Recent data suggest that vascular endothelial growth factor receptor inhibitor (VEGFRi) can enhance the anti-tumor activity of the anti-programmed cell death-1 (anti-PD-1) antibody in colorectal cancer (CRC) with microsatellite stability (MSS). However, the comparison between this combination and standard third-line VEGFRi treatment is not performed, and reliable biomarkers are still lacking. We retrospectively enrolled MSS CRC patients receiving anti-PD-1 antibody plus VEGFRi (combination group, n=54) or VEGFRi alone (VEGFRi group, n=32), and their efficacy and safety were evaluated. We additionally examined the immune characteristics of the MSS CRC tumor microenvironment (TME) through single-cell and spatial transcriptomic data, and an MSS CRC immune cell-related signature (MCICRS) that can be used to predict the clinical outcomes of MSS CRC patients receiving immunotherapy was developed and validated in our in-house cohort. Compared with VEGFRi alone, the combination of anti-PD-1 antibody and VEGFRi exhibited a prolonged survival benefit (median progression-free survival: 4.4 vs. 2.0 months, P=0.0024; median overall survival: 10.2 vs. 5.2 months, P=0.0038) and a similar adverse event incidence. Through single-cell and spatial transcriptomic analysis, we determined ten MSS CRC-enriched immune cell types and their spatial distribution, including naive CD4⁺ T, regulatory CD4⁺ T, CD4⁺ Th17, exhausted CD8⁺ T, cytotoxic CD8⁺ T, proliferated CD8⁺ T, natural killer (NK) cells, plasma, and classical and intermediate monocytes. Based on a systemic meta-analysis and ten machine learning algorithms, we obtained MCICRS, an independent risk factor for the prognosis of MSS CRC patients. Further analyses demonstrated that the low-MCICRS group presented a higher immune cell infiltration and immune-related pathway activation, and hence a significant relation with the superior efficacy of pan-cancer immunotherapy. More importantly, the predictive value of MCICRS in MSS CRC patients receiving immunotherapy was also validated with an in-house cohort. Anti-PD-1 antibody combined with VEGFRi presented an improved clinical benefit in MSS CRC with manageable toxicity. MCICRS could serve as a robust and promising tool to predict clinical outcomes for individual MSS CRC patients receiving immunotherapy.
Humans ; Colorectal Neoplasms/drug therapy* ; Male ; Female ; Immunotherapy ; Middle Aged ; Aged ; Tumor Microenvironment/immunology* ; Retrospective Studies ; Microsatellite Instability ; Transcriptome ; Single-Cell Analysis ; Programmed Cell Death 1 Receptor/immunology* ; Gene Expression Profiling ; Immune Checkpoint Inhibitors/therapeutic use* ; Adult ; Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors*

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*

The chemical constituents of the petroleum ether extract derived from the 90% ethanol extract of Elephantopus scaber were investigated. By silica gel column chromatography, C_(18), MCI column chromatography and semi-preparative high performance liquid chromatography, ten compounds were isolated. Their structures were identified as 3β-hydroxy-6β,7β-epoxytaraxeran-14-ene(1), 3β-hydroxyolean-12-en-28-oic acid(2), D-friedoolean-14-ene-3β,7α-diol(3), 3β-hydroxy-11α-methoxyolean-12-ene(4), 3β-hydroxyolean-11,13(18)-diene(5), 11α-hydroxy-β-amyrin(6), betulinic acid(7), 3β-hydroxy-30-norlupan-20-one(8), 6-acetonylchelerythrine(9), and 4',5'-dehydrodiodictyonema A(10) by analysis of the 1D NMR, 2D NMR, MS, and IR spectral data. Among them, compound 1 was a new triterpene and other compounds except compounds 2 and 7 were isolated from this plant for the first time.
Triterpenes/isolation & purification* ; Drugs, Chinese Herbal/isolation & purification* ; Molecular Structure ; Asteraceae/chemistry* ; Chromatography, High Pressure Liquid ; Magnetic Resonance Spectroscopy

