Autophagy in allografts rejection: A new direction?

Despite the introduction of new and effective immunosuppressive drugs, acute cellular graft rejection is still a major risk for graft survival. Modulating the dosage of immunosuppressive drugs is not a good choice for all patients, new rejection mechanisms discovery are crucial to limit the inflammatory process and preserve the function of the transplant. Autophagy, a fundamental cellular process, can be detected in all subsets of lymphocytes and freshly isolated naive T lymphocytes. It is required for the homeostasis and function of T lymphocytes, which lead to cell survival or cell death depending on the context. T cell receptor (TCR) stimulation and costimulator signals induce strong autophagy, and autophagy deficient T cells leads to rampant apoptosis upon TCR stimulation. Autophagy has been proved to be activated during ischemia-reperfusion (I/R) injury and associated with grafts dysfunction. Furthermore, Autophagy has also emerged as a key mechanism in orchestrating innate and adaptive immune response to self-antigens, which relates with negative selection and Foxp3(+) Treg induction. Although, the role of autophagy in allograft rejection is unknown, current data suggest that autophagy indeed sweeps across both in the graft organs and recipients lymphocytes after transplantation. This review presents the rationale for the hypothesis that targeting the autophagy pathway could be beneficial in promoting graft survival after transplantation.

MicroPET imaging of tumor angiogenesis and monitoring on antiangiogenic therapy with an (18)F labeled RGD-based probe in SKOV-3 xenograft-bearing mice.

So far, there is no satisfactory imaging modality to monitor antiangiogenesis therapy of ovarian cancer noninvasively. The aim of this study was to evaluate the effectiveness and sensibility of an (18)F labeled Arg-Gly-Asp (RGD) peptide in imaging and monitoring antiangiogenic responds in SKOV-3 xenograft-bearing mice. (18)F-FB-NH-PEG4-E[PEG4-c(RGDfK)]2 (denoted as (18)F-RGD2) was synthesized and employed in this study. Mice bearing ovarian cancer SKOV-3 tumors were used for biodistribution and microPET imaging studies compared with (18)F-FDG imaging. Animals were treated with low-dose paclitaxel and the effect of paclitaxel therapy on (18)F-RGD2 accumulation was investigated. Microvascular density (MVD) of SKOV-3 tumors was detected to assess the reliability of (18)F-RGD2 in antiangiogenesis monitoring. Biodistribution studies for (18)F-RGD2 revealed favorable in vivo pharmacokinetic properties, with significant levels of receptor-specific tumor uptake determined via blocking studies. MicroPET imaging results demonstrated high contrast visualization of SKOV-3 tumors. And tumor to background ratio (T/NT) of (18)F-RGD2 uptake was significantly higher than that of (18)F-FDG. Studies on antiangiogenic therapy demonstrated percentage of injected dose per gram of tissue (%ID/g) tumor uptake of (18)F-RGD2 which was obviously decreased in the treatment group than the control group, especially at 60 min (by 31.31 ± 7.18 %, P = 0.009) and 120 min (by 38.92 ± 8.31 %, P < 0.001) after injection of (18)F-RGD2. MVD measurement of SKOV-3 tumors confirmed the finding of the biodistribution studies in monitoring antiangiogenesis therapy. (18)F-RGD2, with favorable biodistribution properties and specific affinity, is a promising tracer for tumor imaging and monitoring antiangiogenesis therapy in ovarian cancer SKOV-3 xenograft-bearing mice.

Non-invasive imaging of allogeneic transplanted skin graft by 131I-anti-TLR5 mAb.

Although (18)F-fluorodeoxyglucose ((18)F-FDG) uptake can be used for the non-invasive detection and monitoring of allograft rejection by activated leucocytes, this non-specific accumulation is easily impaired by immunosuppressants. Our aim was to evaluate a (131)I-radiolabelled anti-Toll-like receptor 5 (TLR5) mAb for non-invasive in vivo graft visualization and quantification in allogeneic transplantation mice model, compared with the non-specific radiotracer (18)F-FDG under using of immunosuppressant. Labelling, binding, and stability studies were performed. BALB/c mice transplanted with C57BL/6 skin grafts, with or without rapamycin treatment (named as allo-treated group or allo-rejection group), were injected with (131)I-anti-TLR5 mAb, (18)F-FDG, or mouse isotype (131)I-IgG, respectively. Whole-body phosphor-autoradiography and ex vivo biodistribution studies were obtained. Whole-body phosphor-autoradiography showed (131)I-anti-TLR5 mAb uptake into organs that were well perfused with blood at 1 hr and showed clear graft images from 12 hrs onwards. The (131)I-anti-TLR5 mAb had significantly higher graft uptake and target-to-non-target ratio in the allo-treated group, as determined by semi-quantification of phosphor-autoradiography images; these results were consistent with ex vivo biodistribution studies. However, high (18)F-FDG uptake was not observed in the allo-treated group. The highest allograft-skin-to-native-skin ratio (A:N) of (131)I-anti-TLR5 mAb uptake was significantly higher than the ratio for (18)F-FDG (7.68 versus 1.16, respectively). (131)I-anti-TLR5 mAb uptake in the grafts significantly correlated with TLR5 expression in the allograft area. The accumulation of (131)I-IgG was comparable in both groups. We conclude that radiolabelled anti-TLR5 mAb is capable of detecting allograft with high target specificity after treatment with the immunosuppressive drug rapamycin.

