Protective role of programmed death 1 ligand 1 (PD-L1)in nonobese diabetic mice: the paradox in transgenic models

OBJECTIVE—Coinhibitory signals mediated via programmed death 1 (PD-1) receptor play a critical role in downregulating immune responses and in maintaining peripheral tolerance. Programmed death 1 ligand 1 (PD-L1), the interacting ligand for PD-1, widely expressed in many cell types, acts as a tissue-specific negative regulator of pathogenic T-cell responses. We investigated the protective potential of PD-L1 on autoimmune diabetes by transgenically overexpressing PD-L1 in pancreatic β-cells in nonobese diabetic (NOD) mice.RESEARCH DESIGN AND METHODS—We established an insulin promoter–driven murine PD-L1 transgenic NOD mouse model to directly evaluate the protective effect of an organ-specific PD-L1 transgene against autoimmune diabetes. Transgene expression, insulitis, and diabetic incidence were characterized in these transgenic NOD mice. Lymphocyte development, Th1 cells, and regulatory T-cells were analyzed in these transgenic mice; and T-cell proliferation, adoptive transfer, and islet transplantation were performed to evaluate the PD-L1 transgene–mediated immune-protective mechanisms.RESULTS—The severity of insulitis in these transgenic mice is significantly decreased, disease onset is delayed, and the incidence of diabetes is markedly decreased compared with littermate controls. NOD/SCID mice that received lymphocytes from transgenic mice became diabetic at a slower rate than mice receiving control lymphocytes. Moreover, lymphocytes collected from recipients transferred by lymphocytes from transgenic mice revealed less proliferative potential than lymphocytes obtained from control recipients. Transgenic islets transplanted in diabetic recipients survived moderately longer than control islets.CONCLUSIONS—Our results demonstrate the protective potential of transgenic PD-L1 in autoimmune diabetes and illustrate its role in downregulating diabetogenic T-cells in NOD mice.Programmed death 1 (PD-1) is an immunoreceptor of the CD28/CTLA-4 family whose expression is induced in activated T- and B-cells and in macrophages (1,2). PD-1 has two cytoplasmic tyrosine motifs: one an immunoreceptor tyrosine-based inhibition motif and the other an immunoreceptor tyrosine-based switch motif (ITSM). On interaction of PD-1 with its ligands PD-L1 (B7-H1) or PD-L2 (B7-DC), the tyrosine-phosphorylated ITSM of PD-1 recruits a src homology 2 domain–containing tyrosine phosphatase 2, which mediates the dephosphorylation signaling and reduces lymphocyte activation (3). PD-1^−/− mice on different genetic backgrounds develop distinct autoimmune phenotypes, such as lupus-like glomerulonephritis/arthritis in C57Bl/6 (B6) mice or anticardiac troponin I–mediated dilated cardiomyopathy in Balb/c mice (4,5). These observations indicate that PD-1 is a critical negative regulator of lymphocyte activation and that the phenotype of PD-1 deficiency–induced autoimmunity is highly influenced by other genetic factors.Murine PD-L1 is expressed on many cell types, including stromal cells within many organs, but PD-L2 expression is much more restricted, occurring mainly in dendritic cells, activated monocytes, and macrophages (6,7). Although accumulating data indicate that both PD-L1/PD-1 and PD-L2/PD-1 signals can suppress T-cell proliferation and effector function by blocking cell cycle progression and cytokine production, signaling through PD-L1 interaction is more potent than that through PD-L2 (8). This is consistent with the observations that cytokine production, cytotoxic activity, and clonal expansion were significantly enhanced in T-cells and antigen-presenting cells from PD-L1^−/− mice, compared with cells from wild-type or PD-L2^−/− mice (9,10). Moreover, the PD-L1^−/− mice revealed an increased susceptibility to the induction of autoimmune diseases, such as experimental allergic encephalomyelitis (9), strongly suggesting a protective role of tissue PD-L1 in the maintenance of immune tolerance. Furthermore, treatment of nonobese diabetic (NOD) mice with a combination of agonistic PD-L1.Ig fusion protein and monoclonal antibodies (mAbs) to CD154 induced long-term islet allograft survival, whereas the inhibition of PD-L1–mediated signals by blocking antibody exacerbated autoimmune diabetes (11). Based on these findings, we hypothesize that transgenic expression of PD-L1 in an islet-specific manner may help in preventing T-cell–mediated islet destruction in NOD mice.To investigate the preventive and/or therapeutic potential of PD-L1 in autoimmune diabetes, we generated transgenic NOD mice overexpressing PD-L1 under control of an insulin promoter. Although a recent report demonstrated that local expression of transgenic PD-L1 on β-cells of B6 mice unexpectedly promotes organ-specific autoimmunity and transplant rejection (12), we hypothesized that overexpression of PD-L1 on islet cells in NOD mice would enhance inhibitory signaling through the PD-1–PD-L1 interaction and protect islet cells from lymphocyte attack. Our results demonstrate that transgenic PD-L1 on islet cells significantly ameliorates the severity of insulitis and incidence of diabetes in NOD mice. Interestingly, our results also indicate that local transgene expression not only protects islets in situ but also mediates a peripheral tolerance. Moreover, transgenic islets transplanted in diabetic recipients survived moderately longer than control islets. Overall, we demonstrate for the first time the preventive potential of transgenic PD-L1 in autoimmune diabetes and provide a theoretical basis for organ-specific genetic manipulation for disease prevention.     

