Down-Regulation of Donor Kupffer Cell B7 Expression Reduced Recipient Lymphocyte Activation and Secretion of Interleukin-2 In Vitro.

BACKGROUND: Kupffer cell (KC), a kind of important antigen-presenting cell in liver, play an important role in the process of acute rejection after liver transplantation. The aim of this study was to investigate effect of suppression of donor KC B7 expression on recipient lymphocyte activation and secretion of interleukin-2 (IL-2) in vitro. METHODS: Liver ex vivo perfusion with collagenase IV and density-gradient centrifugation were used to isolate donor Lewis rat KCs. The interference fragments of the B7 molecule were designed to construct RNA interference vector pSilencer 3.1H1-Neo-B7 that was transfected into KCs of donor rat. Reverse-transcription polymerase chain reaction was used to detect the changes in the expression of B7 molecules in KCs. The transfected KCs were divided into 3 groups: A, control group; B, empty vector group; and C, RNA interference group. The lymphocytes of recipient Brown Norway (BN) rats were isolated and cocultured with the cells in the 3 groups. Enzyme-linked immunosorbent assay was used to detect the content of IL-2 in the culture supernate. Methylthiazolyl tetrazolium assay was used to detect the proliferation of lymphocytes. RESULTS: The yield rate of KCs was 5 × 10(7), and the cell viability was >98%. RNA interference vector had been successfully constructed and identified by means of enzyme digestion and sequencing. The expression of B7 in KCs decreased by 22% after RNA interference (P < .01). After coculturing with lymphocytes of BN rats, compared with the control group, the decreased expression of B7 significantly inhibited the activation and proliferation of lymphocytes as well as the secretion of IL-2 by lymphocytes. The proliferation of lymphocytes in recipient BN rats decreased by 49% (P < .01), and the secretion of IL-2 in the culture supernate decreased by 67% (P < .01). CONCLUSIONS: This study successfully constructed a B7 RNA interference vector, and applied it to assessing reduction of B7 expression in donor KCs. RNA interference significantly suppressed the activation of recipient T lymphocytes and secretion of IL-2 via the CD28/B7 costimulatory pathway and may induce immune tolerance in liver transplants.

PD-1/PD-L1 costimulatory pathway-induced mouse islet transplantation immune tolerance.

BACKGROUND: Programmed death-1/PD-1 ligand-1 (PD-1/PD-L1) costimulatory signals may play an important role in T-cell-induced immune response. The aim of this study is to investigate the role of the PD-1/PD-L1 costimulatory pathway on immunotolerance induction in mouse pancreatic islet transplantation. METHODS: Full-length mouse PD-L1 cDNA was subcloned into pShuttle-GFP-CMV(-) shuttle plasmid. The product was cut by certain restriction endonuclease and ligated with pAdxsi vector. The adenovirus bone plasmid was transformed into DH5α-competent bacteria. After linearization, the recombined adenovirus DNA was transfected into 293 cells for package and amplification. Streptozotocin was injected intraperitoneally into C57BL/6 (H-2(b)) mouse to induce diabetic model recipient. Recipients were randomly divided into 3 groups. Group A was the control. Group B and group C were injected with Ad-EGFP and Ad-PD-L1 through the tail vein, respectively, 1 day before islet transplantation. The 300 to 400 islets of DBA/2 (H-2(d)) were transplanted into the renal subcapsular space of the diabetic model recipient. We monitored and analyzed the blood glucose levels and the survival time of grafts after transplantation. RESULTS: Recombinant adenovirus Ad-PD-L1 had high efficiency expression of PD-L1 in recipient mouse. The blood glucose concentration of mice in the Ad-PD-L1 gene treatment group was obviously lower than that of the control and Ad-EGFP treatment groups and was stable and kept within the normal range at post-transplant 21 days. The survival time of grafts in the Ad-PD-L1 group (27.6 ± 3.5 days) was significantly longer than in the control (7.8 ± 0.33 days) and Ad-EGFP groups (7.6 ± 0.59 days), P < .01. Mixed lymphocyte response showed a specific decrease reaction of recipient lymphocyte vs donor lymphocyte. Flow cytometry detection showed that unsplit cells occupied 90% of recipient mouse lymphocytes, but unsplit cells among normal C57BL/6 mouse lymphocytes without Ad-PD-L1 gene treatment were 51 in the control group. The differences between them were significant (P < .01). CONCLUSION: Recombinant adenovirus Ad-PD-L1 has been successfully constructed. In mouse pancreatic islet transplantation, it can suppress the activation of recipient T lymphocyte through the PD-1/PD-L1 costimulatory pathway, and significantly prolong the survival time of grafts.

