COX-2 and iNOS are critical in advanced glycation end product-activated chondrocytes in vitro.

BACKGROUND: The advanced glycation end products (AGEs) accumulate in joints of osteoarthritis patients. This study aimed to investigate the roles of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) pathways in AGE-mediated cartilage damage. MATERIALS AND METHODS: Methylglyoxal-modified albumin was used as the source of AGE. Porcine and human chondrocytes were prepared from the joint cartilage of pigs and osteoarthritis patients. The activation of COX-2, iNOS, nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and protein kinases was determined by Western blotting, kinase assay, electrophoretic mobility shift assay (EMSA) or transfection assay. Prostaglandin E(2) (PGE(2)) and NO concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and Griess reaction respectively. The enzymatic activity of COX was determined by measuring the conversion of arachidonic acid to PGE(2). The release of sulphated glycosaminoglycan and the intensity of Safranin O staining were used to measure cartilage degradation. RESULTS: AGE potently induced COX-2-PGE(2) and iNOS-NO activation in porcine and human chondrocytes. Meanwhile, the upstream molecules regulating COX-2/iNOS activation, such as AP-1, NF-kappaB, extracellular signal regulated protein kinase (ERK) and c-jun N-terminal kinase (JNK), were activated by AGE. Although AGE could not activate p38 directly, by measuring COX enzyme activity, the inhibition of p38 resulted in suppressing AGE-induced conversion of arachidonic acid to PGE(2). Furthermore, successful blockage of either COX-2 or NOS activity significantly reduced AGE-mediated proteoglycan release and cartilage degradation. CONCLUSIONS: This study highlights the significance of COX-2 and iNOS pathways in AGE-mediated OA pathogenesis and their potential as therapeutic targets that are beyond pain killing for OA treatment.

Western and Chinese antirheumatic drug-induced T cell apoptotic DNA damage uses different caspase cascades and is independent of Fas/Fas ligand interaction.

Spontaneous or therapeutic induction of T cell apoptosis plays a critical role in establishing transplantation tolerance and maintaining remission of autoimmune diseases. We investigated the mechanisms of apoptosis induced by Chinese and Western antirheumatic drugs (ARDs) in human T cells. We found that hydroxychloroquine, Tripterygium wilfordii hook F, and tetrandrine (Tet), but not methotrexate, at therapeutic concentrations can cause T cell death. In addition, Tet selectively killed T cells, especially activated T cells. Although ARD-induced cytotoxicity was mediated through apoptotic mechanisms, Fas/Fas ligand interaction was not required. We further demonstrated that the processes of phosphatidylserine externalization and DNA damage along the ARD-induced T cell apoptotic pathway could operate independently, and that selective inhibition of DNA damage by caspase inhibitors did not prevent T cells from undergoing cell death. Moreover, we found that Tet- and Tripterygium wilfordii hook F-induced T cell DNA damage required caspase-3 activity, and hydroxychloroquine-induced T cell DNA damage was mediated through a caspase-3- and caspase-8-independent, but Z-Asp-Glu-Val-Asp-fluomethyl ketone-sensitive, signaling pathway. Finally, the observation that ARD-induced activation of caspase-3 in both Fas-sensitive and Fas-resistant Jurkat T cells indicates that Fas/Fas ligand interaction plays no role in ARD-induced T cell apoptosis. Our observations provide new information about the complex apoptotic mechanisms of ARDs, and have implications for combining Western and Chinese ARDs that have different immunomodulatory mechanisms in the therapy of autoimmune diseases and transplantation rejection.

Infection of human dendritic cells by dengue virus causes cell maturation and cytokine production.

Dengue virus (DV) infection is a major problem in public health. It can cause fatal diseases such as Dengue hemorrhagic fever and Dengue shock syndrome. Dendritic cells (DC) are professional APCs required for establishing a primary immune response. Here, we investigated the role of human PBMC-derived DC in DV infection. Using different techniques, including plaque assay, flow cytometry analysis, nested RT-PCR, and confocal microscope and electron microscope examinations, we show that DV can enter cultured human DC and produce virus particles. After entrance, DV could be visualized in cystic vesicles, vacuoles, and the endoplasmic reticulum. The DV-infected DC also showed proliferation and hypertrophy of the endoplasmic reticulum as well as the swollen mitochondria. In addition, the DV-stimulated DC could express maturation markers such as B7-1, B7-2, HLA-DR, CD11b, and CD83. Furthermore, the infection of DC by DV induced production of TNF-alpha and IFN-alpha, but not IL-6 and IL-12. Although DC underwent spontaneous apoptosis in the absence of feeding cytokines, this process appeared to be delayed after DV infection. Our observations provide important information in understanding the pathogenesis of DV infection.

