[Expression of TNF-alpha signaling adapter proteins in peripheral blood mononuclear cells in lupus nephritis patients of different TCM asthenia syndromes].

OBJECTIVE: To investigate the mRNA expressions of the TNF adapter proteins, including TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMCs) of lupus nephritis (LN) patients of various TCM asthenia syndromes. Methods Fifty-one inpatients with LN were differentiated according to TCM syndrome differentiation, 13 cases of yin-deficiency with inner heat syndrome (A); 26 cases of both qi-yin deficiency syndrome (B), 12 cases of Pi-Shen yang-deficiency syndrome (C). Peripheral venous blood samples from the 51 LN patients and 17 healthy subjects were collected to separate PBMCs. The mRNA expressions of TNF adapter molecules (TRADD, FADD, RIP-1 and TRAF-2), as well as Caspase-3 and interleukin-1beta (IL-1beta) were analyzed by quantitative real-time PCR and the differences among them were compared. RESULTS: (1) As compared with the healthy subjects, expression of TRADD mRNA in patients of syndrome A, B and C was lowered to 0.54, 0.32, and 0.38-fold, respectively (P < 0.05, P < 0.01), showing insignificant difference among the three syndromes; (2) FADD mRNA lowered to 0.79, 0.62, and 0.72-fold respectively, only with significance shown in syndrome B (P < 0.05); (3) RIP-1 mRNA lowered to 0.79, 0.50, and 0.60-fold respectively with significance shown in syndrome B and C (P < 0.01, P < 0.05), and insignificant difference was shown among the three syndromes; (4) TRAF-2 lowered to 0.70, 0.52, and 0.50-fold respectively (P < 0.01, P < 0.01, P = 0.07), significance shown in syndrome B and C (P < 0.01), but with insignificant difference among the three; (5) Caspase-3 elevated in all patients of the three syndromes (all P < 0.01); (6) IL-1beta in syndrome A was apparently lower ed to the normal range and also lower than that in the other two syndromes (both P < 0.05). CONCLUSIONS: Expressions of TRADD, FADD, RIP-1 and TRAF-2 mRNA decreased in all the patients of various TCM asthenia syndromes, the decrement in patients of syndrome B and C was lesser than that in syndrome A. These abnormal low expressions of signal proteins might be the substantial bases for asthenia syndromes of LN patients, and the apoptotic signal mediated by them may involve in the formation of asthenia syndrome in LN.

Protein expression and purification of human Zbtb7A in Pichia pastoris via gene codon optimization and synthesis.

Human Zbtb7A was proved to be an important molecular switch in oncogenesis. However, it is difficult to obtain its protein expression in prokaryotic system, due to high G+C content and rare codons in zbtb7a gene. Therefore, to further research the function and application of this protein, we optimized its coding sequence according to the codon bias of Pichia pastoris, synthesized the sequence with two-step PCR and confirmed the accuracy by DNA sequencing. The assembled fragment was introduced into P. pastoris expression vector pPIC9K and the resultant plasmid pPIC9K-zbtb7a-his(6) was transformed into the P. pastoris strain GS115 by electroporation. The products of the transformants induced by methanol were analyzed by 10% SDS-PAGE and identified by Western Blot assay. The expression conditions of the selected transformant were optimized. Additionally, a two-step purification protocol was applied to purify the recombinant protein. The results showed that the synthetic coding sequence of human Zbtb7A was successfully obtained and inserted into pPIC9K vector. Human Zbtb7A protein was expressed in P. pastoris and identified by western blot. The optimal conditions for its expression in P. pastoris were under a final concentration of 1% methanol and a time-course of 4d. Through the two-step purification, Zbtb7A protein was purified in high purity and its production reached up to as high as 18 mg/L. These results indicated that an effective procedure for expressing and purifying human Zbtb7A in P. pastoris was established.

[Clinical diagnosis and treatment of BK virus infection in renal transplant recipients].