OBJECTIVE: Emerging evidence suggests that exposure to ultrafine particulate matter (UPM, aerodynamic diameter < 0.1 µm) is associated with adverse cardiovascular events. Previous studies have found that Shenlian (SL) extract possesses anti-inflammatory and antiapoptotic properties and has a promising protective effect at all stages of the atherosclerotic disease process. In this study, we aimed to investigated whether SL improves UPM-aggravated myocardial ischemic injury by inhibiting inflammation and cell apoptosis. METHODS: We established a mouse model of MI+UPM. Echocardiographic measurement, measurement of myocardialinfarct size, biochemical analysis, enzyme-linked immunosorbent assay (ELISA), histopathological analysis, Transferase dUTP Nick End Labeling (TUNEL), Western blotting (WB), Polymerase Chain Reaction (PCR) and so on were used to explore the anti-inflammatory and anti-apoptotic effects of SL in vivo and in vitro. RESULTS: SL treatment can attenuate UPM-induced cardiac dysfunction by improving left ventricular ejection fraction, fractional shortening, and decreasing cardiac infarction area. SL significantly reduced the levels of myocardial enzymes and attenuated UPM-induced morphological alterations. Moreover, SL significantly reduced expression levels of the inflammatory cytokines IL-6, TNF-α, and MCP-1. UPM further increased the infiltration of macrophages in myocardial tissue, whereas SL intervention reversed this phenomenon. UPM also triggered myocardial apoptosis, which was markedly attenuated by SL treatment. The results of in vitro experiments revealed that SL prevented cell damage caused by exposure to UPM combined with hypoxia by reducing the expression of the inflammatory factor NF-κB and inhibiting apoptosis in H9c2 cells. CONCLUSION Overall, both in vivo and in vitro experiments demonstrated that SL attenuated UPM-aggravated myocardial ischemic injury by inhibiting inflammation and cell apoptosis. The mechanisms were related to the downregulation of macrophages infiltrating heart tissues.
Animals ; Apoptosis/drug effects* ; Particulate Matter/adverse effects* ; Mice ; Male ; Inflammation/drug therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Mice, Inbred C57BL ; Myocardial Ischemia/drug therapy* ; Cell Line

Background::Homoharringtonine (HHT) is an effective anti-inflammatory, anti-viral, and anti-tumor protein synthesis inhibitor that has been applied clinically. Here, we explored the therapeutic effects of HHT in a mouse heart transplant model.Methods::Healthy C57BL/6 mice were used to observe the toxicity of HHT in the liver, kidney, and hematology. A mouse heart transplantation model was constructed, and the potential mechanism of HHT prolonging allograft survival was evaluated using Kaplan–Meier analysis, immunostaining, and bulk RNA sequencing analysis. The HHT-T cell crosstalk was modeled ex vivo to further verify the molecular mechanism of HHT-induced regulatory T cells (Tregs) differentiation. Results::HHT inhibited the activation and proliferation of T cells and promoted their apoptosis ex vivo. Treatment of 0.5 mg/kg HHT for 10 days significantly prolonged the mean graft survival time of the allografts from 7 days to 48 days ( P <0.001) without non-immune toxicity. The allografts had long-term survival after continuous HHT treatment for 28 days. HHT significantly reduced lymphocyte infiltration in the graft, and interferon-γ-secreting CD4 + and CD8 + T cells in the spleen ( P <0.01). HHT significantly increased the number of peripheral Tregs (about 20%, P <0.001) and serum interleukin (IL)-10 levels. HHT downregulated the expression of T cell receptor (TCR) signaling pathway-related genes ( CD4, H2-Eb1, TRAT1, and CD74) and upregulated the expression of IL-10 and transforming growth factor (TGF) -β pathway-related genes and Treg signature genes ( CTLA4, Foxp3, CD74, and ICOS). HHT increased CD4 + Foxp3 + cells and Foxp3 expression ex vivo, and it enhanced the inhibitory function of inducible Tregs. Conclusions::HHT promotes Treg cell differentiation and enhances Treg suppressive function by attenuating the TCR signaling pathway and upregulating the expression of Treg signature genes and IL-10 levels, thereby promoting mouse heart allograft acceptance. These findings may have therapeutic implications for organ transplant recipients, particularly those with viral infections and malignancies, which require a more suitable anti-rejection medication.

