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Family Study of TAP Gene Polymorphism and HLA-TAP Haplotypes in Koreans.

Immune Network.2002;2(4):248-255. doi:10.4110/in.2002.2.4.248

BACKGROUND: TAP1 and TAP2 are two ABC transporter genes located within the class II region of the human MHC. Their protein products form a heterodimer whose function is to transport peptides from the cytoplasm into the endoplasmic reticulum. This study was performed to examine the polymorphism of TAP genes and the distribution of HLA-TAP haplotypes in the Korean population through family analysis. METHODS: The subjects used in this study were 50 healthy Korean families consisting of 233 individuals. TAP1 (codons 333 and 637) and TAP2 (codons 379, 565, 577, 651, 665, and 687) typings were carried out by the PCR-restriction fragment length polymorphism (RFLP) method. HLA-DRB1 and DQB1 genotyping results from a previous study were used for HLA-TAP haplotype analysis. RESULTS: The number (gene frequency) of TAP1 and TAP2 alleles detected were 3 for TAP1 (A 81.5%, B 17.0%, and C 1.5%) and 8 for TAP2 (A1 32.0%, A2 12.5%, B 34.0%, Bky2 6.5%, C 7.0%, D 3.0%, E 4.5%, and G 0.5%). Eleven TAP1-TAP2 haplotypes were observed with frequency 1%, among which 4 haplotypes (A-B, B-A1, A-Bky2, and C-E) showed weak but significant positive linkage disequilibrium (P<0.05). When DRB1-DQB1 haplotypes were extended to TAP1 and TAP2 loci, much diversification of haplotypes was observed: 19 different DRB1- DQB1 haplotypes formed 58 different haplotypes extended to TAP1 and TAP2 loci. These results add more evidence to the view that recombination hotspot is present within and around TAP gene region. CONCLUSION: The allele frequencies of TAP1 and TAP2 genes and the distribution of TAP1-TAP2 and HLA-TAP haplotypes were studied in Koreans based on a family study.
Alleles ; Cytoplasm ; Endoplasmic Reticulum ; Gene Frequency ; Haplotypes* ; HLA-DRB1 Chains ; Humans ; Linkage Disequilibrium ; Peptides ; Recombination, Genetic

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Polymorphisms in the TNF-alpha Gene and Extended HLA and TNF-alpha Haplotypes in Koreans.

Yoon June PARK ; Hye Jin PARK ; Myoung Hee PARK

Immune Network.2002;2(4):242-247. doi:10.4110/in.2002.2.4.242

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is known to play an important role in various conditions such as inflammation, autoimmunity, apoptosis, insulin resistance and sleep induction. Five single nucleotide polymorphisms (SNPs) have been known to affect the transcriptional activities of TNF-alpha: 1,-031T/C, -863C/A, -857C/T, -308G/A and -238G/A. METHODS: We have investigated 5 SNPs of the promoter region of TNF-alpha gene, the distribution of 5-locus TNF-alpha haplotypes, and their haplotypic associations with previously typed HLA-A, -B and -DRB1 loci in 107 healthy unrelated Koreans. TNF-alpha SNPs were typed using PCR-single-strand conformation polymorphism (SSCP) and PCR-restriction fragment length polymorphism (RFLP) methods. RESULTS: The allele frequencies of -1,031C, -863A, -857T, -308A, and -238A, which are known as the high-producer-type, were 19.3%, 15.9%, 14.0%, 5.9%, and 2.9%, respectively. The frequency of -308A allele, known to be associated with autoimmune diseases, was 5.9% in Koreans which was lower than Caucasians (14-17%) and somewhat higher than Japanese (1.7%). Five most common TNF-alpha haplotypes (-1,031/ -863/ -857/ -308/ -238) comprised over 95% of total haplotypes: TCCGG (58.4%), CACGG (14.8%), TCTGG (13.7%), TCCAG (5.3%), and CCCGA (3.1%). Strong positive associations (P<0.001) were observed between TCCGG and B62; between CACGG and B51, DRB1*0901; between TCTGG and B35, B54, B59, DRB1* 1201; and between TCCAG and A33, B58, DRB1*0301, DRB1*1302. Five most common extended haplotypes (>3%) comprised around 16% of total haplotypes: A33-B58- TCCAG-DRB1*1302, A24-B52-TCCGG-DRB1*1502, A33-B44-TCCGG-DRB1*1302, A24- B7-TCCGG-DRB1*0101, and A11-B62-TCCGG-DRB1*0406. The distribution of extended HLA and TNF-alpha haplotypes showed that most of HLA haplotypes were almost exclusively associated with particular TNF-alpha haplotypes. CONCLUSION: The results obtained in this study would be useful as basic data for anthropologic studies and disease association studies in Koreans.
Alleles ; Apoptosis ; Asian Continental Ancestry Group ; Autoimmune Diseases ; Autoimmunity ; Gene Frequency ; Haplotypes* ; HLA-A Antigens ; Humans ; Inflammation ; Insulin Resistance ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Tumor Necrosis Factor-alpha*