OBJECTIVE: To explore methods of clinical diagnosis and treatment of BK virus (BKV) infection in renal transplant recipients. METHODS: Urine samples were collected from 227 renal transplant recipients who had undergone renal transplantation at most 48 months to detect the decoy cells. Samples of urine and peripheral blood (BP) were collected to undergo real-time PCR to detect the BKV DNA. Part of the renal-recipients received graft biopsy. The recipients with BKV viruria or viremia were divided into 2 groups: intervention group and control group. The 51 patients of the intervention group had the doses of cyclosporine A (CsA) reduced (n=19), had their doses of FK506 reduced (n=22), or underwent replacement of FK506 with CsA (n=10). And other 29 patients in the control group did not receive any intervention. Acute rejection was intensively monitored. The amount of decoy cells, and BKV load in the urine and PB samples were measured again after 3 months. RESULTS: The positive rates of urine decoy cell, BKV viruria, and viremia in all patients were 33.0%, 33.5%, and 15.4% respectively. In the intervention group, the median levels of decoy cells in urine, and of BKV DNA in urine and PB before intervention were 5.0/10 HP, 1.50 x 10(4) copies/ml and 0 copy/ml respectively ; and the median levels of decoy cell in urine, and of BKV DNA in urine and PB were all 0 after intervention, all significantly lower than those before intervention (all P < 0.01). In the control group, the median levels of decoy cells in urine, and of BKV DNA in urine and PB were 6.0 /10 HP, 0.79 x 10(4) copies/ml, and 0 copy/ml respectively before observation, and the median level of BKV load in urine ofter observation was 2.21 x 10(4) copies/m, significantly higher than that before observation (P < 0.01), however, the median levels of decoy cells in urine and of BKV DNA in PB were 5.0 /10 HP and 0 copy/ml respectively, not significantly different from those before observation ( both P > 0.05). The differences between the levels of urine decoy cells, urine BKV DNA level and blood BKV DNA level of the intervention group were all significantly greater than those of the control group (Z = -2.749, -5.089, -1.996; P = 0.006, 0.000, 0.046 respectively). And during the intervention no acute rejection was observed. Four cases of BKVAN were diagnosed. Treatment of immunosuppression reduction showed effectiveness in 4 BKVAN recipients. The levels of decoy cells in urine, and BKV load in urine and in PB samples were all decreased. The graft functions were improved. CONCLUSION: Urine cytology is very convenient, useful and sensitive for the evaluation and followup of renal transplant patients, and can reflect renal histological presentation indirectly. Also BKV DNA detection in the urine and peripheral blood is important to screen the evidence of BK reaction in order to prevent irreversible graft damage of BKVAN. The treatment of immunosuppressant reduction and replacement of FK506 with CsA are effective in BKV infection recipients at the early stage.

Decreased expressions of the TNF-alpha signaling adapters in peripheral blood mononuclear cells (PBMCs) are correlated with disease activity in patients with systemic lupus erythematosus.

Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine. Systemic lupus erythematosus (SLE) is an autoimmune and inflammatory disease. However, until now, the expression and pathophysiological role of TNF adapters in SLE have been poorly understood. This study aims to investigate the expression of mRNA for the TNF adapter proteins including TNF receptor-associated death domain (TRADD) protein, Fas-associated death domain (FADD) protein, receptor-interacting protein 1 (RIP-1), and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMCs) from patients with SLE and to explore the relationship between the expression of these adapters and the SLE disease activity. PBMCs were isolated from the venous blood of 51 SLE patients and 17 healthy subjects. The expression of mRNA for TNF adapter molecules such as TRADD, FADD, RIP-1, and TRAF-2 in PBMCs were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. There were constitutive expressions of mRNA for TRADD, FADD, RIP-1, and TRAF-2 in PBMCs from healthy subjects. The expression of mRNA for all the adapter molecules significantly decreased in PBMCs from patients with SLE, which were 0.38-, 0.69-, 0.59-, and 0.55-fold, respectively, compared to those of control subjects (P < 0.05). The expression of Caspase 3 was significantly increased in SLE patients (P < 0.01); however, the expression of IL-1beta was not significantly different between SLE and control subjects. The expression of TRADD, FADD, RIP-1, and TRAF-2 in PBMCs from patients with SLE were negatively correlated with SLEDAI, the correlation coefficient of which was -0.285, -0.280, -0.307, and -0.298, respectively (P < 0.05). The expression of mRNA for TNF adapter molecules TRADD, FADD, RIP-1, and TRAF-2 decreased significantly in PBMCs from patients with SLE, and the expression of these adapters were negatively correlated with the SLE activity index. These abnormalities may be involved in the immunopathogenic injury mediated by the aberration TNF-alpha signaling pathway in SLE.

