Expression of programmed death ligand-1 (PD-L1) in metastatic and postchemotherapy viable testicular germ cell tumors

IntroductionChemotherapy for testicular germ cell tumors (GCT) is highly effective, with few patients who do not respond. Clinical studies to evaluated novel treatments are challenging given the rarity of these patients. Therefore, we sought to evaluate PD-L1 staining on metastatic and postchemotherapy viable testicular GCTs as a surrogate for potential benefit for immunotherapy targeting the PD-1/PD-L1 axis.MethodsEthics research committee approval for this retrospective study was obtained by four participating institutions (CHU de Québec, St. Joseph's Health Care, Halifax Health Science Centre, Johannes Gutenberg University). Patients with viable metastatic testicular GCTs pathology samples were included. Patients with pure teratoma were excluded. PD-L1 staining with the 22C3 clone was evaluated on samples with >100 viable tumor cells using the combined positive score (CPS).ResultsFrom 51 patients identified at participating institutions, 24 postchemotherapy and 18 chemotherapy-naive metastatic samples were available for PD-L1 staining, with 9 matched prechemotherapy samples and 7 matched orchiectomy pathology samples, respectively. The median CPS score was 55.6 (IQR 16–100) for all metastatic samples, 44.9 (IQR 13–100) for postchemotherapy metastatic samples, and 68.8 (IQR 38–100) for chemotherapy-naïve metastatic samples, with the median number of viable tumor cells at 545, 500, and 550, respectively. Differences were not significant between chemotherapy-naïve and postchemotherapy samples (P = 0.07), though among non-seminoma GCT metastatic samples, CPS scores were significantly lower postchemotherapy (P = 0.02). Significant differences among postchemotherapy metastatic tumors were also seen according to predominant subtype, with lower CPS scores for predominant yolk sac and higher values for predominant seminoma and choriocarcinoma. In 7 patients with matched specimens pre- and postchemotherapy, a significant increase in CPS was observed for seminoma (26.7 vs. 81.7, P = 0.045), but not nonseminoma GCTs. Comparing all chemotherapy naïve-samples, PD-L1 expression was higher in metastatic samples versus testicular samples (mean CPS 68.8 vs. 39.8, P = 0.02). This was also seen in matched chemotherapy-naïve samples (mean CPS 77.9 vs. 33.1, P = 0.01).ConclusionOur results suggest that most patients with refractory GCTs postchemotherapy will not benefit from PD-1/PD-L1 immunotherapy. However, the high PD-L1 expression in patients with predominant or pure seminoma post-chemotherapy suggests this may represent a subgroup for whom further trials may be considered.     

Altered bile acid metabolism in nonobese, spontaneously diabetic (NOD) mice

Cholesterol and bile acid metabolism was examined in nonobese, spontaneously diabetic (NOD) female mice before and after the development of diabetes. After the development of glucosuria, the plasma and liver cholesterol levels, gallbladder bile weight after 5-h fasting, biliary cholesterol, phospholipid and bile acid concentrations, the lithogenic index, the pool size of bile acids, and fecal sterol excretion markedly increased, but fecal bile acid excretion and fractional turnover rates for the cholic acid and chenodeoxycholic acid groups decreased. The distribution percentage of bile acids in the small intestine did not change, but it increased in the gallbladder and decreased in the large intestine. One striking finding was a change in the bile acid composition: increases were recorded in cholic and deoxycholic acids while decreases occurred in bile acids derived from chenodeoxycholic acid, such as beta-muricholic and ursodeoxycholic acids in the bile and alpha-muricholic, beta-muricholic, omega-muricholic, and hyodeoxycholic acids in the feces. Therefore, the cholic acid group/chenodeoxycholic acid group (CA/CDCA) ratio increased in the bile, feces, and small and large intestines after the development of diabetes. These changes were very similar to those observed in alloxan-treated mice, suggesting that the changes found in NOD mice are caused by insulin deficiency.     

Alpha-1 antitrypsin treatment of spontaneously diabetic nonobese diabetic mice receiving islet allografts