Monitoring Early-Stage Acute Rejection by Imaging CXCR3-Positive Cell Infiltration: Evaluation of ¹²5Iodine-Labeled CXCL10.

OBJECTIVES: It has been reported that CXCR3 is related to inflammatory cell infiltration. The purpose of this study was to investigate iodine-125-labeled CXCL10, a ligand of CXCR3, as a tracer targeting CXCR3 to detect acute rejection in a mouse skin transplant model. MATERIALS AND METHODS: The isograft and allograft skin models were established with BALB/c and C57BL/6 mouse skin, respectively, as donors and BALB/c mice as recipients. We used reverse transcriptase-polymerase chain reaction and immunochemistry staining to test CXCR3 expression. ¹²5I-labeled CXCL10 was produced with the iodogenic method. Allograft/isograft mice were examined with whole body autoradiography and ex vivo biodistribution after tail vein injection of ¹²5I-labeled CXCL10 on day 8 posttransplant. RESULTS: CXCR3 expression was higher in allograft tissue than in isograft control. ¹²5I-labeled CXCL10 was prepared with high specificity and affinity. Biodistribution results showed higher ¹²5I-labeled CXCL10 uptake in allograft tissue. The target-to-nontarget ratio was 3.01 ± 0.25 at 24 hours, a result higher than that shown in the isograft group. Pharmacokinetic analyses of ¹²5I-labeled CXCL10 showed that distribution half-life was 0.34 hour and the elimination half-life was 9.83 hours. Dynamic whole body autoradiography images of ¹²5I-labeled CXCL10 showed excellent graft visualization in the allograft compared with the isograft group at all checking points, with visualization much more obvious at 12 and 24 hours. CONCLUSIONS: These data suggest that CXCR3 is a promising imaging target for immune cell infiltration in early-stage acute rejection and ¹²5I-labeled CXCL10 can successfully image acute rejection with good pharmacokinetics.

Autophagy is essential for flavopiridol­induced cytotoxicity against MCF­7 breast cancer cells.

Flavopiridol (FP) exerts antitumoral effects by triggering tumor cell cycle arrest and cytotoxicity in human breast cancer cell lines. The potent antitumor activity of FP is through its inhibition of cyclin­dependent kinases; however, this may not be the only mechanism of action. The present study aimed to investigate whether FP is able to induce autophagy and to examine the effects of autophagy on cell death in FP­treated MCF­7 human breast cancer cells. MCF­7 cells were treated with either FP alone or FP in combination with chloroquine (CQ). Expression levels of autophagy­related protein LC3B­II and p62/sequestosome 1 (SQSTM1) were used to monitor autophagic flux. MCF­7 cells were transfected with autophagy­related 5 (ATG5) small interfering (si)RNA to block autophagy. Cell viability and cell cycle status were determined. Following incubation with FP, MCF­7 cells exhibited significantly higher autophagy compared with untreated control cells, and the level of autophagy is comparable with cells under rapamycin induction, which was verified by immunodetection of LC3B­II and p62/SQSTM1 expression and inhibition by CQ. The addition of CQ treatment or ATG5­siRNA transfection against autophagy components attenuated the cytotoxic effects of FP treatment of MCF­7 cells. Furthermore, this autophagy inhibition did not impair the FP­induced cell cycle arrest. These results revealed that autophagy may be involved in FP­induced MCF­7 cell death and autophagy inhibition enhanced the tumor cell pro­survival ability. It is possibly that potential autophagy regulatory drugs may be used as a chemotherapy adjuvant.

Down-regulating cyclin-dependent kinase 9 of alloreactive CD4+ T cells prolongs allograft survival.

