Tumor necrosis factor-n (TNF - alpha) involved in the pathogenesis of multiple sclerosis and contribute to the degeneration of oligodendrocytes as well as neurons. TNF - alpha is produced by miocroglia and astrocytes, which also produce hormones and cytokines that influence its biological activity. Astrocytes, the major glial cells in the CNS, are capable of producing TNF - alpha at both the mRNA and protein levels in response to interleukine-1 (IL-1) or TNF - alpha. Two immunosuppressive cytokines, transforming growth factor - beta (TGF - beta) and IL-10, have been shown to influence glial cell function. TGF - beta can modulate the activity of glial cells by inhibiting interferon-gamma (IFN - gamma) induced expression of class II major histocompatibility complex (MHC) molecules on astrocytes and microglia. To explore the role of astrocytes in the production of TNF - alpha, astrocytes were pretreated with IL-10 or TGF - beta and then stimulated with IL-1p to determine their effects on TNF - alpha production. The secretion of TNF - alpha by human fetal astrocytes was markedly inhibited by TGF - beta at a low concentration. In contrast IL-10 had no effect on TNF - alpha mRNA level. These results show that TGF - beta may regulate the expression of TNF - alpha in activated human fetal astrocytes.
Astrocytes* ; Cytokines ; Gene Expression* ; Humans* ; Interferon-gamma ; Interleukin-10 ; Major Histocompatibility Complex ; Microglia ; Multiple Sclerosis ; Necrosis ; Neuroglia ; Neurons ; Oligodendroglia ; RNA, Messenger ; Transforming Growth Factors

Poliovirus Sabin 1 strain has its own special features that make it a particularly attractive live recombinant mucosal vaccine vehicle. Sabin 1 cDNA was manipulated to have multiple cloning site and viral specific 3C-protease cutting site at the N-terminal end of the polyprotein, named RPS-vax system HIV-1 V3- and principal neutralizing domain (PND)-concatamers were successfully cloned into the multiple cloning site of the vector system and produced expected chimeric viruses by transfection of their RNA transcripts into HeLa cells. These chimeric viruses have shown to express introduced HIV-1 subgenome concatamers efficiently during their replication in the infected HeLa cells. Expressed proteins were confirmed to retain the wild type structures at least in parts. Replication capacity of the chimeric viruses was slightly lower than that of wild type Sabin 1 likely to be due to delay in processing steps during their replication. Differing from the virulent Mahoney vectors, the rec-Sabin 1 chimeric viruses maintained the foreign gene stably during the serial passages. These chimeric viruses have also shown to be able to induce specific humoral immunity to the introduced vaccine proteins when inoculated into the poliovirus receptor-expressing transgenic (Tg-PVR) mice. Antiserum obtained from the immunized transgenic mice showed to have neutralizing capacity to HIV-1 in vitro. These results strongly suggest that the chimeric viruses expressing HIV-1 vaccine epitopes can be used as a good live mucosal vaccine candidate against AIDS.
Animals ; Clone Cells ; Cloning, Organism ; DNA, Complementary ; Epitopes ; HeLa Cells ; HIV* ; HIV-1* ; Humans* ; Immunity, Humoral ; Mice ; Mice, Transgenic ; Poliovirus* ; RNA ; Serial Passage ; Transfection

A putative gamma herpesvirus, termed human herpesvirus 8 (HHV-8), discovered in recent years, has been implicated as a possible etiologic agent for Kaposi`s sarcoma (KS). In South Korea, the incidence of KS in HIV seropositive individuals is very low. The cause of its rarity as compared with other countries is unclear. The objective of this study was performed to determine the prevalence of infection with HHV-8 and to clarify the cause of low incidence of KS in Korean populations including HIV seropositive individuals. The study population was composed of 200 blood donors, 220 voluntary visitors for sexual transmitted infection (STI)-testing in the public health centers, and 214 HIV-seropositive individuals. For the detection of HHV-8 antibodies, all blood samples were tested using Advanced Biotechnologies Inc`s enzyme-linked immunosorbent assay (ELISA) kits and the reactive samples were retested using Biotrin International SARL`s immunofluorescent assay (IFA). Also, we investigated the seroprevalence of Cytomegalovirus (CMV), Varicella-Zoster virus (VZV) and Epstein-Barr Virus (EBV) in order to get more information of HHV-8 and other human herpesviruses transmission in Korea. The prevalence of specific IgG to HHV-8 among HIV seropositive individuals was 7.0% {95% confidential interval: 4.0-11.3%}. The specific antibody to HHV-8 could be detected only in HIV seropositive men. The prevalences of antibodies to other human herpesviruses unlike HHV-8 were very high even in blood donors. These observations strongly suggest that the rarity of KS in this country may be caused by very low prevalence of HHV-8.
Antibodies ; Biotechnology ; Blood Donors ; Cytomegalovirus ; Enzyme-Linked Immunosorbent Assay ; Herpesviridae ; Herpesvirus 3, Human ; Herpesvirus 4, Human ; Herpesvirus 8, Human* ; HIV ; Humans* ; Immunoglobulin G ; Incidence ; Korea* ; Male ; Prevalence ; Public Health ; Sarcoma* ; Sarcoma, Kaposi ; Seroepidemiologic Studies*

