Mutations in the embB Locus among Korean Clinical Isolates of Mycobacterium tuberculosis Resistant to Ethambutol

Resistance of Mycobacterium tuberculosis to ethambutol (EMB) has been assigned to an operon, embCAB, which has been proposed to be a structural gene for mycobacterial arabinosyl transferases. Recently, genetic events resulting in structural mutations at embB have been proposed as major contributors to the EMB-resistance of isolates whose minimum inhibitory concentration (MIC) level is higher than 20 microgram/ml. On the contrary, isolates with a MIC level lower than 20 microgram/ml do not seem to contain any sequence alterations. In this study, in an effort to understand the role of embB mutations at a low-level of EMB resistance, we investigated the sequence polymorphisms of clinical isolates whose MIC levels are lower than 10 microgram/ml. Accordingly, the sequence alterations of a 312-bp region of the embB gene containing the 306th codon, which has been assigned as a hot-spot for EMB-resistance related mutations, were determined for 21 EMB-resistant and 5 EMB-susceptible clinical isolates. In brief, among 21 EMB- resistant isolates examined, 12 (57.1%) contained mutations in embB (10 at the 306th codon and 2 at other sites), and the remaining isolates 9 contained no mutations in any region of embB. The observed mutations included M306V, M306I, and M306L substitutions that have been reported previously. However, 3 were novel types, which included M306T, A313G and Y322C, D331Y double substitutions. On the other hand, all of the EMB-susceptible isolates were found to be free of mutations. In conclusion, our findings suggest that sequence polymorphism of embB may play a pivotal role in the EMB- resistance of M. tuberculosis.

Prevalence of Antibodies to PPD and Lipoarabinomannan of Mycobacterium tuberculosis among Patients with an Indication of Fine Needle Aspiration Biopsy

Recent increase in the incidence of lung cancer often makes it difficult to differentiate between lung cancer and tuberculosis (TB), due to their radiologic similarities. Fine needle aspiration biopsy (FNAB) has been widely employed for the diagnosis of lung cancer and TB, but the diagnostic accuracy of TB is not high enough. As a rapid screening test for tuberculosis, we evaluated serological tests using Mycobacterium tuberculosis PPD and lipoarabinomannan (LAM) antigens. A total of 95 patients with indication of FNAB cytology from initial CT findings were enrolled. 25 patients had TB, 76 thoracic malignancy, and six (7.9%) of the lung cancer patients also had TB, indicating much higher prevalence of TB in thoracic tumor patients. Antibodies to PPD were elevated in 18 (72.0%) of 25 TB patients and in 22 (31.4%) of 70 patients with thoracic malignancy. In contrast, only 3 (4.7%) of 64 healthy controls aged 40 or above were seropositive to PPD antigen. The prevalence of anti-PPD antibodies in thoracic tumor patients was therefore significantly greater than that amongst the healthy controls (p 0.001, chi-square test). However, no significant difference in the prevalence of anti-LAM antibodies was found between study subjects and controls. This study demonstrates that thoracic tumor patients have significantly elevated antibodies to PPD; therefore, high anti-PPD seroreactivity in thoracic tumor patients should be cautiously interpreted. A longitudinal investigation on seropositive thoracic tumor patients is required to determine the role of the serological test for TB in lung cancer patients.

Effects of IL-12 DNA Vaccine on Reactivation of Mycobacterium tuberculosis in Cornell Model

No Abstract Available.

Diversity of Humoral Immune Responses to Recombinant Proteins of Brucella abortus Among Residents in Cheju Province

No Abstract Available.

Diverse Expression of NK Cell Receptor between Fetal Thymocytes and Fetal Liver Lymphocytes from the Same Individuals / 대한면역학회지

Fetal thymus may be the organ for NK cell maturation, but the in vivo evidences are few, Here, by analyzing NK cell receptor, we present that NK cells develop in fetal thymus and fetal liver and that NK cell receptor appears earlier than the expression CD16 or CD56. Moreover, the finding that the repertoire of NK cell receptor is different between fetal thymus and fetal liver lymphocytes suggests that the environmental factors may influence the NK cell receptor repertoire during NK cell maturation.

