Overexpression of Stat3 increases circulating cfDNA in breast cancer.

PURPOSE: Current studies on circulating cell-free DNA (cfDNA) have been focusing on its potential as biomarkers in liquid biopsy by detecting its content or genetic and epigenetic changes for the evaluation of tumor burden and therapeutic efficacy. However, the regulatory mechanism of cfDNA release remains unclear. Stat3 has been documented as an oncogene for the development and metastasis of breast cancer cells. In this study, we investigated whether Stat3 affects the release of cfDNA into blood and its association with the number of circulating tumor cells (CTCs). METHODS: The cfDNA level in plasma of patients with breast cancer and healthy volunteers were determined by quantitative real-time PCR. Three mouse breast cancer models with different Stat3 expression were generated and used to established three breast cancer orthotopic animal models to examine the effect of Stat3 on cfDNA release in vivo. Stat3 mediated Epithelial-mesenchymal phenotype transition of CTCs was determined by immunofluorescence assay and Western blot assay. RESULTS: The data showed that Stat3 increased circulating cfDNA, which is correlated with the increased volume of primary tumors and number of CTCs, accompanied with the dynamic EMT changes regulated by Snail induction. Furthermore, the high level of total circulating cfDNA and Stat3-cfDNA in patients with breast cancer were detected by quantitative real-time PCR using GAPDH and Stat3 primers. CONCLUSION: Our results suggested that Stat3 increases the circulating cfDNA and CTCs in breast cancer.

Zenglv Fumai Granule protects cardiomyocytes against hypoxia/reoxygenation-induced apoptosis via inhibiting TRIM28 expression.

Myocardial ischemia/reperfusion (MIR) injury, which occurs following acute myocardial infarction, can cause secondary damage to the heart. Tripartite interaction motif (TRIM) proteins, a class of E3 ubiquitin ligases, have been recognized as critical regulators in MIR injury. Zenglv Fumai Granule (ZFG) is a clinical prescription for the treatment of sick sinus syndrome, a disease that is associated with MIR injury. The present study aimed to investigate the effect of ZFG on MIR injury and to determine whether ZFG exerts its effects via regulation of TRIM proteins. In order to establish an in vitro MIR model, human cardiomyocyte cell line AC16 was cultured under hypoxia for 5 h and then under normal conditions for 1 h. Following hypoxia/reoxygenation (H/R) treatment, these cells were cultured with different ZFG concentrations. ZFG notably inhibited H/R-induced cardiomyocyte apoptosis. The expression levels of four TRIM proteins, TRIM7, TRIM14, TRIM22 and TRIM28, were also detected. These four proteins were significantly upregulated in H/R-injured cardiomyocytes, whereas their expression was inhibited following ZFG treatment. Moreover, TRIM28 knockdown inhibited H/R-induced cardiomyocyte apoptosis, whereas TRIM28 overexpression promoted apoptosis and generation of reactive oxygen species (ROS) in cardiomyocytes. However, the effects of TRIM28 overexpression were limited by the action of ROS inhibitor N-acetyl-L-cysteine. In addition, the mRNA and protein levels of antioxidant enzyme glutathione peroxidase (GPX)1 were significantly downregulated in H/R-injured cardiomyocytes. TRIM28 knockdown restored GPX1 protein levels but had no effect on mRNA expression levels. Co-immunoprecipitation and ubiquitination assays demonstrated that TRIM28 negatively regulated GPX1 via ubiquitination. In sum, the present study revealed that ZFG attenuated H/R-induced cardiomyocyte apoptosis by regulating the TRIM28/GPX1/ROS pathway. ZFG and TRIM28 offer potential therapeutic options for the treatment of MIR injury.

Effect and Safety of Guanxinning Tablet () for Stable Angina Pectoris Patients with Xin (Heart)-Blood Stagnation Syndrome: A Randomized, Multicenter, Placebo-Controlled Trial.

