BACKGROUND: T cell dysfunction, which includes exhaustion, anergy, and senescence, is a distinct T cell differentiation state that occurs after antigen exposure. Although T cell dysfunction has been a cornerstone of cancer immunotherapy, its potential in transplant research, while not yet as extensively explored, is attracting growing interest. Interferon regulatory factor 4 (IRF4) has been shown to play a pivotal role in inducing T cell dysfunction. METHODS: A novel ultra-low-dose combination of Trametinib and Rapamycin, targeting IRF4 inhibition, was employed to investigate T cell proliferation, apoptosis, cytokine secretion, expression of T-cell dysfunction-associated molecules, effects of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways, and allograft survival in both in vitro and BALB/c to C57BL/6 mouse cardiac transplantation models. RESULTS: In vitro , blockade of IRF4 in T cells effectively inhibited T cell proliferation, increased apoptosis, and significantly upregulated the expression of programmed cell death protein 1 (PD-1), Helios, CD160, and cytotoxic T lymphocyte-associated antigen (CTLA-4), markers of T cell dysfunction. Furthermore, it suppressed the secretion of pro-inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17. Combining ultra-low-dose Trametinib (0.1 mg·kg -1 ·day -1 ) and Rapamycin (0.1 mg·kg -1 ·day -1 ) demonstrably extended graft survival, with 4 out of 5 mice exceeding 100 days post-transplantation. Moreover, analysis of grafts at day 7 confirmed sustained IFN regulatory factor 4 (IRF4) inhibition, enhanced PD-1 expression, and suppressed IFN-γ secretion, reinforcing the in vivo efficacy of this IRF4-targeting approach. The combination of Trametinib and Rapamycin synergistically inhibited the MAPK and mTOR signaling network, leading to a more pronounced suppression of IRF4 expression. CONCLUSIONS Targeting IRF4, a key regulator of T cell dysfunction, presents a promising avenue for inducing transplant immune tolerance. In this study, we demonstrate that a novel ultra-low-dose combination of Trametinib and Rapamycin synergistically suppresses the MAPK and mTOR signaling network, leading to profound IRF4 inhibition, promoting allograft acceptance, and offering a potential new therapeutic strategy for improved transplant outcomes. However, further research is necessary to elucidate the underlying pharmacological mechanisms and facilitate translation to clinical practice.
Animals ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Interferon Regulatory Factors/metabolism* ; Heart Transplantation/methods* ; T-Lymphocytes/immunology* ; Sirolimus/therapeutic use* ; Pyridones/therapeutic use* ; Graft Survival/drug effects* ; Pyrimidinones/therapeutic use* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Male ; Signal Transduction/drug effects*

Acute pancreatitis (AP) is a life-threatening gastrointestinal disorder for which no effective pharmacological treatments are currently available. One of the pharmacological targets that merits further research is the neurokinin 1 receptor (NK1R), which is found on pancreatic acinar cells and responds to the neuropeptide substance P (SP) that participates in AP. Although a few studies have stated the involvement of SP/NK1R in neurogenic inflammation in AP development, the regulatory mechanism remains unclear. In this study, we found that following activation of NK1R by SP, β-arrestin1, a scaffold protein of NK1R, down-regulated transcription of Adss, Adsl, and Ampd in the purine nucleotide cycle, thereby inhibiting mitochondrial function through fumarate depletion. Interestingly, we identified magnolol as a new and natural NK1R inhibitor with a non-nitrogenous biphenyl core structure. It exhibited a beneficial effect on AP by restoring purine nucleotide cycle metabolic enzymes and fumarate levels. Our study not only provides new therapeutic strategies, leading compounds, and drug translation possibilities for AP, but also provides important clues for the study of downstream mechanisms driven by SP in other diseases.