Aspirin differentially regulates endotoxin-induced IL-12 and TNF-alpha production in human dendritic cells.

OBJECTIVE: In the development of autoimmune diseases, dendritic cells (DC) play critical roles. Here, we examined the effect of aspirin on lipopolysaccharide (LPS)-induced DC activation. METHODS: The monocyte-derived DC were established. The cytokine production was measured by ELISA, reverse transcriptase/polymerase chain reaction, or intracellular staining analyzed by flow cytometry. The expression of cell surface molecules was determined by flow cytometry. RESULTS: Aspirin inhibited LPS-induced DC maturation and costimulatory molecules expression. Aspirin, at therapeutic concentrations, also decreased LPS-induced IL-12 and IL-10 production. In contrast, the LPS-induced TNF-alpha production was enhanced by aspirin. The differential effects of aspirin on IL-12 and TNF-alpha production may not be due to down-regulation of cyclooxygenase activities. CONCLUSION: The various effects of aspirin on LPS-stimulated DC may influence the understanding of the diverse immunomodulatory mechanisms of this anti-inflammatory drug.

Plant alkaloid tetrandrine and its analog block CD28-costimulated activities of human peripheral blood T cells: potential immunosuppressants in transplantation immunology.

BACKGROUND: T lymphocyte activation mediated by CD28 costimulation plays a critical role in graft rejection. Plant alkaloid tetrandrine, purified from a Chinese antirheumatic herb, is a potent immunosuppressant. Here, we examined its effects on several CD28-costimulated T-cell activities. In addition, such effects were readily compared with the effects of three tetrandrine analogs. METHODS: T lymphocytes were purified from whole blood by negative selection. The stimuli that mimic CD28 costimulation included both anti-CD3 + anti-CD28 monoclonal antibody and PMA+anti-CD28 monoclonal antibody. The determination of CD28-costimulated cell proliferation was performed by tritium uptake, cytokine production by ELISA, cell surface interleukin 2Ra and CD69 expression by flow cytometry, and mixed leukocyte reaction by tritium uptake. Drug cytotoxicity was determined by trypan blue exclusion, propidium iodide staining, and MTT colorimetric assays. RESULTS: Tetrandrine inhibited CD28-costimulated T-cell proliferation and cytokine production through a mechanism different from that of cyclosporine. In addition, tetrandrine down-regulated both T helper 1 and T helper 2 cytokine production in CD4+ and CD8+ T-cell subpopulations. By examining cytokine production and T-cell activation marker expression, we further demonstrated that, among tetrandrine and its analogs tested, dauricine was the most potent suppressor of CD28-costimulated T-cell activities. Furthermore, the different immunosuppressive activities of these compounds were not associated with their cytotoxic capacities. Finally, the unparalleled inhibitory potency of dauricine on both mixed leukocyte reaction and CD28-costimulated T-cell proliferation suggests that dauricine preferentially targeted CD28-costimulated T-cell activities. CONCLUSIONS: This is the first report to show that tetrandrine and its analogs potently inhibited both PMA+CD28-costimulated and CD3 + CD28-costimulated activation of human peripheral blood T cells. Based upon their structural similarity and different immunosuppressive potency, these in vitro data also provide very useful information for further identification and development of more potent and less toxic immunosuppressants to achieve transplantation success.

Plant alkaloid tetrandrine downregulates protein kinase C-dependent signaling pathway in T cells.

Tetrandrine, a purified traditional Chinese medicinal herb that acts as an immunosuppressant and a Ca2+ channel blocker, has been clinically used to treat patients with arthritis, silicosis and hypertension. Since T cells play a critical role as autoreactive and pathogenic population in autoimmune diseases, in this study, we examined the immunosuppressive effect of tetrandrine on human peripheral blood T cells. We showed that tetrandrine inhibited phorbol 12-myristate 13-acetate (PMA) + ionomycin-induced T cell proliferation, interleukin-2 secretion and the expression of the T cell activation antigen, CD71. Further investigation of the molecular mechanism demonstrated that tetrandrine inhibited the expression of the protein kinase C-dependent interleukin-2 receptor alpha chain and CD69 but not the expression of the Ca2+-dependent CD40 ligand and CD69. Interestingly, when tetrandrine and cyclosporin A were added together, significant synergism in the suppression of T cell activation was observed. Moreover, of the several tetrandrine analogues studied, hernandezine was the most potent inhibitor of protein kinase C signaling events. These results also suggest that the protein kinase C-inhibitory capacity of tetrandrine and its analogues may not be associated with their function as Ca2+ channel blockers. Lastly, we showed that, within therapeutic concentrations, tetrandrine and its analogues could induce cellular apoptosis, which is defective in autoimmune diseases. In conclusion, our findings provide novel information about the molecular mechanism of the immunosuppressive effect of tetrandrine and its analogues in human peripheral blood T cells.