OBJECTIVE: To investigate the effect and safety of Guanxinning Tablet (, GXN) for the treatment of stable angina pectoris patients with Xin (Heart)-blood stagnation syndrome (XBSS). METHODS: One hundred and sixty stable angina pectoris patients with XBSS were randomly assigned to receive GXN (80 cases) or placebo (80 cases, Guanxinning simulation tablets, mainly composed of lactose), 4 tablets (0.38 g/tablet), thrice daily for 12 weeks. After treatment, an exercise stress test (treadmill protocol), Chinese medicine (CM) syndrome score, electrocardiogram (ECG), and nitroglycerin withdrawal rate were evaluated and compared in the patients between the two groups. Meanwhile, adverse events (AEs) were evaluated during the whole clinical trial. RESULTS: Compared with the control group, the time extension of exercise duration in the GXN group increased 29.28 ±17.67 s after treatment (P>0.05); moreover, the change of exercise duration in the GXN group increased 63.10 ±96.96 s in subgroup analysis (P<0.05). The effective rates of angina pectoris, CM syndrome and ECG as well as nitroglycerin withdrawal rate were 81.33%, 90.67%, 45.76%, and 70.73%, respectively in the GXN group, which were all significantly higher than those in the control group (40.58%, 75.36%, 26.92%, 28.21%, respectively, P<0.05). CONCLUSION: GXN was a safe and effective treatment for stable angina pectoris patients with XBSS at a dose of 4 tablets, thrice daily.

Photosynthetic fluorescence characteristics of six macroalgae species in seaweed beds of Gouqi Island, Zhejiang, China.

The study of photosynthetic fluorescence characteristics of algae is important for the analy-sis of photosynthesis and carbon sequestration of algae. In July 2017, six common species of macroalgae found in Gouqi seaweed beds were collected, including Ulva pertusa, Cladophora stimpsonii, Grateloupia livida, Sargassum thunbergii, Polysiphonia urceolata, and Hizikia fusifarme. In the field, the maximal quantum yieids of photosystem2(Fv/Fm) and rapid curves (RLCs) were mea-sured by using pulse-amplitude modulated fluorometer (Diving-PAM). The results showed that the measured maximal quantum yields of U. pertusa, C. stimpsonii, G. livida, S. thunbergii, P. urceolata, and H. fusiforme were 0.702, 0.704, 0.457, 0.618, 0.421 and 0.567, respectively. The Fv'/Fm' of six species were in order of C. stimpsonii>U. pertusa>S. thunbergii＞H. fusiforme＞G. livida＞P. urceolata. The difference between each species and significant difference was found in U. pertusa, C. stimpsonii, and H. fusiforme. H. fusiforme, S. thunbergii and U. pertusa had higher Pm and α than other species, indicating their higher photosynthetic capacity and better adaptation in higher light condition. However, G. livida had higher α but lower Ik, indicating G. livida had higher photosynthetic capacity in low light condition. In a word, differences of photosynthetic capacity and light intense tolerance between the three phyla of macroalgae were found and we suggested H. fusiforme, S. thunbergii and U. pertusa had stronger photosynthetic capacity and light intense tolerance. Our results could provide theoretical basis for the seaweed bed conservation and carbon sequestration of macroalgae.

Stat3/Oct-4/c-Myc signal circuit for regulating stemness-mediated doxorubicin resistance of triple-negative breast cancer cells and inhibitory effects of WP1066.

Doxorubicin (Dox) is widely used in the treatment of triple-negative breast cancer cells (TNBCs), however resistance limits its effectiveness. Cancer stem cells (CSCs) are associated with Dox resistance in MCF-7 estrogen receptor positive breast cancer cells. Signal transducer and activator of transcription 3 (Stat3) may functionally shift non-CSCs towards CSCs. However, whether Stat3 drives the formation of CSCs during the development of resistance in TNBC, and whether a Stat3 inhibitor reverses CSC-mediated Dox resistance, remains to be elucidated. In the present study, human MDA-MB-468 and murine 4T1 mammary carcinoma cell lines with the typical characteristics of TNBCs, were compared with estrogen receptor-positive MCF-7 cells as a model system. The MTT assay was used to detect cytotoxicity of Dox. In addition, the expression levels of CSC-specific markers and transcriptional factors were measured by western blotting, immunofluorescence staining and flow cytometry. The mammosphere formation assay was used to detect stem cell activity. Under long-term continuous treatment with Dox at a low concentration, TNBC cultures not only exhibited a drug-resistant phenotype, but also showed CSC properties. These Dox-resistant TNBC cells showed activation of Stat3 and high expression levels of pluripotency transcription factors octamer-binding transcription factor-4 (Oct-4) and c-Myc, which was different from the high expression of superoxide dismutase 2 (Sox2) in Dox-resistant MCF-7 cells. WP1066 inhibited the phosphorylation of Stat3, and decreased the expression of Oct-4 and c-Myc, leading to a reduction in the CD44-positive cell population, and restoring the sensitivity of the cells to Dox. Taken together, a novel signal circuit of Stat3/Oct-4/c-Myc was identified for regulating stemness-mediated Dox resistance in TNBC. The Stat3 inhibitor WP1066 was able to overcome the resistance to Dox through decreasing the enrichment of CSCs, highlighting the therapeutic potential of WP1066 as a novel sensitizer of Dox-resistant TNBC.

