutionary maintenance of genetic diversity. Because recently developed molecular based HLA typing has several advantages like improved specimen stability and increased resolution of HLA types, the association between HLA alleles and a given disease could be more accurately quantified. Here, in this review, we have collected HLA association data on some autoimmune diseases, infectious diseases, cancers, drug responsiveness and other diseases with unknown etiology in Koreans and attempt to summarize some remarkable HLA alleles related with specific diseases.

Frequency of Killer Cell Immunoglobulin-like Receptors (KIRs) in Korean Patients with Chronic HCV Infection

Natural killer (NK) cells play an important role in innate immunity, especially in the response to viral infections, such as hepatitis C virus (HCV). Killer cell immunoglobulin-like receptors (KIRs) are the primary receptors of NK cells that mediate innate immunity. KIRs are also involved in acquired immunity, because some KIRs are expressed on the surface of certain subsets of T cells. In this study, the frequency of KIR genes, HLA-C allotypes, and combinations of KIR genes with their HLA-C ligands were evaluated in two different groups of the Korean population: controls and patients with chronic HCV infection. The study population consisted of 147 Korean patients with chronic HCV infection. The frequency of KIR2DS2 in patients with chronic HCV infection was 9.5% which was significantly lower than 19.5% of the control (P < 0.01). However, there were no significant differences in the frequency of other KIR genes, HLA-C allotypes or different combinations of KIR genes with their HLA-C ligands. This study can contribute to the further prospective study with a larger scale, suggesting the assumption that KIR2DS2 might aid in HCV clearance by enhancing both the innate and acquired immune responses of people in Korea.

Common variants at the promoter region of the APOM confer a risk of rheumatoid arthritis

Although the genetic component in the etiology of rheumatoid arthritis (RA) has been consistently suggested, many novel genetic loci remain to uncover. To identify RA risk loci, we performed a genome-wide association study (GWAS) with 100 RA cases and 600 controls using Affymetrix SNP array 5.0. The candidate risk locus (APOM gene) was re-sequenced to discover novel promoter and coding variants in a group of the subjects. Replication was performed with the independent case-control set comprising of 578 RAs and 711 controls. Through GWAS, we identified a novel SNP associated with RA at the APOM gene in the MHC class III region on 6p21.33 (rs805297, odds ratio (OR) = 2.28, P = 5.20 x 10(-7)). Three more polymorphisms were identified at the promoter region of the APOM by the re-sequencing. For the replication, we genotyped the four SNP loci in the independent case-control set. The association of rs805297 identified by GWAS was successfully replicated (OR = 1.40, P = 6.65 x 10(-5)). The association became more significant in the combined analysis of discovery and replication sets (OR = 1.56, P = 2.73 +/- 10(-10)). The individuals with the rs805297 risk allele (A) at the promoter region showed a significantly lower level of APOM expression compared with those with the protective allele (C) homozygote. In the logistic regressions by the phenotype status, the homozygote risk genotype (A/A) consistently showed higher ORs than the heterozygote one (A/C) for the phenotype-positive RAs. These results indicate that APOM promoter polymorphisms are significantly associated with the susceptibility to RA.

Recipient's Killer Cell Immunoglobulin-like Receptor Genotype and Human Leukocyte Antigen C Ligand Influence the Clinical Outcome following Living Donor Liver Transplantation

PURPOSE: The design of this study was to determine the most influential factor(s) on post-transplant immunological consequences, particularly with regard to the role of killer cell immunoglobulin-like receptors (KIRs) and their ligands (type I human leukocyte antigen (HLA)) in unstable liver function. METHODS: Retrospectively collected data from 319 recipients undergoing adult living donor liver transplantation (LDLT) using a right lobe graft between January 2002 and August 2008 were analyzed. Patients were categorized according to the serum alanine transaminase (ALT) pattern; stable ALT pattern was defined as ALT pattern during 3 months post-transplantation, except for initial 2 weeks post-transplantation, in which 2 times or less additional elevation(s) of serum alanine transaminase (ALT) (> or =80 IU/L) were observed. When a serum ALT pattern showed fluctuating and/or unpredictable nature, it was defined as an unstable pattern. In addition, genetic information of KIRs and HLA-C allotypes received from 68 recipients and 59 donors was analyzed by way of polymerase chain reaction using sequence-specific primers (PCR-SSP) to determine the factor(s) influencing a serum ALT pattern. RESULTS: Among 319 LDLT recipients included in this study, the actual incidences of AR and unstable ALT pattern were 13.4% (43/319) and 42.3% (135/319), respectively. Unstable ALT pattern correlated with poorer survival following LDLT than stable pattern (P<0.000). Genetically, unstable ALT pattern was related to recipients carrying KIR2DL2(+)/KIR2DS2(+) combined with the heterogeneous HLA-C allotype (HLA-C1/C2), (relative risks 45.0, 95% confidence interval 2.160~937.321; P=0.013). CONCLUSION: This study indicates that, when performing LDLT, pretransplant determination of recipient's KIRs and HLA-C allotypes may be beneficial in coping with post-transplant immunological circumstances.

Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3+CCR4-CD4+ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-gamma, TNF-alpha, and IL-2 in CXCR3+CCR4-CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4+ or CXCR3+CCR4-CD4+ T cells with DC (CD4+/DC or CXCR3+CD4+/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-alpha and LPS (mDC). Although there was no significant difference between the effects of the CXCR3+CCR4-CD4+ and CD4+ T cells on DC phenotype expression, CXCR3+CD4+/DC in CTL culture were able to expand number of CD8+ T cells and increased frequencies of IFN-gamma secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3+CCR4-CD4+ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.

The Relationship between the p27 Expression of Papillary Thyroid Cancer Cells Obtained by FNA and Cervical Lymph Node Metastasis / 대한내분비외과학회지

PURPOSE: The p27 gene as a tumor suppressor gene is associated with colorectal cancer, gastric cancer and breast cancer. Some studies have shown a relationship between the underexpression of p27 and lymph node metastasis in papillary thyroid carcinoma. The aim of this study is to evaluate the relationship between a p27 expression of the papillary thyroid cancer cells obtained by fine needle aspiration (FNA) and cervical lymph node metastasis. METHODS: This study included 60 patients with papillary thyroid cancer and who underwent total thyroidectomy or lobectomy. Central lymph node dissection was routinely done. Out of these patients, 30 patients underwent a FNA procedure during the operation. Immunohistochemical staining for p27 antibody was performed on the papillary thyroid cancer tissues and cells. RESULTS: Cervical lymph node metastasis is correlated with the tumor size and lymphovascular invasion (P<0.001). The underexpression of p27 for the papillary thyroid cancer tissues and cells was associated with lymph node metastasis (P=0.009). CONCLUSION: An evaluation of the p27 expression for the papillary thyroid cancer cells obtained by FNA may be useful as a predictor for lymph node metastasis before surgery.

Susceptibility of Leukemia according to the Genotype of Minor Histocompatibility Antigens in a Korean Population

BACKGROUND: In the search for susceptibility genes responsible for leukemia, genetic studies involving HLA association have been in progress extensively since the first report on its effect on the disease. Here we investigated the genetic associations of different leukemias with 4 autosomal mHags, HA-1, -2, -8 and HB-1. In particular, HB-1 is one of the leukemia-associated minor histocompatibility antigens (mHags) that is significantly expressed by Epstein-Barr virus-transformed- and tumor cells of all B lineage acute lymphoblastic leukemia (ALL). METHODS: A simultaneous genotyping method using PCR sequence-specific primers against HA-1, -2, -8 and HB-1 was developed, and their allelic frequencies in 139 healthy controls and 36 leukemia patients were observed. To compare genotype, phenotype, and gene frequencies of mHags with healthy controls, leukemia patients were classified into sub groups of ALL, acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). RESULTS: The genotype frequencies of HA-1, -2 and -8 were not significantly different from healthy controls in every group of leukemia patients. However, the HB-1 H genotype was significantly increased in leukemia patients (P=0.03, OR=1.82, CI=1.08~3.06), particularly in AML (P=0.01, OR=2.4, CI=1.21~4.76) as compared with healthy controls. CONCLUSION: Our results suggested that the genotype of HB-1 H may be associated with leukemia, particularly with AML. In further study, it is necessary to confirm the association of HB-1 with other leukemias in a larger group of patients, and to identify the underlying mechanism of HB-1 responsible for the occurrence of leukemia.