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Isolation of Mouse Ig Heavy and Light Chain Genomic DNA Clones, and Construction of Gene Knockout Vector for the Generation of Humanized Xenomouse.

Hee Kyung LEE ; Sang Hoon CHA

Immune Network.2002;2(4):233-241. doi:10.4110/in.2002.2.4.233

BACKGROUND: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome. METHODS: Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain lamda genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse. RESULTS: Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress. CONCLUSION: Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea.
Animals ; Antibodies ; Antibodies, Monoclonal ; Biotechnology ; Brain ; Clone Cells* ; DNA* ; Embryonic Stem Cells ; Gene Knockout Techniques* ; Gene Targeting ; Gene Transfer Techniques ; Genes, Immunoglobulin ; Genetic Engineering ; Genome ; Genomic Library ; Homologous Recombination ; Human Body ; Humans* ; Hybridomas ; Indicators and Reagents ; Korea ; Mass Screening ; Mice* ; Mice, Knockout ; Mice, Transgenic ; Polymerase Chain Reaction ; Rodentia

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The Association of HLA-DRB1 and DQB1 Alleles and a Study of Anticardiolipin Antibody and Anti-beta2 Glycoprotein I Antibody in Korean SLE Patients.

Sang Gon LEE ; Hoon Suk CHA ; Yoon Sun YANG

Immune Network.2002;2(4):227-232. doi:10.4110/in.2002.2.4.227

BACKGROUND: Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by diverse clinical manifestations and autoantibody production, which is known to be strongly influenced by genetic factors. Previous studies have revealed the associations of SLE with HLA class II alleles and antiphospholipid antibody system (anticardiolipin antibody (aCL) and anti-beta2 glycoprotein I antibody (anti-beta2 GPI)). Therefore, we studied the associations of HLA class II alleles with SLE and antiphospholipid antibody system. METHODS: The genotyping for HLA-DRB1 and DQB1 alleles were performed in 61 SLE patients and 100 controls by the polymerase chain reaction (PCR)-sequence specific oligonucleotide probe method. ELISA tests for aCL and anti-beta2 GPI were performed in 39 of the 61 SLE patients. The results were evaluated statistically by Chi-square test. RESULTS: The frequencies of the HLA-DRB1*15 and DQB1*06 in SLE patients were significantly higher than those in controls. HLA-DRB1*12 was significantly lower in SLE patients than controls. Nine of 39 patients were positive for aCL (IgG) and three were positive for aCL (IgM). One of 39 patients were positive for anti-beta2 GPI (IgG) and none of them positive for anti-beta2 GPI (IgM). Association of aCL with HLA class II alleles was not observed in our study. CONCLUSION: According to our results, it was found that HLA-DRB1*15 and DQB1*06 were associated with genetic susceptiblility and DRB1*12 was associated with resistance to SLE in Korean population. No Association of aCL with HLA class II alleles was observed and the positive rate for anti-beta2 GPI was very low.
Alleles* ; Antibodies, Anticardiolipin* ; Antibodies, Antiphospholipid ; Autoimmune Diseases ; Enzyme-Linked Immunosorbent Assay ; Glycoproteins* ; HLA-DR Antigens ; HLA-DRB1 Chains* ; Humans ; Lupus Erythematosus, Systemic ; Polymerase Chain Reaction

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Serum Levels of Type 2 Chemokines in Lepromatous Leprosy Patients.