CDK9 (Cyclin-dependent kinase 9)/Cyclin T1/RNA polymerase II pathway has been demonstrated to promote the development of several inflammatory diseases, such as arthritis or atherosclerosis, however, its roles in allotransplantation rejection have not been addressed. Here, we found that CDK9/Cyclin T1 were apparently up-regulated in the allogeneic group, which was positively correlated with allograft damage. CDK9 was inhibited obviously in naive splenic CD4+ T cells treated 6 h with 3 µM PHA767491 (a CDK9 inhibitor), and adoptive transfer of these CD4+ T cells into allografted SCID mice resulted in prolonged survival compared with the group without PHA767491 pretreated. Decelerated rejection was correlated with enhanced IL-4 and IL-10 production and with decreased IFN-γ production by alloreactive T cells. More interestingly, we found that CDK942, not CDK955,was high expressed in allorejection group, which could be prominently dampened with PHA767491 treatment. The expression of CDK942 was consistent with its downstream molecule RNA polymerase II. Altogether, our findings revealed the crucial role of CDK9/Cyclin T1/Pol II pathway in promoting allorejection at multiple levels and may provide a new approach for transplantation tolerance induction through targeting CDK9.

Anti­migratory effect of rapamycin impairs allograft imaging by 18F­fluorodeoxyglucose­labeled splenocytes.

Tracking lymphocyte migration is an emerging strategy for non­invasive nuclear imaging of allografts; however, its clinical application remains to be fully demonstrated. In the present study, the feasibility of using rapamycin­treated 18F­fluorodeoxyglucose (18F­FDG)­labeled splenocytes for the in vivo imaging of allografts was evaluated. C57BL/6 skin was heterotopically transplanted onto non­obese diabetic/severe combined immunodeficient recipient mice. BALB/c 18F­FDG­labeled splenocytes with or without rapamycin pretreatment (designated as FR and FC cells, respectively) were transferred into recipient mice 30 days later. Imaging of radiolabeled cells in the skin grafts was conducted through in vivo dynamic whole­body phosphor­autoradiography and histological analysis. Notably, rapamycin impaired the migration of 18F­FDG­labeled splenocytes to the graft. At all time points, the radioactivity of allografts (digital light units/mm2) was significantly lower in the group that received FR cells, compared with the group that received FC cells (P<0.01). Furthermore, the peak allograft to native skin ratio was 1.29±0.02 at 60 min for the FR group and 3.29±0.17 at 30 min for the FC group (P<0.001). In addition, the in vivo radioactivity of the allografts was observed to be correlated with the transferred cells, which were observed histologically (r2=0.887; P<0.0001). Although 18F­FDG­labeled splenocytes migrated to the allograft, imaging of these cells may not be possible in the presence of rapamycin.

Noninvasive allograft imaging of acute rejection: evaluation of (131)I-anti-CXCL10 mAb.

The purpose of this study was to investigate the use of iodine-131-labeled anti-CXCL10 mAb as tracer targeted at CXCL10 to detect acute rejection (AR) with mice model. Expression of CXCL10 was proved by RT-PCR, ELISA, and immunochemistry staining. All groups were submitted to whole-body autoradioimaging and ex vivo biodistribution studies after tail vein injection of (131)I-anti-CXCL10 mAb. The highest concentration/expression of CXCL10 was detected in allograft tissue compared with allograft treated with tacrolimus and isograft control. Tacrolimus could obviously inhibit the rejection of allograft. Allograft could be obviously imaged at all checking points, much clearer than the other two groups. The biodistribution results showed the highest uptake of radiotracer in allograft. T/NT (target/nontarget) ratio was 4.15 ± 0.25 at 72 h, apparently different from allograft treated with tacrolimus (2.29 ± 0.10), P < 0.05. These data suggest that CXCL10 is a promising target for early stage AR imaging and (131)I-CXCL10 mAb can successfully image AR and monitor the effect of immunosuppressant.

Diversity of RGD radiotracers in monitoring antiangiogenesis of flavopiridol and paclitaxel in ovarian cancer xenograft-bearing mice.