Alpha-1 antitrypsin (AAT) is a serine protease inhibitor able to prevent diabetes onset in nonobese diabetic (NOD) mice and to prolong islet allograft survival in a nonautoimmune murine model. In this study, we explored the effect of chronic administration of human AAT (hAAT) on allogeneic (C57BL/6) islet graft survival in spontaneously diabetic female NOD mice. Mice received intraperitoneal treatment with saline, Prolastin (1 or 2 mg/mouse) or Aralast (2 mg/mouse) on days -1, 0, 3, 6, and 9. Saline-treated mice rejected the grafts 10.0 +/- 2.5 days after transplantation (n = 9). Prolastin 1 mg (n = 9) and 2 mg (n = 3) resulted in rejection on 8.7 +/- 1.4 (not significant) and 13.0 +/- 4.3 days (P < .03), respectively. Aralast-treated mice showed prolongation of graft survival (13 +/- 5.9 days; n = 5; P < .03). Notably, repeated administrations of either hAAT formulation led to sudden death of a proportion of treated animals. Collectively, our preliminary data indicate that prolongation of islet allograft survival in the stringent autoimmune diabetic NOD mouse model can be achieved with hAAT monotherapy. The death of a proportion of treated animals may be consequent to immunization to hAAT and lethal hypersensitivity. Interestingly, this phenomenon was not observed in a non-autoimmune mouse strain (C57BL/6) despite extended hAAT treatment (>100 days).     

Transfer of autoimmune diabetes mellitus with splenocytes from nonobese diabetic (NOD) mice

The nonobese diabetic (NOD) mouse, a model of human type I diabetes, develops insulitis beginning at 4-6 wk of age. By 30 wk of age, 72% of females and 39% of males develop spontaneous diabetes, apparently because of an overwhelming autoimmune response to the insulin-producing beta-cells within the islets. To identify the immune mechanism responsible for destruction of beta-cells in the NOD mouse, we developed an adoptive transfer protocol that induces diabetes in NOD mice at an age when spontaneous diabetes is rarely observed. Splenocytes from overtly diabetic NOD mice were unable to transfer diabetes to very young (less than or equal to 6 wk) irradiated NOD mice but effectively transferred diabetes to irradiated NOD mice greater than 6 wk of age. In such transfers, overt diabetes was induced within 12-22 days in greater than 95% (79/82) of the recipients. Thus, transfer of splenocytes to young mice induces them to become diabetic at a higher frequency and at a younger age than their untreated littermates. Equally successful transfers with as few as 5 X 10(6) spleen cells have been performed in male and female NOD mice, even though males display a lower spontaneous incidence of diabetes than females. Splenocytes obtained from diabetic mice maintained on insulin for up to 2 mo also transferred diabetes. Because NOD mice display increasing levels of insulitis with age, spleen cells obtained from nondiabetic NOD mice of different ages were tested for their ability to transfer diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of renal AGE-receptor genes in NOD mice: possible role in nonobese diabetic renal disease

BACKGROUND: Nonobese diabetic mice (NOD) are prone to glomerular pathology, which is accelerated with the onset of diabetes. Advanced glycation end product (AGE) interactions with AGE-receptors (AGE-Rs) in kidneys can contribute to glomerular injury and diabetic nephropathy (DN). The significant elevation in kidney AGE deposits noted in prediabetic NOD mice suggested that delayed AGE turnover in this model may contribute to its propensity toward DN. METHODS: To explore whether excess tissue AGE was linked to altered AGE-R status in the kidney, mRNA/protein expression, and of several AGE-Rs [AGE-R1, AGE-R2, AGE-R3, scavenger receptor II (ScR-II), and receptor for AGE (RAGE)], was determined in renal cortex and in mesangial cells (MCs) isolated from ND-, D-NOD, and ILE mice (N = 20 per group). Ligand binding, receptor site number, and affinity were determined in MCs from the same mouse groups. RESULTS: Prediabetic NOD kidney AGE-R1 mRNA and protein level were threefold lower than that of ILE mice (P < 0.01), while AGE-R3 mRNA was enhanced by twofold (P < 0.05) and AGE-R2, RAGE, and ScR-II mRNA remained close to normal (ILE). The onset of diabetes in NOD mice, while enhancing AGE-R1 mRNA expression by approximately twofold, failed to raise it above the normal (ILE) level, despite increases in tissue, and serum AGE. The latter was associated with higher elevation in AGE-R3 (sixfold, P < 0.05), RAGE (twofold, P = NS), and ScR-II mRNA (2. 8-fold, P = NS) above control. MCs from prediabetic NOD mice showed a threefold lower level of AGE-R1 mRNA (P < 0.02 vs. ILE) and AGE-R1-protein, and AGE-binding activity (<40% of control ILE). In contrast, AGE-R3 mRNA was enhanced (twofold), while AGE-R2 showed no change. Cultured ND-NOD MCs displayed only one fourth of the AGE-binding sites/cell present on ILE MCs (1.6 x 10(6) vs. 6.6 x 10(6), P < 0.05), which after the onset of diabetes rose to the normal range (7.0 x 10(6)/cell), but failed to exceed it. CONCLUSIONS: Reduced AGE-R1 gene expression in this strain may contribute to delayed AGE removal from and early AGE deposition in kidney tissues. This may act as a trigger for those AGE-R genes involved in growth-promoting changes, leading to DN in this strain.     