Wook LEW ; Koichiro NAKAMURA ; Yayoi TADA ; Ho KWAHCK ; Soo Kyoung CHANG ; Kunihiko TAMAKI

Immune Network.2002;2(4):223-226. doi:10.4110/in.2002.2.4.223

BACKGROUND: The type 2 deviated immunological state is predominant in lepromatous leprosy. Erythema nodosum leprosum (ENL) is an immune-complex mediated reaction that typically occurs in lepromatous leprosy. To date, the serum levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-2 receptor, IL-10, IL-1beta, IL-1 receptor antagonist and monocyte chemoattractant protein-1 (MCP-1) were reported to be higher in lepromatous leprosy. TNF-alpha is also known to be higher in ENL, which is reduced after thalidomide treatment. However the serum type 2 chemokine levels in lepromatous leprosy patients have not been reported. METHODS: The serum levels of the type 2 chemokines such as thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and eotaxin together with IL-12 and IL-10 in the sera from leprosy patients were detected using an enzyme-linked solvent assay (ELISA) method. RESULTS: The Serum TARC, MDC, eotaxin, IL-10 and IL-12 levels in lepromatous leprosy patients were not significantly different from the normal control levels. The serum levels were not significantly different between the paucibacillary group and multibacillary group. The serum TARC or MDC levels in the ENL patients were more reduced after a treatment containing thalidomide. CONCLUSION: The type 2 chemokines are not related to the severity of lepromatous leprosy. The larger reducing effect of the TARC or MDC levels in ENL patients by a treatment containing thalidomide suggests the potential role of these chemokines in the development of ENL and the therapeutic mechanism of thalidomide.
Chemokine CCL17 ; Chemokine CCL2 ; Chemokine CCL22 ; Chemokines* ; Erythema Nodosum ; Humans ; Interleukin-1 ; Interleukin-10 ; Interleukin-12 ; Interleukins ; Leprosy ; Leprosy, Lepromatous* ; Receptors, Interleukin-10 ; Thalidomide ; Tumor Necrosis Factor-alpha

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The Differential Staging of Murine Thymic Lymphoma Cell Lines, Scid.adh, R1.1 and EL-4.

Jong Seok CHAE ; Hae Jung KIM ; Weon Seo PARK ; Youngmee BAE ; Kyeong Cheon JUNG

Immune Network.2002;2(4):217-222. doi:10.4110/in.2002.2.4.217

BACKGROUND: Scid.adh is a recently developed murine thymic lymphoma cell line, which has been used as in vitro model for the study of double negative stage III thymocytes. In this study, we compared the expression profile of a number of genes and proteins, which are tightly related to T cell development and apoptosis, in thymic lymphoma cell lines, R1.1, EL-4, and Scid.adh for the developmental staging. METHODS: We examined the expression of development marker genes and proteins in three lymphoma cell lines by flow cytometry and RT-PCR. In addition, the expression of apoptosis-related molecules including bcl-2, bax and Fas was also investigated. RESULTS: As previously reported, Scid.adh cell line expressed CD8 and CD25 but not TCR alpha chain, while R1.1 cells expressed TCR alpha chain and both CD4 and CD8 transcripts. These suggest that R1.1 might be in double positive stage, and low level of CD44 expression and the absence of CD25 support this suggestion. In contrast, EL-4 cells showed high level of TCR alpha chain transcript, and low-level of CD4 expression, suggesting that EL-4 is in more mature stage than R1.1. Further, this suggestion was supported by the lack of mT-20 in EL-4 cells, which is expressed in the immature thymocytes, and Scid.adh and R1.1 cell lines, but not in the terminally differentiated thymocytes and peripheral T cells. Among the apoptosis-related gene, transcripts of bcl-2 gene were detected in both R1.1 and EL-4 but not in Scid.adh cells, while bax was expressed in all cell lines. Fas expression was the highest in EL-4 cells and low in Scid.adh cell line. CONCLUSION: R1.1 cell may represent double positive stage, and EL-4 is more differentiated cell line. In addition, Scid.adh and EL-4 cell lines are suspected to be useful for the study of function of bcl-2 family and Fas during the thymocyte development, respectively.
Apoptosis ; Cell Line* ; Flow Cytometry ; Genes, bcl-2 ; Humans ; Lymphoma* ; T-Lymphocytes ; Thymocytes

