This study aimed to evaluate the feasibility of CD72 as a complementary CD19-independent B-lineage gating marker for longitudinal measurable residual disease (MRD) surveillance in relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) following CD19 CAR-T therapy. Correlation analyses were performed in 66 B-ALL samples to compare MRD detection using CD72, CD19, and cytoplasmic CD79a. CD72 expression specificity was further evaluated in 129 leukemia patients. In addition, 129 patients with R/R B-ALL treated with autologous CD19 CAR-T therapy in registered clinical trials (ChiCTR-IIh-16008711; NCT03173417) between January 2021 and December 2022 were retrospectively analyzed, with follow-up continued until January 2025. CD72 gating showed excellent concordance with both CD19- and cCD79a-based strategies for MRD assessment. CD72 expression demonstrated high specificity in B-ALL, with a positivity rate of 95.77%, compared with 29.27% in AML and 23.53% in T-ALL. All 129 heavily pretreated patients achieved MRD-negative CR at day 28 after CAR-T infusion and subsequently underwent allo-HSCT, with a median interval of 54 days (range, 40-338). A total of 16 patients experienced MRD relapse during follow-up, including four clinically confirmed CD19-negative relapses that retained CD72 expression. Patients with pre-CAR-T MRD ≤1% showed earlier B-cell recovery than those with MRD >1% (median 30 [18-45] vs. 32 [26-79] days, p = 0.028). The MRD ≤1% cohort demonstrated significantly improved 3-year overall survival compared with the MRD >1% cohort (88.1% vs. 69.2%, p = 0.014). The 3-year cumulative incidence of MRD relapse was significantly lower in the MRD ≤1% cohort than in the MRD >1% cohort (3.96% vs. 17.95%, p = 0.019), while non-relapse mortality was also numerically lower in the MRD ≤1% cohort (5.92% vs. 17.95%, p = 0.053). Multivariate analysis identified KMT2A rearrangement, IKZF1 mutation, TP53 mutation, and elevated pre-CAR-T MRD as independent predictors of inferior outcomes. CD72 represents a feasible complementary B-lineage marker for longitudinal MRD surveillance following CD19 CAR-T therapy. Retention of CD72 expression in clinically confirmed CD19-negative relapses supports its potential utility when CD19 expression is lost after targeted therapy.

Diabetic wounds struggle to heal because of ROS-mediated immune dysfunction, in which the mitochondrial metabolic reprogramming of macrophages is critical for inflammation resolution and tissue repair. Here, we innovatively combine the antioxidant astaxanthin (Ast) and the metabolic activator metformin (Met) via pH-responsive dynamic Schiff base linkages to form amphiphilic Ast-PEG-Met conjugates, which self-assemble into nanomicelles (NMs). This strategy effectively improves the solubility of Ast and enables synergistic delivery of the two complementary drugs. NMs target macrophage mitochondria, where Ast scavenges mtROS and Met activates energy metabolism pathways in a synergistic manner. The two drugs synergistically activate the AMPK-PPARγ axis and promote mitophagy, thereby restoring mitochondrial function and regulating immunometabolic reprogramming. For on-demand release, the NMs were further encapsulated into a ROS-responsive self-healing hydrogel crosslinked by dynamic phenylboronate ester bonds. This system responds to high levels of ROS and an acidic wound microenvironment with favorable biocompatibility. In a diabetic full-thickness skin defect model, this integrated platform markedly accelerated wound healing, promoted collagen deposition and angiogenesis, and alleviated chronic inflammation. This work provides a promising combined strategy involving antioxidant-metabolic regulation for chronic diabetic wound therapy via a dual-responsive hydrogel drug delivery system.