Inhibition of the JNK/MAPK signaling pathway by myogenesis-associated miRNAs is required for skeletal muscle development.

Skeletal muscle differentiation is controlled by multiple cell signaling pathways, however, the JNK/MAPK signaling pathway dominating this process has not been fully elucidated. Here, we report that the JNK/MAPK pathway was significantly downregulated in the late stages of myogenesis, and in contrast to P38/MAPK pathway, it negatively regulated skeletal muscle differentiation. Based on the PAR-CLIP-seq analysis, we identified six elevated miRNAs (miR-1a-3p, miR-133a-3p, miR-133b-3p, miR-206-3p, miR-128-3p, miR-351-5p), namely myogenesis-associated miRNAs (mamiRs), negatively controlled the JNK/MAPK pathway by repressing multiple factors for the phosphorylation of the JNK/MAPK pathway, including MEKK1, MEKK2, MKK7, and c-Jun but not JNK protein itself, and as a result, expression of transcriptional factor MyoD and mamiRs were further promoted. Our study revealed a novel double-negative feedback regulatory pattern of cell-specific miRNAs by targeting phosphorylation kinase signaling cascade responsible for skeletal muscle development.

Comprehensive Genomic Characterization of RNA-Binding Proteins across Human Cancers.

RNA-binding proteins (RBPs) regulate the expression of thousands of transcripts, and some are reported to be involved in human tumorigenesis. However, little is known about the dysregulation of RBPs at the genomic level in human cancers. Here, we conducted comprehensive analyses for expression, somatic copy number alteration (SCNA), and mutation profiles of 1,542 RBPs in ∼7,000 clinical specimens across 15 cancer types. We identified markedly dysregulated RBPs and found that downregulation was a predominant pattern in cancer. Combined with recurrent SCNA data, we identified 76 RBPs as potential drivers. We also discovered a set of 139 RBPs that were significantly mutated in cancers. We confirmed the oncogenic property of six RBPs in colorectal and liver cancer cell lines by using in vitro functional experiments. Our study highlights the potential roles of RBPs in carcinogenesis and lays the groundwork to better understand the functions and mechanisms of RBPs in cancer.

The lncRNA HOTAIRM1 regulates the degradation of PML-RARA oncoprotein and myeloid cell differentiation by enhancing the autophagy pathway.

Increasing evidence has indicated that long noncoding RNAs (lncRNAs) are of great importance in different cell contexts. However, only a very small number of lncRNAs have been experimentally validated and functionally annotated during human hematopoiesis. Here, we report an lncRNA, HOTAIRM1, which is associated with myeloid differentiation and has pivotal roles in the degradation of oncoprotein PML-RARA and in myeloid cell differentiation by regulating autophagy pathways. We first revealed that HOTAIRM1 has different variants that are expressed at different levels in cells and that the expression pattern of HOTAIRM1 is closely related to that of the PML-RARA oncoprotein in acute promyelocytic leukemia (APL) patients. We further revealed that the downregulation of HOTAIRM1 could inhibit all-trans retinoic acid (ATRA) -induced degradation of PML-RARA in APL cells and repress the process of differentiation from promyelocytic to granulocytic cells. More importantly, we found that HOTAIRM1 regulates autophagy and that autophagosome formation was inhibited when HOTAIRM1 expression was reduced in the cells. Finally, through the use of a dual luciferase activity assay, AGO2 RNA immunoprecipitation and RNA pull-down, HOTAIRM1 was revealed to act as a microRNA sponge in a pathway that included miR-20a/106b, miR-125b and their targets ULK1, E2F1 and DRAM2. We constructed a human APL-ascites SCID mouse model to validate the function of HOTAIRM1 and its regulatory pathway in vivo. This is the first report showing that a lncRNAs regulates autophagy and the degradation of the PML-RARA oncoprotein during the process of myeloid cell differentiation blockade, suggesting that lncRNAs may be the potential therapeutic targets for leukemia.

