[Clinical value of ATP determination in CD4+ cells of patients with cytomegaloviral pneumonia after kidney transplantation].

OBJECTIVE: To explore the clinical value of determination of ATP levels in CD4(+) cells of patients with cytomegaloviral pneumonia after kidney transplantation. METHODS: Twenty-eight patients with cytomegaloviral pneumonia following kidney transplantation and 30 healthy volunteers were enrolled in this study. ATP-bioluminescence assay (ATP-CVA) was used to assess the immune response of CD4(+) cells to phytohemagglutinin (PHA) stimulation in the normal volunteers and the recipients (before and at 1, 2, and 4 weeks after renal transplantation, before and at 2 and 4 week after the treatment). RESULTS: ATP concentration in CD4(+) cells of the recipients was 402-/+58 ng/ml before the operation, significantly lower than that in normal volunteers (458-/+196 ng/ml, P<0.05), and reached the lowest level in the first week after operation especially in the recipients with antibody-inducing therapy; ATP level increased slowly since week 2 post-operation, but still remained significantly lower than the preoperative by the fourth week (266-/+87 ng/ml, P<0.05), especially in the recipients receiving antibody-inducing therapy. In the event of cytomegaloviral pneumonia, ATP level underwent a mild reduction to 152-/+78 ng/ml in comparison with the postoperative level at the first week (P>0.05), and was significantly lower than preoperative level (P<0.01); the decrease was especially obvious during the exacerbation of the condition. ATP level then increased slowly after effective treatment, but was still lower than the preoperative level at 4 weeks after the operation (336-/+92 ng/ml, P<0.05). CONCLUSION: The determination of ATP level in CD4(+) cells allows more accurate assessment of the cellular immunity in the renal transplant recipients with cytomegaloviral pneumonia to help in the clinical treatment of the patients.

[Comparison of the surgical approaches for nephrectomy in living related donors].

OBJECTIVE: To compare the clinical effects and graft outcomes of 4 surgical approaches for nephrectomy in living related kidney donors. METHODS: Between June, 2004 and June, 2007, 119 living related kidney donors underwent nephrectomy via different surgical approaches, and their clinical data were retrospectively analyzed. Of these donors, 22 received retroperitoneal open nephrectomy, 21 had retroperitoneoscopic nephrectomy, 13 had hand-assisted laparoscopic nephrectomy, and 63 underwent transperitoneal open nephrectomy. The operating time, warm ischemia time of the graft, renal graft artery and vein lengths, reduction rate of recipient serum creatinine in the first 3 days after renal transplantation, mean hospital stay and complications of the donors were compared between the 4 surgical approaches. RESULTS: Open surgeries were associated with significantly shorter operating time (P=0.0033) and warm ischemia time of the graft (P=0.0001), longer hospital stay (P=0.0000), higher hospital expenses (P=0.0000), faster postoperative reduction of recipient serum creatinine (P=0.0001), and longer renal artery and vein lengths (P=0.0000 on the left and P=0.0001 on the right) than laparoscopic surgeries. In the laparoscopic surgery group, subcutaneous emphysema occurred in 1 case, DGF in 2 cases, and lumbar vein hemorrhage in 2 cases for which open surgery was performed. In the open surgery group, only one case required reoperation due to adrenal gland hemorrhage. All the kidney grafts were successfully harvested without other complications observed in the donors. CONCLUSIONS: Both open and laparoscopic surgeries are safe for nephrectomy in living related kidney donors, and the selection of the surgical approaches depends on the kidney and donor conditions and the surgical proficiency of the surgeons.

[Etiologic investigation of chronic granulomatous inflammation of hand by polymerase chain reaction and DNA sequencing].

OBJECTIVE: To explore the causative pathogens in littoral hand infections which exhibited chronic granulomatous inflammation, the relationship between chronic granulomatous inflammation and mycobacteria and to discuss the prospects of PCR in clinical application for diagnosis of granulomatous inflammation. METHOD: With 16S-rDNA as the target sequence, Nest-PCR was used to detect mycobacteria directly from 37 cases of chronic granulomatous inflammations, and identified them by gene sequencing. RESULTS: Twenty-four of 37 cases were positive for mycobacteria by Nest-PCR, in which 17 were M.marinum, 1 M.chelonae, 2 M.avium, 2 M.kansasii, and 2 M.tubercular through gene sequencing. CONCLUSIONS: Nest-PCR combining gene sequencing proved to be a liable and sensitive method to detect Non-tubercular mycobacteria (NTM) in fresh tissue. NTM is the major factor of hand specific chronic infections other than tubercular. Pathological changes are difficult to differentiate TB from NTM and bacterial evidence was necessary.

