BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

Objective To explore the role and mechanism of mammalian target of rapamycin (mTOR) in oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in vascular smooth muscle cells (VSMCs). Methods A model of oxLDL-induced VSMC ferroptosis was established. VSMCs were co-treated with either the mTOR inhibitor rapamycin or the autophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP), followed by detection of autophagy and ferroptosis-related indexes. Quantitative real-time PCR and Western blot were used respectively to analyze the expression of mTOR, glutathione peroxidase 4 (GPX4), sequestosome 1 (p62), and microtubule-associated protein 1 light chain 3 (LC3). Flow cytometry was employed to assess VSMC death. C11 BODIPY fluorescent staining was used to measure cellular lipid peroxidation levels. Colorimetric assays were performed to determine the contents of malondialdehyde (MDA), ferrous ion (Fe²⁺) and glutathione (GSH). Results oxLDL significantly upregulated mTOR expression in VSMCs, while increasing p62 expression and reducing LC3 expression, thereby suppressing VSMC autophagy. Compared with oxLDL treatment alone, rapamycin co-treatment reversed oxLDL-induced VSMC ferroptosis, as characterized by reduced VSMC death, increased GPX4 expression and GSH contents, along with decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Similarly, CCCP co-treatment activated autophagy characterized by reduced p62 expression and elevated LC3 expression, which subsequently alleviated oxLDL-induced ferroptosis, showing reduced VSMC death, increased GPX4 expressions and GSH contents, and decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Moreover, mTOR inhibition by rapamycin significantly reversed the oxLDL-induced upregulation of p62 and downregulation of LC3. Conclusion mTOR may promote oxLDL-induced VSMC ferroptosis by suppressing autophagy.
Ferroptosis/drug effects* ; Lipoproteins, LDL/metabolism* ; TOR Serine-Threonine Kinases/physiology* ; Autophagy/drug effects* ; Muscle, Smooth, Vascular/metabolism* ; Animals ; Rats ; Myocytes, Smooth Muscle/cytology* ; Cells, Cultured ; Lipid Peroxidation/drug effects* ; Sequestosome-1 Protein/genetics* ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism* ; Microtubule-Associated Proteins/genetics* ; Sirolimus/pharmacology*

Children undergoing hematopoietic stem cell transplantation(HSCT)often experience car-diopulmonary dysfunction,reduced muscle strength,and other complications due to the disease itself and treatment adverse effects.Multiple studies indicate that exercise intervention can promote immune reconstitu-tion,improve physiological functions,and enhance quality of life;however,insufficient exercise remains preva-lent among these children.This review summarizes the current status,influencing factors,exercise outcomes,and management approaches of exercise interventions for pediatric HSCT recipients,aiming to provide refer-ences for developing exercise intervention research suited to China's healthcare context.

Peripheral perfusion index(PPI)is an objective and reliable parameter reflecting the peripheral micro-circulation through the ratio of local pulsatile to non-pulsatile blood flow.PPI is widely used in the early identifica-tion of hypotension,guiding volume management,assessing vascular tone,assisting mechanical ventilation settings,early identification of postoperative complications and poor prognosis of critically ill patients.