Respiratory syncytial virus (RSV) and Influenza virus are the most common pathogen for causing severe upper respiratory infection in all age groups. A multiplex reverse transcription polymerase chain reaction (RT-PCR) has been developed to detect and subtype influenza A (H3N2 and H1N1), B virus and RSV simultaneously in one tube reaction. Amplification with primers derived from conserved sequences within the nucleocapsid for RSV and hemagglutinin subunit for Influenza A (H3N2 and H1N1) and B viruses yielded a 384 bp, a 300 bp, a 236 bp and a 151 bp, respectively. Assay specificity was confirmed by pulse field gel electrophoresis and autosequencing method. Assay sensitivity was 3 PFU/ml of RSV, 22 PFU/ml, 45 PFU/ml of Influenza type A (H3N2 and H1N1) and 6.6 PFU/ml of Influenza B virus by plaque assay. A rapid and sensitive detection method of a one-tube with multiplex RT-PCR capable of identifying more than one viral template as well as synchronizing reverse transcription and PCR had the potential to produce considerable savings of time and cost effectiveness in the diagnostic laboratory.
Conserved Sequence ; Cost-Benefit Analysis ; Electrophoresis ; Hemagglutinins ; Herpesvirus 1, Cercopithecine ; Humans* ; Income ; Influenza B virus ; Influenza, Human* ; Nucleocapsid ; Orthomyxoviridae* ; Polymerase Chain Reaction* ; Respiratory Syncytial Virus, Human* ; Respiratory Syncytial Viruses ; Reverse Transcription* ; Sensitivity and Specificity

A total of 152 strains of Streptococcus pyogenes were isolated from patients with pharyngitis, scarlet fever, skin infection, or invasive streptococcal infections in Seoul, Korea from January 1988 to December 1999. All isolates were epidemiologically characterized to decide phenotypes by T protein serotype and serum opacity factor (OF) detection. Genetic diversity of the isolates were analyzed by emm genotyping and pulsed-field gel electrophoresis (PFGE). T protein serotype showed 17 kinds in distribution and T12 (40.1% of study strains), T4 (19.1%), and T1 (7.9%) were the prevalent ones. When sources of S. pyogenes isolates were analyzed by T serotype distribution, T12 type was predominant in pharyngitis and skin infection isolates which contributed to 30 strains (49.2%) and 11 strains (18.0%), respectively. When T serotype of S. pyogenes isolates were analyzed by emm genotype distribution, of the 61 isolates of T12 type, 48 strains (78.7%) belonged to the emm type 12 (M12) and of the 29 isolates of T4 type, 27 strains (93.1%) belonged to the emm genotype 4 (M4). PFGE of genomic DNA of different emm genotype (emm12, emm4 and emm1) showed distinctive patterns. When the DNA of same emm gene type isolates were analyzed genetic relatedness by PFGE pattern, emm4, emm1, and emm12 types showed over 90%, 75%, and 70% of genetic similarity, respectively. Therefore, it was suggested that these emm genotype isolates were closely related genetically whereas among the isolates of other emm genotypes showed less than 30% of genetic similarity. Show genotypes are more diverse in comparison with phenotypes. In even epidemiologically unrealated isolates, genetic subtypes appeared correlated. The phenotypic and genotypic analysis used in the study were discriminative and appropriate for epidemiological study of S. pyogenes.
DNA ; Electrophoresis, Gel, Pulsed-Field ; Epidemiologic Studies ; Genetic Variation ; Genotype ; Humans ; Korea* ; Pharyngitis ; Phenotype ; Scarlet Fever ; Seoul* ; Skin ; Streptococcal Infections ; Streptococcus pyogenes* ; Streptococcus*