Accelerated diabetes in rat insulin promoter-tumor necrosis factor-alpha transgenic nonobese diabetic mice lacking major histocompatibility class II molecules

The major predisposing genetic component in type 1 diabetes maps to the major histocompatibility complex locus in both mice and humans. To verify the HLA class II association with disease pathogenesis, we adopted the transgenic approach. Expression of HLA-DQ8, the molecule showing the strongest association with human type 1 diabetes, in the diabetes-predisposing milieu of NOD mice in the absence of the endogenous class II molecule I-A(g7) did not render susceptibility to type 1 diabetes. To study if providing a local proinflammatory environment would lead to diabetes in these mice, Abeta(o).NOD.DQ8 were bred with C57BL/6 mice expressing tumor necrosis factor (TNF)-alpha in the beta-cells of the islets of Langerhans. Surprisingly, although diabetes was evident in the F1 intercross expressing rat insulin promoter (RIP)-TNF, offspring lacking either endogenous or transgenic class II molecules developed accelerated diabetes with high frequency in both sexes. Moreover, expression of any functional class II molecule seemed to confer significant protection from diabetes in this model. Thus, neonatal expression of TNF-alpha in an islet-specific manner bypassed the requirement of CD4(+) T-cells and resulted in diabetes that could be mediated by CD8(+) T-cells. We also show for the first time that diabetes in NOD.RIP-TNF mice can occur independent of inheritance of NOD-derived idd1.     

A protective role for programmed death 1 in progression of murine adriamycin nephropathy

Programmed death 1 (PD-1) is a novel member of the CD28/cytotoxic T-lymphocyte-associated protein-4 superfamily, which plays an important role in the regulation of activated T cells. However, it is not clear how PD-1 is expressed in normal and diseased kidney, nor if it has a role in progression of chronic renal disease. PD-1 expression and the effect of monoclonal anti-PD-1 antibody (Ab) were examined in murine adriamycin nephropathy (AN). BALB/c mice were divided into three groups: (a) normal mice, (b) adriamycin (ADR) with control immunoglobulin (Ig)G (ADR-IgG), and (c) ADR with anti-PD-1 Ab (ADR-Ab). AN was induced by a single intravenous injection of ADR. Anti-PD-1 Ab was given by intraperitoneal injection on alternate days from day 0 to day 10, or to day 18. Animals were killed at week 4. Renal function, histological change, and cytokine expression were examined. PD-1 mRNA was detected in kidney tissue of mice with AN in a dose- and time-dependent manner. PD-1 was mainly expressed on injured tubule cells and some interstitial cells, which co-stained with alpha-smooth muscle actin in AN, but not in normal kidney. Anti-PD-1 treatment up to day 18, but not to day 10, worsened glomerular and tubulointerstitial injury. The ratio of urinary protein/creatinine was significantly higher in ADR-Ab mice than ADR-IgG mice. The number of macrophages was significantly increased in ADR-Ab mice compared with ADR-IgG mice. Blockade of PD-1 worsened progressive renal histopathological and functional injury in murine AN. This suggests a possible protective role for PD-1 in chronic renal disease, and its potential as a treatment to slow disease progression.     

Effect of diet on incidence of diabetes in nonobese diabetic mice

Nonobese diabetic/Lt mice exhibit a diabetes incidence greater than 70% in females at 30 wk of age. In studies designed to see whether increased dietary carbohydrate, fat, or protein influenced the severity or age at onset of the syndrome, we fed semipurified AIN-76 diet adulterated with increased amounts of these ingredients. Surprisingly, all AIN-76-based diets greatly reduced the expected incidence of diabetes at 30 wk. In addition, a hypoallergenic infant formula, Pregestimil, containing casein hydrolysate in place of protein, completely prevented diabetes up to 1 yr of age. To assess how dietary components might modulate the diabetes incidence, we adulterated standard AIN-76 diet with skim milk, gluten, brewer's yeast, or a natural-ingredient rodent open-formula mouse diet (Old Guilford 96 [OG96]. No increase in diabetes incidence was seen with skim milk (10%) or wheat gluten (10%), whereas brewer's yeast (10%) and OG96 (25%) added to AIN-76 increased the incidence compared to mice fed OG96 only. The diabetogenic factor or factors in OG96 could be extracted by chloroform plus methanol (2:1), leaving little activity in the residue. We conclude that diet is a critical factor in diabetes development and that unknown chloroform-methanol-soluble substances in natural-ingredient chow not found in semipurified diets can enhance the development of diabetes in genetically susceptible mice.     