Objective:The mouse kidney transplantation model presents challenges in terms of surgical difficulty and low success rate,making it difficult to master.This study aims to provide a crucial model for transplantation immunology research by modifying and developing novel techniques for mouse kidney transplantation. Methods:A total of 57 pairs of mice were used to establish and compare the modified and innovative surgical techniques for mouse kidney transplantation.Three different surgical models were established,including the abdominal suture technique for orthotopic kidney transplantation,the abdominal cuff technique for orthotopic kidney transplantation,and the cervical cuff technique for ectopic kidney transplantation.BALB/c or C57BL/6 male mice,aged 8 to 12 weeks and weighed 20 to 25 g with specified pathogen free-grade were served as the donor mice or the recipient mice.The surgical technique characteristics,key surgical times,complications,and pathological examination in the early postoperative period were summarized and compared. Results:Three different surgical models of mouse kidney transplantation were successfully established.The comparison of warm ischemic time for the 3 groups of mice showed no statistical significance(P=0.510 4).The abdominal suture group had the shortest total operation time of the donor compared with the abdominal cuff group and the cervical cuff group[(18.3±3.6)min vs(26.2±4.7)min and(22.8±2.5)min;both P<0.000 1].There was a significant difference in cold ischemia time among the 3 groups(all P<0.000 1),with(60.8±4.1)min in the cervical cuff group,(43.3±5.0)min in the abdominal suture group,and(88.8±6.7)min in the abdominal cuff group.Due to different anastomosis methods,the cervical cuff group had the shortest time[(17.6±2.7)min],whereas the abdominal cuff group had the longest time[(38.8±5.4)min].The total operation time for the recipients showed significant differences(P<0.000 1),with the abdominal suture group having the shortest time[(44.0±6.9)min],followed by the cervical cuff group[(64.1±5.2)min],and the abdominal cuff group[(80.0±6.0)min]being the longest.In the 32 mice of the abdominal suture group,there were 6 with intraoperative bleeding,including 1 arterial intimal injury bleeding and 5 with bleeding after vessel opening.Six mice had ureteral complications,including ureteral bladder anastomotic stenosis,necrosis,and renal pelvis dilation.Two mice had postoperative abdominal infections.In the abdominal cuff group,there was no intraoperative bleeding,but 6 mice showed mild arterial stenosis and 5 showed venous stenosis,4 arterial injury,4 arterial thrombosis,and 2 ureteral complications.No postoperative infections occurred in the mice.In the cervical cuff group,no intraoperative bleeding,arterial intimal injury,arterial/venous stenosis,or thrombosis were found in 13 mice.Five mice had ureteral complications,including ureteral necrosis and infection,which were the main complications in the cervical cuff group.The renal function in mice of the 3 groups remained stable 7 days after surgery.Hematoxylin and eosin staining and periodic acid-Schiff staining showed no significant differences in terms of acute rejection among the 3 surgical methods(all P>0.05). Conclusion:All 3 surgical methods are able to successfully establish mouse kidney transplantation models,with no significant differences observed in the short-term graft survival and acute rejection.The modified abdominal suture technique and abdominal cuff technique have their respective advantages in research applications.The novel cervical cuff technique for ectopic kidney transplantation model is relatively simple to be prepared and causes less trauma to the mice,providing more options for studies involving xenotransplantation,secondary transplantation,and local lymphatic drainage.However,the difficulty in harvesting the donor kidney and the high incidence of ureteral infections need further validation in long-term survival.This study holds important reference value for choosing the type of mouse kidney transplantation model for different research needs.