Mechanism of immunosuppression of the antirheumatic herb TWHf in human T cells.

OBJECTIVE: To investigate the immunosuppressive mechanism of Tripterygium wilfordii Hook-F (TWHf) in human T cells. TWHf, a traditional Chinese medicinal herb for rheumatoid arthritis, has been shown to inhibit the function of immune effector cells such as neutrophils, macrophages, and B lymphocytes. METHODS: T cell survival was evaluated with trypan blue exclusion assay, morphologic changes with Wright's stain, the induction of endonuclease activity with DNA fragmentation assay, and the subdiploid DNA content with flow cytometry. T cell activation was measured with interleukin 2 (IL-2) ELISA and the expression of several surface molecules with flow cytometry. RESULTS: At high dosages, TWHf caused inhibition of T cell proliferation and this mechanism was mediated through the induction of apoptosis. TWHf, in noncytotoxic dosages, was as potent as cyclosporin A and more potent than prednisolone and cyclophosphamide in inhibiting IL-2 production from activated T cells. TWHf also inhibited both phorbol 12-myristate 13-acetate induced IL-2Ralpha expression and ionomycin induced CD40 ligand expression. TWHf did not reverse downregulated expression of CD3 and CD4 by phorbol ester stimulation. CONCLUSION: This is the first evidence that the immunosuppressive mechanism of TWHf in T cells was mediated through both downregulation of T cell receptor signaling pathway and induction of cellular apoptosis, which is defective in autoimmune diseases.

Development of prostate-specific antigen promoter-based gene therapy for androgen-independent human prostate cancer.

PURPOSE: The goal of this study is to develop a tissue-specific toxic gene therapy utilizing the prostate specific antigen (PSA) promoter for both androgen-dependent (AD) and androgen-independent (AI) PSA-secreting prostate cancer cells. Ideally this gene therapy would be effective without the necessity of exposing the target cells to circulating androgens. MATERIALS AND METHODS: An AI subline of LNCaP, an AD PSA-secreting human prostate cancer cell line, C4-2, was used in this study. Castrated mice bearing C4-2 tumors secrete PSA. A transient expression experiment was used to analyze the activity of two PSA promoters, a 5837 bp long PSA promoter and a 642 bp short PSA promoter, in C4-2 cells. A recombinant adenovirus (Ad-PSA-TK) carrying thymidine kinase under control of the long PSA promoter was generated. The tissue-specific activity of Ad-PSA-TK was tested in vitro and in vivo. RESULTS: The long PSA promoter had superior activity over short PSA promoter, and higher activity in C4-2 cells than in LNCaP cells. High activity of Ad-PSA-TK was observed in C4-2 cells in an androgen deprived condition. In vitro, Ad-PSA-TK was further demonstrated to induce marked C4-2 cell-kill by acyclovir in medium containing 5% FBS. No cell-kill was observed in control WH cells (a human bladder cancer cell line). In vivo, Ad-PSA-P-TK with acyclovir significantly inhibited subcutaneous C4-2 tumor growth and PSA production in castrated animals. CONCLUSION: The 5837 bp long PSA promoter was active in the androgen free environment and could be used to target both androgen-dependent and independent PSA-producing prostate cancer cells in vitro, and prostate tumors in castrated hosts.

Characterization and distribution of insertion sequence IS1239 in Streptococcus pyogenes.

The human pathogenic bacterium Streptococcus pyogenes causes pharyngitis, acute rheumatic fever, glomerulonephritis and toxic-shock-like syndrome. The bacterium synthesizes several extracellular products, including the recently described streptococcal superantigen SSA, a molecule that shares considerable homology with several Staphylococcus aureus enterotoxins. While studying allelic variation at the ssa locus, six isolates expressing serotypes M4, M23, M33, M41, M43, and provisional type PT4854, were identified that had PCR products about 40-bp larger than expected, and one isolate (M15) had an amplified fragment that was more than 1-kb larger than expected. All six isolates have a 34-bp insert located 103 bp 5' of the ssa start codon. The larger product is a result of a 1110-bp insertion at the analogous location. The complementary strand of this insert has a 981-bp open reading frame that potentially encodes a 326-amino-acid polypeptide with substantial homology to the Escherichia coli IS30 transposase. Results of Southern blot analysis showed that at least twelve copies of the sequence are present in the serotype M15 S. pyogenes isolate. This element, designated IS1239, is the first simple insertion sequence described in group-A streptococci. Results of PCR screening showed that 26 of 78 (33%) S. pyogenes isolates expressing distinct M protein serotypes contained sequences with homology to IS1239, which means that the element is widely distributed in the species.

Epidemiology of penicillin-resistant Neisseria gonorrhoeae isolated in Taiwan, 1960-1990.