OBJECTIVE: To explore the clinical phenotype and genetic characteristics of a child with neurodevelopmental disorder caused by homozygous frameshift variant of the TRAPPC6B gene, and to provide reference for the diagnosis of the disease. METHODS: A child with neurodevelopmental disorder caused by homozygous variant of TRAPPC6B gene who was admitted to the Children's Hospital Affiliated to Zhengzhou University in March 2023 due to "inability to stand and walk independently at 1 year and 3 months old" was selected as the study object. The clinical data were collected by retrospective analysis method. Target region high-throughput sequencing was carried out on the child and parental peripheral blood samples, and candidate variant was verified by Sanger sequencing and bioinformatic analysis. The pathogenicity of variant was rated according to the Standards and Guidelines for the Interpretation of Sequence Variants released by American College of Medical Genetics and Genomics (ACMG) (hereinafter referred to as ACMG guidelines). The study has been approved by the Medical Ethics Committee of the Children's Hospital Affiliated to Zhengzhou University (Ethic No.2022-K-L025). RESULTS: The child was a 1-year-and-3-months-old boy whose parents were sib mating. The child presented with global developmental delay, microcephaly and short stature. MRI showed poor white matter myelination, abnormal signals of bilateral periventricular white matter and bilateral external sac, thin corpus callosum, and widening of the third ventricle. Genetic testing revealed that the TRAPPC6B gene of the child had a homozygous variant of c.240_241delAA (p.Q80Hfs*34), which was inherited from his parents. According to the ACMG guidelines, this variant was judged to be potentially pathogenic (PVS1_Strong+PM2_Supporting+PM3_Supporting), resulting in premature occurrence of terminator codons and a change in the three-dimensional structure of protein. The variant was located in the functional domain, which may directly affect the functional domain of the protein, resulting in functional domain defects. CONCLUSION The frameshift variation of TRAPPC6B gene c.240_241delAA (p.Q80Hfs*34) has not been reported, which may be the genetic cause of neurodevelopmental disorders in child in this study. These findings expand the variation spectrum of TRAPPC6B gene and provide basis for genetic counseling and prenatal diagnosis of this family.
Humans ; Infant ; Male ; Frameshift Mutation ; Homozygote ; Neurodevelopmental Disorders/genetics* ; Phenotype

OBJECTIVE: To explore the clinical phenotypes and genetic characteristics of a pedigree with Distal arthrogryposis type 5D (DA5D) caused by compound heterozygous variants in the ECEL1 gene. METHODS: A child (proband) diagnosed with DA5D and his family members (proband's parents and sister) who was admitted to the Department of Rehabilitation Medicine of Henan Children's Hospital in July 2022 due to "multiplex distal arthrogryposis" were enrolled into this study. Clinical data of the proband were collected and peripheral blood samples were obtained from the proband and members of his family about 3 mL. Trio-whole genome sequencing (trio-WGS) was carried out to detected the genetic variations of the proband and his family members. The candidate's pathogenic gene variants were screened and analyzed by Genome Aggregation Database (gnomAD) and other databases. The screened variants were annotated for clinical phenotypes using databases like the Online Mendelian Inheritance in Man (OMIM). The pathogenicity of the candidate variants was predicted by bioinformatics tools such as Provean. Based on the guidelines of the American College of Medical Genetics and Genomics (ACMG), pathogenicity ratings were conducted for variant sites. The protein conservation and mutation structure prediction of ECEL1 protein among species were carried out though MEGA-X and PyMOL. The research protocol of this study was reviewed by the Ethics Committee of Henan Provincial Children's Hospital (Approval No. 2023-H-H01), and informed consent for clinical research was obtained from the guardians of the probands. RESULTS: The proband had multiplex distal arthrogryposis involving hands, feet, knees, and ankles, and had right ptosis, micrognathia, low auricular position, and upturned nose. The parents and sister both had normal phenotypes. Trio-WGS and Sanger sequencing revealed that the child had compound heterozygous variants of paternal c.1742_c.1743insT and maternal c.2314T>G, for which the father and sister were carriers of the c.1742_c.1743insT heterozygous variant and the mother was carrier of c.2314T>A. Neither mutation site has been reported. According to guidelines of ACMG, the c.1742_c.1743insT variant was classified as likely pathogenic (PSV1+PM2_Supporting), and c.2314T>G was classified as uncertain (PM2_Supporting+PM3+PP3). The results of conserved analysis of amino acid residue sequences of ECEL1 protein showed that the missense mutation of the maternal c.2314T>G (p.Cys772Gly) was highly conserved among humans and other seven species. The protein structure prediction revealed that the c.1742_c.1743insT frameshift mutation led to the protein truncation, and the c.2314T>G missense mutation resulted in the failure of forming 1 disulfide bond. CONCLUSION The compound heterozygous variants of ECEL1 gene were considered to be pathogenic for this DA5D patient, which have expanded the mutational spectrum of the ECEL1 gene and provided a reference for clinical diagnosis as well as genetic counseling for this family.
Humans ; Pedigree ; Arthrogryposis/genetics* ; Male ; Female ; Heterozygote ; Phenotype ; Mutation ; Child ; Metalloendopeptidases