Objective:To investigate the effects of high-flux versus low-flux hemodialysis on end-stage renal disease in older adults and evaluate its effects on myocardial injury indexes and micro-inflammatory response indexes. Methods:Seventy-two patients with ESRD who received treatment in Affiliated Hospital of Shaoxing University from January 2019 to January 2020 were included in this study. They were randomly assigned to receive either low-flux hemodialysis (control group, n = 36) or high-flux hemodialysis (observation group, n = 36). All patients received 6 months of treatment. Micro-inflammatory response indexes and renal function indexes pre- and post-treatment, and reverse reactions were observed in each group. Results:Serum interleukin-6, tumor necrosis factor α, and high-sensitivity C-reactive protein levels post-treatment in the observation group were (7.16 ± 1.32) ng/L, (2.10 ± 0.36) pg/L, (2.20 ± 1.06) mg/L respectively, which were significantly lower than those in the control group [(10.45 ± 1.42) ng/L, (5.22 ± 0.65) pg/L, (3.84 ± 1.57) mg/L, t = 10.19, 25.19, 5.19, all P < 0.001]. Serum parathyroid hormone, B-type natriuretic peptide, and cardiac troponin T levels post-treatment in the observation group were (520.36 ± 95.65) pmol/L, (0.45 ± 0.10) μg/L, (15.05 ± 6.37) ng/L, respectively, which were significantly lower than those in the control group [(830.25 ± 102.35) pmol/L, (0.85 ± 0.13) μg/L, (30.25 ± 6.59) ng/L, t = 13.27, 14.63, 9.95, all P < 0.001]. The total response rate was significantly higher in the observation group than in the control group [75.00% (27/36) vs. 47.22% (19/36), χ2 = 3.85, P < 0.05]. The overall incidence of adverse reactions was significantly lower in the observation group than in the control group [19.44% (7/36) vs. 41.67% (15/36), χ2 = 4.18, P < 0.05). Conclusion:High-flux hemodialysis can highly improve the control effect of micro-inflammatory responses and alleviate myocardial injury in older adult patients with end-stage renal disease. High-flux hemodialysis is more effective and safer than low-flux hemodialysis.

Objective:To analyze the influence of clinicopathological features on the prognosis of anorectal malignant melanoma (AMM), and to establish a more accurate prognosis prediction model.Methods:From January 1, 2006 to December 31, 2018, at Fudan University Shanghai Cancer Center, the data of 89 patients diagnosed with AMM and underwent operation were retrospectively analyzed. The Cox proportional hazard regression model was used to analyze factors affecting the prognosis. Backward elimination was used to select variables, Nomogram prognosis prediction model was established and verified internally, and the consistency index was calculated.Results:Among 89 AMM patients, 65 (73.0%) were female, 78(87.6%) were <70 years old, and the most common tumor location was the rectum (48.3%, n=43), followed by the anal canal (31.5%, n=28) and the anorectal canal (20.2%, n=18). Thirty-eight (42.7%) patients directly received abdominal-perineal resection (APR), 37 patients (41.6%) received interferon-based immunotherapy, and 11 patients (12.4%) received both systemic chemotherapy and immunotherapy. The 3-year disease-specific survival (DSS) rate was 41.6%(37/89), and the 5-year DSS rate was 31.5%(28/89). The results of multivariate Cox analysis showed that age ≥70 years old and T2 stage were risk factors of the prognosis of AMM patients (hazard ratio ( HR)=11.29, 4.83; 95% confidence interval ( CI) 2.89 to 44.13, 1.66 to 14.11; both P<0.01), while neurovascular invasion, immunotherapy treatment, systemic chemotherapy combined with immunotherapy, APR after extensive local resection, directly APR and APR combined with inguinal lymphadenectomy were protective factors of the prognosis ( HR=0.09, 0.23, 0.10, 0.13, 0.26, 0.02; 95% CI 0.02 to 0.34, 0.10 to 0.57, 0.02 to 0.49, 0.03 to 0.52, 0.08 to 0.90, 0.00 to 0.27; all P<0.05). The Nomogram model was further established with age, gender, tumor location, T stage, distant metastasis, medication chemotherapy and surgical treatment. The results of the Nomogram model internal verification indicated that the accuracy of the model in predicting 1-year, 3-year and 5-year DSS was good, and the consistency index was 0.749, which was significantly higher than the consistency index of traditional TNM stage (0.607). Conclusions:Most AMM patients are <70 years old, and the majority of them are female. The common location of AMM is rectum, and many patients receive immunotherapy. Age ≥70 years old and T2 stage are risk factors affecting the prognosis of AMM patients. Neurovascalar invasion, immunotherapy based comprehensive treatment, APR after extensive local resection, directly APR and APR combined with inguinal lymphadenectomy are protective factors of the prognosis. Nomogram prognosis prediction model established based on the clinicopathological features and treatment of AMM patients has higher accuracy and clinical reference value than the traditional TNM stage system.