It was found that pressure can promote the regeneration and repair of cartilage defects based on bone marrow mesenchymal stem cells (BMSCs). Since the compressive microenvironment of the cartilage in vivo may change with different movement, the mechanobiological effects of different compressive condition on BMSCs, especially the impact for its chondrogenic differentiation and influence on the cartilage microenvironment, is what we concerned about. Rat BMSCs were cultured and subjected to various types of pressure stimulation for 1 h. The Cell Counting Kit-8 (CCK-8) assay was used to analyze cell proliferation, flow cytometry was employed to assess the cell cycle and apoptosis, confocal microscopy was used to observe the cytoskeleton, and transmission electron microscopy was performed to examine the cellular ultrastructure. RT-PCR was used to identify chondrogenic differentiation markers. Apoptotic vesicles derived from BMSCs were isolated by ultracentrifugation, and differentially expressed microRNAs in these vesicles under − 40 kPa compression were identified by transcriptome sequencing. Specific pressure conditions promoted the proliferation of BMSCs, with dynamic pressure showing a stronger proliferative effect than static pressure. Higher static negative pressure (− 40 kPa) significantly increased the spreading area of BMSCs. Dynamic pressure is stronger than static pressure in promoting cytoskeletal rearrangement, stress fiber formation, and cartilage marker expression in BMSCs. Flow cytometry and transmission electron microscopy results show that both − 40 kPa static and 90 kPa dynamic pressures promote BMSCs apoptosis to some extent. Under − 40 kPa static negative pressure, the differentially expressed microRNAs in BMSCs-derived apoptotic vesicles are involved in stem cell maintenance and chondrogenic proliferation processes. Static negative pressure (− 40 kPa) induces apoptosis in bone marrow mesenchymal stem cells (BMSCs). Notably, compared to chemical induction with staurosporine (STS), BMSCs subjected to − 40 kPa mechanical stimulation display distinct microRNA expression profiles within apoptotic vesicles, specifically enriched in microRNAs implicated in stem cell fate determination and cartilage regeneration. These findings offer valuable insights into biomechanical strategies for optimizing tissue-engineered cartilage repair.

Abstract Background and Aims This study developed and validated a novel immune status evaluation system utilizing multiplex cytokine profiling to objectively assess post-CD19 CAR-T therapy immune responses and facilitate early identification of cytokine release syndrome (CRS). Particularly by quantifying the effects of various cytokines and proposing a formulaic algorithm, it provides an effective tool for the simplified, scientifically rigorous, and rapid assessment of high-volume data. Methods The specimens were from 111 patients with refractory/relapsed B-cell acute lymphoblastic leukemia (B-ALL) treated with CD19 CAR-T therapy at Lu Daopei Hospital since March 2020. The cohort comprised a training set (n=34) for developing a cytokine-based scoring system and a validation set (n=77) to evaluate clinical concordance. Serial measurements of 24 cytokines (IFN-γ, IL-1β, IL-2, IL-6, IL-10, IL-12p70, TNFα, TNFβ, IL-4, IL-5, IL-8, IL-17A, IL-17F, IL-22, IL-2RA, MCP-1, GM-CSF, IL-15, Granzyme B, REG3a, ST-2, TNFRI, Elafin and MIP-1α) were performed in the training set at six timepoints: pre-CAR-T treatment (day 0), and days 4, 7, 14, 21, and 28 post-treatment. The validation set underwent cytokine profiling at days 0 and 7. Results Our study showed that peak cytokine levels occurring at 7-10 days post-treatment, followed by progressive decline as CAR-T/tumor cell interactions diminished and tumor clearance advanced. This reduction coincided with decelerated expansion of CAR-T, CD3+, and CD8+ T-cell populations, with cytokine concentrations returning to near-baseline by approximately day 30. For the convenience of clinical application, we developed a cytokine scoring system for immune status assessment. The 24 factors were divided into four weight levels based on their contribution to predicting CRS: Class I cytokines (IFN-γ, IL-2, IL-6, IL-10, ST-2, IL-8, GM-CSF) weighted at 2.0; Class II (IL-2RA, IL-17F, REG3a, IL-1β, MCP-1, TNFRI) at 1.0; Class III (IL-4, IL-5, IL-22, IL-15, IL-12p70) at 0.5; and Class IV (Elafin, TNFα, Granzyme B) at -1.0. A cytokine is assigned a weight point if its peak value exceeds three times the threshold. The results for each cytokine are summed to obtain a total score. Following targeted therapy, an immune status score ≤ 8 points is classified as CRS Grade 0-1; a score > 8 points and < 18 points is classified as CRS Grade 2; and a score ≥ 18 points is classified as CRS Grade 3-4. Clinical validation demonstrated overall sensitivity of 89.61%, specificity of 96%, positive predictive value (PPV) of 95.83%, and negative predictive value (NPV) of 90%. Crucially, for distinguishing Grade 0-1 CRS (outpatient-manageable) from ≥Grade 2 CRS (requiring hospitalization per ASTCT criteria), the system achieved enhanced performance: 91.55% sensitivity, 99% specificity, 98.48% PPV, and 92% NPV, confirming its utility in guiding critical clinical decisions. Conclusions This standardized system provides an objective, high-throughput tool for immune monitoring and early intervention, with significant implications for clinical decision support in cellular immunotherapy.

