The cytolytic activity of CD4' T cells, both human and murine, has been clearly demonstrated in the immune response to mycobacterial infection and suggested to play a significant role in the protection and immunopathology. However, Uttle is known about the differentiation of CD4' CTL. In order to address this issue, we examined the influences of some factors on the generation of CD4' CTL specific to mycobacterial antigens. After 7 days' stimulation of PBMCs from healthy tuberculin reactors with mycobacterial antigens, the cytolytic activity of purised CD4' T cells toward autologous macrophages infected with mycobacteria was measured by Cr release assay. First, we found that both of live M. tubeiculosis and soluble antigens (ST-CF) induced the cytolytic activity of CD4' T cells, although the inducibility of the former was slightly greater than the latter. Second, the cytolytic activity was maximally induced at the relatively low antigen concentration (0.2:1 bacteria:monocyte ratio or 0.5 mg/ml of ST-CF). Finally, in the presence of increasing amounts of neutralizing anti-IL-12 or anti-IFN-r MoAb, the cytolytic activity of CD4+ T cells was decreased in a dose-dependent manner. These results suggest that low dose of antigen, its particulate type give mycobacteria), IL-12, and IFN-r give some positive signals for the generation of CD4+ CTL.
Humans ; Interleukin-12 ; Macrophages ; T-Lymphocytes* ; Tuberculin

Apoptosis is a distinct mechanism of cell death involved in many physiological and pathological processes. Various stimuli, including phagocytosis of bacteria, can induce apoptosis. As the cells proceed through apoptosis, functional activities decline in accord with phenotypic changes. However, decline in functional activities does not mean instantaneous shut-down of all functions, which is rather the characteristic of necrosis. Phagocytosis and oxidative burst are two of the major tasks of mloid cells for engulfment and killing of microbes. It was reported that the myeloid cells which phagocytosed bacteria underwent apoptosis, rendering resolution of acute inflammation. On the contrary, it was known that phagocytosis of latex beads did induce apoptosis. However, we found phagocytosis of latex beads within the apoptotic cell fraction. Thus we investigated whether phagocytosis of latex beads induced apoptosis or apoptotic cells phagocytosed the beads. We used human promyelocytic cell line HL-60 cultured for 4 days in RPMI1640 supplemented with 10% fetal bovine serum and 1 uM all-trans retinoic acid for phagocytic assay. Phagocytic activity was analyzed by flow cytometry after shaking incubation of HL- 60 cells (5 x 10 cells/ml) with fluorochrome-cougated latex beads for 1 hour at 37C followed by elimination of the un-phagocytosed beads by centrifugation on the density of fetal bovine serum. Apoptotic cells were identified as subdiploid fraction by staining the cells with DNA-dye. To investigate whether phagocytosis of latex beads leads to apoptosis or apoptotic cells phagocytose the beads, the cells wbich had phagocytosed the beads were sequentially analyzed before and after 1, 3, 6, 12 and 24 hours of incubation. On the other hand, the apoptotic cell fraction was sorted to be analyzed for phagocytic activity. The sorted cells were also analyzed by chemiluminescence assay for capability of oxidative burst by stimulation with PMA (5 mM). The results showed little increase in the apoptotic fraction among phagocytic cells during incubation up to 24 hours. Rather the sorted apoptotic cells did phagocytose latex beads. But the sorted cells did not show any capability of oxidative burst. Taken these results into consideration, the apoptotic cells seemed to be on the way of dying process in which oxidative burst was lost while phagocytic activity remained. Thus it was suggested that the primitive function of phagocytosis remained longer in the cells proceeding through apoptosis, while oxidative bunt, requiring mitochondrial function, was lost earlier.
Apoptosis ; Bacteria ; Cell Death ; Cell Line ; Centrifugation ; Flow Cytometry ; Hand ; Homicide ; Humans ; Inflammation ; Luminescence ; Microspheres ; Myeloid Cells ; Necrosis ; Pathologic Processes ; Phagocytes ; Phagocytosis ; Respiratory Burst ; Tretinoin