Mycobacterium tuberculosis infected macrophages can become ineffective at activating CD4+ T cells through presentation of peptide antigens by MHC class II, possibly contributing to the ability of M tuberculosis to persist despite the presence of an intact immune system. Presentation of lipid antigens may help to overcome this problem. CD1 represents the key component of an MHC independent pathway for presentation nonpeptide lipid antigens to T cells. The 38 kDa glycolipoprotein antigen of M. tuberculosis is actively secreted. The antigen induces strong antibody and T-cell responses and provided partial protection against M. tuberculosis infection in mice when it is administered either entrapped in biodegradable microparticles or in the form of a DNA vaccine. But an selective anergy to stimulation with peptide of the 38 kDa was observed in the majority of tuberculosis patients. An 38 kDa antigen has been isolated by affinity chromatography using a monoclonal antibody. This antigen contains some immunosuppressive cell wall associated antigens such as lipoarabinomannan. Therefore, we purified the 38 kDa glycolipoprotein from the culture filtrate of M tuberculosis H37Rv by ammonium sulfate precipitation (55~80%), hydroxylapatite and DEAE-Sephacel column. The purified antigen showed three major bands on isoelectric focusing gel, and two-dimensional electrophoresis (2-DE) analysis of this antigen revealed five distinct spots of the 38 kDa molecular mass. One of five spots had a N-terminal sequence identical to that of the 38 kDa glycolipoprotein (pstS-1). Other protein spots could not determine sequences. An antiserum against the recombinant 38 kDa antigen of M tuberculosis reacted strongly with the purified the 38 kDa antigen.
Ammonium Sulfate ; Animals ; Cell Wall ; Chromatography, Affinity ; DNA ; Durapatite ; Electrophoresis ; Humans ; Immune System ; Isoelectric Focusing ; Macrophages ; Mice ; Mycobacterium tuberculosis* ; Mycobacterium* ; T-Lymphocytes ; Tuberculosis

In this study, we investigated interleukin (IL)-18 and IL-12 following in vitro stimulation with either the 30-kDa or purified protein derivative (PPD) antigens (Ag) of pleural mononuclear cells from 12 cases of tubercular pleurisy (TB-PMC) and 8 cases of malignant pleurisy (MG-PMC). Ag-stimulated TB-PMC produced significantly more IL-12 than did MG-PMC and the levels correlated with those of IFN - gamma. Although elevated IL-18 levels were found in freshly isolated pleural fluids, in vitro IL-18 production in response to either Ag was dramatically decreased in TB-PMC. Pro-IL-18 mRNA was detected before and after Ag stimulation in TB patients. Supernatants from the Ag-stimulated TB-PMC significantly suppressed IL-18 production in normal peripheral blood mononuclear cells (PBMC) and primary malignant cells over an 18 h incubation period. In addition, this suppressive activity was not inactivated by either heat or trypsin. Our findings imply that modulation of IL-12 and IL-18 levels may contribute to the Th1 elevation induced in human TB-P VIC by the 30-kDa and PPD antigens.
Hot Temperature ; Humans ; Interleukin-12* ; Interleukin-18* ; Interleukins ; Mycobacterium tuberculosis ; Pleurisy ; RNA, Messenger ; Trypsin ; Tuberculosis, Pleural

Six strains of Vibrio parahemolyticus isolated from diarrheal patients and the 12 strains from sea water were serotyped and analyzed for biochemical characteristics, antibiotics sensitivity and detection of toxR, gyrB, tdh, and trh genes. Arbitrarily-primed polymerase chain reaction method were performed on the 6 strains from patients and the following results were obtained. 1. The Vibrio parahemolyticus isolated from patients were belonged to 5 different serotypes: 04:K8, 04:KUT, 06: K18, 010:K71 and 03:K6, but those isolated from sea water were belonged to 5 different serotypes: O1:KUT, 02:KUT, 03:K45, 04:K37 and OUT:KUT. All strains explained have different serotypes depending on the different source, 2. Three serotype (04:K8, 04:KUT, 06:K18) isolated from patients were positive for the urease hydrolysis, whereas only one strain of serotype O1:KUT isolated from sea water was positive to the same. Furthermore, the serotype 06:K18 (1 strain) was positive for the fermentation of dulcito1. Both toxR and gyrB genes were detected from all strains isolated. 3. As for control the 2 strains of serotype 03:K6 and 6 strains isolated from patients, serotype 03:K6 were resistant to oxacillin, penicillin, vancomycin. All strains were sensitive to chloramphenicol and tetracycline yet the antibiogram type showed 6 groups from I to VI. 4. DNA probe hybridization method was used to detect genes. The trh1 was detected both from serotype 04:KUT and 06:K18 isolated from patients and the trh2 was also detected from one strain from each 010:K71 and O1:KUT isolated from patients and sea-water respectively, The tdh gene only was detected from two strains of 03:K6 isolated from patients of 1998. The tdh, trh 1 and trh2 were not detected from 7 strains out of 12 strains isolated from sea water whereas the production titer of TDH isolated from patients showed from 2048 times to 4096 times. 5. Four strains of the serotype 03:K6 isolated from Korea, India and Japan as well as 3 strains from Korean patients were tested by AP-PCR to classify serotypes. As for its result the amplicon showed the same in the 4 strains of the serotype 03:K6 whereas the four strains of different serotype from patients are so difference as to explain no inter- relations at all. The result explains that the serotype 03:K6 is the same genes regardless from where it is isolated.
Anti-Bacterial Agents ; Chloramphenicol ; DNA ; Fermentation ; Humans ; Hydrolysis ; India ; Japan ; Korea ; Microbial Sensitivity Tests ; Oxacillin ; Penicillins ; Polymerase Chain Reaction ; Seawater* ; Tetracycline ; Urease ; Vancomycin ; Vibrio parahaemolyticus* ; Vibrio* ; Virulence Factors* ; Virulence*

