| 1. |
- Chen, Daxin, et al.
(author)
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Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins
- 1999
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In: Transplantation. - : Ovid Technologies (Wolters Kluwer Health). - 0041-1337. ; 68:6, s. 832-839
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Journal article (peer-reviewed)abstract
- Background. Thrombotic vascular occlusion resulting in infarction occurs during hyperacute rejection of allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. A similar process is also found in disorders of diverse etiology including atherosclerosis, vasculitis, and disseminated intravascular coagulation. Methods. We have previously constructed two membrane-tethered anticoagulant fusion proteins based on human tissue factor pathway inhibitor and the leech anticoagulant hirudin and demonstrated their functional efficacy in vitro. These constructs have now been modified by the addition of a P-selectin sequence to the cytoplasmic tail to localize them in Weibel-palade bodies. They have been transfected into Weibel-palade body-positive endothelial cells isolated from the inferior vena cava of normal pigs. Results. In resting endothelial cells, fusion protein expression colocalized with P-selectin and was confined to Weibel-palade bodies. These cells had a procoagulant phenotype in recalcified human plasma. However, after activation with phorbol ester the anticoagulant proteins were rapidly relocated to the cell surface where they specifically inhibited the clotting of human plasma. Conclusions. Novel anticoagulant molecules may prove useful therapeutic agents for gene therapy in thrombotic disease and postangioplasty or for transgenic expression in animals whose organs may be used for clinical xenotransplantation. Expression in vascular endothelial cells may be regulated by inclusion of P-selectin cytoplasmic sequence, to restrict cell surface expression to activated endothelium.
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| 2. |
- Qi, Zhongquan, et al.
(author)
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Single dose anti-CD4 monoclonal antibody for induction of tolerance to cardiac allograft in high- and low-responder rat strain combinations
- 1997
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In: Transplant Immunology. - 0966-3274. ; 5:3, s. 204-211
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Journal article (peer-reviewed)abstract
- Repeated administration of monoclonal antibodies (mAb) directed against the CD4 lymphocyte receptor may induce specific, long-lasting unresponsiveness to fully MHC-mismatched cardiac allografts in rats without additional immunosuppression. We assessed the effect of a single dose of murine anti-rat depleting anti-CD4 mAb (OX-38) on allograft survival in high- and low-responder rat strain combinations. Isogenic strains of DA (RT1(av1)), PVG (RT1(c)), AUG (RT1(c)), and WF (RT1(u)) rats were used. Recipients in antibody treated groups were given one dose of 5 mg/kg OX-38 mAb on the day of transplant, a dose which was shown to effectively deplete (or block) circulating CD4+ T cells. Other groups were treated for 10 days with cyclosporin A (CsA) and/or Linomide, a novel immunomodulator, which is the first compound able to fully eliminate the effect of CsA in the rat cardiac allograft model. The DA strain was identified as a low-responder to the allogeneic haplotype RT1(c) (PVG or AUG), but not to RT1(u) (WF), and developed true tolerance following RT1(c) grafting and OX-38 or low-dose CsA (5 mg/kg) induction, as verified by the response to retransplantation of a graft from the same donor strain or a third-party challenge. PVG recipients of DA grafts were characterized by high response and only modest (OX-38; median 9.5 days) or moderate (CsA; 23.5 days) prolongation of graft survival. Contrasting graft survival results were obtained in the low-responder combination, either very early rejection (at 10 days) or permanent graft survival (> 100 days). Linomide challenge affected CsA treatment in the high-responder combination but not tolerance induction in the low-responder combination, or the effect of OX-38. It was concluded that in rat heart transplantation a single-dose anti-CD4 mAb therapy may induce permanent donor-specific unresponsiveness in a low-responder strain combination, and that anti-CD4 mAb seems to be unique among immunosuppressive agents while being resistent to challenge by Linomide.
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| 3. |
- Riesbeck, Kristian, et al.
