BACKGROUND: Renal osteodystrophy (ROD) is a skeletal pathology associated with chronic kidney disease-mineral and bone disorder (CKD-MBD) that is characterized by aberrant bone mineralization and remodeling. ROD increases the risk of fracture and mortality in CKD patients. The underlying mechanisms of ROD remain elusive, partially due to the absence of an appropriate animal model. To address this gap, we established a stable mouse model of ROD using an optimized adenine-enriched diet and conducted exploratory analyses through ribonucleic acid sequencing (RNA-seq). METHODS: Eight-week-old male C57BL/6J mice were randomly allocated into three groups: control group ( n = 5), adenine and high-phosphate (HP) diet group ( n = 20), and the optimized adenine-containing diet group ( n = 20) for 12 weeks. We assessed the skeletal characteristics of model mice through blood biochemistry, microcomputed tomography (micro-CT), and bone histomorphometry. RNA-seq was utilized to profile gene expression changes of ROD. We elucidated the functions of differentially expressed genes (DEGs) using gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA). DEGs were validated via quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: By the fifth week, adenine followed by an HP diet induced rapid weight loss and high mortality rates in the mouse group, precluding further model development. Mice with optimized adenine diet-induced ROD displayed significant abnormalities in serum creatinine and blood urea nitrogen levels, accompanied by pronounced hyperparathyroidism and hyperphosphatemia. The femur bone mineral density (BMD) of the model mice was lower than that of control mice, with substantial bone loss and cortical porosity. ROD mice exhibited substantial bone turnover with an increase in osteoblast and osteoclast markers. Transcriptomic profiling revealed 1907 genes with upregulated expression and 723 genes with downregulated expression in the femurs of ROD mice relative to those of control mice. Pathway analyses indicated significant enrichment of upregulated genes in the sphingolipid metabolism pathway. The significant upregulation of alkaline ceramidase 1 ( Acer1 ), alkaline ceramidase 2 ( Acer2 ), prosaposin-like 1 ( Psapl1 ), adenosine A1 receptor ( Adora1 ), and sphingosine-1-phosphate receptor 5 ( S1pr5 ) were successfully validated in mouse femurs by qRT-PCR. CONCLUSIONS Optimized adenine diet mouse model may be a valuable proxy for studying ROD. RNA-seq analysis revealed that the sphingolipid metabolism pathway is likely a key player in ROD pathogenesis, thereby providing new avenues for therapeutic intervention.
Animals ; Mice ; Chronic Kidney Disease-Mineral and Bone Disorder/genetics* ; Male ; Disease Models, Animal ; Mice, Inbred C57BL ; Sphingolipids/metabolism* ; Transcriptome/genetics* ; Signal Transduction/genetics* ; X-Ray Microtomography ; Adenine

Tumor-associated macrophages(TAMs)play a pivotal role in the tumor microenvironment and have traditionally been clas-sified into M1 and M2 phenotypes;however,recent studies have revealed considerable internal heterogeneity among these macro-phages,which requires a more detailed classification.T cells play a key role in tumor immunity,and their population dynamics and functional states have a direct impact on therapeutic outcomes.The interactions between TAMs and various T cell subsets,including CD4+T cells,regulatory T cells,and CD8+T cells,have a guiding significance for tumor immunotherapy.Such interactions between TAMs and T cells involve a variety of molecules and signaling pathways,such as programmed death receptor 1 and its ligand,as well as various cytokines and their receptors.Furthermore,exogenous factors,including chemoradiotherapy and infections,can also influence these interactions.Research on the interactions between different macrophage populations and T cell subsets within the tumor immune microenvironment can help to understand immune status and mechanism in lesions,thereby providing new targets and strategies for tu-mor immunotherapy.