Enhancement of Proliferation and Antigen Presentation of Human B Cells in Vitro by K562 Cells Expressing CD40L

BACKGROUND: CD40-activated B (CD40-B) cells might be an attractive source of autologous antigen-presenting cells (APCs) for immunotherapy due to the convenience to obtain from peripheral blood and expand in vitro. Moreover, CD40-B cells were found to be comparable with DCs in their capacity to raise antigen-specific CD8+ T cells. Here, we have established K562 cells expressing CD40L to expand CD40-activated B cells used for APCs. METHODS: After activation of B cell by K562/CD40L, CD40-B cells were examined by counting B cell numbers. Surface expression of CD54, CD80, CD86 and HLA class II was measured by flow cytometry. The CD40-B cells were tested for its function as APC by mixed lymphocyte reactions (MLR) and by induction of T cell responses specific for pp65 peptide in vitro. RESULTS: The expansion of B cells by K562/CD40L increased about 6-folds compared with anti-CD40 or K562. Furthermore, the expression of CD54, CD80, CD86 and HLA class II was up-regulated by K562/CD40L. B cells by K562/CD40L showed comparable antigen presentation activity with mature DCs as shown in MLR, INF-gamma ELISPOT assay. CONCLUSION: These results suggest that K562/CD40L could be used to generate activated B cells as potent APCs which could be useful for cellular vaccination and adoptive immunotherapy.

Killer Cell Immunoglobulin-like Receptor (KIR) Analysis in Adult Korean Patients with Acute Myeloid Leukemia

BACKGROUND: The prevalent natural killer (NK) cells induce alloreaction against leukemic cells during post-transplant. NK cell alloreactivity depends on the compatibility of killer cell immunoglobulin-like receptors (KIR) epitopes for graft-versus-host disease. Genotypic expressions of inhibitory or activating KIR in patients with acute myelogenous leukemia (AML) and their HLA-matched sibling donors, as a model for Korean KIR haplotype diversity and NK alloreactivity, were investigated. METHODS: Ninety-two patients in complete remission and their 76 HLA-matched sibling donors were enrolled in this study. All the patients were scheduled to receive allogeneic hematopoietic stem cell transplantations (HSCT). KIR PCR-SSP typing was performed for 19 different kinds of KIR genes and pseudogenes. The PCR data representing the KIR genotypes from both the patients and donors were compared. RESULTS: We found 43 Korean KIR haplotypes. Thirty-three variable haplotypes for the AML patients, in addition to 25 haplotypes for the normal HSCT donors, were demonstrated. Of note, the expressions of specific genes such as 2DL2 (P=0.026), 2DS2 (P=0.042), and 2DS4 (P=0.037) revealed remarkable differences between the patients and the normal donors. Korean HLA-identical sibling pairs showed 38% KIR matches in terms of the gene content and allelic polymorphism. Although the KIR gene content was the same between the patients and the donors, 40% of those matched pairs of patients and donors showed allelic polymorphism, specifically in the context of 2DL5 and 2DS4 genes. CONCLUSION: These results indicate that the expressions of donor inhibitory and activating repertoire of KIR genotypes, even in the HLA-matched sibling setting, are unique parameters to be considered when we perform allogeneic sibling HSCT.

Measurement of HIV-1-specific T Cell Immunity by Using Recombinant Gag Protein / 감염과화학요법

BACKGROUND: HIV-specific immunity, such as strong CD4+ helper and CD8+ cytotoxic T lymphocytes (CTL) responses, develops soon after HIV infection, but usually it can not control HIV replication which ultimately results in severe immune deficiency. HIV-specific CTLs, which are induced by HIV-specific CD4+ helper responses, are the key to cellular immune control of HIV. Measurement of HIV-1-specific CTLs using recombinant Gag protein may be very useful, because it is not restricted by HLA haplotype of the infected individual. MATERIALS AND METHODS: Enzyme-linked immunospot (ELISPOT) assays by using recombinant Gag protein were performed to evaluate HIV-1-specific gamma-interferon cellular responses of 25 HIV-1 infected Korean patients, who had been treated at least for the prior 12 months with highly active antiretroviral therapy at Catholic University Kangnam St. Mary's Hospital. RESULTS: The study group consisted of 25 chronically HIV-infected individuals with a median age of 51 years. The median CD4 counts were 556/mm3 (range:369-994/mm3) and HIV RNA titers were < 25 copies/mL (range: <25-180copies/mL). HIV-1-specific ELISPOT assay results range from 0 to 49 SFCs/2 x 10(5) PBMCs (median, 23.5 SFCs/2 x 10(5) PBMCs). CMV pp65-specific ELISPOT assay results range from 5 to 591 SFCs/2 x 10(5) PBMCs (median, 34 SFCs/2 x 10(5) PBMCs). There was no correlation between CD4 counts and HIV-1-specific SFCs measured by ELISPOT using recombinant protein. CONCLUSION: ELISPOT assays by using recombinant Gag protein may be considerable value in the assessment of cell-mediated immunity of HIV-1 infected patients.