(author)
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Ciprofloxacin induces an immunomodulatory stress response in human T lymphocytes
- 1998
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In: Antimicrobial Agents and Chemotherapy. - 0066-4804. ; 42:8, s. 1923-1930
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Journal article (peer-reviewed)abstract
- Exposure of cells to adverse environmental conditions invokes a genetically programmed series of events resulting in the induction of specific genes. The fluoroquinolone antibiotic ciprofloxacin has recently been reported to upregulate interleukin-2 (IL-2) gene induction. In the present investigation, the effect of ciprofloxacin at supratherapeutic concentrations on immediate-early (<2 h) gene expression in primary human peripheral blood lymphocytes was studied with Northern blots. In addition, transcriptional activity of IL-2 and metallothionein enhancer and promoter regions and transcription factors AP-1, NF-κB, and NF-AT were analyzed by chloramphenicol acetyltransferase (CAT) and electrophoretic mobility shift assays, respectively. The concentration of c-fos, c-jun, c-myc, junB, and fra-1 mRNAs was increased in activated peripheral blood lymphocytes incubated with ciprofloxacin compared to that in untreated controls. Ciprofloxacin increased CAT activity in stimulated lymphocytes transfected with plasmids containing either the IL-2 or metallothionein enhancer. Furthermore, among the transcription factors tested, AP-1 activity was increased in stimulated purified T helper lymphocytes incubated with ciprofloxacin compared to drug- free controls. Taken together, ciprofloxacin increased the levels of immediate-early transcripts, enhanced IL-2 and metallothionein promoter induction, and upregulated AP-1 concentrations in primary lymphocytes, reflecting a program commonly observed in mammalian stress responses.
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| 4. |
- Riesbeck, Kristian
(author)
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Cisplatin at clinically relevant concentrations enhances interleukin-2 synthesis by human primary blood lymphocytes
- 1999
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In: Anti-Cancer Drugs. - 0959-4973. ; 10:2, s. 219-227
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Journal article (peer-reviewed)abstract
- Cytotoxic drugs influence the expression of certain genes in cancer cells. Cisplatin has recently been shown to modulate interleukin (IL)-1 and tumor necrosis factor (TNF)-α production in macrophages. In this study, we wanted to investigate whether cisplatin interferes with the IL-2, IL-2 receptor (IL-2R), interferon (IFN)-γ, and TNF-α expression in phytohemagglutin-stimulated human peripheral blood lymphocytes. IL-2 was analyzed in a bioassay, while IFN-γ and TNF-α were measured by ELISA. Northern blots were performed to quantify steady-state cytokine mRNA levels. Furthermore, T cell subsets and IL-2R surface expression were analyzed by means of flow cytometry. A maximum stimulatory effect on IL-2 production (1.8-fold increase) was observed with cisplatin at 5-10 μM while IFN-γ and TNF-α synthesis and IL-2R density were unaffected. However, cisplatin-treated cells displayed enhanced IL-2, IL-2R, IFN-γ and TNF-α mRNA levels compared to drug-free controls. Cisplatin did not prolong cytokine mRNA half-life as revealed with the transcriptional inhibitor actinomycin D. In contrast to an inhibited growth of CD4+ T lymphocytes, CD3+CD8+ cell density was unaffected at intermediate cisplatin concentrations (10 μM). Bleomycin, carboplatin, doxorubicin, novobiocin or etoposide, which were included for comparison, did not interfere with IL-2 expression. Our data imply that cisplatin most likely stimulated cytokine transcription via a putative stress induced signaling pathway.
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| 5. |
- Riesbeck, Kristian, et al.
(author)
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Destructive knee joint infection caused by Peptostreptococcus micros : Importance of early microbiological diagnosis
- 1999
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In: Journal of Clinical Microbiology. - 0095-1137. ; 37:8, s. 2737-2739
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Journal article (peer-reviewed)abstract
- Peptostreptococcus micros is a commensal of the oral cavity and the genitourinary tract that rarely causes serious infections. A case of a destructive knee joint infection with rapid progress caused by P. micros is presented. The significance of the microbiological findings was initially not acknowledged, which contributed to a nonsuccessful clinical outcome.