[Half-dose Zenapax for acute rejection prevention after renal transplantation].

OBJECTIVE: To investigate the efficacy and safety of half-dose Zenapax for prevention of acute rejection after renal transplantation. METHODS: According to the immunosuppressive regimen and renal function after transplantation, patients were divided into 4 groups, namely groups A, B, C, and D of 90, 73, 11 and 13 patients, respectively. Blood creatinine measured 1 week after operation was <176.6 micromol/L in groups A and B, and was >353 micromol/L in groups C and D. Patients in groups A and C were given 25 mg Zenapax (0.5 mg/kg) and MMF 0.75 g before operation, and those in groups B and D had only MMF of 0.75 g. All patients were given Pred, CsA and MMF after operation, and the rejection episodes, the time of acute rejection onset, the rate of rejection reversal and complications were analyzed in the time period of 6 months after operation. RESULTS: After the operation, 13 patients (14.4%) developed acute rejection in group A, 18 (24.6%) in group B, 6 (54.5%) in group C and 7 (53.8%) in group D (P<0.01). The incidence of acute rejection in group B was significantly lower than that in groups C and D groups (P<0.01), and the latter two groups had similar incidence. The time of acute rejection onset ranged from 3 to 9 days postoperatively (mean 6.2-/+3.2 days) in group A, significantly delayed as compared with that in group B (range 2-8 days, mean 4.7-/+3.1 days), group C (range 2-7 days, mean 4.3-/+4.2 days) and group D group (range 2-9 days, mean 3.9-/+3.5 days), but the time was similar between groups B, C, and D (P>0.05). All acute rejection cases in group A was reversed, and the rate of reversal was 88.9% (16/18) in group B, 83.3% in group C, and 71.4% in group D. No significant differences were noted in such complications as infection, vascular injuries or gastrointestinal reactions between the 4 groups (P>0.05). CONCLUSION: Zenapax at the dose of 25 mg can safely decrease the risk of acute rejection in patients with good postoperative renal function recovery, but dose not seem effective in patients with delayed graft function recovery.

[Expression of IgG receptor on human umbilical vein endothelial cells stimulated with TNF-alpha and IFN-gamma].

AIM: To detect the expression of IgG receptors (FcgammaR) on cytokine-stimulated human umbilical vein endothelial cells (HUVECs). METHODS: By using ELISA, immunocytochemical staining, immunofluorescent staining and RT-PCR, the expression and subtypes of FcgammaR were detected. RESULTS: Non-stimulated HUVECs expressed very low level of FcgammaRIIa. FcgammaRIIa mRNA was dramatically up-regulated upon 24 hour stimulation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma(IFN-gamma). ELISA results indicated that the expression of FcgammaRIIa increased 16 folds after stimulation with TNF-alpha and IFN-gamma for 3 days (P<0.01). Immunofluorescent staining showed that FcgammaRIIa was expressed on the surface of the stimulated HUVECs. CONCLUSION: TNF-alpha and IFN-gamma could increase FcgammaRIIa expression on HUVECs. The enhanced expression of FcgammaRIIa may mediate the deposition of immune complexes to blood vessels under vasculitic conditions.

[Management of urinary obstruction following renal transplantation: report of 16 cases].

OBJECTIVE: To explore the diagnosis and treatment of urinary obstruction involving the transplanted kidney. METHODS: A retrospective analysis was performed in 16 cases of urinary obstruction involving the transplanted kidney, including 5 cases of ureteral calculi, 6 vesicoureteral anastomotic stricture, 2 pyeloureteral junction stricture after transplantation, 1 ureter necrosis due to graft rejection, and 2 infection surrounding the renal graft and ureter end necrosis. RESULTS: Only one patient had the renal graft removed due to massive hemorrhage in an open surgery for correction of urinary obstruction, and the renal function of the graft was preserved in all the other cases after endoscopic or open surgeries. In the follow-up for 0.5 to 3 years after the second surgery, serum creatinine of the patients were maintained within the range of 90-150 micromol/L, without further renal enlargement or exacerbation of renal retention shown by B-mode ultrasonography. CONCLUSION: Urinary obstruction after renal transplantation is a difficult surgical complication, which can be managed by endoscopic or open surgeries depending on the causes of the obstruction.

[Differential analysis of tyrosine-phosphorylated proteins in human hepatocellular carcinoma cell lines with different metastasis potentials].

