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- Johansson, RK, et al.
(författare)
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Expression of nitric oxide synthase in bladder smooth muscle cells: Regulation by cytokines and L-arginine
- 2002
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Ingår i: Journal of Urology. - 1527-3792. ; 168:5, s. 2280-2285
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: The expression and regulation of the different isoforms of nitric oxide synthase (NOS) in bladder smooth muscle cells are controversial and to our knowledge have not yet been studied systematically. Therefore, the expression and regulation of NOS were studied in rat bladder smooth muscle cells after stimulation with cytokines, lipopolysaccharide and L-arginine. Materials and Methods: Primary cell cultures were prepared from rat bladders. The expression of NOS mRNA was examined by reverse transcriptase-polymerase chain reaction and inducible NOS (iNOS) protein expression was studied by Western blot analysis and immunohistochemistry. Nitrite accumulation in the culture medium was determined by the Griess assay. The expression of iNOS was also studied immunohistochemically in whole bladder strips stimulated by cytokines. Results: NOS mRNA expression was not detected in unstimulated cells. Stimulating bladder smooth muscle cells with a cytokine mixture of interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta induced iNOS mRNA and protein expression. The combination of interleukin-1beta plus tumor necrosis factor-alpha appeared to be crucial for iNOS induction in bladder smooth muscle cells. Exposing bladder smooth muscle cells to lipopolysaccharide did not induce iNOS. Adding L-arginine increased nitrite accumulation in cytokine mixture stimulated bladder smooth muscle cells, while iNOS positive cells were detected in the smooth muscle layer of cytokine mixture stimulated bladder strips. Conclusions: NOS was not detected in unstimulated bladder smooth muscle cells. However, bladder smooth muscle has the potential to express iNOS when exposed to cytokines known to be produced during urinary tract infection.
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| 6. |
- Poljakovic, Mirjana, et al.
(författare)
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Escherichia coli-induced inducible nitric oxide synthase and cyclooxygenase expression in the mouse bladder and kidney
- 2001
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Ingår i: Kidney International. - : Elsevier BV. - 1523-1755 .- 0085-2538. ; 59:3, s. 893-904
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The host response to urinary tract infection includes the production of different inflammatory mediators. We investigated the cellular localization and time course of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) expression in the mouse bladder and kidney after bacterial infection. METHODS: Experimental urinary tract infection in mice was established by intravesical inoculation of a clinical uropathogen Escherichia coli (E. coli) AD 110. Urine was collected at 6-, 12-, 24-, and 72-hours postinstillation, and the nitrite concentration was determined. The induction of iNOS and COX-2 was studied by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Nitrite levels in the urine had increased threefold at 6 and 12 hours postbacterial instillation. Bladders from mice instilled with AD 110, but not with phosphate-buffered saline, showed a large number of iNOS-- and COX-2--expressing inflammatory cells. The inflammatory cell activation peaked at 6 and 12 hours postinstillation and had vanished by 72 hours. iNOS expression was detected in some urothelial cells after 24 and 72 hours, but COX-2 expression was not detected. In the kidney, infection activated an iNOS and COX-2 response, as shown by immunoreactivity in inflammatory cells at all time points. A strong epithelial iNOS response was observed in the renal pelvis at 12, 24, and 72 hours postinstillation, but COX-2 was not detected. Enhanced tissue expression of iNOS and COX-2 after bacterial instillation was also demonstrated by RT-PCR. CONCLUSIONS: E. coli AD 110 induced expression of iNOS and COX-2 in the urinary tract. Inflammatory cells expressed both iNOS-and COX-2, but epithelial cells expressed only iNOS and with a later onset than in the inflammatory cells. This suggests that the epithelial iNOS response is not caused by direct bacterial activation, but more likely is by mediators involved in the inflammatory response.
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| 7. |
- Poljakovic, Mirjana, et al.
(författare)
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Human renal epithelial cells express iNOS in response to cytokines but not bacteria.
- 2002
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Ingår i: Kidney International. - : Elsevier BV. - 1523-1755 .- 0085-2538. ; 61:2, s. 444-455
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Epithelial cells form the mucosal barriers that prevent the entry of mucosal pathogens, and respond to bacterial infections by producing various host defense molecules. In this study, we examined the inducible nitric oxide synthase (iNOS) response of primary human renal tubular epithelial cells (HRTEC) following infection with uropathogenic Escherichia coli Hu734, or stimulation with lipopolysaccharide (LPS) or cytokines. METHODS: Induction of iNOS was examined by RT-PCR, Western blot, immunohistochemistry and nitrite measurements. The effects of endogenously produced nitric oxide (NO), and exogenously applied DETA/NO, SIN-1 and H2O2 on cell viability were analyzed using a respiration assay. RESULTS: HRTEC did not produce NO following infection with E. coli Hu734, LPS alone, or in combination with interferon-gamma (IFN-gamma), even though these agents caused a marked increase in iNOS expression by RAW 264.7, a macrophage cell line. In contrast, iNOS protein and mRNA expression by HRTEC increased after exposure to a cytokine mixture consisting of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. This was due to the combination of IL-1beta and IFN-gamma, but the individual cytokines had no effect. Inducible NOS-expressing cell cultures showed reduced viability, and this effect was inhibited with the NOS inhibitor L-NMMA in RAW 264.7 cells, but not in HRTEC. HRTEC were more sensitive to oxidative stress induced by H2O2 than to nitrogen stress induced by DETA/NO. CONCLUSIONS: We conclude that uropathogenic E. coli that attach to HRTEC fail to directly activate iNOS expression, and that iNOS expression during bacterial infection is more likely to result from stimulation by local cytokines such as IL-1beta and IFN-gamma.