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| 6. |
- Riesbeck, Kristian, et al.
(author)
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Endothelial cells expressing an inflammatory phenotype are lysed by superantigen-targeted cytotoxic T cells
- 1998
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In: Clinical and Diagnostic Laboratory Immunology. - 1071-412X. ; 5:5, s. 675-682
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Journal article (peer-reviewed)abstract
- The objective of this study was to investigate whether the superantigen staphylococcal enterotoxin A (SEA), which binds to HLA class II and T-cell receptor Vβ chains, can direct cytotoxic T cells to lyse cytokine-stimulated endothelial cells (EC). In addition, we wanted to determine whether SEA- primed cytotoxic T cells could be targeted to EC surface molecules as a means of a novel cancer immunotherapy. Human umbilical vein EC (HUVEC), dermal microvascular EC (HMVEC), or the EC line EA.hy926 stimulated with gamma interferon (IFN-γ) or tumor necrosis factor alpha (TNF-α) displayed upregulated HLA class II and adhesion molecule (CD54 and CD106) expression, respectively. SEA-primed T cells induced a strong cytotoxicity against IFN- γ,- and TNF-α-activated EA.hy926 which had been preincubated with SEA. Blocking of CD54 completely abrogated the T-cell attack. SEA-D227A, which has a mutated class II binding site, did not promote any cytotoxicity. A strong lysis was observed when a fusion protein consisting of protein A and SEA- D227A was added together with T cells to TNF-α-induced EA.hy926 and HUVEC precoated with monoclonal antibodies (MAb) directed against HLA class I, CD54, or CD106 molecules. Finally, an scFv antibody fragment reactive with an unknown EC antigen was fused with SEA-D227A. Both EA.hy926 and HMVEC were efficiently lysed by scFv-SEA-D227A-triggered cytotoxic T cells. Taken together, superantigen-activated T-cell-dependent EC killing was induced when EC expressed an inflammatory phenotype. Moreover, specific MAb targeting of the superantigen to surface antigens induced EC lysis. Our data suggest that directed T-cell-mediated lysis of unwanted proliferating EC, such as those in the tumor microvasculature, can be clinically useful.
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| 7. |
- Riesbeck, Kristian, et al.
(author)
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Enhancement of the immunosuppressive effect of cyclosporin A by ciprofloxacin in a rat cardiac allograft transplanation model
- 1995
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In: Transplant International. - 1432-2277. ; 8:2, s. 96-102
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Journal article (peer-reviewed)abstract
- Ciprofloxacin hyperinduces interleukin-2 production in stimulated human and mouse lymphocytes. In this study, an enhanced and prolonged interleukin-2 response was also detected in polyclonally stimulated rat splenocytes in the presence of ciprofloxacin (5–80mgrg/ml) compared to control cells without any antibiotic. Ciprofloxacin was able to counteract the immunosuppressive effect of 10ng/ml cyclosporin A (CyA) but did not interfere with higher CyA concentrations. In parallel, ciprofloxacin did not influence thymidine uptake in mixed lymphocyte reactions in the presence of CyA. To obtain an in vivo application of these findings, graft survival was studied by performing rat cardiac allograft transplantations in the presence or absence of CyA. Brown Norway rats served as donors and Wistar Furth rats as recipients. Ciprofloxacin was injected intraperitoneally either at a high-dose regimen (240 mg/kg per 24h) into rats every 8th h starting 1 day before transplantation until day 21 or graft loss, or it was injected at a low and clinically relevant dose regimen (45mg/kg per 24h) until day 9. CyA was administered orally (10mg/kg per 24h) from day 1 through day 9. Ciprofloxacin given alone at a high-dose regimen resulted in a median graft survival of 14.8 days, which was significantly longer than graft survival in rats without treatment (median 8.0 days). A low-dose regimen of ciprofloxacin alone did not affect graft survival. Ciprofloxacin at a highdose regimen combined with CyA prolonged graft survival to a median of 24.0 days compared to 20.5 days with CyA alone. Ciprofloxacin administered in the drinking water (200mg/kg per 24h) until day 9 in addition to CyA did not affect graft survival. However, when the same dose regimen was used in experiments with PVG rats as donors and Wistar/Kyoto as recipients, graft survival was significantly prolonged to a median of 45 days. Ciprofloxacin, given orally without the addition of CyA, did not influence graft survival in either of the two strain combinations. Thus, our data show that ciprofloxacin has no negative impact on heart graft survival rats. It remains to be clarified whether ciprofloxacin influences graft survival in humans.