OBJECTIVES: To compare expressions of tyrosine-phosphorylated proteins in different hepatocellular carcinoma cell lines with different metastasis potential and to screen key molecules associated with HCC metastasis and recurrence. METHODS: Using two-dimensional electrophoresis, Western blotting and MALDI-TOF-MS/MS, we analyzed tyrosine-phosphorylated protein profiles of Hep3B, MHCC97L and MHCC97H, HCC cell lines with different metastasis potentials. RESULTS: 10 spots were detected in Hep3B, 19 in MHCC97L and 17 in MHCC97H. Seventeen significantly different phosphotyrosine proteins in gel were identified by MALDI-TOF-MS/MS, including Annexin I. CONCLUSION: The changed expression of tyrosine-phosphorylated proteins is associated with HCC metastasis and recurrence.

Identification of a novel nucleus protein involved in the regulation of urokinase in 95D cells.

The urokinase-type plasminogen activator (uPA) plays an important role in cellular invasion. By using the downstream part of a 74 bp DNA region called the cooperation mediator (COM) of the uPA promoter as a bait sequence in the yeast one-hybrid screen, a gene called PBK1 was previously cloned from the cDNA library of the 95D lung cancer cell strain. In this study, the intracellular distribution of PBK1 was studied by using the transient transfection of pEGFP-C3-PBK1, and PBK1 was found to be localized in the nucleus. Co-transfection of pEGFP-C3-PBK1 and the deletion mutants of the pGL3-uPA promoter indicated that PBK1 can increase the uPA promoter activity by about 25% and this effect is uPA enhancer-dependent. Western blotting and Enzyme-linked immunoadsordent assay further confirmed that PBK1 can upregulate the expression of uPA. Our results suggest that PBK1 is involved in the regulation of uPA expression, which might provide a new clue to further understanding the regulation mechanism of uPA expression.

Interaction between plasminogen activator inhibitor type-2 and pre-mRNA processing factor 8.

The plasminogen activator inhibitor type-2 (PAI-2) dependent apoptosis protection is due to the 33 amino acids fragment located between helix C and D of PAI-2, this fragment may interact with some unknown intracellular proteins. In this study we used the fragment between helix C and D of PAI-2 as a bait to perform a yeast two-hybrid screen using a cDNA library constructed with HeLa cells during apoptosis, and retrieved a clone encoding 94 amino acid residues of C-terminus of pre-mRNA processing factor 8 (PRPF8). Co-immunoprecipitation experiments confirmed that PAI-2 could interact with PRPF8 in vivo. PAI-2 could bind PRPF8 C-terminal in both the inside and outside of nuclear. These results suggested that the interaction between these two proteins might not be involved in the apoptosis process.

Antisense Tiam1 down-regulates the invasiveness of 95D cells in vitro.

As a specific guanine nucleotide exchange factor of Rac1, Tiam1 (T-lymphoma invasion and metastasis inducing protein 1) is involved in a number of cellular events, such as cytoskeleton reorganization, cell adhesion, and cell migration. Since Tiam1 was implicated in the invasion and metastasis of T-lymphoma cells and breast tumor cells, we compared the expression level of Tiam1 in two human giant-cell lung carcinoma cell strains with high or low metastasis potential, and found that Tiam1 expression level in high-metastatic 95D cells was higher than that in low-metastatic 95C cells. To further confirm the role of Tiam1 in invasion and metastasis, we constructed the antisense Tiam1 expression plasmid (pcDNA3-anti-Tiam1), which was transfected into 95D cells. A stable transfected clone with decreased Tiam1 expression was screened and selected for further research. Transwell assay showed that down-regulation of endogenous Tiam1 by anti-Tiam1 can reduce the in vitro invasiveness of 95D cells. Our results suggested that Tiam1 signaling contributed to the invasion and metastasis of the human giant-cell lung carcinoma cells.

A novel gene delivery system targeting urokinase receptor.

Recombinant proteins that combine different functions required for cell targeting and intra-cellular delivery of DNA present an attractive approach for the development of nonviral gene delivery vectors. Here, we described a novel protein termed ATF-lys10 which facilitated cell-specific gene transfer via receptor-mediated endocytosis. ATF-lys10 was composed of the amino-terminal fragment of urokinase and ten lysines at the carboxyl terminus. Bacterially expressed ATF-lys10 protein existed in soluble form, and had antigenicity of human urokinase. Purified ATF-lys10 specifically bound to uPAR-expressing cells and formed protein-DNA complexes with plasmid pGL3-control. After neutralization of excess negative charge with poly-L-lysine, these complexes served as a specific gene delivery vector for uPAR-expressing cells. Lyso-somotropic compounds, such as chloroquine, drastically increased the ATF-lys10 mediated gene delivery efficiency. Our results suggest that the recombinant protein ATF-lys10 with the properties of DNA binding and tumor cell targeting represents a promising method for gene transfer and expression in tumor cells.