Abstract Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for various hematologic malignancies. However, post-transplant diarrhea is a frequent and clinically significant complication. Among its leading causes, graft-versus-host disease (GVHD) and infection often present with overlapping gastrointestinal symptoms, yet require different treatment approaches. Prompt and accurate differentiation between these etiologies is therefore critical for guiding therapeutic decision-making and improving patient outcomes. This study evaluated weekly monitoring of lymphocyte subsets and cytokines starting from white blood cell engraftment to develop a predictive model for differentiating infection- and GVHD-associated diarrhea after allo-HSCT. Methods We retrospectively analyzed 58 patients who underwent endoscopic evaluation with biopsy for post-HSCT diarrhea at Beijing Lu Daopei Hospital between April 2021 and December 2024. Based on histopathology, 34 were diagnosed with GVHD and 24 with infection. Lymphocyte subsets and cytokine levels were monitored weekly following white blood cell engraftment, including the week prior to diarrhea onset. Patients were follow-up continued until May 31, 2025. Results 1、Clinical characteristics did not differ significantly between the GVHD group (n=34) and infection group (n=24), including pre-transplant disease status, transplant type, conditioning regimen, ATG formulation, stem cell source, third-party donor use, graft cell dose, and cGVHD incidence(p all ＞0.05). Overall survival was comparable between groups (P=0.408). GVHD-associated diarrhea occurred earlier (median onset: 31 vs. 58 days) and lasted longer (86 vs. 28 days) than infection-related diarrhea. Frequency and volume of diarrhea were similar between groups (χ²=1.501, P=0.139; χ²=0.858, P=0.394). Both groups commonly presented with rash and hemorrhagic cystitis; however, hematochezia (26.5% vs. 8.3%) and hyperbilirubinemia (20.6% vs. 4.2%) were more frequent in the GVHD group. 2、At one week prior to diarrhea onset, the GVHD group showed significantly higher levels of Reg3α (2205.74 pg/mL [1174.11–3293.06] vs. 1333.70 [1058.83–1634.65], P=0.037), CD3⁺ T% (76.13% [47.45–84.27] vs. 50.33% [34.09–63.18], P=0.005), CD8⁺ T% (48.29±19.33 vs. 34.53±19.88, P=0.011), naive CD4⁺ T% (2.28% [0.75–5.73] vs. 1.06% [0.30–2.19], P=0.021), and CD4⁺ Th2% (40.18±21.11 vs. 27.77±20.61, P=0.030). Conversely, the GVHD group had lower NK cell proportion (17.80% [9.70–40.13] vs. 36.66% [15.46–46.78], P=0.042) and NK cell count (48.00/μL [22.25–73.25] vs. 80.00/μL [39.00–164.75], P=0.021). 3、Based on t-test or M-U test results, effect sizes for intergroup differences at one week prior to diarrhea onset were summarized using a forest plot. CD3⁺T%, CD8⁺ T%, absolute NK cell count (cells/μL), and the proportion of naïve CD4⁺T cells among total CD4⁺T cells demonstrated moderate effect sizes in distinguishing GVHD- from infection-associated diarrhea. Reg3αlevels and NK% showed small effect sizes. The direction of these effect sizes was consistent with the differences identified in comparative analyses.Using cytokine and immunophenotypic data from 1 week prior to diarrhea, a bivariate LASSO model incorporating NK cell count and CD4⁺CD28⁺% was constructed, yielding an AUC of 0.799. The optimal cutoff value was 0.408, with sensitivity 0.708 and specificity 0.882. 4、To improve diagnostic accuracy, a multivariable predictive score (PS) was developed using significant features from weeks 4, 5, and pre-diarrhea via LASSO selection. The final model incorporated 9 parameters:PS=2.6714+(CD8+T%)*(-0.0116)+NK*0.0049+(CD4+CD28+T%)*(-0.0217)+(CD4+Th2%)*(-0.0256)+(TNFRIweek4)*(-0.0005)+(NKweek4)*(0.0006)+(GM-CSFweek5)*(-1.6570)+(NK week5)*(0.0038)+(CD3+CD38+T% week5)*(0.0243).Multivariate logistic regression showed that each 1-unit increase in PS was associated with a 5.133-fold higher odds of infection-related diarrhea (OR=5.133; 95% CI: 2.47–14.71; P<0.001). The model demonstrated excellent discriminatory power (AUC=0.908), with an optimal cutoff of 0.669 (sensitivity: 0.875; specificity: 0.794). The multi-timepoint model outperformed single-timepoint predictions. Conclusion Immune profiling before diarrhea onset enables differentiation between GVHD and infection. A multi-timepoint model showed strong diagnostic accuracy (AUC=0.908) and may support clinical decision-making.