[Expression of tumor necrosis factor-alpha signaling adapter proteins in peripheral blood mononuclear cells from patients with systemic lupus erythematosus].

OBJECTIVE: To investigate the mRNA expression of the tumor necrosis factor (TNF)-adapter proteins, TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor-interacting protein 1 (RIP-1), and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE), and to explore the relationship between the expression of these adaptors and the SLE disease activity. METHODS: PBMC were isolated from venous blood of 51 SLE patients and 17 healthy subjects. The mRNA expression of TRADD, FADD, RIP-1, TRAF-2, Caspase 3, and interleukin (IL)-1beta were analyzed by quantitative real time RT-PCR. Disease activity was measured using the SLE Disease Activity Index (SLEDAI). RESULTS: All healthy subjects showed mRNA expressions of TRADD, FADD, RIP-1, and TRAF-2. The mRNA expression levels of TRADD, FADD, RIP-1, and TRAF- in the PBMC from the patients were 0.38, 0.69, 0.59, and 0.55 tomes that from the control subjects (all P < 0.05). The expression levels of these 4 adapters of the SLE patients with the SLEDAI >/= 10 were significantly lower than those of the SLE patients with the SLEDAI < 10 (all P < 0.05). The Caspase3 mRNA expression of the SLE patients was significantly higher than that of the healthy controls (P < 0.01); however, the IL-1beta mRNA expression was not significantly different between the SLE and control subjects. The mRNA expression levels of TRADD, FADD, RIP-1 and TRAF-2 in the PBMC from the SLE patients were all negatively correlated with the SLE activity index with the coefficient correlation of -0.285, -0.280, -0.307, and -0.298 respectively (all P < 0.05). CONCLUSION: The mRNA expression levels of the TNF adapter molecules, such as TRADD, FADD, RIP-1, and TRAF-2, decrease significantly in the PBMC from the SLE patients, and are negatively correlated with the SLE activity index. These abnormalities may participate in the immunopathogenic injury mediated by the aberration TNFalpha signaling pathway in SLE.

Tacrolimus is an alternative therapy option for the treatment of adult steroid-resistant nephrotic syndrome: a prospective, multicenter clinical trial.