Transfection of CD40Ig into liver prevents acute rejection in rat liver transplantation.

OBJECTIVE: To investigate the effect of CD40Ig upon acute rejection of rat liver transplants. METHODS: Thirty-two orthotopic liver transplants were performed using Lewis to BN rats with "the two-cuff technique". The rats were randomly divided into three groups. Group A served as controls (n = 10); group B (n = 11) and group C (n = 11); Lipofectamine2000-pcDNA3.1 or Lipofectamine2000-pcDNA3.1. CD40Ig complex was injected into Lewis portal vein ex vivo before cold storage of the liver. On the fifth day after transplantation, three rats in each group were killed to study the pathological changes and TUNEL immune histochemistry performed to examine CD40Ig expression. Lymphocytes were obtained from the spleen. The mixed lymphocyte reaction (MLR) was performed to determine tolerance and sheep anti-human immunoglobulin G (IgG)-FITC-labeled T cells counted by flow cytometry. Postoperative survival times of rats in each group were recorded. The pathological changes of dead rats were observed. RESULTS: The mean survival times of group A and B were 11.00 +/- 4.28 and 12.75 +/- 5.57 days, respectively. There were serious acute rejections in allograft liver in groups A and B. Apoptosis index was 33.67 +/- 5.69 versus 39.00 +/- 5.29. Group C mean survival time was 41.25 +/- 13.70 days (P < .01). Immune histochemistry showed CD40Ig-positive elements in the allograft liver, which revealed light acute rejection and apoptosis index was 0.27 +/- 0.21 (P < .01). The part of the allografted liver in a dead rat showed light acute rejection while the others displayed chronic rejection. Recipients were specifically tolerant to donors in the MLR assay. The IgG-FITC-labeled T cells accounted for 11.57% of all T cells in group C. CONCLUSIONS: CD40Ig transfection inhibited T-cell costimulatory pathway, prevented acute rejection, and prolonged graft survival.

Evaluation of MAGE-1 and MAGE-3 as tumour-specific markers to detect blood dissemination of hepatocellular carcinoma cells.

The members of MAGE gene family are highly expressed in human hepatocellular carcinoma (HCC). In the present study, we tested the tumour-specific MAGE-1 and MAGE-3 transcripts in the peripheral blood of HCC patients by nested RT-PCR to detect the circulating tumour cells and evaluate their potential clinical implication. Of 30 HCC patients, the positive rate of MAGE-1 and MAGE-3 transcripts was 43.3% (13 out of 30) and 33.3% (10 out of 30) in PBMC samples, whilst the positive rate was 70% (21 out of 30) and 53.3% (16 out of 30) in the resected HCC tissue samples, respectively. The positivity for at least one MAGE gene transcript was 63.3% (19 out of 30) in PBMC samples of HCC patients and 83.3% (25 out of 30) in the resected HCC tissue samples. MAGE-1 and/or MAGE-3 mRNA were not detected in the PBMC of those patients from whom the resected HCC tissues were MAGE-1 or MAGE-3 mRNA negative, nor in the 25 PBMC samples from healthy donors. The detection of MAGE transcripts in PBMC was correlated with the advanced stages and tumour size of the HCC, being 82.4% (14 out of 17) in tumour stages III and IVa, 56.6% (five out of nine) in stage II, and null (nought out of four) in stage I. The serum alpha-FP in 33.3% (10 out of 30) of HCC patients was normal or slightly elevated (< 40 ng ml(-1)). However, six of these 10 patients (alpha-FP < 40 ng ml(-1)) were MAGE-1 and /or MAGE-3 mRNA positive in their PBMC. The follow-up survey of MAGE mRNA in PBMC was performed in 12 patients. Seven patients with persistent MAGE-1 and/or MAGE-3 mRNA positive or from negative turned to positive died because of metastasis and/or recurrence. In striking contrast, all four patients with MAGE-1 and/or MAGE-3 mRNA from positive turned to negative and one patient with persistent MAGE-3 transcript negative are alive after last test. Collectively, detection of MAGE transcripts with follow-up survey in PBMC is a feasible and reliable assay for the early prediction of the relapse and prognosis of the HCC patients.