Clinical isolates of Enterobacteriaceae (189 Klebsiella, 61 Enterobacter, 32 Serratia, 19 E. coli, 7 Proteus, and 3 Citrobacter) from one university hospital were epidemiologically analyzed by using transferable R plasmids resistant beta-lactam antibiotics including broad-spectrum cephalosporins. About 30% of E. cloacae and S. marcescens and about 5% of K. pneumoniae were resistant to one or more broad-spectrum j3-lactam antibiotics including cefotaxim, ceftazidime, aztreonam, or cefoxitin but all isolates of E. aerogenes, K oxytoca, and P. mirabilis were susceptible. Thirty-six conjugative R plasmids including 8 plasmids resistant expanded-spectrum cephalosporins were obtained from multiple resistant K. pneumoniae (19), E. cloacae (9), E. coli (4), and C. freundii (1). Thirty-one plasmids were subjected to R plasmid analysis and classified 20 different plasmid types. Among them 5, 2, and 2 plasmids belong to 3 different types respectively showed identical molecular size, endonuclease fragment pattern by Southem hybridization pattern by TEM-1 probe, pI value by isoelctric focusing, and also identical antibiogram and biotype of wild strains harboring plasmids. But all of plasmids resistant to cefotaxim, ceftazidime, aztreonam or cefoxitin showed different palsmid anlysis patterns. These results indicate that the epidemic strains of 3 clonal types had been present in this hospital and anlysis using transferable R plasmid and bla gene can be used to discriminate multi-resistant clinical isolates of Enterobacteriaceae.
Anti-Bacterial Agents* ; Aztreonam ; Cefotaxime ; Cefoxitin ; Ceftazidime ; Cephalosporins ; Cloaca ; Dermatoglyphics* ; Enterobacter ; Enterobacteriaceae* ; Klebsiella ; Microbial Sensitivity Tests ; Mirabilis ; Plasmids* ; Pneumonia ; Proteus ; R Factors ; Serratia

This study describes the purification and characterization of type II restriction endonuclease of Helicobacter pylori in order to understand the DNA restriction and modification of H. pylori. H. pylori cell extract was subjected to polyethyleneimine treatment, salt precipitation, heparine-sepharose column chromatography, and fast protein liquid chromatography (FPLC) using Resource Q column and Mono Q column to purify the type II restriction endonuclease. Hpy51-I was characterized to recognize the sequneces 5`-GT(G/C)AC-3`, yielding 5-base 5` protruding ends. The restriction sequence was identical to that of Tsp 45 I. The enzyme exhibited its maximal activity in the presence of 10-20 mM LaCl, but was inhibited completely in the presence of more than 80 mM NaCl. The enzyme showed its maximal activity in the presence of 1-10 mM MgC1(2). The optimal pH and temperature for enzyme activity was pH 9.0 and 37 degrees C, respectively. MnC1(2) could not substitute for MgC1(2) in reaction mixture. And addition of j3-mercaptoethanol and bovine serum albumin in reaction mixture led to loss of enzyme activity of Hpy51-I. The whole cell extract of H. pylori strain 51 was confirmed to carry the enzyme activity for methylation of Hpy51-I-recognised sequence. Hpy51-I digested genomic DNAs of enteric bacteria to less than I kb while it could not cut the genomic DNAs of H. pylori isolates. In this study, the type II restriction enzyme (Hpy51-I) of H. pylori was identified and characterized its biochemical properties, demonstrating that Hpy51-I might be one of the barriers for preventing the introduction of foreign DNAs into H. pylori.
Chromatography ; Chromatography, Liquid ; DNA ; DNA Restriction Enzymes* ; Enterobacteriaceae ; Helicobacter pylori* ; Helicobacter* ; Hydrogen-Ion Concentration ; Methylation ; Polyethyleneimine ; Serum Albumin, Bovine