BACKGROUND: The optimal therapy for adult steroid-resistant nephrotic syndrome (SRNS) remains a therapeutic challenge. We investigated the efficacy and safety of tacrolimus as a promising regimen in Chinese adult patients. METHODS: A prospective, multicenter trial was conducted in 9 nephrology centers from 2006 to 2008, in patients with SRNS (defined as failure to respond to 1 mg/kg/day of prednisone for 8, and 16 weeks, in focal segmental glomerulosclerosis). Patients were treated with tacrolimus (TAC) plus prednisone for 12 months. TAC dose was titrated to achieve a target trough blood concentration of 5-10 ng/ml for the first 6 months and 4-6 ng/ml for the subsequent 6 months. The primary outcomes included complete or partial remission [complete remission (CR): proteinuria <0.3 g/24 h, with serum albumin ≥ 3.5 g/dl and stable renal function; partial remission (PR): proteinuria between 0.3 and 3.5 g/24 h and a decrease of at least 50 % from the baseline level, with serum albumin ≥ 3.0 g/dl and stable renal function]. Secondary end-points included relapse rate, changes of clinical parameters (proteinuria, serum albumin, and lipid profile) and adverse events. RESULTS: Twenty-four patients with SRNS were enrolled. After 6 months of therapy, CR was achieved in 58.3 % of patients and PR in 16.7 %, yielding a final response rate of 75.0 %. The decrease in proteinuria was 43.1 ± 17.5 % after the first month of treatment (P < 0.001). Complete or PR was achieved in 6 of 8 patients with minimal change disease, 4 of 6 patients with mesangioproliferative glomerulonephritis (MsPGN), 6 of 7 patients with focal segmental glomerulosclerosis (FSGS), and all 2 patients with IgA nephropathy. Two patients (1 with MsPGN and 1 with FSGS) experienced relapses during the subsequent 6 months of follow-up. Adverse events included infection, hand tremor, diarrhea, acute reversible or persistent nephrotoxicity. CONCLUSIONS: In conjunction with prednisone, TAC may be an alternative therapeutic regimen for adult SRNS patients. However, adverse events in these patients should be carefully monitored, especially at the beginning of treatment. Randomized controlled trials with longer follow-up are warranted.

Outcomes of tacrolimus therapy in adults with refractory membranous nephrotic syndrome: a prospective, multicenter clinical trial.

INTRODUCTION: The treatment of adult refractory idiopathic membranous nephropathy with steroid and other immunosuppressant-resistant nephrotic syndrome can be a significant challenge. The authors investigated the efficacy and safety of tacrolimus (TAC) as a promising regimen. METHODS: A prospective, multicenter trial was conducted in 9 nephrology centers from 2006 to 2008. Fourteen patients were enrolled. In conjunction with prednisone, TAC was started at 0.05 mg/kg/d, titrated to achieve a trough blood level of 5 to 10 ng/mL for the first 6 months, then reduced to 4 to 6 ng/mL for the subsequent 6 months. The primary endpoints included complete or partial remission. Secondary endpoints included relapse, change of clinical parameters and adverse events. RESULTS: After 12 months, complete remission was achieved in 35.7% of patients and partial remission in 42.9%, yielding a response rate of 78.6%. Proteinuria, serum albumin, cholesterol, triglyceride and low-density lipoprotein were improved significantly (P < 0.001, P < 0.001, P = 0.002, P = 0.01, P = 0.004, respectively). Proteinuria and serum albumin were significantly improved (42.0% ± 13.2%, P = 0.02; 15.2% ± 4.5%, P = 0.01, respectively) even after the first month of treatment. One patient relapsed during the subsequent 6 months of follow-up. Adverse events included 2 cases of infection and 1 case each of hyperglycemia, hand tremor, sudden death (nondrug related) and diarrhea. CONCLUSIONS: TAC plus prednisone may be an alternative therapeutic option for steroid and general immunosuppressant-resistant membranous nephrotic syndrome patients, with a favorable safety profile. However, given the limitation of a small number of patients in this trial, further study with a larger number and longer follow-up is needed.

[Clinical diagnosis of BK virus infection in renal transplant recipients].

OBJECTIVE: To explore the clinical diagnosis of BK virus (BKV) infection in renal transplant recipients. METHODS: Urine and peripheral blood samples were taken from 234 renal transplant recipients for BKV detection with cytological test and real-time PCR. RESULTS: The occurrence rate of urine decoy cells, BKV viruria and viremia in these patients was 33.3 %, 33.3% and 16.2%, respectively, and the median level of urine decoy cells was 6/10 HPF, with the median level of urine and peripheral blood BKV of 7.62 x 10(3) copy/ml and 7.61 x 10(3) copy/ml, respectively. The positivity rate of BKV in the urine samples were significantly higher than that in peripheral blood samples (P=0.000). The amount of decoy cells was related to BKV load in the urine samples (gamma=0.59, P=0.000), but the BKV load in the urine samples was not related to that in peripheral blood samples (P=0.14). CONCLUSION: Renal transplantation is associated with increased BKV shedding, indicating the necessity of BKV monitoring in renal transplant recipients with urine cytology, which is convenient and sensitive and indicates renal histological changes indirectly. Urine and peripheral blood BKV DNA detection is of value in identifying BKV activation to prevent irreversible graft damage of BKV-associated nephropathy.