Postharvest diseases significantly affect the quality of fresh-cut Mesembryanthemum crystallinum L. gamma-aminobutyric acid (GABA) is a non-protein amino acid that plays a crucial role in the plant's disease resistance process as an endogenous metabolic product and signaling molecule. This study found that exogenous treatment of ice plant with GABA has a positive effect on inhibiting postharvest diseases by regulating the activity of antioxidant enzymes and activating the reactive oxygen species (ROS) pathway, thereby protecting cells from oxidative damage. Concurrently, the addition of exogenous GABA activates the salicylic acid pathway and promotes the biosynthesis of phenylpropanoids, thus enhancing the plant's defense against pathogenic microorganisms. This research proves that GABA could be used as a green preservative to limit postharvest disease losses in fresh-cut ice plants caused by Fusarium sp.

Approximately 30% of acute myeloid leukemia (AML) patients express CD7 on their myeloblasts. We have previously demonstrated that scFv-based “naturally selected” CD7 CAR-T (NS7CAR-T) therapy shows significant efficacy with a favorable safety profile in T-cell lymphoid malignancies. Here we derived dual nanobody-based dVHH NS7CAR-T cells that have superior CD7 binding specificity, affinity to their scFv-based counterparts and improved proliferative capability. In this phase I clinical trial, we evaluated the efficacy and safety of dVHH NS7CAR-T cells in patients with CD7-positive refractory/relapsed (r/r) AML. A cohort of ten patients received dVHH NS7CAR-T cells across two dosage levels of 5×105/kg and 1×106/kg. Before enrollment, patients had undergone a median of 8 (range: 3-17) prior lines of therapy. Seven patients had prior transplants. Following NS7CAR-T cell infusion, 7/10 (70%) patients achieved complete remission (CR). The median observation time was 178 days (28-776 days). Among the seven patients who achieved CR, 3 who relapsed from prior transplants underwent a second allogeneic hematopoietic stem cell transplant (allo-HSCT). One patient remained leukemia-free on day 401, and the other two died on day 241 and day 776 from non-relapse-related causes. Three CR patients without consolidative allo-HSCT relapsed within 90 days. All the nonresponders and relapsed patients had CD7 loss. The treatment was well-tolerated, with 80% experiencing mild cytokine release syndrome and none had neurotoxicity. This trial underscores the potential promising treatment of dVHH NS7CAR-T in providing clinical benefits with a manageable safety profile to CD7-positive AML patients, warranting further investigation. NCT04938115

CD7 Chimeric Antigen Receptor-T cell (CAR-T) therapy demonstrates efficacy in relapsed/refractory (R/R) acute T-lymphoblastic leukaemia (ALL)T-ALL/lymphoblastic lymphoma (LBL), but concerns about T-cell depletion and severe immunodeficiency persist. We compared infection rates and immune cell subsets in 60 R/R T-ALL/LBL patients receiving naturally selected CD7 CAR-T (NS7CAR-T) with 60 R/R B-ALL patients undergoing CD19 CAR-T. Infections were monitored from infusion until allogeneic haematopoietic stem cell transplantation (HSCT) or up to 3 months. Overall infection rates did not significantly differ between groups (36.67% vs. 24.56%, p = 0.24), although the incidence of early immune effector cell-associated haematotoxicity (ICAHT) grade III-IV was higher in the CD7 CAR-T group than in the CD19 CAR-T group (33.9% vs. 16.7%, p = 0.03). Post-CD7 CAR-T infusion analysis showed a significant decline in CD7(+) T cells and an increase in non-CAR-T-derived CD7(-) T cells, particularly non-CAR-T cells, which rose to a median proportion of 84.4% (range: 22.1%-99.9%) by day 28; meanwhile, CD7(-) natural killer (NK) cells approached nearly 100% following the depletion of CD7(+) NK cells. This study indicates that while CD7 CAR-T therapy significantly reduces CD7(+) T cells, it does not lead to increased short-term infection rates. The notable expansion of non-CAR-T-derived CD7(-) T and NK cells helps preserve immune function, highlighting distinct therapeutic mechanisms between CD7 CAR-T and CD19 CAR-T due to their different lineage restrictions.