To study the gyrA mutations of E. coli from clinical specimens, 410 strains were isolated from 1994 to 1997 in Kyungpook National Vniversity hospital. Antimicrobial susceptibility tests, PCR and sequencing of gyrA, and in vitro induction of quinolone resistance were done. The frequency of quinolone resistant E. coli strains increased constantly during 1994 through 1996. Quinolone-resistant strains were more often resistant to unrelated antibiotics than quinolone-susceptible strains (chi-square test, p<0.05). All of the randomly selected 55 quinolone- resist#ant strains were highly resistant to nalidixic acid (NAL) but had low level resistance to fluoroquinolones. All of the 55 quinolone-resistant strains showed an amino acid substitution of Ser -> Leu (TCG -> TIG) at codon 83. In addition, four different types of amino acid substitution affecting codon 87 (Asp) were detected, 1) type I: Asn (GAC -> AAC); 2) type II: Tyr (GAC -> TAC); 3) type III: Oly (GAC -> GGC); 4) type IV: His (GAC -> CAC). The mutation of type IV has not been reported previously in quinolone-resistant E. coli strains. It is thought that the specific amino acid substitution probably affects minimum inhibitory concentrations (MIC) of quinolones because the MICs of ciprofloxacin, norfloxacin, and ofloxacin in type II were significantly higher than those of type I. By in vitro induction, MICs to quinolone-susceptible strains resulted in the increase in the MICs of all quinolones tested by 2- to 2048-fold. The induced mutants by quinolones had amino acid substitutions at codon 83, SerLeu or Asp87Asn, Gly or Tyr. Alteration of Ser83 results in the most effective increase in the MIC of quinolone such as NAL and alterations of Asp87 result in the effective increase of MIC of fluoroquinolone. These results suggest that the continuous use of quinolones might induce the specific amino acid substitution at gyrA.
Amino Acid Substitution ; Anti-Bacterial Agents ; Ciprofloxacin ; Codon ; Escherichia coli* ; Escherichia* ; Fluoroquinolones ; Gyeongsangbuk-do ; Microbial Sensitivity Tests ; Nalidixic Acid ; Norfloxacin ; Ofloxacin ; Polymerase Chain Reaction ; Quinolones

A recent study showed that comparative sequence analysis of rpoB DNAs could reveal natural relationships in genus Mycobacterium [J Clin Microbiol. 37 (6). 1999]. rpoB DNAs showed interspecies variation and intraspecies conservation, Based on these data, we developed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) protocols which enable species differentiation in genus Mycabacterium. When this assay was applied to 24 clinical isolates identified as M. avium complex (MAC) by biochemical test, these were successfully differentiated into M. avium and M. intracellulare. These results were concordant with those obtained by 16s rDNA analysis. It is the first report that PCR-SSCP analysis of rpoB DNA could be used for species differentiation of MAC strains.
DNA* ; DNA, Ribosomal ; Mycobacterium avium Complex* ; Mycobacterium avium* ; Mycobacterium* ; Polymerase Chain Reaction* ; Polymorphism, Single-Stranded Conformational* ; Sequence Analysis

Invasion of enteric bacteria, such as Salmonella and invasive E. coli, into intestinal epithelial cells induces proinflammatory gene responses and finally epithelial cell apoptosis. In this study, we asked whether invasive bacterial infection of human intestinal epithelial cells could upregulate cyclooxygenase-2 (COX-2) gene expression and whether increased COX-2 expression could influence intestinal epithelial cell apoptosis. Expression of COX-2 mRNA and prostaglandin (PG) E production were upregulated in HT-29 colon epithelial cells which were infected with S. dublin or invasive E. coli, as examined by quantitative RT-PCR and radioimmunoassay. Inhibition of COX-2 expression and PGE2 production using NS-398, a specific COX-2 inhibitor, showed a significant increase af epithelial cell apoptosis and caspase-3 activation in HT-29 cells infected with invasive bacteria. However, the addition of valerylsalicylate, a specific COX-1 inhibitor, did not change apoptosis in S. dublin-infected HT-29 cells. These results suggest that upregulated COX-2 expression and PGE2 production in response to invasive bacterial infection could contribute to host defense by inhibiting apoptosis of intestinal epithelial cells.
Apoptosis* ; Bacteria ; Bacterial Infections* ; Caspase 3 ; Colon ; Cyclooxygenase 2 ; Dinoprostone ; Enterobacteriaceae ; Epithelial Cells* ; Gene Expression ; HT29 Cells ; Humans ; Prostaglandin-Endoperoxide Synthases* ; Radioimmunoassay ; RNA, Messenger ; Salmonella