[Therapeutic effect of sirolimus against chronic allograft nephropathy in kidney transplant recipients].

OBJECTIVE: To investigate the efficacy and safety of sirolimus in management of chronic allograft nephropathy (CAN). METHODS: A retrospective study was conducted involving 31 CAN patients followed up since March 2002, who experienced a change from a calcineurin inhibitor (CNI)-based regimen to a SRL-based regimen. Serum creatinine (Cr) in these patients was compared before and after the regimen change, and the adverse events associated with SRL were analyzed. RESULTS: Till March 2007 when the study closed, 15 patients reached the primary endpoint for resuming dialysis, 8 had improved and 8 had stable renal function. In patients with high Cr(0)(> or =3 mg/L, n=12), 9 resumed dialysis and 2 had improved renal function, but one of the patients with renal improvement eventually died due to infection; in the patients with low Cr(0)(<3 mg/L, n=19), 5 resumed dialysis, 8 had stable renal function and 6 had improved renal function, showing significant difference between the 2 groups (P=0.003). Altogether 14 patients reached the secondary endpoint for ceasing SRL for severe infection (5 patients, of whom 4 resumed dialysis and 1 died of infection) or adverse events associated with SRL (9 patients, of whom 4 resumed dialysis, 2 had stable and 3 had improved renal function). Hyperlipidemia (51.6%), leukocytopenia (41.9%), mouth ulcer (29.0%) and liver function lesion (16.1%) were the commonest adverse events in these patients, and totalling 13 severe adverse events were recorded, including 2 fatal cerebral hemorrhage, 3 fatal infection episodes, and 8 pulmonary and urinary infections that require hospitalization. CONCLUSION: Conversion from a CNI-based to SRL-based regimen can be effective for some CAN cases, especially for those with Cr(0) below 3 mg/L. Attention must be given to adverse events like hyperlipidemia and leukocytopenia, as well as the related cerebral vascular accidents and infections.

[Characteristics of neoplasm occurrence of renal allograft recipients and their prognosis].

BACKGROUND & OBJECTIVE: Renal allograft recipients are more likely to develop neoplasm than general population because of long-term immunosuppressive treatment and concurrent infections. This study was designed to analyze the clinical features of neoplasm occurrence of renal allograft recipients, and the effect of radical surgery (RS) on their prognosis. METHODS: Records of 2 160 renal allograft recipients treated in our center from Oct. 1987 to Apr. 2003 were retrospectively studied. The time to neoplasm development, pathologic type of tumor, patients' survival time were analyzed to explore the clinical features of neoplasm developing after kidney transplantation. Recipients developed neoplasms were divided into RS group and non-RS group according to their treatment pattern. The effect of RS on patients' survival was estimated. RESULTS: A total of 33 patients developed neoplasms after transplantation. Among them,11(33.3%) developed neoplasms in digestive system. The median survival time of RS group (10 patients) was 41.5 months, that of non-RS group (23 patients) was 6.0 months. The 20-month survival rate of RS group was 70.0%, while that of non-RS group was 13.0%. CONCLUSIONS: Renal allograft recipients are more likely to develop neoplasm than general population. Moreover, their main malignancies are liver cancer, skin cancer, lymphoma and thyroid carcinoma, which differ from those observed in general population. Early diagnosis and treatment, especially feasible RS, will improve short-term outcome, while long-term therapeutic effect needs to be further observed.