[Effect of cimetidine on wedged hepatic venous pressure in cirrhotic patients].

Basing on our supposition that "humoral mechanism" played an important role in the pathogenesis of portal hypertension due to cirrhosis, antagonists to some of these humoral substances (HS) would have a lowering effect on the portal pressure in cirrhotic patients. The wedged hepatic venous pressure (WHVP) was used as an indicator in reflecting the changes of portal pressure. Cimetidine was given intravenously to 8 cirrhotic patients, an average lowering of 0.72 kPa (7.3 cm H2O) of WHVP was observed. This was of clinical significance as compared with the previous results of splenorenal shunting operations.

Effect of cimetidine on wedged hepatic venous pressure in cirrhotic patients.

According to our supposition that "humoral mechanism" plays an important role in the pathogenesis of portal hypertension due to cirrhosis, antagonists to some of humoral substances would lower the portal pressure in cirrhotic patients. Wedged hepatic venous pressure (WHVP) was used as an indicator for changes of portal pressure. Cimetidine was given intravenously to 8 cirrhotic patients, in whom an average lowering of 0.72 kPa (7.3 cm H2O) of WHVP was observed subsequently. This change was of clinical significance as compared with the previous results of splenorenal shunting operations.

Changes of blood humoral substances in experimental cirrhosis and their effects on portal hemodynamics.

The changes of humoral substances in the blood of cirrhotic rats were studied together with their effects on portal hemodynamics at different stages during the development of cirrhosis. The profiles of humoral substances and hemodynamics in two different cirrhotic rat models were also investigated. During the development of cirrhosis, glucagon increased markedly in all stages, histamine and vasoactive intestinal polypeptide (VIP) increased in the early stage, serotonin (5-HT) and somatostatin (SS) increased in the middle and late stages. There were different patterns of humoral substances in different cirrhotic models. Glucagon was the main humoral substance elevated in CCL4 induced cirrhosis, but histamine and 5-HT were mainly elevated in the blood in thioacetamide (TAA) induced cirrhosis. The hemodynamics altered differently in different stages during the development of cirrhosis and differently in the two cirrhotic rat models. Exchange transfusions between normal and cirrhotic rats resulted in an elevation of portal flow in normal rats, but no such changes were found after exchange pressure and an increase of portal blood transfusions between normal rats. The relationship between the humoral substances and portal hemodynamics is discussed. The results of this study strongly support the hypothesis of "humoral mechanism" in the pathogenesis of portal hypertension due to cirrhosis.

Reversal of mesangial enlargement in rats with long-standing diabetes by whole pancreas transplantation.

An important unanswered question about clinical use of pancreas transplantation is: can pancreas transplants reverse or, at least, stabilize well-established lesions of insulin-dependent diabetes mellitus (IDDM)? To answer this question, we performed whole pancreas transplantations in 190 highly inbred rats 6, 9, 12, 15, 18, and 21 mo after induction of diabetes mellitus (DM) with alloxan. We then studied the effect on renal mesangial enlargement (ME) for 24 mo after onset of DM by a quantitative morphologic technique in which camera lucida tracings of the mesangium were made at X 1250 and were analyzed using an electronic planimeter connected to a calculator/computer. A pretransplant kidney biopsy was obtained so that the rats served as their own controls. In addition, studies were performed for 28 mo in 57 untreated diabetic controls and in 55 nondiabetic controls. Monthly metabolic studies showed that whole pancreas transplantation maintained very tight, lifelong metabolic control of diabetes. Kidney sections obtained for 2 yr from diabetic controls and for 21 mo from diabetic rats before transplantation showed highly significant increases in total mesangial area, nuclear-free mesangial area, and percentage of glomerular area occupied by nuclear-free mesangial area. Pancreas transplantation consistently produced a highly significant reversal of well-established ME, regardless of when it was performed.(ABSTRACT TRUNCATED AT 250 WORDS)