In the tumor microenvironment(TME),the metabolic reprogramming of macrophages and T cells plays a critical role in the formation of an immunosuppressive state and tumor immune escape.Polyamines are key signaling molecules and precursors for biosyn-thesis,and the metabolic state of polyamines profoundly regulate the functions of tumor cells and immune cells.Cytokines and other substances within the TME induce the expression of enzymes related to polyamine metabolism,drive the metabolic reprogramming of polyamines,and thus influence macrophage polarization and T cell differentiation;meanwhile,the metabolic reprogramming of poly-amines leads to the competitive depletion of polyamine resources within the TME,promote tumor cells to optimize their metabolic strat-egies for survival advantages,and inhibit the function of immune cells.Therefore,inhibitors of polyamine metabolism may be used as antitumor drugs by regulating metabolic reprogramming.

Immunotherapy,represented by immune checkpoint inhibitors,has been widely used in patients with non-small cell lung cancer(NSCLC).With remarkable therapeutic efficacy,immunotherapy has been recommended by multiple domestic and international guidelines and expert consensuses.However,there are still some controversial clinical issues worth discussing in the current immuno-therapy of NSCLC,such as perioperative immunotherapy selection,immune resistance mechanisms and treatment strategies after drug resistance,the evaluation of immunotherapy efficacy,the management of adverse immune reactions,immunotherapy strategies for driver gene-positive patients,immunotherapy selection in special populations,and other immunotherapy-related clinical issues of con-cern.This article presents and discusses seven hot issues in NSCLC immunotherapy,aiming to provide a reference for tumor clinicians.

Objective:To investigate the clinical features, treatment strategies and prognosis of adult thrombotic thrombocytopenic purpura (TTP) patients and improve the clinicians' understanding of TTP.Methods:It was a case series analysis study. The clinical data of TTP patients admitted to ZhongDa Hospital affiliated to Southeast University from August 2013 to November 2024 were retrospectively collected. The clinical manifestations, laboratory tests, treatment methods and prognosis of TTP patients were analyzed. Kaplan-Meier method and multivariate Cox proportional hazards regression model were utilized to assess the association between rituximab treatment and survival outcomes.Results:The study included 24 TTP patients, with age of (58.38±15.03) years (21 to 87 years), 14 females (58.33%) and 10 males (41.67%). The first symptoms were often neurological abnormalities (lethargy, coma, sudden glossolalia or unconsciousness (10 patients, 41.67%). Five patients (20.83%) had the quinary syndrome, including fever, microangiopathic hemolytic anemia, thrombocytopenia, renal insufficiency and neurological symptoms, and 13 patients (54.17%) had the triad syndrome, including neurological syndromes, microangiopathic hemolytic anemia and thrombocytopenia. Twenty-three patients (95.83%) had anemia. Twenty patients (83.33%) presented with neurological abnormalities, among which 10 patients died of neurological events. Renal insufficiency occurred in 14 patients (58.33%). Nine patients (37.50%) presented with large areas of skin ecchymosis. Except for 1 patient complicating with lung adenocarcinoma and 1 patients complicating with bone metastasis tumor, the other patients had no active tumors. All 24 patients had PLASMIC scores ≥ 4 points, of which 11 patients (45.83%) had PLASMIC scores ≥ 6 points. Fourteen patients (58.33%) received the treatment for plasma exchange, and 7 patients (29.17%) did not undergo plasma exchange and received component transfusion and glucocorticoids therapy with poor prognosis due to rapid disease progression, old age or severe disease. Furthermore, 3 patients (12.50%) were only treated with component transfusion and glucocorticoids therapy for economic reasons, and died shortly after hospital discharge. Eight patients received plasma exchange, glucocorticoids combined with rituximab, of which one died, four survived, and three were lost to follow-up. Finally, fifteen patients (62.50%) died, 4 patients survived, and 5 patients were lost to follow-up (still alive before hospital discharge). Kaplan-Meier survival analysis demonstrated that mortality in the rituximab group was significantly lower than that in the non-rituximab group (Log-rank test, χ2=13.185, P<0.001). Multivariate Cox proportional hazards regression analysis further confirmed that no receiving rituximab was an independent correlated factor of death ( HR=10.453, 95% CI 1.309-83.436, P=0.027). Conclusions:TTP usually starts with neurological symptoms, and can affect multiple systems. The patients with neurological abnormalities have a poor prognosis. The patients with TTP have a rapid disease progression and a high mortality rate. Rapid identification and timely treatment are crucial for improving the prognosis of TTP. Combining rituximab based on plasma exchange and glucocorticoids may reduce mortality of TTP patients.