Transcriptional regulation of urokinase receptor in high- (95D) and low-metastatic (95C) human lung cancer cells.

To study the transcriptional regulation of urokinase receptor (uPAR) in high- (95D) and low-metastatic (95C) human lung cancer cells, we performed PCR to amplify 2238 bp uPAR promoter from 95C and 95D cells. According to the results of sequencing, five different bases are found in uPAR promoter between 95C and 95D cells. The results of luciferase activity assay showed that these differences have no significant effect on the uPAR promoter activity. Based on a normal uPAR promoter, progressive truncated mutants were constructed. The transient transfection/reporter assay showed that the promoter region from -136 to +9 may interact with relevant nuclear factors, which result in different levels of uPAR expression between 95C and 95D cells.

Interaction of plasminogen activator inhibitor-2 and proteasome subunit, beta type 1.

The apoptosis protection by plasminogen activator inhibitor -2(PAI-2) is dependent on a 33 amino acids fragment between helix C and D of PAI-2 which is probably may be due to the interaction of PAI-2 with unknown intracellular proteins. In this study we used the fragment between helix C and D of PAI-2 as bait to screen a HeLa cells cDNA library constructed during apoptosis in a yeast two-hybrid system and retrieved a clone that encodes 241 amino acids of proteasome (prosome, macropain) subunit, beta type 1(PSMbeta1) which plays important roles in NF-kappaB activation. GST-pulldown experiments confirmed the interaction between PAI-2 and PSMB1 in vitro. These data suggest that the antiapoptosis activity of PAI-2 is probably related to its interaction with PSMbeta1.

[Surgical techniques for the ureterointestinal anastomosis in continent urinary diversion].

OBJECTIVE: To compare the complications of direct and antirefluxing techniques of ureterointestinal anastomosis in continent urinary diversion. METHODS: Sixty-three patients underwent continent urinary diversion. Twenty-four patients were treated by the direct ureteroenteric anastomosis and the others treated by the antirefluxing technique. The follow up studies included following-up the information of ureteric stricture, ureteric reflux, renal function and acute urinary infection. It was assessed for 3 months to 6 years with a mean follow up of 26 months after operation. RESULTS: Of 78 ureters reimplanted using antirefluxing technique. A total of 12 ureters had anastomotic stricture formation postoperatively. Only one of 48 ureters reimplanted using direct anastomoses had anastomotic stricture. The difference between the direct and antirefluxing technique groups was remarkable (chi2 = 4.375, P < 0.05). Furthermore, there was no significant difference between the direct and antirefluxing technique groups in regard to ureteric reflux, renal function and acute urinary infection. CONCLUSIONS: Antirefluxing anastomoses resulted in obviously higher rate of ureterointestinal anastomotic stricture in comparison with the direct anastomosis. The direct ureteroenteric anastomosis may be the suitable choice for patients undergoing continent urinary diversion.

[Application of multihole U-shaped drainage stent in complex posterior urethra surgery].

OBJECTIVE: To study a method for using a new drainage stent following complex posterior urethral operation. METHODS: Fifty-five patients,15 of whom had complex posterior urethrorectal fistula, 35 had complex posterior stricture or atresia, and 5 had bladder exstrophy, received surgical treatment, after which multihole U-shaped drainage stent was applied. RESULTS: All the patients were normal in micturition and no complications occurred during the follow-up period lasting for 1 to 10 years. CONCLUSION: Multihole U-shaped drainage stent performs the functions of both stenting and drainage, and is applicable in complex posterior urethral surgery.

[Biological function of fusion protein ATF-PAI2CD].