Nasopharyngeal carcinoma--review of the molecular mechanisms of tumorigenesis

Nasopharyngeal carcinoma (NPC) is a head and neck cancer rare throughout most of the world but common in certain geographic areas, such as southern Asia. While environmental factors and genetic susceptibility play important roles in NPC pathogenesis, the Epstein-Barr virus in particular has been implicated in the molecular abnormalities leading to NPC. There is upregulation of cellular proliferation pathways such as the Akt pathway, mitogen-activated protein kinases, and the Wnt pathway. Cell adhesion is compromised due to abnormal E-cadherin and beta-catenin function. Aberrations in cell cycle are due to dysregulation of factors such as p16, cyclin D1, and cyclin E. Anti-apoptotic mechanisms are also upregulated. There are multiple abnormalities unique to NPC that are potential targets for novel treatments.     

Novel SLA-DQ alleles and their recombinant molecules in xenogeneic stimulation of human T cells

MHC class II antigens DR and DQ are essential for graft rejection both in allo- and xeno-transplantation. The antigens, especially the DQA and DQB gene-coencoded DQ molecules, are also involved in transplantation tolerance induced by activation of regulatory T cells. Here we report six novel DQ alleles from three properly inbred Chinese pig strains Gz, Bm and Yn. In our study, cDNA of swine leukocyte antigen (SLA)-DQA and -DQB were amplified by RT-PCR and sequenced for each strain. The ORF-containing SLA-DQA and -DQB genes are composed of 768 (or 765) and 786 nucleotides, encoding antigen molecules of 255 (or 254) and 261 amino acid residues, respectively. Sequences of both SLA-DQA and -DQB alleles showed disparities when compared either among the three pig strains or with available SLA data, which allows our novel alleles receiving their accession numbers from GenBank. The sequence analysis further revealed a phylogenic connection of our SLA-DQ alleles with SLA-DQ(c) haplotype. In addition, the homologies of MHC DQ or DQ-like molecules between Chinese pigs (SLA) and human (HLA) are higher than those between pigs and mice (H-2). By co-transfection of Bm pig DQA and DQB genes into L929 cells, the Bm-DQ heterodimer-expressed cells could effectively stimulate the human lymphoproliferation in presence of human APCs with a mean stimulation index (SI) 9.9+/-1.4. This functional assay indicated that our recombinant DQ antigens are capable of initiating human lymphoproliferation in a xeno-MLR.     

Effects of the Antioxidants Lycium Barbarum and Ascorbic Acid on Reperfusion Liver Injury in Rats

Objective

Ischemia/reperfusion (I/R) of the rat liver can induce liver injury through mechanisms involving oxidative and nitrosative stresses. In this study we examined the effects of antioxidants Lycium barbarum (LB) and ascorbic acid on I/R-induced liver injury in rats.

Methods

Liver ischemia was induced by clamping the common hepatic artery and portal vein of rats for 40 minutes. Thereafter, flow was restored with reperfusion for 90 minutes. Blood samples collected before ischemia and after reperfusion were analyzed for alanine transaminase (ALT), lactic dehydrogenase (LDH), hydroxyl radical, and nitric oxide (NO) levels. Pharmacologic interventions included administration of ascorbic acid (100 mg/kg, i.p., 1 hour before I/R) or LB, an extract of Gogi berries: 600 mg in 100 mL of drinking water for 2 weeks prior to experimentation.