Objective:To explore the correlation between serum nidogen-2 (NID-2) and thoracic aortic calcification in patients with chronic kidney disease (CKD), and construct a risk prediction model based on NID-2 to evaluate its value in predicting the risk of the severe thoracic aortic calcification and cardiovascular and cerebrovascular events in CKD patients.Methods:It was a prospective cohort study. Patients with CKD at stage 3 to 5D in the Zhongda Hospital Affiliated to Southeast University from January 2022 to January 2023 were enrolled. Syngo.via software was used to evaluate the volume of thoracic aortic calcification, and enzyme-linked immunosorbent assay was employed to determine the level of serum NID-2. According to the volume of thoracic aortic calcification, the patients were divided into three groups: no calcification group, mild calcification group and severe calcification group. The top 25% of the patients were defined as no or mild calcification group, and the latter 75% were defined as severe calcification group. The follow-up period was one year. During the follow-up period, cardiovascular and cerebrovascular events, as well as all-cause death among the enrolled patients were recorded. Logistic regression analysis was used to screen the influencing factors of thoracic aortic calcification. Based on the results of logistic regression analysis, a nomogram prediction model was constructed. The receiver operating characteristic curve (ROC curve), calibration curve, and decision curve were employed to evaluate the discrimination, calibration and clinical practicality of the nomogram model.Results:A total of 132 patients were included, with 91 males (68.94%) and age of (56.51±16.37) years. There were 60 CKD 3-5 stage patients (non-dialysis, 45.45%) and 72 CKD 5D patients (dialysis, 54.55%). Serum ND-2 levels differed significantly among healthy individuals, dialysis patients and non-dialysis patients ( H=70.651, P<0.001). There was no statistically significant difference in serum NID-2 level between the no or mild calcification group and the severe calcification group in dialysis patients ( Z=350.00, P=0.426). The serum NID-2 level in the severe calcification group was significantly higher than that in the no or mild calcification group in non-dialysis patients ( Z=242.00, P=0.019). In non-dialysis patients, there was a statistically significant correlation between serum NID-2 level and volume of thoracic aortic calcification ( r=0.40, P<0.001). In dialysis patients, there was no statistically significant correlation between serum NID-2 level and volume of each segment of thoracic aortic calcification (all P>0.05). The univariate logistic regression analysis showed that, age, hemoglobin, serum albumin, estimated glomerular filtration rate, NID-2, hypertension, type 2 diabetes mellitus and cerebral infarction were correlated factors of thoracic aortic calcification in non-dialysis patients (all P<0.05). Multivariate logistic regression analysis showed that age ( OR=1.22, 95% CI 1.08-1.50, P=0.010) was an independent correlated factor of thoracic aortic calcification in non-dialysis patients. The above related variables of univariate logistic regression analysis were incorporated into a nomogram to construct a predictive model for severe vascular calcification in non-dialysis patients, yielding an AUC of 0.94 (95% CI 0.89-0.99) in ROC curve, with a sensitivity of 83% and a specificity of 95%. A nomogram model based on above variables for predicting cardiovascular and cerebrovascular events in non-dialysis patients demonstrated an AUC of 0.95 (95% CI 0.90-1.00) in ROC curve, with a sensitivity of 95% and a specificity of 87%. Conclusions:In non-dialysis patients, serum NID-2 level in the severe calcification group is significantly higher than that in the no or mild calcification group. The serum NID-2 is a related factor of thoracic aortic calcification and cardiovascular and cerebrovascular events in non-dialysis patients. The nomogram prediction model constructed by combining NID-2 with age, hemoglobin, serum albumin, estimated glomerular filtration rate, hypertension, type 2 diabetes mellitus and cerebral infarction has a high predictive value for the risk of thoracic aortic calcification as well as cardiovascular and cerebrovascular events in non-dialysis patients.