Abstract Objective: To find a strong and applicable panel for T-ALL MRD detection by flow cytometry and find the relation with genetics. Methods: From February 28, 2024 to May 29, 2025, 1025 T-ALL patients were tested for MRD in our Hospital using full-spectrum flow cytometry. A total of 1570 tests were completed. All patients were Chinese, without disabilities, with a male-to-female ratio of 768:257, and a median age of 16 years (1-68). There was 1 Uyghur, 2 Tibetans, 5 Hui people, and 1017 Han people. The detectionpanel was CD99 FITC/cCD3 PE/CD3 BV785/CD48 PECy7/CD4 APC Cy7/CD5 APC R700/CD2 BV605/CD7 APC/CD16 efluor 450/CD56 BV711/TdT BV421/CD45 V500/CD34 PerCP Cy5.5/CD94 BV650/CD8a BV570/TCRγδ BV480. A total of 612 people had detectable genes. Ten cases of complete remission and ten cases of MRD positive were selected for correlation testing between the traditional panel and the full-spectrum panel. Data analysis was performed using Kaluza 2.3.0. Statistical analysis was conducted using SPSS 17.0. 68 cases were implemented in Python. Results: (1) The correlation between the full-spectrum flow cytometry and the traditional flow cytometry was good. (2) Among the 1570 tests, 155 tests from 103 individuals were positive. Among the positive patients, the ratio of male to female was 79:24, with a median age of 21 years (ranging from 4 to 68 years). There was 1 Tibetan, 1 Hui, and 101 Han. The median tumor burden was 1.66% (ranging from 0.002% to 94.15%). (3) 68 MRD positive cases had genetics results, 33 had WT1, 2 had EVI, 13 were positive for SIL::TAL1, 1 for PCM1::JAK2, 4 for SET::CAN, 6 for MLL::AF6, 1 for MBNL1::TAL1, 1 for KMT2A::PAP1GDS1, 2 for DIAPH1::PDGFB, and 3 for CALM::AF10. One case of EVI and 6 cases of WT1 had values below the reference range. (4) Among the 52 cases with CD7 negative after CD7 CAR-T, 43 were MRD negative and 9 were MRD positive. Among the MRD positive patients, 2 were cCD3 negative. Among 52 cases with CD7 negative,cases negative for cCD3, CD2, and CD5 was 0. (5) Among the 6 cases with lost cCD3, 2 cases were negative for both CD2 and CD5. (6) The relationship between genetics and immunophenotype: the probability of co-expression of CD7 and CD99 was the highest in the MLL::AF6, CALM::AF10, and EVI1 genetic abnormality groups, which was 100% (P < 0.001). The genotypes that were prone to cCD3 weakening or partial loss were PCM1::JAK2 (100%, 2/2), SET::CAN (75%, 3/4), MLL::AF6 (67%, 2/3), DIAPH1::PDGFB (50%, 1/2), EVI1 (50%, 1/2), WT1 (40%, 14/35), and CALM::AF10 (33%, 1/3). On the MRD recognition markers, strong expression of CD99 and weakened CD48 were common abnormalities. PCM1::JAK2, SET::CAN, and WT1 were prone to losing the characteristic of strong expression of CD99. The probability of weakened or negative CD48 was 100% for MLL::AF6 (6/6), SET::CAN (4/4), CALM::AF10 (3/3), MBNL1::TAL1 (1/1),and KMT2A::PAP1GDS1 (1/1). It was 64% for SIL::TAL1, and 40% for WT1. The positive rate of CD34 in all T-ALL cases was only 19%, while CALM::AF10 and EVI1 was 100% positive, MLL::AF6 was 50%, WT1 was 23%, SET::CAN was 25%. CD45dim/CD7bri is the most commonly used gating method, but should exclude NK cell interference. The probability of CD56 positivity was 67% for CALM::AF10 and 20% for WT1. The positive rate of CD3 was 79% (11/14) for SIL::TAL1, 50% for DIAPH1::PDGFB (1/2) and EVI1 (1/2), 46% (16/35) for WT1, and 25% (1/4) for SET::CAN. MLL::AF6 has a 100% positive rate for CD7briCD99briCD48-CD56-CD34+, theoretically providing the highest degree of identification. Next is EVI1, which is 100% positive for CD7briCD99briCD48+CD56-CD34+ because CD48 could not provide contribution. CALM::AF10 has a 