Measurement of HIV-1-specific T Cell Immunity by Using Recombinant Gag Protein / 감염과화학요법

BACKGROUND: HIV-specific immunity, such as strong CD4+ helper and CD8+ cytotoxic T lymphocytes (CTL) responses, develops soon after HIV infection, but usually it can not control HIV replication which ultimately results in severe immune deficiency. HIV-specific CTLs, which are induced by HIV-specific CD4+ helper responses, are the key to cellular immune control of HIV. Measurement of HIV-1-specific CTLs using recombinant Gag protein may be very useful, because it is not restricted by HLA haplotype of the infected individual. MATERIALS AND METHODS: Enzyme-linked immunospot (ELISPOT) assays by using recombinant Gag protein were performed to evaluate HIV-1-specific gamma-interferon cellular responses of 25 HIV-1 infected Korean patients, who had been treated at least for the prior 12 months with highly active antiretroviral therapy at Catholic University Kangnam St. Mary's Hospital. RESULTS: The study group consisted of 25 chronically HIV-infected individuals with a median age of 51 years. The median CD4 counts were 556/mm3 (range:369-994/mm3) and HIV RNA titers were < 25 copies/mL (range: <25-180copies/mL). HIV-1-specific ELISPOT assay results range from 0 to 49 SFCs/2 x 10(5) PBMCs (median, 23.5 SFCs/2 x 10(5) PBMCs). CMV pp65-specific ELISPOT assay results range from 5 to 591 SFCs/2 x 10(5) PBMCs (median, 34 SFCs/2 x 10(5) PBMCs). There was no correlation between CD4 counts and HIV-1-specific SFCs measured by ELISPOT using recombinant protein. CONCLUSION: ELISPOT assays by using recombinant Gag protein may be considerable value in the assessment of cell-mediated immunity of HIV-1 infected patients.

HLA-restricted and Antigen-specific CD8+ T Cell Responses by K562 Cells Expressing HLA-A*0201

BACKGROUND: Identification of antigen-specific T cells has yielded valuable information on pathologic process and the disease state. Assays for quantification of inflammatory cytokines or lytic-granule molecules have been generally used to evaluate antigen specific T cell response, however their applicability have been hampered due to the limited source of autologous antigen-presenting target cells (APC). METHODS: K562, a leukemic cell line deficient of human leukocyte antigen (HLA), was transfected with a gene encoding HLA-A*02 (K562/A*02) and its function as stimulator cells in inducing activation of HLA-matched T cells was evaluated by IFN-gamma enzyme linked immunospot (ELISPOT) assay. RESULTS: The stable transfectant K562/A*02 pulsed with HLA- A*02 restricted peptide could specifically induce IFN-gamma secretion by CD8+ T cells compared to no detectable secretion by CD4+ T cells. However, CD56+ NK cells secreted IFN-gamma in both K562/A*02 with peptide and without peptide. The number of IFN-gamma secreted CD8+ T cells was increased according to the ratio of T cells to K562 and peptide concentration. Formalin-fixed K562/A*02 showed similar antigen presenting function to live K562/A*02. Moreover, K562/A*02 could present antigenic- peptide to not only A*0201 restricted CD8+ T cells but also CD8+ T cells from A*0206 donor. CONCLUSION: These results suggest that K562/A*02 could be generally used as target having specificity and negligible background for measuring CD8+ T cell responses and selective use of K562 with responsder matched HLA molecules on its surface as APC may circumvent the limitation of providing HLA-matched autologous target cells.

Enhancement of Adenoviral Transduction and Immunogenecity of Transgenes by Soluble Coxsackie and Adenovirus Receptor-TAT Fusion Protein on Dendritic Cells

BACKGROUND: Investigating strategy to enhance efficiency of gene transfer via adenovirus is critical to sustain gene expression in targeted cells or tissues to regulate immune responses. However, the use of adenovirus as a gene delivery method has been limited by the native tropism of the virus. In this study, the critical parameter is to improve the efficient binding of viral particles to the plasma membrane prior to cellular uptake. METHODS: Human immunodeficiency virus (HIV-1) trans-acting activator of transcription (TAT), a protein transduction domain, was fused to the ectodomain of the coxsackie-adenovirus receptor (CAR). The CAR-TAT protein was produced from a Drosophila Schneider 2 cells (S2) transfected with CAR-TAT genes. The function of CAR- TAT was analyzed the efficiency of adenoviral gene transfer by flow cytometry, and then immunizing AdVGFP with CAR-TAT was transduced on dendritic cells (DCs). RESULTS: S2 transfectants secreting CAR-TAT fusion protein has been stable over a period of 6 months and its expression was verified by western blot. Addition of CAR-TAT induced higher transduction efficiency for AdVGFP at every MOI tested. When mice were vaccinated with DC of which adenoviral transduction was mediated by CAR-TAT, the number of IFN-gamma secreting T-cells was increased as compared with those DCs transduced without CAR-TAT. CONCLUSION: Our data provide evidence that CAR-TAT fusion protein enhances adenoviral transduction and immunogenecity of transgenes on DCs and may influence on the development of adenoviral- mediated anti-tumor immunotherapy.