Spirochetes were isolated from the midgut of Ixodes persulcatus ticks captured at Chungju, Korea and identified as Borrelia afzelii strains by polymerase chain reaction. To determine the pathogenicity of the B. afzelii strains isolated in Korea, the microbiological and pathological features of Lyme disease were observed in C3H/He mice after intraperitoneal inoculation of the fresh isolate of B. afzelii strain. The results are summarized as follows 1) The Borrelia were detected in the tissues of heart, spleen, kidney, urinary bladder and knee joint within 7 days after inoculation of infection by dark field microscopic examination. The isolation rate from heart, urinary bladder and joint was significantly higher than the rate from spleen, kidney, and blood samples. 2) The Borrelia was detected in heart muscle by indirect immunofluorescent antibody test. 3) Antibody to the Borrelia was detected as early as one week after inoculation. 4) The marked tropism of the Borrelia was observed in myocardial, urinary tract and joint tissue. The main pathological features are inflammation in tissues of heart, kidney, joint and urinary bladder. From these results, the Borrelia afzelii strain isolated in Korea were determined as pathogenic strain.
Animals ; Borrelia burgdorferi Group* ; Borrelia burgdorferi* ; Borrelia* ; Chungcheongbuk-do ; Heart ; Inflammation ; Ixodes ; Joints ; Kidney ; Knee Joint ; Korea* ; Lyme Disease ; Mice ; Myocardium ; Pathology ; Polymerase Chain Reaction ; Spirochaetales ; Spleen ; Ticks ; Tropism ; Urinary Bladder ; Urinary Tract ; Virulence*

Growth under conditions of iron-restriction and the production of siderophore was examined in Vibrio mimicus ATCC 33653. This strain grew and multiplied in the presence of the high-affinity iron chelators ethylenediamine-di (o-hydroxyphenylacetic acid). Chrorne azurol S (CAS) agar and solution were used to detect the production of siderophore under these condition. Siderophore could be detected in the iron-rcstricted culture supernatants. The siderophore was extracted from iron-restricted culture supernatants by phenol-chloroform-ether method and purified by Dowex ion-exchange and Sephadex G-25 gel filtracton chromatography. The purified siderophore was confirmed by paper chromatography and HPLC. The Purified siderophore enhanced the growth of V. mimicus when the bacterium was grown in iron limited medium. Injection of both the siderohore and the bacteria to mice resulted in more rapid death than that of the only bacteria. However, the siderophore did not show lethality to mice and any toxicity to cell line like HeLa and U937.
Agar ; Animals ; Bacteria ; Cell Line ; Chelating Agents ; Chromatography ; Chromatography, High Pressure Liquid ; Chromatography, Paper ; Iron ; Mice ; Vibrio mimicus* ; Vibrio*