Dynamic Changes in Phenotypic Groups in Patients with Stable Angina Pectoris after Treatment with Xinxuekang Capsule: A Randomized Controlled Trial.

OBJECTIVE: To reveal the cutoff point and influencing factors in the dynamic change in phenotypic group in patients with stable angina pectoris (SAP) after Xinxuekang capsule treatment. METHODS: Five hundred and seventy-six SAP patients were randomly assigned to receive Xinxuekang (XXK) capsules or Compound Danshen (CDS) tablets for 8 weeks. Global similarity degree analysis and nonlinear mixed effects modeling (NONMEM) were employed to reveal the cutoff points and influencing factors in dynamic changes in the SAP phenotypic group. The phenotypic group was defined as the six phenotypes in SAP, including angina, choking sensation in the chest, palpitations, dark purple lips, ecchymosis on the tongue, and fine-choppy pulse, which were quantitatively evaluated on Days 0, 14, 28, 42, and 56. RESULTS: Variation in the six individual phenotypes and distribution of the SAP phenotypic profile were similar between the two experimental groups, but cutoff points for changes in the SAP phenotypic group were 7.28 and 10.73 weeks in XXK and CDS groups, respectively. Degree of severity of SAP as well as study site significantly affected the tendency for change in the SAP Xueyu Zheng in both XXK and CDS treatment groups. Different Chinese patent drugs affected the tendency for change in phenotypic group in patients with SAP. XXK was superior to CDS in controlling a clinical phenotypic group. CONCLUSION: Based on global similarity degree analysis and NONMEM, the cutoff point and influencing factors in phenomic variation of SAP may be determined, to improve the development and modification of treatment regimens.

[Effects of enhanced UV-B radiation on terrestrial ecosystem carbon cycle: a review].

As one of the most important phenomena of global climate change, the enhancement of ultraviolet-B radiation (UV-B, 280-320 nm) could have critical impact on the carbon cycle in terrestrial ecosystem. Through the impacts on plant photosynthesis, litter decomposition, and soil respiration, the enhanced UV-B radiation can affect the carbon input, turnover, and output of terrestrial ecosystem. Other climatic factors (ambient CO2 concentration, air temperature, and precipitation) may promote or mitigate the impact of enhanced UV-B radiation on terrestrial ecosystem carbon cycle. This paper introduced the background of UV-B radiation enhancement, reviewed the impacts of enhanced UV-B radiation and its interactions with other climatic factors on terrestrial ecosystem carbon cycle, summarized the existing problems in related researches, and discussed the priorities and directions of future researches.

Collision-induced dissociation studies of Fe(m)O(n) (+) : Bond energies in small iron oxide cluster cations, Fe(m)O(n) (+) (m=1-3, n=1-6).

A variety of iron oxide cluster cations is synthesized in a laser vaporization ion source. The kinetic energy dependence of the collision-induced dissociation (CID) of mass selected Fe(m)O(n) (+) (m=1-3, n=1-6) clusters with Xe is studied in this work using a guided ion beam tandem mass spectrometer. Examination of the general dissociation behavior over a broad collision energy range (0-15 eV) shows that iron oxide clusters can dissociate via evaporation of neutral Fe and O atoms as well as fission by loss of neutral O(2), FeO, FeO(2), Fe(2)O(2), and Fe(2)O(3) fragments. Such fission pathways, which are not observed in the CID studies of pure Fe cluster cations and most other pure transition metal cluster cations, result from the strong iron oxygen bonds. In general, the predominant dissociation pathways are found to correlate with the oxidation state of the iron in the cluster. Thresholds for loss of neutral Fe, O, O(2), FeO, FeO(2), Fe(2)O(2), and Fe(2)O(3) from various iron oxide cluster cations are quantitatively determined. These values are used to determine bond energies and heats of formation for both neutral and cationic iron oxide clusters in this size range.

[Liver transplantation in end-stage liver disease with portal vein thrombosis].