Results

This protocol resulted in elevation of blood concentrations of NO, hydroxyl radical, ALT, and LDH (P < .001) in the I/R-induced liver injury group. Ascorbic acid significantly attenuated the reperfusion liver injury by attenuating hydroxyl radical (P < .01) and NO (P < .05) release. The LB aggravated I/R-induced liver injury by increasing hydroxyl radical release with no effect on NO release.

Discussion and conclusions

This I/R protocol resulted in oxidative and nitrosative stress and liver injury. Ascorbic acid showed significant protective effects on reperfusion liver injury by attenuating hydroxyl radical and NO release. In contrast, LB aggravated liver injury by increasing hydroxyl radical release.     

Color Doppler Ultrasound Detection and Classification of the Tangential Hepatic Vein before Laparoscopic Cholecystectomy

Background: Unexpected fatal bleeding from the gallbladder bed during laparoscopic cholecystectomy is often associated with injury to the middle hepatic vein. This paper studies whether preoperative color Doppler ultrasound is effective in reducing the risk of injury. Also a venous classification is suggested. Methods: Between June 1999 and February 2004, 2,146 patients undergoing laparoscopic cholecystectomy by standard method received preoperative color Doppler ultrasound examinations. The closest distance between the hepatic vein and the gallbladder was studied. Also, cases of liver cirrhosis, number of conversions to open cholecystectomy, intraoperative blood loss, operative time, complications, and hospital stay were recorded (group D). At the end of the study, we retrospectively reviewed the same parameter of another 2,146 patients who received laparoscopic cholecystectomy without preoperative color Doppler ultrasound between the period of March 1995 and June 1999 (group ND). Results: In group D, 108 patients had cirrhosis. Four hundred and ninety-six patients (27 cases of cirrhosis) had a closest distance of 1 mm or less between the vein and the gallbladder. There were two conversions to open cholecystectomy, but none related to gallbladder bed bleeding. In group ND, there were five conversions, including four cases of gallbladder bed bleeding from the middle hepatic vein and one case of severe adhesion. The conversion rate was significantly higher. In group ND, the mean intraoperative blood loss in the cases of liver cirrhosis was significantly greater. Also, the operative time of patients with the closest vein and gallbladder distance of 1 mm or less in group D was significantly longer. Conclusions: Color Doppler ultrasound is an effective method for detecting the presence of potential bleeders. Although the operative time will be a bit longer, the operation can be done under meticulous care and complete preparation, so that the conversion rate and the risk of fatal hemorrhage can be reduced, especially in patients with liver cirrhosis.     

Effect of radial shock wave therapy for carpal tunnel syndrome: A prospective randomized,double‐blind,placebo‐controlled trial

Three recent studies demonstrated the positive effect of extracorporeal shock wave therapy (ESWT) for treating carpal tunnel syndrome (CTS). However, none have entirely proved the effects of ESWT on CTS because all studies had a small sample size and lacked a placebo‐controlled design. Moreover, radial ESWT (rESWT) has not been used to treat CTS. We conducted a prospective randomized, controlled, double‐blinded study to assess the effect of rESWT for treating CTS. Thirty‐four enrolled patients (40 wrists) were randomized into intervention and control groups (20 wrists in each). Participants in the intervention group underwent three sessions of rESWT with nightly splinting, whereas those in the control group underwent sham rESWT with nightly splinting. The primary outcome was visual analog scale (VAS), whereas the secondary outcomes included the Boston Carpal Tunnel Syndrome Questionnaire (BCTQ), cross‐sectional area (CSA) of the median nerve, sensory nerve conduction velocity of the median nerve, and finger pinch strength. Evaluations were performed before treatment and at 1, 4, 8, and 12 weeks after the third rESWT session. A significantly greater improvement in the VAS, BCTQ scores, and CSA of the median nerve was noted in the intervention group throughout the study as compared to the control group (except for BCTQ severity at week 12 and CSA at weeks 1 and 4) (p < 0.05). This is the first study to assess rESWT in a randomized placebo‐controlled trial and demonstrate that rESWT is a safe and effective method for relieving pain and disability in patients with CTS. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:977–984, 2016.     

NO在创伤愈合过程中的作用

创伤修复是损伤组织恢复其完好结构的必需过程，大致分为三个阶段：①局部炎症反应阶段：②细胞增殖分化及肉芽组织形成阶段：③组织重建阶段。有研究发现包括一氧化氮（nitric oxide，NO）在内的小分子自由基对伤口的良好愈合起到了关键作用。现就NO在创伤修复过程中的作用作以阐述。