Objective:To validate the usefulness of copy number variation sequencing (CNV-seq) in detecting the deletion/duplication of the DMD gene in Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients. Methods:One hundred and seventy-seven cases who visited the Department of Medical Genetics, Affiliated Hospital of Kunming University of Science and Technology/the First People′s Hospital of Yunnan Province from April 2018 to November 2023 were collected. All patients had previously accepted multiplex ligation-dependent probe amplification (MLPA) to detect the deletion/duplication of the DMD gene, including 90 cases of normal control with a negative result of MLPA and 87 cases with the deletion or duplication of the DMD gene (61 cases of DMD and 26 cases of BMD). CNV-seq was performed in a single-blind manner to detect DMD gene deletion or duplication for all of 177 cases to obtain the detection efficiency of CNV-seq in comparison with MLPA. Results:Comparing to MLPA, CNV-seq had a coincidence rate of 88.7% (157/177) for detecting DMD gene deletion/duplication, with a sensitivity of 77.0% (67/87), a specificity and a positive predictive value of both 100.0% (90/90 and 67/67, respectively), a negative predictive value of 81.8% (90/110), and a Kappa value of 0.773. Of the 87 patients with the deletion or duplication of the DMD gene, CNV-seq detected 67 cases with DMD gene deletion/duplication, including 62 cases with deletion and 5 cases with duplication, with fragment ranging from 150 to 750 kb. While CNV-seq missed 23.0% (20/87) of positive cases, mainly due to the involved fragments spanning only 1 to 4 exons, and with a variation size less than 50 kb, below the resolution (100 kb) of CNV-seq. The detection rate of CNV-seq in BMD cases (84.6%, 22/26) was a little higher than that in DMD cases (73.8%, 45/61), but there was no significant difference between 2 subgroups ( χ2=1.211, P=0.271). The results of CNV-seq in normal controls were all negative, and consistent with the results of MLPA. Conclusion:CNV-seq can detect 77.0% (67/87) of deletion/duplication of the DMD gene in patients with DMD/BMD, while the deletion/duplication less than 100 kb may be inevitably unidentified, therefore it is recommended as an assistant screening technique in prenatal diagnosis for DMD gene deletion or duplication.

Objective:To investigate the clinical features, treatment strategies and prognosis of adult thrombotic thrombocytopenic purpura (TTP) patients and improve the clinicians' understanding of TTP.Methods:It was a case series analysis study. The clinical data of TTP patients admitted to ZhongDa Hospital affiliated to Southeast University from August 2013 to November 2024 were retrospectively collected. The clinical manifestations, laboratory tests, treatment methods and prognosis of TTP patients were analyzed. Kaplan-Meier method and multivariate Cox proportional hazards regression model were utilized to assess the association between rituximab treatment and survival outcomes.Results:The study included 24 TTP patients, with age of (58.38±15.03) years (21 to 87 years), 14 females (58.33%) and 10 males (41.67%). The first symptoms were often neurological abnormalities (lethargy, coma, sudden glossolalia or unconsciousness (10 patients, 41.67%). Five patients (20.83%) had the quinary syndrome, including fever, microangiopathic hemolytic anemia, thrombocytopenia, renal insufficiency and neurological symptoms, and 13 patients (54.17%) had the triad syndrome, including neurological syndromes, microangiopathic hemolytic anemia and thrombocytopenia. Twenty-three patients (95.83%) had anemia. Twenty patients (83.33%) presented with neurological abnormalities, among which 10 patients died of neurological events. Renal insufficiency occurred in 14 patients (58.33%). Nine patients (37.50%) presented with large areas of skin ecchymosis. Except for 1 patient complicating with lung adenocarcinoma and 1 patients complicating with bone metastasis tumor, the other patients had no active tumors. All 24 patients had PLASMIC scores ≥ 4 points, of which 11 patients (45.83%) had PLASMIC