OBJECTIVE: To summarize the experience in the managements of portal vein thrombosis (PVT) and to evaluate the impact of PVT on intraoperative course and postoperative outcome in liver transplantation. METHODS: Between May 1995 and September 2007, 194 orthotopic liver transplantations were performed, of which 24 cases presented portal vein thrombosis. There were 12 patients with grade I, 9 with grade II, 2 with grade III and 1 with grade IV. The management of PVT depended mainly on its extent. Ligation of the collateral circulation, especially spontaneous or surgical splenorenal shunt, was made as approaches to improve portal flow.Heparin or low-molecule-weight heparin as a prophylactic anticoagulation therapy was maintained during and after operation if prothrombin time is less than eighteen seconds. Follow-up Doppler ultrasonography was used daily in the early postoperative period. Risk factors and variables associated with the transplant and the post-transplant period were analyzed and compared with 170 patients transplanted without PVT. RESULTS: Surgical techniques were eversion thromboendovenectomy in 21 patients with PVT grades I and II, extra-anatomic mesenteric graft in 2 with grade III, and anastomosis to a collateral vein in 1 with grade IV. The study demonstrated more RBC transfusions [(15.2 +/- 11.8) U vs. (8.6 +/- 6.6) U, P = 0.006], longer surgery procedures [(492 +/- 89) min vs. (403 +/- 105) min, P = 0.001] and hospital stay [(32.4 +/- 13.5) d vs. (22.1 +/- 9.1) d, P = 0.001] in the PVT group. However, there were no differences in overall morbidity (58.3% vs. 50.6%, P = 0.478), hospital mortality (8.3% vs.6.5%, P = 0.73) and 1-year survival (87.5% vs. 89.4%, P = 0.778). The incidence of rethrombosis was higher in the PVT group (8.3% vs.1.2%, P = 0.021). Two cases rethrombosis were successfully cured by percutaneous thrombolysis, balloon angioplasty, and stent placement. CONCLUSION: Portal thrombosis is associated with greater operative complexity and rethrombosis, but has no influence on overall morbidity and mortality in liver transplantation.

[Prevention and treatment of biliary complications following orthotopic liver transplantation].

OBJECTIVE: To study the prevention and treatment of biliary complications after orthotopic liver transplantation. METHODS: Clinical data of 183 recipients who had received liver transplantation between May 1995 and December 2006 were retrospectively analyzed. RESULTS: Biliary complications occurred in 15 patients (15/183, 8.2%). The incidence for short-term and long-term complication were 6.0% (11/183) and 2.2% (4/183) respectively. No biliary complications was due to hepatic artery thrombosis(HAT). Four cases who received PTC(percutaneous transhepatic cholangiography) with stent insertion,8 cases who received ERCP( endoscopic retrograde cholangiopancreatography) with stent insertion and 1 who received Roux-en-Y choledochojejunostomy for anastomotic stricture were successfully cured. Two cases required relaparotomy died for fungus infection eventually. The mortality due to biliary complications was 1.1%. CONCLUSIONS: The rapid combined abdominal organ harvesting technique could shorten the ischemia time and ameliorate the injury due to vascular and bile duct variances, which could reduce the incidence of biliary complication. PTC and (or) ERCP combined with stent insertion were main procedure for biliary complications not related to HAT after liver transplantation.

Retinoic acid downregulates microRNAs to induce abnormal development of spinal cord in spina bifida rat model.

OBJECTS: MicroRNAs have been found in the developing central nervous system, but little is known about their functions in development, especially in the abnormal development of spinal cord in spina bifida. To this end, we have studied the mechanism of microRNAs involved in the morphogenesis of the spinal cord in all-trans-retinoic acid (RA)-treated spina bifida rat fetus. MATERIALS AND METHODS: Timed-pregnant rats were gavage-fed RA, and embryos were obtained on 13.5, 15.5, 17.5, and 19.5 days. MicroRNAs' expression profile was analyzed by Northern blot. In situ apoptosis detection and microRNA in situ hybridization methods on sections of paraffin-embedded tissues were employed to explore the mechanism. CONCLUSION: Administration of RA reduced the size of the spinal cord, probably as a consequence of increased cell death. There is a dramatic decrease in the expression of miR-9/9*, miR-124a and miR-125b, and Bcl2 and P53 as well in the sacral cord from E13.5 to E19.5 days post coitum. Our data showed that expression of these microRNAs was dysregulated in RA-treated spinal cord during embryonic development, suggesting that they may be involved in the development of the spinal cord.