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| 8. |
- Riesbeck, Kristian, et al.
(author)
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Expression of hirudin fusion proteins in mammalian cells : A strategy for prevention of intravascular thrombosis
- 1998
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In: Circulation. - 0009-7322. ; 98:24, s. 2744-2752
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Journal article (peer-reviewed)abstract
- Background - Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation. Methods and Results - An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti- hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4-P- selectin fusion proteins accumulated in adrenocorticotropic hormone- containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane. Conclusions - Hirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis.
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| 9. |
- Riesbeck, Kristian, et al.
(author)
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Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface
- 1997
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In: Thrombosis and Haemostasis. - 0340-6245. ; 78:6, s. 1488-1494
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Journal article (peer-reviewed)abstract
- Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation.
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| 10. |
- Östraat, Ö., et al.
(author)
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Thalidomide prolonged graft survival in a rat cardiac transplant model but had no inhibitory effect on lymphocyte function in vitro
- 1996
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In: Transplant Immunology. - 1878-5492. ; 4:2, s. 117-125
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Journal article (peer-reviewed)abstract
- The effects of thalidomide on in vitro interleukin 2 (IL-2) production and thymidine uptake by human peripheral blood lymphocytes or rat splenocytes were investigated. Phytohaemagglutinin-stimulated human lymphocytes were incubated in the presence of thalidomide added at culture initiation. No immunosuppressive effect of thalidomide was observed in these experiments. Primary human mixed lymphocyte cultures treated with thalidomide for 6 days were also unaffected. A microsomal rabbit liver homogenate was prepared for metabolizing thalidomide. Stimulated lymphocytes secreted significantly more IL-2 in the presence of microsomal-treated thalidomide than did controls. The effect of thalidomide was then studied either as single therapy or in combination with cyclosporin A (CyA) in a rat allograft cardiac transplantation model. In addition, T cell subsets were analysed by flow cytometry in untransplanted rats treated with thalidomide. Treatment was given as induction therapy from the day of transplantation until day 9. Graft survival in rats treated with thalidomide was significantly prolonged compared to the untreated group. No difference in graft survival was detected between rats treated with thalidomide or CyA only. Graft survival was found to be slightly prolonged in rats given thalidomide and CyA in combination compared to rats treated with CyA alone. In untransplanted rats given thalidomide a decrease of CD4 positive T cells was detected on days 3 and 5. The T helper/cytotoxic-suppressor cell ratio was significantly diminished but, after 1 week of treatment, values for T cell subsets had almost returned to baseline levels. No inhibitory effect was obtained when phytohaemagglutinin-stimulated rat splenocytes were cultured with metabolized thalidomide.In summary, the ability of thalidomide to improve allograft survival in a solid organ transplant model was verified. The occurrence of thalidomide-induced changes in T cell subset ratios was demonstrated. In in vitro studies, however, there was no decrease but an increase in IL-2 production, and no change in thymidine uptake. The mechanism responsible for the immunosuppressive effect of thalidomide remains to be elucidated.
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