OBJECTIVE To investigate the in vitro inhibitory mechanism of berberine on the proliferation of tumor stem cells and evaluate its in vivo safety. METHODS Flow cytometry was used to select tumor stem cells from mouse skin melanoma B16F10 cells； CD44， CD133， Nanog homologous box protein （NANOG） and octamer-binding transcription factor 4 （OCT4） were used as indicators to characterize tumor stem cells. Tumor stem cells were divided into control group， all-trans retinoic acid （ATRA） group， and berberine group， and the CCK-8 method was used to detect the effects of berberine on the viability of tumor stem cells； flow cytometry was adopted to detect cell apoptotic rate， the proportion of CD44+/CD133+ and the positive cell rate of sex determining region Y box protein 2 （SOX2）； the morphological changes of tumor balls were recorded after treatment with berberine； the morphology of cell pyroptosis in each group was recorded， and the release rate of lactate dehydrogenase （LDH） was detected； Western blot assay was adopted to detect the expressions of pyroptosis-related protein gasdermin E （GSDME）， GSDME- N， caspase-3 and cleaved caspase-3. Preliminary evaluation of in vivo safety of berberine was conducted by using zebrafish embryo toxicity experiments. RESULTS Compared with B16F10 cells， the proportion of CD44+/CD133+ cells in tumor stem cells and the fluorescence intensity of NANOG and OCT4 were significantly increased （P＜0.000 1）. The half-inhibitory concentration of berberine to tumor stem cells was 50.98 μmol/L. Compared with the control group， the apoptotic rate of cells in the berberine group was significantly increased， while the proportion of CD44+/CD133+ cells and the rate of SOX2 positive cells were reduced significantly （P＜0.000 1）； tumor stem cell spheroids were atrophied， with partial cell death. After treatment with berberine， tumor stem cells exhibited swelling in their outermost layer， the release rate of LDH of cells was significantly increased and the release rate of LDH increased with increasing dose； the protein expressions of GSDME-N and cleaved-caspase-3 of cells in berberine 20， 40 μmol/L groups were significantly increased， and the protein expressions of GSDME and caspase-3 were significantly reduced （except for berberine 20 μmol/L group， P＜0.05）. The embryonic development of zebrafish treated with berberine was almost unaffected， and the survival rate of embryo reached 100%， with no obvious abnormalities observed. CONCLUSIONS Berberine has good activity against the proliferation of tumor stem cells， and its mechanism of action may be related to activating GSDME and promoting cell pyroptosis； berberine has good in vivo safety.

ObjectiveTo employ the effective components and antiarrhythmic mechanism of Wenxin Granules （WXKL） by cell membrane chromatography （CMC） and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF/MS）， combined with network pharmacology. MethodIn this study， the CMC/UPLC-Q-TOF/MS technique was employed to identify the components in WXKL that could specifically bind to myocardial cell membranes. By utilizing databases such as SwissTarget Prediction and GeneCards， the targets of WXKL's effective components and arrhythmia-related targets were mined. Cytoscape software was used to construct a "component-target-disease" network. Gene ontology（GO） function and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment analyses were carried out， and molecular docking of key components and targets was performed. Finally， further verification was conducted through in vivo experiment of rats. ResultA total of 39 effective components were identified in WXKL. These included 13 components derived from Panax notoginseng， 15 components from Codonopsis pilosula， seven components from Glycyrrhizae Radix et Rhizoma， one component from Succinum， one component from Polygonatum odoratum， one component shared by both P. odoratum and C. pilosula， and one component shared by both Panax notoginseng and C. pilosula. Network pharmacology predicted that WXKL had 16 core antiarrhythmic targets and 79 related pathways， mainly involving adrenergic signaling in cardiomyocytes， cyclic guanosine monophosphate （cGMP）/protein kinase G （PKG）， calcium signal， cyclic adenosine monophosphate （cAMP）， interleukin （IL）-17， mitogen-activated protein kinase （MAPK）， and tumor necrosis factor （TNF） signaling pathways. The results of in vivo experiment of rats showed that WXKL significantly improved the expression of β₁-adrenergic receptor （β₁-AR）， cAMP， TNF-α， and calcium voltage-gated channel subunit alpha 1C （CACNA1C）. ConclusionWXKL can exert its antiarrhythmic effects through multiple components， multiple targets， and multiple pathways. This study provides a scientific basis for explaining the potential pharmacodynamic substance foundation and mechanism of action of traditional Chinese medicine in treating arrhythmia.