scores ≥ 6 points. Fourteen patients (58.33%) received the treatment for plasma exchange, and 7 patients (29.17%) did not undergo plasma exchange and received component transfusion and glucocorticoids therapy with poor prognosis due to rapid disease progression, old age or severe disease. Furthermore, 3 patients (12.50%) were only treated with component transfusion and glucocorticoids therapy for economic reasons, and died shortly after hospital discharge. Eight patients received plasma exchange, glucocorticoids combined with rituximab, of which one died, four survived, and three were lost to follow-up. Finally, fifteen patients (62.50%) died, 4 patients survived, and 5 patients were lost to follow-up (still alive before hospital discharge). Kaplan-Meier survival analysis demonstrated that mortality in the rituximab group was significantly lower than that in the non-rituximab group (Log-rank test, χ2=13.185, P<0.001). Multivariate Cox proportional hazards regression analysis further confirmed that no receiving rituximab was an independent correlated factor of death ( HR=10.453, 95% CI 1.309-83.436, P=0.027). Conclusions:TTP usually starts with neurological symptoms, and can affect multiple systems. The patients with neurological abnormalities have a poor prognosis. The patients with TTP have a rapid disease progression and a high mortality rate. Rapid identification and timely treatment are crucial for improving the prognosis of TTP. Combining rituximab based on plasma exchange and glucocorticoids may reduce mortality of TTP patients.

Objective:To explore the correlation between serum nidogen-2 (NID-2) and thoracic aortic calcification in patients with chronic kidney disease (CKD), and construct a risk prediction model based on NID-2 to evaluate its value in predicting the risk of the severe thoracic aortic calcification and cardiovascular and cerebrovascular events in CKD patients.Methods:It was a prospective cohort study. Patients with CKD at stage 3 to 5D in the Zhongda Hospital Affiliated to Southeast University from January 2022 to January 2023 were enrolled. Syngo.via software was used to evaluate the volume of thoracic aortic calcification, and enzyme-linked immunosorbent assay was employed to determine the level of serum NID-2. According to the volume of thoracic aortic calcification, the patients were divided into three groups: no calcification group, mild calcification group and severe calcification group. The top 25% of the patients were defined as no or mild calcification group, and the latter 75% were defined as severe calcification group. The follow-up period was one year. During the follow-up period, cardiovascular and cerebrovascular events, as well as all-cause death among the enrolled patients were recorded. Logistic regression analysis was used to screen the influencing factors of thoracic aortic calcification. Based on the results of logistic regression analysis, a nomogram prediction model was constructed. The receiver operating characteristic curve (ROC curve), calibration curve, and decision curve were employed to evaluate the discrimination, calibration and clinical practicality of the nomogram model.Results:A total of 132 patients were included, with 91 males (68.94%) and age of (56.51±16.37) years. There were 60 CKD 3-5 stage patients (non-dialysis, 45.45%) and 72 CKD 5D patients (dialysis, 54.55%). Serum ND-2 levels differed significantly among healthy individuals, dialysis patients and non-dialysis patients ( H=70.651, P<0.001). There was no statistically significant difference in serum NID-2 level between the no or mild calcification group and the severe calcification group in dialysis patients ( Z=350.00, P=0.426). The serum NID-2 level in the severe calcification group was significantly higher than that in the no or mild calcification group in non-dialysis patients ( Z=242.00, P=0.019). In non-dialysis patients, there was a statistically significant correlation between serum NID-2 level and volume of thoracic aortic calcification ( r=0.40, P<0.001). In dialysis patients, there was no statistically significant