Expression of the 38 kDa Protein of Mycobacterium tuberculosis in M . bovis BCG and Use in the Serodiagnosis of Tuberculosis

The 38 kDa protein of Mycobacterium tuberculosis, which was known previously as antigen 5, has been extensively used in the serodiagnosis of tuberculosis. In an attempt to develop and evaluate a serodiagnostic test using the antigen, we expressed the 38 kDa protein in BCG and its seroreactivity was compared to that expressed in Escherichia coli. The coding region of the 38 kDa protein was amplified by PCR, and the gene was cloned into a Mycobacterium-E. coli shuttle expression vector pYMC-his and pQE30 expression vector and expressed in BCG and E. coli, respectively. Both recombinant 38 kDa proteins showed strong seroreactivity against pooled serum from tuberculosis patients. There was no significant difference in seroreactivity between the two recombinant antigens in sera from the far advanced tuberculosis patients. However, of 25 tuberculosis patients graded as ""minimal"" by chest X-ray, 5 (20.0%) were seropositive by r38 kDa expressed in E. coli, while 8 (32.0%) by that expressed in BCG. Likewise, higher seroreactivity by r38 kDa expressed in BCG was found in sera from the moderately advanced tuberculosis. This study thus indicates that the recombinant 38 kDa expressed in BCG is more effective than that expressed in E. coli in detecting antibodies to the native 38 kDa protein of M. tuberculosis in sera from minimally affected tuberculosis patients.

Molecular analysis of HLA-DR gene expression induced by IFN-gamma in malignant melanoma cell lines

Human leukocyte antigen (HLA) class II molecules are polymorphic cell surface glycoproteins that are crucial for the cellular interaction in immune response. The expression of class II molecules is regulated in a tissue-specific and cytokine-inducible manner, and is mainly restricted to the antigen presenting cells. However, some tumor cells also express class II molecules, and in some class-II-negative tumor cells, class II expression is inducible by interferon (IFN)-gamma. However, their expression varies, even though the tumor cells originate from the same histological origin; some tumor cells show strong expression, others show weak or no expression. To determine whether this differential expression of class II molecules on tumor cells is transcriptionally regulated, FACS analysis and Northern hybridization were performed using a panel of melanoma cell lines, IGR3, Malme-3M, SK-Mel-24, and SK-Mel-28 to analyze the cell surface expression and mRNA transcription rate of HLA-DR before and after treatment with IFN-gamma. FACS analysis showed that before IFN-gamma treatment, IGR3 and Malme-3M cells barely expressed HLA-DR. On the contrary, almost all of the SK-Mel-24 cells (> 90%) and a relatively high rate (> 50%) of SK-Mel-28 cells expressed HLA-DR. After IFN-gamma treatment, HLA-DR expression was induced in Malme-3M cells and SK-Mel-28 cells which displayed elevated levels of HLA-DR expression in a time-dependent manner. However, IGR3 cells never responded to IFN-gamma. Northern analysis showed that treatment with IFN-gamma led to the steady-state mRNA augmentation of the HLA-DR gene in Malme-3M and SK-Mel-28, whereas in IGR3, IFN-gamma did not augment the transcriptional rate of the HLA-DR gene. To further clarify this differential modulation, sequencing analysis of PCR product of the HLA-DR proximal promoter region was done, since the transcription rate of the class II gene is controlled by the well-conserved proximal promoter region. Six independent clones from PCR products of the HLA-DRA proximal promoter region and 16 clones from PCR products of the HLA-DRB proximal promoter region were isolated from the above cell lines and sequenced. Comparison of the nucleotide sequences of all 6 clones of DRA promoter showed that the sequences are extremely similar in both regulatory sequences and their intervening sequences. Sixteen clones of HLA-DRB promoter showed sequence variations such as substitution and insertion/deletion, and these 16 clones could be further grouped into 6 homologues with sequence homology. These data established that the melanoma cell lines studied here showed a differential susceptibility to IFN-gamma on the modulation of HLA-DR molecules, that this modulation is transcriptionally regulated, and that the difference in promoter activity by sequence variation might contribute to such a differential transcriptional regulation at the promoter level.

