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*

OBJECTIVES: The traditional processing method for paraffin-embedded tissues is time-consuming, while the ultrasonic rapid processing method has a short processing time. However, its effects on tissue proteins, DNA, and RNA remain unclear. This study aims to evaluate the effects of the ultrasonic rapid processing method on proteins, DNA, and RNA in paraffin-embedded tissues through hematoxylin and eosin (HE) staining, immunohistochemical staining, and molecular pathological detection. METHODS: Surgical specimens from patients with breast cancer, colorectal cancer, lung cancer, signet-ring cell gastric cancer, liver cancer, and other tumors were selected. Two tissue blocks (1 to 3 mm in diameter) were obtained from each specimen (previously processed and diagnosed by routine pathology). One block was assigned to the control group (traditional processing method), and the other was the experimental group (ultrasonic rapid processing method). Via HE staining, immunohistochemical staining, DNA quality fragment analysis, fluorescent in situ hybrid for HER2 gene expression test, second-generation sequencing for EGFR and ALK gene mutation test, real-time reverse transcription PCR (real-time RT-PCR) for prognosis detection of breast cancer etc, the difference between 2 groups was compared, and further impact of the ultrasonic rapid processing method was analyzed. RESULTS: Compared with the control group, the ultrasound-assisted rapid method efficiently completed fixation, dehydration, clearing, and paraffin embedding, significantly reducing sample preparation time before pathological diagnosis. Results of HE staining, immunohistochemistry, DNA fragment analysis, fluorescence in situ hybridization for HER2 gene, next-generation sequencing for EGFR and ALK gene, and real-time RT-PCR for breast cancer prognosis were entirely consistent with those of the control group. CONCLUSIONS The ultrasonic rapid processing method can quickly and effectively shorten the time for specimen processing before pathological diagnosis, and will not affect the DNA, RNA and proteins of the specimens. It can meet the subsequent HE staining, immunohistochemistry and molecular pathological detection.
Humans ; Paraffin Embedding/methods* ; Female ; RNA/analysis* ; DNA/analysis* ; Breast Neoplasms/pathology* ; Neoplasms/genetics* ; Ultrasonics/methods* ; Proteins/analysis*

Background and aim:Few studies have reported hepatitis B surface antigen(HBsAg)kinetics after nucleos(t)ide analog(NA)discontinuation in patients with noncirrhotic chronic hepatitis B(CHB).The study specifically investigated long-term HBsAg kinetics after NA discontinuation. Methods:Between January 2014 to January 2024,this study prospectively enrolled 106 outpatients with noncirrhotic CHB who met the discontinuation criteria after NA consolidation treatment.Demographic,clinical,and laboratory data were collected and analyzed after NA discontinuation. Results:Ninety-six patients who finished 5 years of follow-up were included.HBsAg remained unde-tectable in 29 patients with end of treatment(EOT)HBsAg negativity.Among 67 patients with EOT HBsAg positivity,HBsAg seroclearance occurred in 12(17.9％)patients with an estimated annual inci-dence of HBsAg seroclearance of 3.6％.Patients with EOT HBsAg levels of ≤1000 IU/mL had a higher HBsAg seroclearance rate than those with EOT HBsAg levels of＞1000 IU/mL(33.3％vs.5.4％).The pro-portion of patients with HBsAg ≤1000 IU/mL increased during follow-up.Logistic regression analysis indicated that the EOT HBsAg level was an independent factor for HBsAg seroclearance and an HBsAg level decline exceeding 1 log10 IU/mL.The optimal EOT HBsAg cutoff for both HBsAg seroclearance and an HBsAg level decline exceeding 1 log10 IU/mL was 359 IU/mL. Conclusions:Patients with EOT HBsAg negativity experienced no relapse and maintained HBsAg sero-clearance during 5 years of follow-up after NA discontinuation.A higher HBsAg seroclearance rate can be obtained in patients with EOT HBsAg levels of ≤1000 IU/mL during 5 years of follow-up after NA discontinuation.Close monitoring and proper NA retreatment are recommended to guarantee the safety of NA discontinuation.

Objective:To investigate the expression of heat shock protein 90 (HSP90) and the relationship between the expression of HSP90 and the clinicopathological features or prognosis in patients with lung adenocarcinoma (ADC).Methods:The paraffin specimens of 193 patients with lung adenocarcinoma and 53 non cancerous lung tissues (bullae and bronchiectasis) resected in Xiangya Second Hospital of Central South University were analyzed retrospectively. The expression of HSP90 in the tissue chip was detected by immunohistochemical streptavidin-perosidase (SP) method by high-throughput tissue chip, and the relationship between its expression level and the clinicopathological characteristics of patients was analyzed; Kaplan Meier survival curve was used to analyze the difference between different expression levels of HSP90 and the overall survival time of patients with lung adenocarcinoma.Results:The positive expression of HSP90 in lung adenocarcinoma was significantly higher than that in non cancerous lung tissue ( P<0.001), the expression level of HSP90 in clinical stage Ⅲ patients was higher than that in clinical stage Ⅰ-Ⅱ patients ( P=0.008), and the expression level of HSP90 in patients with lymph node metastasis was higher than that in patients without lymph node metastasis ( P=0.024); The 10-year survival rate of lung adenocarcinoma patients with high expression of HSP90 was significantly lower than that of patients with low expression of HSP90 ( P=0.001). The 10-year survival rate of lung ADC patients with stage Ⅲ and lymph node metastasis was significantly lower than that of patients with stage Ⅰ-Ⅱ and no lymph node metastasis ( P<0.05). Multiple Cox proportional hazard regression analysis further identified that lung ADC patients with overexpression of HSP90 had a poor prognosis ( P=0.010). Conclusions:HSP90 might play an important part in the development and progression of lung ADC and might act as a novel prognostic marker for patients with lung ADC.