Unexpected sensitivity of nonobese diabetic mice with a disrupted poly(ADP-Ribose) polymerase-1 gene to streptozotocin-induced and spontaneous diabetes

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that consumes NAD in response to DNA strand breaks. Its excessive activation seems particularly deleterious to pancreatic beta-cells, as exemplified by the complete resistance of PARP-1-deficient mice to the toxic diabetes induced by streptozotocin. Because of the possible implication of this enzyme in type 1 diabetes, many human trials using nicotinamide, an inhibitor of PARP-1, have been conducted either in patients recently diagnosed or in subjects highly predisposed to this disease. To analyze the role of this enzyme in murine type 1 diabetes, we introgressed a disrupted PARP-1 allele onto the autoimmune diabetes-prone nonobese diabetic (NOD) mouse strain. We showed that these mice were protected neither from spontaneous nor from cyclophosphamide-accelerated diabetes. Surprisingly they were also highly sensitive to the diabetes induced by a single high dose of streptozotocin, standing in sharp contrast with C57BL/6 mice that bear the same inactivated PARP-1 allele. Our results suggest that NOD mice are characterized not only by their immune dysfunction but also by a peculiarity of their islets leading to a PARP-1-independent mechanism of streptozotocin-induced beta-cell death.     

Prevention of autoimmune diabetes by FTY720 in nonobese diabetic mice

BACKGROUND: FTY720 prevents allograft rejection with remarkable potency without inducing generalized immunosuppression. We determined the effect of FTY720 on development of autoimmune diabetes in nonobese diabetic (NOD) mice. METHODS: NOD mice were given FTY720 (0.5 mg/kg, orally) five times per week starting from 4 weeks of age. RESULTS: Daily FTY720 prevented development of diabetes in 15 of 16 treated mice, whereas 70% of untreated NOD mice became diabetic by 35 weeks of age. Withdrawal of FTY720 at 35 weeks of age led to development of diabetes within 2 weeks in five mice, whereas the remaining mice maintained diabetes-free conditions for up to 44 weeks of age. No side effect of the drug was seen throughout the treatment period. FTY720 also prevented cyclophosphamide-induced diabetes in NOD mice. CONCLUSIONS: FTY720 is a safe and benign therapeutic agent that may be used chronically in prediabetic individuals.     

Development and function of diabetogenic T-cells in B-cell-deficient nonobese diabetic mice

Insulin-dependent diabetes (type 1 diabetes) in the NOD mouse is a T-cell-mediated autoimmune disease. However, B-cells may also play a critical role in disease pathogenesis, as genetically B-cell-deficient NOD mice (NOD.microMT) have been shown to be protected from type 1 diabetes and to display reduced responses to certain islet autoantigens. To examine the requirements for B-cells in the development of type 1 diabetes, we generated a B-cell-naive T-cell repertoire by transplantation of NOD fetal thymuses (FTs) into NOD.scid recipients. Surprisingly, these FT-derived NOD T-cells were diabetogenic in 36% of NOD.scid recipients, despite the absence of B-cells. In addition, T-cells isolated from NOD.microMT mice were diabetogenic in 22% of NOD.scid recipients. Together, these results indicate that B-cells are not an absolute requirement for the generation or effector function of an islet-reactive T-cell repertoire in NOD mice. We suggest that conditions favoring rapid lymphocyte expansion can reveal autoreactive T-cell activity and precipitate disease in genetically susceptible individuals.     

Mapping non-class II H2-linked loci for type 1 diabetes in nonobese diabetic mice

Major histocompatibility complex (MHC) plays a largely predominant role in the genetic predisposition to type 1 diabetes, in both humans and rodents. While class II loci have long been recognized as essential, they do not fully explain the MHC-linked genetic component of type 1 diabetes. In the present study, using new NOD congenic strains harboring defined chromosomal segments from C57BL/6 mice, we circumscribed three distinct loci influencing murine type 1 diabetes and tightly linked to but separated from the class II region. Our findings might guide the search for additional HLA-linked loci in human type 1 diabetes.     

趋化素样因子超家族6与程序性死亡配体1在胶质瘤中的表达及其临床意义

目的:检测趋化素样因子超家族6(CMTM6)及程序性死亡配体1(PD-L1)在胶质瘤组织中的表达,并分析两种蛋白的表达相关性及其对预后的影响。方法:收集郑州大学第五附属医院神经外科收治的胶质瘤患者76例与同时期脑外伤开颅患者(对照组)40例,留取术中组织标本,通过免疫组织化学法(IHC)和蛋白质印迹法(Western ...