Monophosphoryl lipid A (MPL) upregulates major histocompatibility complex (MHC) class I expression by increasing interferon-gamma (IFN-gamma)

Tumor immunity is primarily mediated by cells as CD8+ cytotoxic T lymphocytes (CTL) recognize tumor antigen by MHC class I molecules. But most tumors are associated with a decreased expression of MHC class I to escape the antitumor immunity of the host. Our previous data have demonstrated that MPL has an antitumor effect on metastatic lung cancer of B16 melanoma with enhancing cytotoxicity due to increase of IFN-gamma and IL-2, and decrease of IL-4, which indicates the stimulation of type 1 helper T cells (Th1). To determine the effects of MPL, IFN-gamma, TNF-alpha, and IL-1 alpha on MHC class I expression of B16 melanoma cells, we evaluated the expression of MHC class I molecules with treatments of MPL, IFN-gamma, TNF-alpha, and IL-1 alpha by flow cytometry. The supernatant of MPL-treated spleen cells in vitro upregulated the expression of MHC class I molecules of B16 melanoma cells compared to the control supernatant of spleen cells. The MHC class I expression of B16 melanoma cells treated with IFN-gamma, but not TNF-alpha or IL-1 alpha, increased in a time-dependent manner. In conclusion, MPL upregulated MHC class I expression of B16 melanoma cells by activating spleen cells via IFN-gamma. These data suggest that increased IFN-gamma by MPL is responsible for the upregulation of MHC class I expression to augment cytotoxicity. Therefore, we suggest that MPL could play an important role in immunotherapy.

Factors affecting transformation efficiency of BCG with a Mycobacterium-Escherichia coli shuttle vector pYUB18 by electroporation

BCG has been one of the vehicles for multi-recombinant vaccine. However, low transformation efficiency of BCG with plasmid DNA hampered studies involving expression of foreign antigens in BCG. In an effort to determine the optimal conditions, this study was initiated to investigate factors involved in the transformation of BCG with a Mycobacterium-Escherichia coli shuttle vector, pYUB18, by electroporation. Mycobacterium bovis BCG (strain 1173P2) was grown in Middlebrook (M) 7H9 broth containing albumin-dextrose-catalase and 0.05% tween 80, and transformed BCG was grown in M7H10 agar containing kanamycin for counting viable cells. Pretreatment of BCG with 10 mM CaCl2 improved the transformation efficiency, but overnight incubation of BCG with 1% glycine did not. The transformation efficiency in BCG also varied depending on voltage, resistance, and DNA concentration. The maximum transformation efficiency was obtained when the infinity resistance, 12.5 Kv/cm, and 100 ng of DNA were used, and reached 1.4 x 10(5) CFU/microgram of plasmid DNA, which is about 3-100 times greater than those from previous reports. The transformation conditions described in this study, therefore, will give us a better position for employing BCG as a vehicle for developing multi-recombinant vaccines.

Expression of p58 in Fetal Thymocytes and Fetal Liver Lymphocytes / 대한면역학회지

Bipotent progenitors for T and natural killer (NK) lymphocytes are thought to exist among early precursor thymocytes or liver lymphocytes. The identification of such a progenitor population or mature NK cells in such organs remains undefined. Here we report the identification of a novel receptor of NK cells, p58 (HLA class I-specific inhibitory receptors), in fetal thymocytes and fetal liver lymphocytes. Our finding suggests the NK cells mature in the developmental stage during feta1 ontogeny. Flow cytometric analysis revealed p58 positive cells in thymocytes or in fetal liver lymphocytes and reverse transcription PCR also showed amplification of p58 RNA. The result of single stranded conformational polymorphism (SSCP) showed it discriminates one or two base pair differences of the p58 gene. Although the question still remains as to whether the expression of p58 is due to the NK cells or natural T cells, it is clear the p58 is expressed in fetal thymocytes or liver lymphocytes. And SSCP analysis using appropriate sets of primers used in this study, is helpful to study the diversity of p58.