The epidemiology of penicillin-susceptible Neisseria gonorrhoeae isolated in Taiwan from 1960 to 1990 is summarized. The isolation of N. gonorrhoeae less sensitive to penicillin (i.e., with intrinsic resistance) was first reported in 1960. The rate at which organisms less sensitive to penicillin (MIC, greater than or equal to 0.5 microgram/mL) were isolated increased to 17%, 50%, 80.1%, and 88.8% in 1967, 1975, 1984, and 1990, respectively. Penicillinase-producing N. gonorrhoeae (PPNG) first appeared in Taiwan in late 1976, and the first six strains of PPNG isolated were from United States military servicemen who had relocated from Southeast Asia. The percentage of PPNG strains rose to 37.82% in 1982, and has remained high (50%-62%) since 1983. In the present study, resistance of N. gonorrhoeae to spectinomycin (MIC, greater than 32 micrograms/mL), third-generation cephalosporins (MIC, greater than 4 micrograms/mL), or quinolones (MIC, greater than 4 micrograms/mL) has not been found. Strains requiring arginine, hypoxanthine, and uracil for growth, which frequently cause disseminated gonococcal infections, were not isolated. PPNG strains tended to be of the prototrophic auxotype (55%); non-PPNG strains were mostly of the proline-requiring auxotype (48.8%). Two kinds of R plasmids were isolated in the PPNG strains: the 4.4-MD Asian type (82%-95%) and the 3.05-MD Toronto type (5%-18%). All of the PPNG strains possessing Toronto R plasmid were of the same auxotype/serotype (prototrophic/IB). Evidence suggests that the Asian-type R plasmid was imported into Taiwan in 1976, while the Toronto-type R plasmid may have first emerged in Taiwan in 1983.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of plasmid pattern in paediatric intensive care unit outbreaks of nosocomial infection due to Enterobacter cloacae.

Sequential outbreaks of nosocomial infection due to multiply-resistant Enterobacter cloacae occurred in September 1987, and between December 1988 and January 1989, in a paediatric intensive care unit. A total of eight neonates were affected and most had received ventilatory support. Initially, we were unable to determine whether the two outbreaks were caused by the same strain of E. cloacae. After applying plasmid profile analysis to identify epidemic strains, we established that the strain from the first outbreak was different from the second outbreak strain, as each had its own plasmid pattern. During the second outbreak, an environmental bacteriological survey was carried out. We found that the distilled water containers were contaminated with E. cloacae which had the same plasmid profile. After changing the distilled water containers and by reinforcement of aseptic techniques, the nosocomial outbreak was terminated.

[Epidemiological characterization of Neisseria gonorrhoeae isolates from Taiwan. Part II: Distribution of auxotyping, serogrouping and plasmid profile].

Epidemiology of gonococcal infection in Taiwan was investigated. Six hundred twelve isolates from 7 cities in 1983-1984 were examined for auxotyping by Hendry and Stewart's method, for serotyping by coagglutination of monoclonal antibodies with the antigenic specificity of gonococcal protein I molecules and for plasmid profile by the alkaline quick method. The results are described below: (1) Thirty-six auxotypes were found. Prototropic (Prototype) 45.4% (284/612) and Proline type (Pro) 38.7% (237/612) were the two dominant auxotypes. Arg- Hyp- Ura- which was thought to be correlated with disseminated gonococcal infection was not found. (2) Fifty-five percent penicillinase producing Neisseria gonorrheal (PPNG) were prototype and 48.8% non-PPNG were Pro. (3) Sixteen serotypes were identified among 56 strains. There was 17.8 (10/56 which belonged to the IA group and 82.2% (46/56) to the IB group. (4) Five molecular weight plasmids were found. They were 2.6 Mdal, 3.05 Mdal, 4.7 Mdal, 7.8 Mdal and 24.5 Mdal. (5) There were 7 plasmid profiles including 2.6 Mdal (26.4%); 2.6 Mdal +24.5 Mdal (23.2%); 2.6 Mdal+7.8 Mdal+24.5 Mdal (0.4%); 2.6 Mdal+4.7 Mdal (8.9%); 2.6 Mdal+4.7 Mdal+24.5 Mdal (38.2%); 2.6 Mdal+4.7 Mdal+24.5 Mdal (0.2%) and 2.6 Mdal+3.05 Mdal+24.5 Mdal (2.4%). (6) All the isolates harbored cryptic 2.6 Mdal plasmid. Eighty three percent of the PPNG and 48.6% of the non-PPNG harbored 24.5 Mdal plasmid. Twelve out of 305 PPNG isolates were "Toronto" type 3.05 Mdal plasmid. Others were "Asian" 4.7 Mdal plasmid. There were no "African" type 3.2 Mdal plasmids found.