100% positive rate for CD7briCD99briCD48-CD34+, but the CD56 positive rate is 67% (2/3), and it also needs to be differentiated from NK cells, making it an easily identifiable subtype. PCM1::JAK2 and some WT1 subtypes may be relatively more difficult to identify. Conclusion: Full-spectrum flow cytometry can simultaneously perform more than four pan-system markers, theoretically meeting the gating requirements for MRD detection after CAR-T therapy at 98.51%. However, to increase detection coverage, pan-T markers need to be added. There is a certain correlation between genetics and immunophenotype. Different genotypes benefit from different markers, and full-spectrum flow cytometry can simultaneously perform more marker combinations to maximize coverage of common abnormalities in various genetic subtypes.

ABSTRACT:B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy has demonstrated promising therapeutic efficacy in relapsed or refractory (R/R) multiple myeloma. However, distinct CAR T-cell constructs exhibit varying therapeutic outcomes. As the antigen-recognition domain, nanobodies offer a small, stable, single-domain structure with enhanced affinity and specificity compared with conventional single-chain variable fragments. We explored the use of nanobody-based BCMA(S103) CAR T-cell therapy for R/R plasma cell myeloma. The CAR construct incorporates dual-nanobody variable domain of the heavy chain of heavy chain antibody (VHHs) targeting BCMA. A cohort of 27 patients was treated with S103 CAR T-cell therapy, which included 4 patients of plasma cell leukemia, and 1 patient of anaplastic plasma cell myeloma. Eleven patients had multiple extramedullary lesions, and 11 patients exhibited high-risk genetic abnormalities, including 4 with TP53 mutations. One month after CAR T-cell infusion, the overall response rate (ORR) was 96.3% (26/27), with a complete response (CR) + very good partial response (VGPR) rate of 59.2% (16/27). At the 3-month follow-up, the ORR increased to 100% (27/27), with a CR + VGPR rate of 81.5% (22/27). The median duration of remission was 11 months (range, 2-36 months). The 1-year overall survival rate was 61.1%, and progression-free survival was 57.2%. In conclusion, BCMA CAR T-cell therapy, utilizing dual-nanobody VHHs targeting BCMA, demonstrates a high ORR and manageable safety profile in treating patients with R/R plasmacytic myeloma, including those with high-risk features such as extramedullary lesions, high-risk cytogenetic abnormalities, plasma cell leukemia, or anaplastic plasmacytoma. This trial was registered at www.ClinicalTrials.gov as #NCT04447573.

Osseointegration is the most important factor determining implant success. The surface modification of TiO2 nanotubes prepared by anodic oxidation has remarkable advantages in promoting bone formation. However, the mechanism behind this phenomenon is still unintelligible. Here we show that the nanomorphology exhibited open and clean nanotube structure and strong hydrophilicity, and the nanomorphology significantly facilitated the adhesion, proliferation, and osteogenesis differentiation of stem cells. Exploring the mechanism, we found that the nanomorphology can enhance mitochondrial oxidative phosphorylation (OxPhos) by activating Piezo1 and increasing intracellular Ca2+. The increase in OxPhos can significantly uplift the level of acetyl-CoA in the cytoplasm but not significantly raise the level of acetyl-CoA in the nucleus, which was beneficial for the acetylation and stability of β-catenin and ultimately promoted osteogenesis. This study provides a new interpretation for the regulatory mechanism of stem cell osteogenesis by nanomorphology.