Astrovirus is frequently associated with diarrhea in children. It can not be readily isolated by cell culture, and an electronmicroscope is usually used for detection of this agent. Recently in 1995 a combined method of reverse transcription-polymerase chain reaction (RT-PCR) was designed for easier detection of astrovirus, which is based on the conserved sequence in 3'-end of genomes of the 7 known serotypes of human astrovirus. As of yet there has not been any report of astrovirus data in Korea using the RT-PCR methods. The purpose of this study was to detect astrovirus incidence, severity of symptoms, seasonal variation and coinfection rate with rotavirus in Korean children inpatients with diarrhea. Fecal specimens from 61 young children hospitalized with gasteroenteritis Korea from Jan. 1996 through Mar. 1997. They were examined for astroviurs infection by RT-PCR method. Results are as follows: 1. Astrovirus was detected at 9.8% (6/61) from fecal specimens of children with severe diarrhea by EIA using monoclonal antibody coated plates. 2. Astorvirus was detected at 29.5% (18/61) from fecal specimens of children with severe diarrhea by RT-PCR. 3. The age of the 18 children affected by astrovirus ranged from 2 monthes to 7 years with mean of 3.0 years. 4. Mean hospital stay of the 1S children was 6.1 days. 5. Five (27.8%) astrovirus RT-PCR positive strains were confirmed in November and in December, respectively out of 18 specimens in total. 6. Astrovirus coinfection with rotavirus type G1 was confirmed in 15/16 specimens (93.8%), and with type G2 was in 1/16 specimens (6.3%).
Cell Culture Techniques ; Child* ; Coinfection ; Conserved Sequence ; Diarrhea ; Feces* ; Genome ; Humans ; Incidence ; Inpatients ; Korea ; Length of Stay ; Mamastrovirus ; Polymerase Chain Reaction* ; Reverse Transcription* ; Rotavirus ; Seasons

Eight strains of multidrug-resistant (MDR) Salmonella typhi were isolated from Kyonggi area during January-February,1997. They were resistant to ampiciUin, amoxicillin, carbeniciillin, tetracycline, chloramphenicol, trimethoprim/sulfamethoxazole, trimethoprim. Eight strains had one plasmid respectively which size was approximately M.W 220 kb and showed same restriction pattern by endonuclease HindIII. The plasmid was similar to the plasmid in size that was related to multidrug resistant S. typhi isolated from southeast Asia. It were transferred by conjugation to recipient E, coli K-12 in frequency of 2.43 x10-4 - 1.73 x 10-2 and transconjugant showed same drug-resistant pattem with donor cells. All of 8 strains produced B-lactamase that was assummed to TEM-1 type by isoelectric focusing and PCR.
Amoxicillin ; Asia, Southeastern ; Chloramphenicol ; Deoxyribonuclease HindIII ; Gyeonggi-do ; Humans ; Isoelectric Focusing ; Korea* ; Plasmids* ; Polymerase Chain Reaction ; Salmonella typhi* ; Salmonella* ; Tetracycline ; Tissue Donors ; Trimethoprim

Orientia tsutsugamushi is obligate intracellular bacterium that grows within the cytoplasm of the eukaryotic host cells. Therefore capability of the attachment, entry into the host cell and intracellular survival should be critical process for oriential infection. In this study we investigated the cellular invasion mechanism of Orientia tsutsugamushi and the role of transmembrane heparan sulfate proteoglycan, which binds diverse components at the cellular microenvironment and is implicated as host cell receptors for a variety of microbial pathogens. First of all Orientia tsutsugamushi can invade a wide range of nonprofessional phagocytic cells including fibroblast, epithelial cells a#nd endothelial cells of various host species, including B and T lymphocytes. Thus, it was postulated that the attachment of O. tsutsugamushi requires the recognition of ubiquitous surface structures of many kinds of host cells. Treatments with heparan sulfate and heparin inhibited the infection of Orientia tsutsugamushi in dose-dependent manner for L cell, mouse fibroblast, whereas other glycosaminoglycans such as chondroitin sulfate had no effect. Collectively, these findings provide strong evidence that initial interaction with heparan sulfate proteoglycan is required for the oriential invasion into host cells.
Animals ; Cellular Microenvironment ; Chondroitin Sulfates ; Cytoplasm ; Endothelial Cells ; Epithelial Cells ; Eukaryotic Cells* ; Fibroblasts ; Glycosaminoglycans ; Heparan Sulfate Proteoglycans ; Heparin ; Heparitin Sulfate ; Mice ; Orientia tsutsugamushi* ; Phagocytes ; T-Lymphocytes