INTRODUCTION: Although encouraging results had been shown in antiangiogenesis therapy monitoring, the underlying mechanism of RGD radiotracer accumulation needs to be further illustrated. This study was aimed to investigate the diversity of RGD radiotracers in monitoring antiangiogenic agent's effects and the underlying mechanism in ovarian cancer-bearing mice with a new agent flavopiridol compared with paclitaxel. METHODS: Ovarian cancer SKOV-3 xenograft-bearing mice were established and divided into three groups, flavopiridol, paclitaxel and control. Flavopiridol (5mg/kg body weight) and paclitaxel (20mg/kg body weight) were administered every 3 days for 16 days. Tumor growth and proliferation were monitored by caliper measurements and immunofluorescence staining. Antiangiogenic effects were determined by tumor microvessel density (MVD) in vivo and by endothelial cell tube formation assay in vitro, respectively. (99m)Tc-3P-RGD2 was prepared, and its biodistribution studies were carried out. The effect of antiangiogenesis therapy on integrin αvß3 expression was studied by immunohistochemical staining and flow cytometry. RESULTS: Both paclitaxel and flavopiridol therapy could apparently inhibit tumor growth and proliferation, and antiangiogenic effects of therapy were validated in vivo and in vitro. However, compared with the control group, ID%/g tumor uptake of (99m)Tc-3P-RGD2 showed a significant decrease at 2 hours (by 39.96%±8.23%, P=0.044) and at 4 hours (by 35.76%±11.42%, P=0.024) post injection in the paclitaxel-treated group, but a slight increase of tumor uptake in the flavopiridol-treated group at 2 hours (by 4.42%±0.24%, p=0.898) and at 4 hours (by 12.2%±1.84%, P=0.702). The further studies indicated flavopiridol therapy has a dual-effect, reducing integrin αvß3 expression on endothelial cells due to the reduction of tumor MVD and up-regulating the integrin αvß3 expression on tumor cells. CONCLUSIONS: There is diversity in evaluating antiangiogenic response when using (99m)Tc-3P-RGD2, which may be an important reminder in future clinical applications of RGD radiotracers as a strategy for antiangiogenesis therapy response monitoring.

Non-invasive imaging of Toll-like receptor 5 expression using 131I-labeled mAb in the mice bearing H22 tumors.

Toll-like receptor 5 (TLR5) is overexpressed in several cancers and metastases, and presents an enticing target for molecular imaging of primary tumors. In the present study, 131I-anti-TLR5 monoclonal antibody (mAb) was evaluated for its use as a novel radiotracer for imaging hepatocarcinoma in mice bearing H22 tumors. The expression of TLR5 was analyzed by quantitative polymerase chain reaction and immunohistochemistry. The anti-TLR5 mAb and isotype immunoglobulin G (IgG) were radiolabeled with iodine-131 by the Iodogen method. The in vitro stability of iodinalized probes was determined in serum or saline for a series of times, and then evaluated with radio-thin-layer chromatography. The biodistribution study and autoradiography were performed in H22 tumor-bearing mice. It was found that H22-xenografted tumor tissue exhibited a higher level of TLR5 expression compared with normal liver tissues. 131I-anti-TLR5 mAb and 131I-IgG were obtained subsequent to purification, with high radiochemical purity (>95%), and remained stable for 48 h in human serum. The target-to-non-target ratio in the 131I-anti-TLR5 mAb group was significantly higher compared with the 131I-IgG group. The biodistribution study and autoradiography demonstrated that 131I-anti-TLR5 mAb was specifically retained in hepatocarcinoma with a high tumor uptake. Altogether, these results show that 131I-anti-TLR5 mAb is capable of detecting lesions in a TLR5-expressing tumor, with high target selectivity, and may offer a promising agent for hepatocarcinoma diagnosis and encourage further investigation.

Basic fibroblast growth factor upregulates survivin expression in hepatocellular carcinoma cells via a protein kinase B-dependent pathway.

Basic fibroblast growth factor (bFGF) plays an important role in tumor angiogenesis. Several studies have reported that bFGF may influence cell apoptosis through different signaling pathways. The aim of the present investigation was to study the effect of bFGF on the activities of protein kinase B (PKB)/survivin and cell apoptosis in hepatocellular carcinoma cells (Bel-7402). We treated Bel-7402 cells with bFGF and wortmannin [phosphatidylinositol 3-kinase (PI3K)-specific inhibitor] separately to observe the expression of PKB and survivin detected with RT-PCR and western blotting. The cell cycle and apoptosis were assayed with flow cytometry. We found a significant increase in PKB expression in the group treated with 25 ng/ml bFGF for 10 min (P<0.05), and this effect was significantly inhibited by pretreatment with wortmannin (200 nM) for 1 h. After treatment with 10 ng/ml bFGF, the expression of survivin mRNA in Bel-7402 cells increased significantly, and reached the peak at 16 h (P<0.05); however, this effect could be significantly inhibited by pretreatment with wortmannin (200 mM) in a time-dependent manner. Following incubation with 25 ng/ml bFGF for 10 min, the apoptosis rate and M phase were significantly decreased and S phase cells increased compared with the wortmannin (200 nM)-treated group. When this group was pretreated with wortmannin (200 nM) for 1 h, the apoptosis rate and S phase were significantly increased, M phase cells decreased. The results revealed that wortmannin could induce high apoptosis rates in hepatocellular carcinoma cells, and bFGF could inhibit the cell apoptosis induced by wortmannin. These findings indicate that bFGF could rapidly activate the PKB activities, enhance the expression of survivin and the proliferation of hepatocellular carcinoma cells via the PI3K pathway, thus it may serve as a novel molecule for early targeting therapy of hepatocellular carcinoma.