Objective:To investigate the effect of mechano-growth factor（MGF）on osteoclast activity and its mechanism.Methods:The RAW264.7 precursor osteoclast cell line was cultured with 25 ng/ml macrophage-colony stimulating factor（M-CSF）and 30 ng/ml receptor activator of NF-κB ligand（RANKL），and identified by tartrate resistant acid phosphatase（TRAP）staining after 7 days of culture. Western blot anslysis was used to determine the effect of 45 ng/ml MGF on the phosphoinositide-3-kinase/protein kinase B（PI3K/AKT）signaling pathway in separated osteoclasts，including levels of AKT，phosphorylation（p）-AKT，lactation mammalian target of rapamycin（mTOR），p-mTOR and TRAP at 0，4，8 and 12 hours. Real-time fluorescence quantitative PCR was used to expressions of TRAP in osteoclasts at 0，4，8 and 12 hours. The PI3K/Akt phosphorylation inhibitor LY294002（20 μmol/L）combined with MGF（45 ng/ml）was used to act on osteoclasts，and expression levels of Akt，p-Akt，mTOR，p-mTOR and TRAP were detected by Western blot at 0，4，8 and 12 hours.Results:After culturing RAW264.7 cells with M-CSF and RANKL for 7 days，a large number of osteoclasts with positive TRAP staining can be obtained. Western blot analysis showed expression levels of Akt and mTOR did not change significantly over time（ P>0.05），expression levels of p-Akt and p-mTOR increased continuously from（2.18±0.34）pg/ml and（0.83±0.10）pg/ml at 0 hour to（3.86±0.36）pg/ml and（1.56±0.19）pg/ml at 12 hours（ P<0.05），and expression level of TRAP decreased significantly over time，from（5.66±0.47）pg/ml at 0 hour to（3.76±0.38）pg/ml at 12 hours（ P<0.05）. Real-time fluorescence quantitative PCR analysis of expression of TRAP in osteoclasts showed that MGF inhibited the expression of TRAP in osteoclasts，which decreased from 1.02±0.06 at 0 hour to 0.53±0.11 at 12 hours（ P<0.05）. After acting LY294002 combined with MGF on osteoclasts，Western blot analysis showed expression levels of Akt and mTOR did not change significantly over time（ P>0.05），expression levels of p-AKT and p-mTOR decreased significantly from（3.28±0.18）pg/ml and（3.29±0.22）pg/ml at 0 hour to（2.06±0.34）pg/ml and（2.04±0.20）pg/ml at 12 hours（ P<0.05），and expression level of TRAP had no significant difference over time（ P>0.05）. Conclusions:MGF inhibits osteoclast activity by inhibiting the expression of TRAP in osteoclasts through PI3K/Akt signaling pathway. LY294002 inhibits the expression of PI3K/Akt signaling pathway in osteoclasts，further verifying the mechanism of MGF inhibiting osteoclast activity，and this finding puts forward new ideas for clinical prevention and treatment of osteoporosis.

BACKGROUND:The ankle joint is capable of flexion and extension, including plantar flexion and dorsiflexion, to act as a support and a lever. An ankle injury, often accompanied by fracture and ligament injury, seriously threatens the ankle joint function. Previous diagnosis of ankle injury mainly relied on clinical signs and X-ray examination. However, X-ray examination is not accurate enough for ankle injury diagnosis because it cannot clearly diagnose damage to the surrounding ligaments, tendons and other soft tissues except for obvious fractures. Mutlisequence and multiplanar MRI is currently the optimal noninvasive method for high-resolution determination of soft tissue deformations, but little has been reported on the diagnostic accuracy of ankle ligament and tendon injury. OBJECTIVE:To observe the diagnostic value of MRI for ligament and tendon injury of the ankle in its normal position, and during complete plantar flexion and dorsiflexion. METHODS:It is a single-center, prospective, diagnostic trial that wil be completed at the First Hospital of Hebei Medical University, China. Sixty cases were recruited, including 30 cases of normal ankle joint and 30 cases of ankle ligament and tendon injury. MRI scans of the ankle joint in normal position, complete plantar flexion and complete dorsiflexion were performed in al the cases, and the multi-position MRI results were compared. The primary outcome measure is the sensitivity of MRI to ligament and tendon injury of the ankle during complete plantar flexion. The secondary outcomes include the specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio of MRI to ligament and tendon injury of the ankle during the complete plantar position as wel as rate of correct diagnosis;specificity and sensitivity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio in normal position or during the complete dorsiflexion as wel as rate of correct diagnosis;the morphology of the ankle on the multi-position MRI. This study design was registered at ClinicalTrial.gov (03049423) on February 8, 2017. This study protocol has been approved by the Medical Ethics Committee of Qinghai University Affiliated Hospital of China (approval No. 2015076) and wil be performed in accordance with the guidelines of the Declaration of Helsinki, formulated by the World Medical Association. Signed informed consent regarding trial procedure and treatment was obtained from each volunteer. DISCUSSION:This study aims to determine the rate of correct diagnosis of ankle ligament and tendon injury using the MRI, to clarify the diagnostic value of MRI for ankle ligament and tendon injury, and to provide a quantitative MRI diagnostic standard for developing a reasonable surgical treatment.