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Suppressed DNA Repair Mechanisms in Rheumatoid Arthritis.

Sang Heon LEE ; Gary S FIRESTEIN

Immune Network.2002;2(4):208-216. doi:10.4110/in.2002.2.4.208

BACKGROUND: Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity. Key members of the MMR system include MutS alpha (comprised of hMSH2 and hMSH6), which can sense and repair single base mismatches and 8-oxoguanine, and MutS beta (comprised of hMSH2 and hMSH3), which repairs longer insertion/deletion loops. METHODS: To provide further evidence of DNA damage, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells (PBC) of RA patients using specific primer sequences for 5 key microsatellites. RESULTS: Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis (OA) tissue. Western blot analysis of the same tissues for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Western blot analysis demonstrated constitutive expression of hMSH2, 3 and 6 in RA and OA FLS. When FLS were cultured with SNAP, the RA synovial pattern of MMR expression was reproduced (high hMSH3, low hMSH6). CONCLUSION: Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.
Arthritis ; Arthritis, Rheumatoid* ; Blood Cells ; Blotting, Western ; DNA Adducts ; DNA Damage ; DNA Mismatch Repair ; DNA Repair* ; DNA* ; Humans ; Microsatellite Instability ; Microsatellite Repeats ; Nitric Oxide ; Nitrogen ; Osteoarthritis ; Oxidative Stress ; Oxygen ; Polymerase Chain Reaction ; S-Nitroso-N-Acetylpenicillamine ; Synovial Membrane ; Tissue Donors

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Production of Plasma Leptin and Expression of Interferon-gamma Inducible Protein-10 (IP-10), Monokine Induced by Interferon-gamma (Mig) and Interleukin-8 (IL-8) mRNA in Kawasaki Disease.

Young Hwan LEE ; Won Duk KIM ; Hee Sun KIM

Immune Network.2002;2(4):202-207. doi:10.4110/in.2002.2.4.202

BACKGROUND: Kawasaki disease is an acute febrile illness with systemic vasculitis which primarily affects children, We examined the production of leptin in plasma and gene expressions of CXC chemokines in peripheral blood mononuclear cells from patients with Kawasaki disease. METHODS: Consecutive 39 samples from 13 patients according to the different clinical stages (acute, subacute, convalescent) of Kawasaki disease were collected. The plasma leptin levels according to clinical stages of Kawasaki disease were examined by ELISA and the expression of IP-10, Mig and IL-8 mRNAs in 39 samples (13 samples of each stage) from 13 cases were examined by RT-PCR. RESULTS: There were not significant changes of plasma leptin levels according to the clinical stages of Kawasaki disease. The mean values of plasma leptin concentrations during each of the stages (n=13, p>0.05, pg/ml) were 335.8+/-549.0 in acute, 358+/- 347.6 in subacute, and 443.6+/-645.9 in convalescent stage. The mRNAs of IP-10, Mig, and IL-8 were expressed in 13/13 (100%), 2/13 (15%), 9/13 (69%) during acute stage, 13/13 (100%), 6/13 (46%), 13/13 (100%) during subacute stage, and 13/13 (100%), 4/13 (31%), 10/13 (77%) during the convalescent stage, respectively. In three patients, the production of leptin and expression of IP-10 mRNA were dramatically decreased according to the process of the clinical stages. In five patients with prominent cervical lymphadenopathy, the expression of IL-8 mRNA during the subacute stage was more elevated than the acute and convalescent stages. CONCLUSION: This data suggests that the production of leptin and the gene expressions of IP-10, Mig and IL-8 seem to have no significant correlation to the clinical stages of Kawasaki disease. However, expression patterns of IP-10, Mig and IL-8 mRNA may be related to the specific clinical manifestations, and the expression of IP-10 may also be correlated to leptin levels with pericardial involvement.
Chemokines, CXC ; Child ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Interferon-gamma* ; Interleukin-8* ; Leptin* ; Lymphatic Diseases ; Mucocutaneous Lymph Node Syndrome* ; Plasma* ; RNA, Messenger* ; Systemic Vasculitis