Evaluation of PCR-SSCP vs. PCR - Sequence Analysis for Detecting Rifampicin Resistance of Mycobacterium tuberculosis Clinical Isolates

In the present study, we made an attempt to compare polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with PCR-direct sequence analysis for their accuracy and sensitivity in detecting resistance to rifampicin (RMP). A total of 32 clinical isolates of Mycobacterium tuberculosis including 22 resistant and 10 sensitive isolates, whose drug susceptibility have been tested by conventional proportion method, were analyzed by using PCR-SSCP and PCR-sequence analysis. Among 22 RMP resistant isolates, 16 isolates showed SSCP profiles different from that of a RMP sensitive control strain, M. tuberculosis H37Rv indicating the possible existence of a sequence alteration in this region of the rpoB gene, while 6 resistant isolates displayed SSCP profiles indistinguishable from the sensitive control strain. On the other hand, all of 10 RMP sensitive isolates showed SSCP profiles similar to that of the sensitive control strain. Therefore, overall agreement rste between conventional proportion method and PCR-SSCP reached 81%. Subsequently, all of 32 clinical isolates were subjected to sequence analysis. The results from the sequence analysis revealed that all of 22 resistant isolates indeed contain mutations in the stretch of 81 bp region of rpoB gene, while none of 10 sensitive isolates contain any sequence alterations. Therefore, this study suggests that PCR-sequence analysis works more efficiently and accurately than PCR-SSCP analysis for rapid screening of RMP-resistant M. tuberculosis clinical isolates.

Construction of a Mycobacterium - Escherichia coli Shuttle Vector and Use in the Expression of Foreign Genes in Mycobacteria

The ability to introduce recombinant DNA molecules back into mycobacteria would greatly increase the potential of molecular genetic approaches for the study of mycobacteria as well as for the use in clinical purposes. We have initiated the construction of vectors that facilitates the introduction of recombinant DNA into mycobacteria. The vector was designed to contain replicons for multiplication in mycobacteria and Escherichia coli, a promoter for gene expression, a drug resistant gene for selecting transformants, and a few restriction enzyme sites for convenient cloning. Constructed Mycobacterium-E. coli shuttle vector named p YMC (hsp60) was shown to transform M. smegmatis at high efficiency and maintain plasmid at stable level. The ability of the vector to express cloned foreign gene was also monitored by measuring the expressed level of luciferase gene which was used as a reporter. High level of luciferase activity in M. smegmatis with pYMC (hsp60:luc) was detected confirming successful construction of Mycobacterium-E. coli shuttle vector.

Detection of Mycobacterium tuberculosis Antigens in Sputum for the Diagnosis of Pulmonary Tuberculosis

As an effort to develop a rapid and sensitive diagnostic test, we produced previously a monoclonal antibody (MAb) specific to the lipoarabinomannan (LAM) antigen and used in a sandwich ELISA for detection of mycobacterial antigens in sputum. In this study, we attempted to improve the antigen detection assay by combination of af5nity-purified antibodies against Mycobacterium tuberculosis soluble antigen and anti-LAM MAb. With the new assay, the LAM antigen was detectable as low as 2 ng/ml, and none of 10 gram-negative and gram-positive organisms gave significant absorbance, thus indicating the specific detection of mycobacterial antigens. Sputum samples from 62 patients who were suspected having tuberculosis and from 37 healthy controls were examined. The sensitivity of the antigen detection assay ranged from 0% in the 1+ culture group to 78.8% in the 3+ culture group. When the results were combined, 15 of 24 culture-positive samples were antigen-positive, thus giving an overall sensitivity of 62.5%. The overall specificity was 96.0%, when all the culture-negative samples were combined. The results thus demonstrate that the antigen detection assay can provide a rapid supplemental information for the diagnosis of pulmonary diagnosis.