Objective To evaluate application of Steinmenn pins to assist reduction in the treatment of unstable intertrochanteric fractures with proximal femoral nail antirotation(PFNA).Methods From February 2010 to June 2013,38 unstable intertrochanteric fractures were treated by us.There were 23 men and 15 women,aged from 32 to 69 years.By Evans-Jensen classification,18 cases were type Ⅲ,13 type Ⅳ and 7 type Ⅴ.They were divided into 2 groups (n =19).Group A received reduction on a traction bed assisted by Steinmenn pins plus PFNA fixation while group B received reduction only on a traction bed plus PFNA fixation.The 2 groups were compared in terms of fracture reduction,operation time,intro-operative blood loss,fracture healing time,and Harris scores one year postoperation.Results According to the evaluation system modified by Baumgaetner et al.,the postoperative quality of fracture reduction was fine in 15 cases and fair in 4 in group A while it was fine in 9 cases,fair in 8 and poor in 2 in group B,showing a significant difference between the 2 groups (P ＜ 0.05).The operation time in group A (50.7 ± 11.9 min) was significantly shorter than in group B (63.4 ± 15.1 min),and the hip joint Harris score (89.4 ±4.4) one year after operation for group A was significantly higher than that for group B (79.6 ±6.4) (P ＜ 0.05).There were no significant differences between the 2 groups regarding intraoperative blood loss,fracture heeling time and follow-up time (P ＞ 0.05).No cases of refracture,delayed union,nonunion,or avascular necrosis of the femoral head were reported.Conclusion In the treatment of unstable intertrochanteric fractures,compared with reduction only on a traction bed plus PFNA fixation,application of Steinmenn pins to assist reduction on a traction bed plus PFNA fixation may lead to better curative efficacy due to its limited invasion,simplicity and beneficial assistance in reduction.

Guided by the demands of society,we propose a new professional training objectives of medical laboratory technology,which is to cultivate comprehensive talents covering the entire industrial chain of medical laboratory technology with profound foundation,broad caliber,high quality and outstanding ability.According to the training objective,we build up 4321 talents training system and try to carry out preliminary practice and exploration on talent cultivation model from the following aspects,such as the construction of curriculum system,the reform of the teaching contents and methods,training of students' professional skills and entrepreneurial innovation spirit.

Objective To investigate the role of colostomy stoma improving the quality of life of patients after the re-turn to society. Methods This study was conducted from February 2009 to February 2012 in our hospital for colostomy surgery in patients with clinical observation in order to improve the quality of life. Results Except for nausea, vomiting, constipation, diarrhea, the QLQ-C30 scores of treatment group after treatment for 6 months were significantly higher than those in control group (P<0.05). Patients in the treatment group on body image, hopes for the future were signifi-cantly higher than those before treatment (P<0.05); Symptom scale and sub entries for the difference between the two groups was significant (P<0.05). Conclusion The implementation of colostomy patients of early stage can enhance the self resistance, reduce the psychological burden of patients, improve the quality of life of patients with colostomy.