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Identification of Functionally Different Rat IgE in RBL-2H3 Exocytosis.

Jin Sub KIM ; Sungae CHO ; Kyoung Hwan JOO ; Joon Sang LEE ; Daniel H CONRAD ; Sung Weon CHO

Immune Network.2002;2(4):195-201. doi:10.4110/in.2002.2.4.195

BACKGROUND: IgE is closely related to the development of allergies. However, the poor relationship between the specific IgE level and the severity of allergic diseases suggests the possibility of functionally different IgE isoforms. With this in mind, rat basophilic leukemia (RBL)-2H3 activation was analyzed with each type of rat IgE for two parameters, exocytosis and IL-4 mRNA production. RBL-2H3 has been well documented in the rat mucosal mast cell line. METHODS: RBL-2H3 cells sensitized with each kind of rat IgE was activated by cross-linking FcRI with B5 (monoclonal anti-rat IgE mouse IgG antibodies). The RBL-2H3 exocytosis was measured by analyzing the beta-hexosaminidase level, and the level of IL-4 mRNA synthesis was analyzed using semi- quantitative RT-PCR. Rat IgE, which was produced by a parasite infection (REP), was prepared using either Paragonimus westermani metacercariae (REP-PW) or Anisakis simplex third stage larvae (REP-AS). A rat IgE prototype of IR162 was prepared by a peritoneal injection of immunocytoma. RESULTS: The level of exocytosis showed a linear relationship with the rat IgE concentration when REP-PW or REP-AS was applied. However, it exhibited a biphasic response with IR162. In addition, the time course of heating at 56oC illustrated the similarity between REP-PW and REP-AS, which differed from that of IR162. In contrast, the level of IL-4 mRNA synthesis in the RBL-2H3 cells with IR162 was comparable to that of either REP-PW or REP-AS. CONCLUSION: These results suggest that functionally different rat IgE isoforms exists in RBL-2H3 exocytosis.
Animals ; Anisakis ; Basophils ; beta-N-Acetylhexosaminidases ; Exocytosis* ; Heating ; Hot Temperature ; Hypersensitivity ; Immunoglobulin E* ; Immunoglobulin G ; Interleukin-4 ; Larva ; Leukemia ; Mast Cells ; Metacercariae ; Mice ; Paragonimus westermani ; Parasites ; Protein Isoforms ; Rats* ; RNA, Messenger

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Psoriasis as a T-cell-mediated Immunologic Disease.

Wook LEW

Immune Network.2002;2(4):189-194. doi:10.4110/in.2002.2.4.189

Although the exact mechanism responsible for the pathogenesis of psoriasis is unclear, interferon-gamma producing type 1 T cells have been reported to play a significant role. Infiltrating activated type 1 T cells in the lesions are believed to be responsible for stimulating keratinocytes, which produce many cytokines and growth factors. The hyperproliferative epidermis is understood to be the result of either the cytokines produced by the intraepidermal T cells or the reactive phenomenon after keratinocyte damage. The microenvironment in psoriatic lesions deviates toward the type 1 status, because of the increased type 1 cytokines and either the decreased or unchanged type 2 cytokines observed in psoriatic lesions. Therefore, this review focused on a T-cell-mediated immunological basis for the current hypothesis of the psoriasis pathogenesis.
Cytokines ; Epidermis ; Immune System Diseases* ; Intercellular Signaling Peptides and Proteins ; Interferon-gamma ; Keratinocytes ; Psoriasis* ; T-Lymphocytes

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