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| 8. |
- Poljakovic, Mirjana
(författare)
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Inducible nitric oxide synthase in experimental urinary tract infection
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Urinary tract infection (UTI) is among the most common bacterial infections in humans and the majority are caused by Escherichia coli (E. coli). Abundant evidence indicates that nitric oxide (NO) produced by the inducible NO synthase (iNOS) plays an important role in host defense against infection. This thesis examines iNOS expression in experimental UTI models. The rat and pig bladder urothelium did not express eNOS, nNOS or iNOS under normal conditions. iNOS expression in the rat bladder urothelium was, however, observed after systemic LPS treatment. In experimental bacterial UTI in mice, inflammatory cells expressed both iNOS and COX-2, but uroepithelial cells expressed only iNOS and with a later onset than observed in the inflammatory cells. Increased urinary nitrite levels in E. coli-infected mice coincided in time with the observed iNOS positive inflammatory cells. No difference in bacterial clearance or persistence was noted in bladders and kidneys of wild-type and iNOS deficient mice. When the effect of NO/peroxynitrite on bacterial viability was investigated, it was noted that a uropathogenic E. coli strain was less sensitive to NO-mediated killing than an avirulent E. coli strain. Isolated human uroepithelial cells did not express iNOS when exposed to uropathogenic E. coli or LPS. A combination of IL-1b, TNFa and IFNg was required for iNOS induction in human uroepithelial cells. The signaling pathways leading to iNOS induction depend on the activation of tyrosine kinases, JAK/STAT, PKC, p38 MAPK and NF-kB. Cytokines, but not bacteria, caused nuclear translocation and binding of NF-kB to the iNOS promoter. P fimbriated bacteria and sphingomyelinase inhibited cytokine-induced iNOS expression. Oxidative stress induced by H2O2 had more profound effects on cell viability in uroepithelial cells than NO and nitrogen species. In summary, our results demonstrate activation of iNOS in inflammatory and uroepithelial cells as part of the host defense against bacterial UTI. Wild-type and iNOS deficient mice were equally susceptible to E. coli-induced UTI. iNOS expression in uroepithelial cells was induced by cytokines but not bacteria. The failure of E. coli to cause iNOS induction may be related to insufficient translocation and binding of nuclear NF-kB. Both bacteria and uroepithelial cells were relatively resistant to NO-mediated cytotoxicity.
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| 9. |
- Poljakovic, Mirjana, et al.
(författare)
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Signalling pathways regulating inducible nitric oxide synthase expression in human kidney epithelial cells
- 2003
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Ingår i: European Journal of Pharmacology. - 0014-2999 .- 1879-0712. ; 469:1-3, s. 21-28
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Tidskriftsartikel (refereegranskat)abstract
- The purpose of this study was to elucidate the signalling pathways involved in the cytokine-activated inducible nitric oxide synthase (iNOS) response in a human kidney epithelial cell line, A498. Unstimulated cells did not express iNOS. Exposure of A498 cells to a cytokine mixture consisting of interferon gamma, interleukin-1 beta and tumor necrosis factor-alpha (TNF-alpha) increased nitrite production, iNOS mRNA and protein expression. Pharmacological inhibition of tyrosine kinases, including janus kinase (JAK2), and protein kinase C (PKC) inhibited cytokine-mediated nitrite production and iNOS protein expression. The involvement of mitogen-activated protein kinases (MAPKs) was investigated. Inhibition of p38 MAPK, but not of an upstream activator of extracellular signal-regulated kinase (ERK), caused a decrease in iNOS expression and nitrite production in response to cytokines. Electrophoretic mobility shift assay of nuclear extract from cytokine-stimulated cells demonstrated a pronounced binding to a nuclear factor kappa B (NF-kappa B) sequence present in the human iNOS promoter. Furthermore, the NF-kappa B inhibitor pyrrolidinedithiocarbamate (PDTC) decreased cytokine-activated iNOS protein expression and nitrite production. The present study has demonstrated that cytokine-stimulated iNOS expression in human kidney epithelial cells involves activation of tyrosine kinases, including JAK2, PKC, p38 MAPK and NF-kappa B.
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