Objective:To analyze the effectiveness and interobserver agreement of the Parer five-tier, the National Institute of Child Health and Human Development (NICHD) three-tier, and the International Federation of Gynecology and Obstetrics (FIGO) three-tier electronic fetal monitoring (EFM) systems in identification of neonatal acidosis during labor.Methods:This retrospective study was conducted on full-term singleton cephalic deliveries with neonatal acidosis (umbilical artery blood gas pH≤7.1) and normal newborns (umbilical artery blood gas pH≥7.2) in the Nanjing Drum Tower Hospital, Nanjing University Medical School from January to December 2020. EFM tracings during the last 30-60 min before delivery were collected. Four obstetricians independently described the features of randomly sorted and coded EFM tracings. Another obstetrician categorized these tracings using the NICHD three-tier, FIGO three-tier, and Parer five-tier evaluation systems based on the features. All researchers were masked to the clinical characteristics and maternal and neonatal outcomes. The sensitivity and specificity for identifying neonatal acidosis, as well as the interobserver agreement, were analyzed for all three systems. Independent sample t-test, Chi-square (or Fisher's exact test) and Mann-Whitney U tests were used for statistical analysis. Inter-group comparisons of sensitivity and specificity between the three evaluation systems were assessed using McNemar's test. The Kappa statistic was used to analyze interobserver agreement. Results:This study included a total of 3 558 cases. After propensity score matching, there were 44 cases of neonatal acidosis and 78 control cases. There were no significant differences in parity, gestational weeks, modes of delivery, placental abruption, or analgesia rates between the two groups. The rates of instrumental vaginal delivery and neonatal intensive care unit (NICU) admission in the acidosis group were significantly higher than those in the control group [15.8% (7/44) vs. 2.6% (2/78), χ2=8.45, P=0.003; 31.8% (14/44) vs. 12.8% (10/78), χ2=8.45, P=0.004], while the umbilical artery blood pH and mean base excess were lower in the acidosis group than in the control group [7.04±0.07 vs. 7.30±0.05, t=4.98; (－12.40±3.32) vs. (－5.64±1.95) mmol/L, t=13.61; both P<0.001]. (2) Using the NICHD three-tier system, 95.5% (42/44) of the acidosis cases and 89.7% (70/78) of the control cases were classified as having category Ⅱ EFM tracings, indicating potential fetal acid-base imbalance; category Ⅲ EFM tracings were only observed in 4.5% (2/44) of the cases in the acidosis group. With the FIGO three-tier system, 81.8% (36/44) of the acidosis cases were categorized as having "pathological" tracings, and with the Parer five-tier system, 86.4% (38/44) of the acidosis cases were correctly classified into the "orange or red" risk zones that indicated acid-base imbalance. Among the control cases, there were 28.2% (22/78) with EFM tracings of "normal patterns" categorized by the FIGO three-tier system, and 41.0% (32/78) classified into the "green or blue" risk zones by the Parer five-tier system, which indicated good fetal conditions. None of the acidosis cases were misdiagnosed as being normal by the Parer five-tier system. (3) Compared with the NICHD three-tier system, both the FIGO three-tier and the Parer five-tier systems showed increased diagnostic sensitivity [4.5% (1.2%- 14.5%) vs. 81.8% (66.8%-89.4%) and 86.4% (71.8%-92.4%)], but decreased specificity [100.0% (95.3%- 100.0%) vs. 87.2% (78.0%-92.9%) and 84.6% (75.0%-91.0%)]. There was no statistically significant difference in the sensitivity or specificity between the FIGO three-tier and Parer five-tier systems for identifying neonatal acidosis ( P=0.727 and 0.791). (4) When reading the tracings of control cases, the total agreement rate for the NICHD three-tier system by different observers was as high as 94.2%, while the total agreement rates for the FIGO three-tier and Parer five-tier systems were 69.7% and 67.7%, respectively. In the interpretation of EFHR tracings for acidosis cases, the interobserver agreement for the Parer five-tier system was excellent [Kappa (95% CI): 0.87 (0.79-0.95)], while both the NICHD three-tier and FIGO three-tier systems showed good agreement [Kappa (95% CI): 0.77 (0.66-0.88) and 0.72 (0.60-0.84)]. Conclusions:The Parer five-tier and the FIGO three-tier systems have higher sensitivity in identifying neonatal acidosis than the NICHD three-tier system, and the Parer five-tier system achieves a higher negative predictive value and a greater agreement in the interpretation of pathological EFM patterns.

The management of surplus drugs is an important part of drug administration. At present, China′s medical institutions are in the initial exploration stage in managing surplus drugs.This study analyzed the causes, safety hazards, management policies, and management problems of surplus drugs in medical institutions, and proposed targeted countermeasures and suggestions, including establishing unified and standardized management methods, consensus or guidelines, optimizing internal management of medical institutions, improving the management awareness of medical staff, and clarifying the benefits of surplus drugs, so as to provide references for medical institutions to manage surplus drugs reasonably.

To standardize the management of surplus drugs, improve the efficiency of medical resource utilization, promote the rational use of medical insurance funds, and reduce the financial burden on patients, a large tertiary hospital implemented a practice for managing surplus drugs starting in May 2023. This practice encompassed multiple aspects, including the establishment of organizational structure, clarification of responsibilities, formulation of billing for fractional doses and a reasonable surplus drugs list, establishment of standardized management processes, and allocation of special funds for surplus drugs. These efforts had initially achieved effective management of surplus drugs. As of November 2023, the management of surplus drugs had benefited 136 908 patients, with an average savings of 873.61 yuan per patient and a cumulative savings of approximately 34.7 million yuan in medical insurance funds. This practice had effectively reduced the wastage of medical resources, and could provide references for promoting standardized management of surplus drugs in medical institutions of China. In the future, the hospital should further expand the coverage of surplus drugs, ensure patients′ rights to informed consent, and establish a comprehensive performance incentive mechanism to promote the sustainable development of surplus drug management.