Prevention and treatment of rejection after simultaneous pancreas-kidney transplantation.

OBJECTIVE: To explore methods of preventing and reversing rejection after simultaneous pancreas-kidney (SPK) transplantation. METHODS: Seventeen patients underwent SPK transplantation from September 1999 to September 2003 were reviewed retrospectively. Immunosuppression was achieved by a triple drug regimen consisting of cyclosporine, mycophenolate mofteil (MMF), and steroids. Three patients were treated with anti-CD3 monoclone antibody (OKT3, 5 mg x d(-1)) for induction therapy for a mean period of 5-7 days. One patients received IL-2 receptor antibodies (daclizumab) in a dose of 1 mg x kg(-1) on the day of transplant and the 5th day posttransplant. One patient was treated with both OKT3 and daclizumab for induction. RESULTS: No primary non-functionality of either kidney or pancreas occurred in this series of transplantations. Function of all the kidney grafts recovered within 2 to 4 days after transplantation. The level of serum creatinine was 94 +/- 11 micromol/L on the 7th day posttransplant. One patient experienced the accelerated rejection, resulting in the resection of the pancreas and kidney grafts because of the failure of conservative therapy. The incidence of the first rejection episodes at 3 months was 47.1% (8/17). Only the kidney was involved in 35.3% (6/17); and both the pancreas and kidney were involved in 11.8% (2/17). All these patients received a high-dose pulse of methylprednisone (0.5 g x d(-1)) for 3 days. OKT3 (0.5 mg x d(-1)) was administered for 7-10 days in two patients with both renal and pancreas rejection. All the grafts were successfully rescued. CONCLUSION: Rejection, particularly acute rejection, is the major cause influencing graft function in SPK transplantation. Monitoring renal function and pancreas exocrine secretion, and reasonable application of immunosuppressants play important roles in the diagnosis and treatment of rejection.

Water adsorption, desorption, and clustering on FeO(111).

The adsorption of water on FeO(111) is investigated using temperature programmed desorption (TPD) and infrared reflection absorption spectroscopy (IRAS). Well-ordered 2 ML thick FeO(111) films are grown epitaxially on a Pt(111) substrate. Water adsorbs molecularly on FeO(111) and desorbs with a well resolved monolayer peak. IRAS measurements as a function of coverage are performed for water deposited at 30 and 135 K. For all coverages (0.2 ML and greater), the adsorbed water exhibits significant hydrogen bonding. Differences in IRAS spectra for water adsorbed at 30 and 135 K are subtle but suggest that water adsorbed at 135 K is well ordered. Monolayer nitrogen TPD spectra from water covered FeO(111) surfaces are used to investigate the clustering of the water as a function of deposition or annealing temperature. Temperature dependent water overlayer structures result from differences in water diffusion rates on bare FeO(111) and on water adsorbed on FeO(111). Features in the nitrogen TPD spectra allow the monolayer wetting and 2-dimensional (2D) ordering of water on FeO(111) to be followed. Voids in a partially disordered first water layer exist for water deposited below 120 K and ordered 2D islands are found when depositing water above 120 K.

[Analysis of rejection after simultaneous pancreas-kidney transplantation].

OBJECTIVE: To explore methods of preventing and reversing rejection after simultaneous pancreas-kidney transplantation (SPK). METHODS: Seventeen patients performed SPK operation from Sep, 1999 to Sep, 2003 were reviewed retrospectively. Immunosuppression was achieved by triple regimen consisting of cyclosporine, mycophenolate mofetil (MMF)/azathioprine and steroid. 2 patients were treated with Dalizumab, the other three patients used OKT3 as immune induction. RESULTS: 1 patient experienced the accelerated rejection, the pancreas and kidney grafts were resected because of failure of conservative therapy. 8 patients experienced renal acute rejection, 2 cases suffered from pancreas acute rejection at the same time. All these patients received daily high dose pulse steroid for 3 days. OKT3 was administered in 2 patients with steroid resistance rejection. All the grafts were successfully rescued. CONCLUSIONS: Reasonable application of immunosuppression after SPK operation and adoption of systemic measures which can reduce sensitivity of high risk receptor before SPK operation are the effective methods of preventing and treating rejection.