To express the fusion protein ATF-PAI2CD (urokinase-type plasminogen activator amino terminal fragment-plasminogen activator inhibitor type 2 with the region inter C and D helices deleted ) gene in E.coli and determine the biological characterization of fusion protein ATF-PAI2CD, the cDNA fragment encoding ATF-PAI2CD was cloned into the expression vector pLY-4 and transformed into E.coli JF1125. After temperature induction, the expression amount of ATF-PAI2CD account for 15% of total bacterial protein. The result was confirmed by Western blot. ATF-PAI2CD protein was isolated and purified by washing and solubilization of inclusion body, renaturation and ion exchange chromatography. The final product displayed a single band with a corresponding molecular weight 62 kD in SDS-PAGE. The purity was over 90%, the protein yield was 50% and the specific activity was 12 000 IU/mg. The PAI activity was measured by chromogenic assay. The purified fusion protein inhibited urokinase-type plasminogen activator as measured by milk-agarose plate assay, and bound to human lung cancer cells via uPA receptor (uPAR), which was confirmed by radio competition experiments. The results indicate that the biological characteristics of ATF-PAI2CD were very similar to those of the wide type PAI-2 (or mutants PAI-2, PAI-2CD) and to pro-uPA in binding to uPAR-bearing cells.

Identification of interaction between PAI-2 and IRF-3.

HeLa cells transfected with plasminogen activator inhibitor-2 ( PAI-2 ) were protected from TNF- alpha-induced apoptosis. The apoptosis protection by PAI-2 is dependent on a 33 amino acids fragment between helix C and D of PAI-2, which may be due to the interaction of PAI-2 with some intracellular proteins. In this study, the yeast two-hybrid system was used to screen a HeLa cells cDNA library constructed during apoptosis with the fragment between helix C and D of PAI-2 as bait. We retrieved a clone that encodes 98 amino acids of C-terminus of interferon regulatory factor-3 (IRF-3). Co-immunoprecipitation experiments confirmed the interaction between PAI-2 and IRF-3 in vivo. IRF-3 belongs to a family of the IRF transcription factors and has been shown to participate in a large number of biological processes. These data suggest that IRF-3 may be involved in the apoptosis protection and antiviral function of PAI-2.

[Screening of proteins interacting with tTG in HeLa cells].

Tissue transglutaminase(tTG) belongs to a class of transglutaminase family which is up-regulated in almost all cells apoptosis and is thought to be closely related to cell apoptosis. To investigate the mechanism of tTG in cell apoptosis, yeast two hybrid system was used to screen HeLa cDNA library. One of the 17 positive clones we have obtained encoded the glutamine-rich carboxyl terminus of TIAR, and this interaction between tTG and TIAR, which was finely regulated by Ca(2+), was proved in vitro by GST pull-down. These findings suggest that tTG might affect the function of TIAR by a calcium-dependent posttranslational modification and the interaction might possibly be involved in the regulation of cell apoptosis.

Interventional therapy for stenosis and aneurysm in renal graft artery: report of 9 cases.

OBJECTIVE: To study the interventional therapy for renal graft artery stenosis and aneurysm patients with renal transplantation to further improve the survival rate of the graft. METHOD: Seven patients with of renal graft artery stenosis received balloon dilatation of the stenotic artery, followed by stent implantation. For renal graft artery aneurysm in another 2 patients, thrombin infusion and stent implantation were respectively performed. RESULTS: The condition was successfully managed in 6 of the 7 patients with renal artery stenosis, whose serum Cr levels dropped to below 106 micromol/L 3 d after the operation. Thrombin infusion in one of the 2 patients with renal artery aneurysm caused thrombus in the renal graft and then aneurysm rupture, resulting in final graft loss. The other aneurysm case was successfully managed with stent implantation. CONCLUSIONS: Interventional therapy as balloon dilatation combined with stent implantation is ideal for treating renal graft artery stenosis, and stent implantation constitutes an important management for artery aneurysm in the renal graft.

[Misdiagnosis of corticomedullary mixed pathological changes in adrenals: report of 4 cases].

Pathological changes usually occur independently in the adrenal cortex and medulla because of their distinct embryonic origins, and changes involving both the cortex and medulla are rare. We report 4 cases of corticomedullary mixed pathological changes adrenal glands. CT scanning of the adrenal glands showed unilateral abnormalities in all the 4 cases, 3 of which were diagnosed as aldosteronism and the other pheochromocytoma before surgery. Unilateral adrenalectomy was performed in the 4 patients 3 being cured and discharged. The other 1 had recurrence 18 months postoperatively with suspected pathological changes on the other side. Subsequent pathological examination confirmed the suspicion in both the cortex and medulla of the other adrenal gland. In cases with clinical presentations as simultaneous onset of aldosteronism and catecholamine responses, pathological changes in both the cortex and medulla of the adrenal glands should be considered. Perioperative management of such cases should be the same as that in cases of catecholamine responses, and the diagnosis relies on histopathological examination.