correlation between serum NID-2 level and volume of each segment of thoracic aortic calcification (all P>0.05). The univariate logistic regression analysis showed that, age, hemoglobin, serum albumin, estimated glomerular filtration rate, NID-2, hypertension, type 2 diabetes mellitus and cerebral infarction were correlated factors of thoracic aortic calcification in non-dialysis patients (all P<0.05). Multivariate logistic regression analysis showed that age ( OR=1.22, 95% CI 1.08-1.50, P=0.010) was an independent correlated factor of thoracic aortic calcification in non-dialysis patients. The above related variables of univariate logistic regression analysis were incorporated into a nomogram to construct a predictive model for severe vascular calcification in non-dialysis patients, yielding an AUC of 0.94 (95% CI 0.89-0.99) in ROC curve, with a sensitivity of 83% and a specificity of 95%. A nomogram model based on above variables for predicting cardiovascular and cerebrovascular events in non-dialysis patients demonstrated an AUC of 0.95 (95% CI 0.90-1.00) in ROC curve, with a sensitivity of 95% and a specificity of 87%. Conclusions:In non-dialysis patients, serum NID-2 level in the severe calcification group is significantly higher than that in the no or mild calcification group. The serum NID-2 is a related factor of thoracic aortic calcification and cardiovascular and cerebrovascular events in non-dialysis patients. The nomogram prediction model constructed by combining NID-2 with age, hemoglobin, serum albumin, estimated glomerular filtration rate, hypertension, type 2 diabetes mellitus and cerebral infarction has a high predictive value for the risk of thoracic aortic calcification as well as cardiovascular and cerebrovascular events in non-dialysis patients.

Objective:To validate the usefulness of copy number variation sequencing (CNV-seq) in detecting the deletion/duplication of the DMD gene in Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients. Methods:One hundred and seventy-seven cases who visited the Department of Medical Genetics, Affiliated Hospital of Kunming University of Science and Technology/the First People′s Hospital of Yunnan Province from April 2018 to November 2023 were collected. All patients had previously accepted multiplex ligation-dependent probe amplification (MLPA) to detect the deletion/duplication of the DMD gene, including 90 cases of normal control with a negative result of MLPA and 87 cases with the deletion or duplication of the DMD gene (61 cases of DMD and 26 cases of BMD). CNV-seq was performed in a single-blind manner to detect DMD gene deletion or duplication for all of 177 cases to obtain the detection efficiency of CNV-seq in comparison with MLPA. Results:Comparing to MLPA, CNV-seq had a coincidence rate of 88.7% (157/177) for detecting DMD gene deletion/duplication, with a sensitivity of 77.0% (67/87), a specificity and a positive predictive value of both 100.0% (90/90 and 67/67, respectively), a negative predictive value of 81.8% (90/110), and a Kappa value of 0.773. Of the 87 patients with the deletion or duplication of the DMD gene, CNV-seq detected 67 cases with DMD gene deletion/duplication, including 62 cases with deletion and 5 cases with duplication, with fragment ranging from 150 to 750 kb. While CNV-seq missed 23.0% (20/87) of positive cases, mainly due to the involved fragments spanning only 1 to 4 exons, and with a variation size less than 50 kb, below the resolution (100 kb) of CNV-seq. The detection rate of CNV-seq in BMD cases (84.6%, 22/26) was a little higher than that in DMD cases (73.8%, 45/61), but there was no significant difference between 2 subgroups ( χ2=1.211, P=0.271). The results of CNV-seq in normal controls were all negative, and consistent with the results of MLPA. Conclusion:CNV-seq can detect 77.0% (67/87) of deletion/duplication of the DMD gene in patients with DMD/BMD, while the deletion/duplication less than 100 kb may be inevitably unidentified, therefore it is recommended as an assistant screening technique in prenatal diagnosis for DMD gene deletion or duplication.