Identification of the CDR3 Gene Sequence on the beta Chain of the T Cell Receptor in T Cell Leukemia Cell Line / 대한면역학회지

In order to develop a method for the detection of minimal residual leukemic disease (MRD) in T cell acute lymphocytic leukemia (T cell ALL), T cell leukemia cell line was used to detect clonal TCR p chain mRNA and to synthesize CDR3 specific oligonucleotide probe. For the Jurkat cell line clonal TCR p chain cDNA was amplified by using RT-PCR with oligonucleotide primer, Vp universal primer. As the result of RT-PCR an approximate 300 bp fragment of the TCR chain was obtained, and the partial identification of the TCR p chain gene and the amino acid sequence of the fragment were done by gene cloning and sequencing. The gene sequence of TCR p obtained was identified as Vp8-Jp1.2-Cp2. Diversity gene segment could not be found. Within the p chain, the CDR3 region was identified as 12 amino acids (SFSTCSANYGYT). It is kown that TCR is expressed in about 40% of the all T cell ALL. However it is not kown what percentage of the TCR p chain mRNA expression translates into the actual TCR molecule. It is not certain how many patients with MRD can be detected by this method used in this study, but this technique might be useful to detect MRD in at least 40% of the patients with T-cell ALL.

Effect of Anti - idiotype Antibody on Anti - DNA Antibody Production by Hybridoma Cells / 대한면역학회지

Anti-idiotype antibody (anti-id Ab) which recognizes idiotope in the variable region of immunoglobulin (Ig) can regulate Ab production by B cells in vivo and in vitro. Although it has been reported that anti-id Ab can suppress IgM production by lymphocytes or hybridoma cells without suppression of cell proliferation, the regulatory mechanism of anti-id Ab is not completely understood. We studied the effects of anti-id Ab on the production of IgG class anti-DNA Ab by hybridoma cells, on the proliferation of cells, and on the transcription levels of Ig genes. In contrast to suppressive effect of anti-id Ab on the production of IgM previously reported by others, stimulatory effects of anti-id Ab on the production of IgG by hybridoma cells as well as the proliferation of these .cells were observed. However, little effect of anti-id Ab on the transcription levels of Ig genes was observed. These results suggest that anti-id Ab can increase Ab production by stimulation of cell proliferation. Furthermore, these results suggest that the effect of anti-id Ab on the production of Ab may be determined by the difference in class of Ab produced by hybridoma cells following the treatment with anti-id Ab.

Detection of Antibodies to Nerve Antigens in Sera from Leprosy Patients and Relevance to the Nerve Damage / 대한면역학회지

No abstract available.

Induction of ICAM-1 and HLA-DR expression by IFN-gamma in malignant melanoma cell lines

Two human malignant melanoma cell lines, Malme-3M and SK-Mel-28, were analyzed for their ability to induce the expression of intercellular adhesion molecule 1 (ICAM-1) and human leukocyte antigen (HLA)-DR molecules on their cell surfaces as well as at the transcriptional level before and after treatment with interferon (IFN)-gamma. Both cell lines demonstrated a high percentage(> 99%) of ICAM-1 expression regardless of IFN-gamma treatment. Before IFN-gamma treatment, Malme-3M cells barely expressed HLA-DR molecules ( 50%) of HLA-DR expression. Both cell lines displayed elevated levels of HLA-DR expression in a time dependent manner after IFN-gamma treatment. However, these two cell lines have been shown to respond differentially to IFN-gamma. The molecular mechanism underlying such a differential behavior was investigated, and HLA-DR gene regulation was studied at the transcriptional level. Treatment with IFN-gamma led to the steady-state mRNA augmentation of the HLR-DR gene. The HLA-DRA mRNA augmentation was similar in both cell lines, whereas in Malme-3M, IFN-gamma did not augment the rate of transcription of the HLA-DRB gene as much as in SK-Mel-28. Data from this study established the fact that the melanoma cell lines displayed a differential susceptibility to IFN-gamma on the modulation of HLA-DR molecules, and this modulation was transcriptionally regulated.