OBJECTIVE: To explore the genetic and clinical characteristics of near-tetraploidy/tetraploidy karyotype (NT/T) in patients with myelodysplastic syndrome (MDS). METHODS: Cytogenetic findings of 1576 inpatients with primary MDS were retrospective analyzed, among which 9 were diagnosed with NT/T. Clinical data including gender, age, morphology, genetic feature and prognosis were analyzed. RESULTS: The prevalence of MDS patients with NT/T (NT/T-MDS) among all cases was 0.57%. Karyotyping analysis suggested that eight MDS patients had sole NT/T, while the remainder one had a complex karyotype. In addition to the typical morphology of MDS, NT/T-MDS had unique morphology including huge blast, double-nuclear cell and irregular nuclear membrane. One NT/T-MDS patient gave up therapy, and the remaining eight underwent the first course of treatment, albeit with poor prognosis. Only one patient had complete remission, one had partial remission, three had no remission; and three had converted to acute myeloid leukemia. CONCLUSION NT/T-MDS is rare and has unique morphology. Generally, NT/T-MDS patients have poor prognosis. However, NT/T cannot be simply classified as high-risk group, but with consideration whether they have affected particular chromosomal structures as well as other clinical data.
Humans ; Karyotype ; Leukemia, Myeloid, Acute/complications* ; Myelodysplastic Syndromes/pathology* ; Prognosis ; Retrospective Studies ; Tetraploidy

Objective:To investigate the mutations of the epidermal growth factor receptor (EGFR) gene and its clinical signifi-cance in non-small cell lung cancer (NSCLC). Methods:The EGFR gene mutations of exons 18 to 21 in NSCLC were detected by us-ing the ADx-ARMS? detection kit method. Results:The total mutation percentage in exons 18 to 21 of the EGFR gene was 45.8%(98/214) in NSCLC. These mutations predominantly occur in exons 19 and 21. EGFR gene mutation percentages were found in exons 18 (0.93%, 2/214), 19 (22.0%,47/214), 20 (2.3%, 5/214), and exon 21 (20.6%, 44/214) in the NSCLC. Two NSCLC cases were identified to have double EGFR gene mutations of exons 19 and 21. EGFR gene mutations were more frequently observed with adenocarcinoma histology (50.3%, 93/185) than with squamous cell carcinoma (17.2%, 5/29) (P=0.001). EGFR gene mutations occur more frequently in NSCLC cases in women than in men (P=0.002). EGFR gene mutations were significantly higher in NSCLC with lymphatic metastasis (66.7%) than in NSCLC without lymphatic metastasis (39.5%) (P<0.05). However, no evident association was found between EGFR gene mutations and age, as well as tumor grade and clinical stage of NSCLC (P>0.05). Conclusion:NSCLC, especially lung adenocar-cinomas, has exhibits frequent EGFR gene mutations in China. EGFR gene mutations in exons 19 and 21, combined with the clinical pathological features of lung cancer, can serve as the molecular marker to evaluate the efficacy of EGFR TKI for NSCLC patients.

OBJECTIVE: To determine the expression of p53 and its clinical significance in HER2-negative breast invasive ductal carcinoma (BIDC). METHODS: The expression of p53, ER and PR in the HER2-negative BIDC was detected by immunohistochemistry and the results were analyzed by SPSS10.0 software packet, chi-square test, spearman's correlation analysis, Kaplan-Meier survival curves and Cox regression analysis. RESULTS: The positive expression of p53 protein in BIDC with pathological grade III was significantly higher than that with grade I (P<0.05), but there was no significant correlation between the expression of p53 and age, clinical stage, or lymph node metastasis status in the BIDC. The positive expression of p53 protein in BIDC with ER-positive was significantly lower than that with ER-negative (P<0.01). The positive expression of p53 protein was significantly lower in BIDC with common expression of ER and PR than that with negative expression of ER or PR (P<0.05). The HER2-negative BIDC patients with p53-positive expression had a lower 5 year survival than those with p53-negative expression. CONCLUSION The positive expression of p53 protein might have significant prognostic value and is an independent prognostic marker in HER2 -negative BIDC.
Breast Neoplasms ; genetics ; metabolism ; Carcinoma, Ductal ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Lymphatic Metastasis ; Prognosis ; Receptor, ErbB-2 ; Tumor Suppressor Protein p53 ; genetics ; metabolism