Objective:To explore the effects of different polymers on in vitro biomimetic mineralization of small intestinal submucosa(SIS)scaffolds,and to evaluate the physicochemical properties and bio-compatibility of the SIS scaffolds.Methods:The SIS scaffolds prepared by freeze-drying method were im-mersed in simulated body fluid(SBF),mineralized liquid containing polyacrylic acid(PAA)and mine-ralized liquid containing PAA and polyaspartic acid(PASP).After two weeks in the mineralized solu-tion,the liquid was changed every other day.SBF@SIS,PAA@SIS,PAA/PASP@SIS scaffolds were ob-tained.The SIS scaffolds were used as control group to evaluate their physicochemical properties and bio-compatibility.We observed the bulk morphology of the scaffolds in each group,analyzed the microscopic morphology by environment scanning electron microscopy and determined the porosity and pore size.We also analyzed the surface elements by energy dispersive X-ray spectroscopy(EDX),analyzed the struc-ture of functional groups by Flourier transformed infrared spectroscopy(FTIR),detected the water ab-sorption rate by using specific gravity method,and evaluated the compression strength by universal me-chanical testing machine.The pro-cell proliferation effect of each group of scaffolds were evaluated by CCK-8 cell proliferation method.Results:Under scanning electron microscopy,the scaffolds of each group showed a three-dimensional porous structure with suitable pore size and porosity,and crystal was observed in all the mineralized scaffolds of each group,in which the crystal deposition of PAA/PASP@SIS scaffolds was more regular.At the same time,the collagen fibers could be seen to thicken.EDX analysis showed that the characteristic peaks of Ca and P were found in the three groups of mineralized scaffolds,and the highest peaks were found in the PAA/PASP@SIS scaffolds.FTIR analysis proved that all the three groups of mineralized scaffolds were able to combine hydroxyapatite with SIS.All the scaf-folds had good hydrophilicity.The compressive strength of the mineralized scaffold in the three groups was higher than that in the control group,and the best compressive strength was found in PAA/PASP@SIS scaffold.The scaffolds of all the groups could effectively adsorb proteins,and PAA/PASP@SIS group had the best adsorption capacity.In the CCK-8 cell proliferation experiment,the PAA/PASP@SIS scaffold showed the best ability to promote cell proliferation with the largest number of living cells observed.Con-clusion:Compared with other mineralized scaffolds,PAA/PASP@SIS scaffolds prepared by mineralized solution containing both PAA and PASP have better physicochemical properties and biocompatibility and have potential applications in bone tissue engineering.

Objective To evaluate the maturity and metabolic status of heterotopic ossification(HO)by single-photon emission computed tomography(SPECT)/CT fusion bone imaging.Methods The clinical and SPECT/CT fusion bone imaging data of 57 patients with HO confirmed by pathology or follow-up were analyzed retrospectively.HO was graded by CT,and the characteristics of radioactive concentration of HO were analyzed.Results Of 57 cases,single lesion in 52 cases,and multiple lesions in 5 cases,with a total of 63 lesions,mostly located in the hip joint(55.6%,35/63)and thigh(19.0%,12/63).There were 41 lesions in the middle stage and 22 lesions in the late stage.In the visual evaluation of SPECT/CT fusion bone imaging,the middle stage lesions were mostly clumps or flakes,with moderate or high radioactive concentration(75.6%,31/41),furthermore,the concentration range was larger than or equal to the total or limited range of CT ossification(21/41,50.1%),with high concentration mainly located in the mixed areas of ossification density.The concentration of the late stage lesions was mostly non-radioactive(72.7%,16/22).Conclusion SPECT/CT fusion bone imaging can show the range,degree and maturity of HO radioactive concentration,and can accurately locate the area with osteoblastic activity,which provides scientific basis for the selection of surgical timing.