Discrepancy in T cell clonal expansions in synovial fluid and peripheral blood from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease involving the synovial membrane of peripheral joints. T cells specific for self antigens may play a critical role. Identification of T cell receptors (TCR) of such specific T cell clones is very important for treatment, prevention and identification of relevant autoantigens. To identify specific T cells, TCR V beta family repertoire and the clonal expansion of T cells were analyzed in this study. The percentage of V beta 5+ or V beta 8+ cells in the synovial fluid mononuclear cells (SFMCs) was similar to that in the peripheral blood mononuclear cells (PBMCs). However, the percentage of DR+ T cells in the SFMCs was higher (p< 0.01). Analyzing the clonality of T cells in 8 V beta families (V beta 1, V beta 5, V beta 8, V beta 14, V beta 16, V beta 17, V beta 18, V beta 20), clonal expansions in CD8+ T cells from the SFMCs were found more frequently than in the PBMCs. The patterns of clonal expansions were discrepant between the SFMCs and the PBMCs even in the same patient, which suggests several inflamed tissue specific T cell clonal expansions in the SFMCs. These T cell clones might be activated by autoantigens which are not identified yet and responsible for the RA pathogenesis.

Detection of Mycobacterium leprae in Skin Biopsy Sepcimens From Leprosy patients by Polymerase Chain Reaction / 대한피부과학회지

BACKGROUND: Polymerase chain reaction(PCR) has brought an oppotunity for rapid detection of Mycobacterium leprae in clinical pecimens for the diagnosis of leprosy. Th DNA segment specific to M. leprae was detectable in a matteir of hours and DNA from one orgnisa appeared positive by PCR. However, the PCR tool has not been evaluated using elinical specimeriis from leprosy patients and controls. OBJECTIVE & METHODS: The primers amplifying 372bp segment of rebetitive sequence of M. leprae DNA were used in PCR. Skin biopsy specimens from 102 leprosy patient, and controls were examined for the presence of M. leprae by PCR and the results were aomared with microscopic and histopathologic findings. RESULTS: 1. As a result, of PCR after DNA preparation of M. leprae, six other mycobacteria, ten other bacteria, and skin from leprosy with five other skin biopsy tissues, 372bp DNA fragment was specifically amplified from M. leprae. 2. Dot blot, hybridization of PCR products showed that the 372bp DNA from skin biopsy specimens were derived from M. leprae. 3. As a result of PCR after DNA preparation of 10-fold diluted M. legrae from mouse footpad, PCR gave a positive result as low as one organism. 4. Of 87 specimens in which acid-fast bacilli were found under microcopic examinations 97% had positive PCR results. 5. Of 97 specimens which hadihistopathologic evidences of leprosy 95% had positive PCR results. 6. Of 15 specimens in which acid-fast bacilli were not found under n!icroscopic examinations 73% had positive PCR results. In three of five cases which had neither histopathologic nor microscopic evidences of leprosy had positive PCR results. CONCLUSION: PCR method amplifying 372bp fragment of repetitive seqi,ence was highly sensitive and specific in detecting M. leprae DNA in skin biopsy specimens, thus may be a useful tool as an additive diagnostic method, espcially for cases where microscopic antihystopathologic findings are not definite.

Nuclear protein binding patterns in the 5'-upstream regulatory elements of HLA class I genes

The expression of MHC class I genes has been thought to be regulated by two major cis-acting regulatory elements. The first region, enhancer A (Enh A) spanning from positions -210 to -165 contains perfect palindrome (PP), TGGGGATTCCCCA. The PP is well-conserved both in mouse and human MHC class I genes, even though the PP is disrupted by 2 bp substitutions (TGAGGATTCTCCA) in HLA-C genes. Three proteins binding to the Enh A of HLA-A and -B locus genes, but very weakly or nearly not to the Enh A of HLA-C locus gene have been identified. To determine functional importance of the PP for binding of trans-acting protein, mutant DNA probes were made by site-directed in vitro mutagenesis and then electrophoretic mobility shift assay was performed. HLA-A mutant DNA probe, in which the PP is disrupted, shows the same nuclear protein binding pattern as that of the HLA-C gene, and HLA-C mutant DNA probe, in which the PP is introduced, shows the same nuclear protein binding pattern as that of the wild type HLA-A gene. These data suggest that the perfect palindrome and its cognate DNA binding nuclear protein play an important role in the HLA class I gene regulation, and thus the lower expression of HLA-C antigen may be ascribed to no or very weak factor binding to the nonpalindromic sequences of HLA-C upstream DNA.