Aim. Fractalkine/CX3CR1 system may contribute to the pathogenesis of renal allograft chronic rejection (CR). Vascular endothelial growth factor (VEGF) is an endothelial mitogen, which shows increased expression in inflammation and vasculopathy. This study sought describe the expression and distribution of Fractalkine/CX3CR1 and VEGF, and their relationship to human renal allograft CR. Methods. Renal tissue from 10 patients with CR was examined for Fractalkine/CX3CR1 and VEGF protein by immunohistochemistry for comparison with patients displaying hyperacute rejection (n=10), acute rejection (n=10), and normal kidneys (n=10). All patients were selected based upon histologically proven diagnoses between 1992 and 2003. Results. Immunohistochemistry revealed that Fractalkine/CX3CR1 were mostly expressed in the tubulointerstitium and tubular epithelial cell basolateral membrane. Some vessels showed positive staining for Fractalkine/CX3CR1 as well as occasionally glomerular parietal wall cells. Among the CR group, VEGF was mostly expressed in tubular epithelium and the tubulointerstitium. A proportion of glomeruli and vessels had positive staining for VEGF, which was up-regulated most strikingly in the interstitial compartment in CR. There was markedly increased expression of Fractalkine/CX3CR1 and VEGF protein in the interstitium of the CR compared with other groups (P<.05). VEGF colocalized with the expression of Fractalkine/CX3CR1. Conclusion. Fractalkine/CX3CR1 and VEGF may play an important role in the development of interstitial fibrosis via mononuclear cell-induced cytokine production and myofibroblast stimulation in CR. Further studies are necessary to identify the role in the pathogenesis of CR.

MYCOPHENOLATE mofetil (MMF) is widely used in organ transplantation combined with other immunosuppressants to prevent acute rejection. 1-3 Because MMF can produce side effects, especially hematological and/or gastrointestinal toxicity, 1-5 therapeutic monitoring is becoming mandatory. Furthermore, there is no study to show the characteristics of MMF pharmacokinetics (PK) in Chinese patients. This study was designed to investigate the relationship between clinical events and the PK of mycophenolic acid (MPA) in Chinese kidney transplant recipients.

Aims. Cytoprotective genes have shown to display potent anti-inflammatory and antiapoptotic functions in endothelial and smooth muscle cells. We investigated whether cytoprotective genes, especially A20, were involved in mycophenolate mofetil (MMF)’s ability to ameliorate transplant arteriosclerosis in an experimental chronic rejection model. Methods. Sprague-Dawley rat renal grafts were orthotopically transplanted into Wistar rats following the procedure of Kamada with our modification. The recipients were divided into three oral treatment groups: (1) vehicle group (cyclosporine [CsA] 10mg/kg•d×10d followed by vehicle), (2) CsA group (CsA 10mg/kg•d×10dd followed by CsA 6mg/kg • d), (3) MMF group (converted from CsA 10mg/kg•d×10d toMMF20mg/kg•d on day 11). At the same time points after transplantation, the rats were sacrificed to harvest the renal allografts. The expression of four cytoprotective genes, A20, heme oxygenase (HO)-1, Bcl-2, and Bcl-XL, was analyzed by quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry. Results. The four-cytoprotective genes were all detected in rat kidney allografts undergoing chronic allograft nephropathy. The expression of A20 in grafted kidneys was significantly higher in the MMF than in the CsA or the vehicle group (P<.01). There was no significant difference between the CsA and the MMF groups in the expression of HO-1, Bcl-2, and Bcl-XL. Conclusions. We demonstrated that MMF improved the expression of A20 in rat kidney allografts undergoing chronic allograft nephropathy. The correlation between MMF and A20 provide an explanation for the mechanism by which MMF ameliorates transplant arteriosclerosis in an experimental animal model of chronic rejection.

To screen different combinations of prostate-specific membrane antigen (PSMA) promoter/enhancer with the strongest transcriptional activity in prostate-specific cells, we used PSMA regulatory elements to control specific expression of the target gene in gene therapy of prostate adenocarcinoma. PSMA promoter and enhancer DNA sequences were amplified from the LNCaP human prostate cancer cell line by polymerase chain reaction, then recombinant plasmids of the enhanced green fluorescent protein (EGFP: pEGFP-PSMAPro, pEGFP-PSMAE-P, pEGFP-PSMAE(r)-P, pEGFPPSMA E(d)-P, and pEGFP-PSMAE(t)-P) were constructed with molecular clonal techniques. At the same time, all experimental cell lines were analyzed for the expression of PSMA with the use of PSMA monoclonal antibody and the ABC immunohistochemical assay kit. After plasmids were transfected via liposome, we observed the expression of the reporter gene (EGFP) under a fluorescent microscope and compared the different levels of EGFP expression with reverse transcriptase polymerase chain reaction and flow cytometry so that we could choose the one with the highest transcriptional activity. Only the LNCaP cell line expressed PSMA positively with immunohistochemical stain. The PSMA promoter/enhancer had transcriptional activity in PSMA(1) cell lines and no activity in PSMA(2) cell lines. PSMAE-P achieved the strongest activity in different PSMA promoter/ enhancer combinations. We confirmed the specific expression of PSMA in prostate cells again. Similarly, transcriptional activity of the PSMA promoter/enhancer was prostate specific. PSMAE-P achieved the strongest transcriptional activity among PSMA promoter/ enhancer combinations, which could be used in advanced research for tissue-specific treatment.

Aims. It is increasingly recognized that expression of cytoprotective genes in grafts can affect the progress of chronic allograft nephropathy (CAN). Little is known about the influence of different immunosuppressive regimens on expression of cytoprotective genes in allografts undergoing CAN. We investigate whether there is difference between rapamycin (Rapa) and FK506 in the expression of cytoprotective genes in rat kidney allografts undergoing CAN. Methods. Sprague-Dawley rat renal grafts were orthotopically transplanted into Wistar rats following the procedure of Kamada with our modification. The recipients were divided into three oral treatment groups: group 1: vehicle group (cyclosporine [CsA] 10mg/kg•d×10 days followed by vehicle); group 2: Rapa group (CsA 10mg/kg•d×10d followed by Rapa 0.8mg/kg•d); group 3: FK506 group (CsA 10mg/kg•d×10d followed by FK506 0.15mg/kg • d). At the same times after transplantation, the rats were sacrificed to harvest the renal allografts. The expression of four cytoprotective genes, A20, heme oxygenase (HO)-1, Bcl-2, and Bcl-X/L were analyzed in these grafted kidneys by quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Results. Four cytoprotective genes were all detected in rat kidney allografts undergoing CAN. The expression of A20 in the Rapa group was significantly higher than that in the FK506 or the vehicle group (P<.05). There was no significant difference between the Rapa group and FK506 group in the expressions of HO-1, Bcl-2, and Bcl-X/L. Conclusions. We demonstrate that various immunosuppressive agents have different effects on the expression of cytoprotective genes.