Analysis of HLA in Patients with Obstructive Sleep Apnea Syndrome / 결핵및호흡기질환

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is believed to have multifactorial causes. The major risk factors for OSAS are obesity, narrowed upper airways, and abnormal cranial-facial structures. A genetic basis for OSAS has been also suggested by reports of families with many members affected. This study analyzed the HLA typing in patients with OSAS to determine the possible role of genetics in OSAS. METHODS: Twenty-five Korean patients with OSAS (1 woman and 24 men; age range 30-66 years) were enrolled in this study. A diagnosis of OSAS was made using full-night polysomnography. The control group consisted of 200 healthy Korean people. Serologic typing of the HLA-A and B alleles was performed in all patients using a standard lymphocyte microcytotoxicity test. Analysis of the polymorphic second exons of the HLA-DRB1 gene was performed using a polymerase chain reaction-sequence specific oligonucleotide probe. RESULTS: The allele frequency of HLA-A11 was significantly lower in patients with OSAS compared with the controls (p45) than in the controls (p<0.05). CONCLUSION: This study revealed an association between OSAS and the HLA-A11 and DRB1*09 alleles as well as association between the disease severity and the HLA-DRB1*08 allele in Korean patients. These results suggest that genetics plays an important role in both the development and the disease severity of OSAS.

Detection of Differentially Expressed Genes in Glioblastoma by Suppression Subtractive Hybridization

OBJECTIVE: A variety of genetic alterations in human glioblastoma comprises signal transduction and cell cycle arrest control of cellular processes. Subtractive hybridization is potentially a faster method for identifying differentially expressed genes associated with a particular disease state. Using the technique of subtraction, we isolated novel genes that are overexpressed in glioblastoma tissue as compared to normal brain tissue. METHODS: We evaluated the differential expression of genes in each of hybridizing tester and driver cDNAs to digested 130 clones. After sequencing of 130 clones and homology search, this study performed to determine mRNA expression of the unknown gene, "clone 47", in brain tissue, glioblasoma, and several cancer cell lines by reverse transcription-polymerase chain reaction (RT-PCR). To test the time course for G0-phase arrest, serum stimulation and expression at various times for RT-PCR performed. RESULTS: We identified 23 novel genes by BLAST of the digested 130 clones. The expressions of "clone 47" mRNA of glioblastoma and several cancer lines were significantly higher than normal brain tissues and several normal cell lines. We confirmed the mRNA expression of "clone 47" was up-regulation for 0.5~1hr of WI-38 cell differentiation. CONCLUSION: The novel gene, "Clone 47" is upregulated in glioblastoma tissue and several cancer cell lines. This gene is time dependent activation during time course of serum stimulation. This result suggests that "clone 47" play a role in brain tumorigenesis and the activation of this "clone 47" may be necessary for the development of cancer.

Enhanced CEA-specific Immune Responses by Tat-LLO Fusion Protein

BACKGROUND: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. However, it is known that the induction of immune response to CEA is very difficult because CEA is a self-antigen expressed in fetal cells and weakly expressed in normal colorectal epithelial cells. To enhance anti-tumor immunity specific for CEA, recombinant CEA protein was modified using listeriolysin O (LLO) for endosomal lysis and transactivator of transcription (Tat) domain for transducing extracellular proteins into cytoplasm. METHODS: After immunization using dendritic cells pulsed with Tat-CEA, both Tat-CEA and LLO, and both Tat-CEA and Tat-LLO, antibody titer to CEA and LLO, cytotoxic T lymphocyte activity and the frequency of IFN-gamma producing T lymphocytes were measured. RESULTS: Immunization using DC pulsed with both Tat-CEA and Tat-LLO protein showed the increasement of production of CEA-specific antibody in serum, cytotoxic T lymphocyte activity, the frequency of IFN-gamma secreting T cells, compared with DC pulsed with both Tat-CEA and LLO. Furthermore the ratio of CD8+ T cell to CD4+ T cell among CEA-specific T cells was increased in group pulsed with both Tat-CEA and Tat-LLO. CONCLUSION: These results suggested that DC vaccine using Tat-LLO could be used for the development of effective immunotherapy for the treatment of tumor.