Based on the androgen receptor(AR)/mammalian target of rapamycin(mTOR)signaling pathway, the effects of Xihuang Pills-medicated serum on the proliferation and apoptosis of prostate cancer LNCaP cells were investigated. The drug-containing serum of SD rats was prepared by intragastric administration of Xihuang Pills suspension. The effects of low-, medium-, and high-dose Xihuang Pills-containing serum on the in vitro proliferation of LNCaP cells were detected by cell counting kit-8(CCK-8). Flow cytometry was used to detect the apoptosis level of LNCaP cells after intervention with different concentrations of Xihuang Pills. Protein expression of cleaved cysteinyl aspartate-specific proteinase caspase-3(cleaved caspase-3), B-cell lymphoma-2(Bcl-2), and AR as well as the phosphorylation level of mTOR protein were detected by Western blot. The results showed that compared with the blank serum, the drug-medicated serum could blunt the activity of LNCaP cells. Low-, medium-, and high-dose Xihuang Pills-containing serum could significantly increase the cell apoptosis rate, increase the expression of cleaved caspase-3 protein, decrease the expression of Bcl-2 protein, reduce the expression of AR protein, and down-regulate the level of phosphorylated mTOR(p-mTOR). To study the effect of Xihuang Pills on the growth of LNCaP cells in vivo, different doses of Xihuang Pills were used to intervene in the subcutaneous graft model in nude mice inoculated with LNCaP cells. The expression levels of AR, mTOR, p-mTOR, Bcl-2, and cleaved caspase-3 were detected by Western blot. The results showed that the volumes of subcutaneous graft tumor in the low-dose, medium-dose, and high-dose Xihuang Pills groups significantly decreased compared with that in the model group. The weight of subcutaneous transplanted tumor in each group with drug intervention was significantly lower than that in the model group. Compared with the model group, the low-dose, medium-dose, and high-dose Xihuang Pills groups showed increased cleaved caspase-3 protein expression, decreased Bcl-2 and AR protein expression, and reduced p-mTOR protein expression. Further experiments showed that AR agonist R1881 could block the anti-proliferation and pro-apoptotic effects of Xihuang Pills. The mechanism of Xihuang Pills against prostate cancer is related to the inhibition of the AR/mTOR signaling pathway, inhibition of LNCaP cell proliferation, and induction of apoptosis in cancer cells.
Humans ; Male ; Mice ; Rats ; Animals ; Caspase 3/metabolism* ; Mice, Nude ; Cell Line, Tumor ; Rats, Sprague-Dawley ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Prostatic Neoplasms/pathology* ; Cell Proliferation ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Mammals/metabolism*

Objective: To analyze the phenotypic-genotypic characteristics of hereditary deafness caused by OTOA gene variations. Methods: Family histories, clinical phenotypes and gene variations of six pedigrees were analyzed, which were diagnosed with hearing loss caused by OTOA gene variations at the PLA General Hospital from September 2015 to January 2022. The sequence variations were verified by Sanger sequencing and the copy number variations were validated by multiplex ligation-dependent probe amplification (MLPA) in the family members. Results: The hearing loss phenotype caused by OTOA variations ranged from mild to moderate in the low frequencies, and from moderate to severe in the high frequencies in the probands, which came from six sporadic pedigrees, among which a proband was diagnosed as congenital deafness and five were diagnosed as postlingual deafness. One proband carried homozygous variations and five probands carried compound heterozygous variations in OTOA gene. Nine pathogenic variations (six copy number variations, two deletion variations and one missense variation) and two variations with uncertain significance in OTOA were identified in total, including six copy number variations and five single nucleotide variants, and three of the five single nucleotide variants were firstly reported [c.1265G>T(p.Gly422Val),c.1534delG(p.Ala513Leufs*11) and c.3292C>T(p.Gln1098fs*)]. Conclusions: OTOA gene variations can lead to autosomal recessive nonsyndromic hearing loss. In this study, the hearing loss caused by OTOA defects mostly presents as bilateral, symmetrical, and postlingual, and that of a few presents as congenital. The pathogenic variations of OTOA gene are mainly copy number variations followed by deletion variations and missense variations.
Humans ; DNA Copy Number Variations ; Hearing Loss, Sensorineural/genetics* ; Deafness/genetics* ; Hearing Loss/genetics* ; Phenotype ; Genotype ; Nucleotides ; Pedigree ; Mutation ; GPI-Linked Proteins/genetics*