Background In the past few years, CD7 became a highly reliable marker of CAR-T therapy treating T cell acute lymphocytic leukemia(T-ALL). In this case, better understanding of CD7 CAR-T therapy on cellular level became meaningful. Aim To figure out the correlation of cytokines level and CAR-T expansion to CRS and outcomes of CD7 CAR-T therapy. Methods Peripheral blood samples were collected from 70 patients treated with CD7 CAR-T from Dec. 2020 to Nov. 2022 in Hebei Yanda Ludaopei Hospital. There were 56 males and 14 females, from 2-year old to 60 with median age 19. Based on criteria of CRS and assessment results, 70 patients were split in different groups. According to CRS level, two groups were set as low (CRS0-1, 56 of 70) group and high group (CRS2-4, 14 of 70). Due to assessment results, 55 of 70 were classified as CR/CRi group, 15 of 70 were Non-CR group. 24 Cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-17F, IL-22,TNFα, TNFβ, IFN-γ, IL-2RA/sCD25, MIP-1α, MCP-1, GM-CSF, IL-15, REG3a, Elafin, ST-2, Granzyme B, TNFRI) were measure with microbeads with 2 lasers 4 colour Calibur flow cytometer. CAR-T cells, target cells(CD7+ cells), T cells subsets(CD3+, CD4+, CD8+, CD8+TCM, CD8+TEM, Treg) were tested with 3 lasers 8-10 colour Canto flow cytometer. All the cellular variations were calculated by comparing value of peak-time to value of D0 which was used as baseline. Time points day0, 4, 7, 11, 14, 21, 30 were monitored after CAR-T infusion. FCM data was analyzed with Diva software. Prism 8 was used for statistic analyse. Results According to patient characteristics (shown in table 1a), no matter under which criteria, patients in all 4 groups had similar median age and transfection efficiency. First of all, based on criteria of CRS level, patients were split into low group and high group(upper table 1a). CAR-T peak time was appeared significantly later(p=0.0274) in high group. Along with that, in high group, CD8+TEM(effect memory T) cells was amplified significantly more than that in low group at peak time point(p=0.0258). Clearance rates of target cells(CD7+) in low group were significantly higher(p=0.003). According to cytokine secretion levels, the level of IL-4(p=0.0183), IL-6(p<0.0001), IL-8(p=0.043), IL-10(p<0.0001), IL-22(p=0.0463), IFN-γ(p<0.0001), sCD25(<0.0001), MCP-1(p=0.0092), IL-15(p=0.0029), Granzyme B(p=0.0027) were significant higher in high group(as shown in figure 1). All median peak time of those 10 cytokines in high group were calculated, from day 10 up to day 15 after CAR-T infusion. According to the time gap between each of 10 cytokines with CAR-T expansion, MCP-1 had the largest time gap, which means MCP-1 reached peak time 9 days earlier than that of CAR-T cells. The median peak-time gap between cytokines level with CAR-T cells expansion was 6 days(showed in table 1b). Secondly, due to criteria of assessment results of CD7 CAR-T therapies, 70 patients were classified as CR/CRi group and Non-CR group (lower table 1a). CAR-T expansion, T cell subsets variation showed no statistical difference between two groups, and only target cells (CD7+) clearance rate was significantly higher in CR group. For all 24 cytokines measured in this study showed no significant differences between both groups. Conclusions Base on our data, IL-4, IL-6, IL-8, IL-10, IL-15, IL-22, IFN-γ, sCD25, MCP-1, and Granzyme B were correlated to severity of CRS after CD7 CAR-T therapy. Those 10 cytokines showed positive correlation to the level of CRS, and reached peak time earlier than that of CAR-T expansion, which means all these 10 cytokines could be used as indicators to predict occurrence and severity of CRS. Concurrence time of peak expansion of CD7 CAR-T cells, and amplification of CD8+TEM cells were significantly related to CRS. For all the patients who reached complete remission or CRi, CD7+ clearance rates were higher with statistical significance. Our results proved potential indicators for CRS prediction and prognosis.

Introduction: Multicolor flow cytometry (MFC) is one of the most widely utilized methods for measurable residual disease (MRD)detection. As the technology advances, full-spectral flow cytometry (FSFC) which allows the simultaneous analysis of multiple markers is promising to improve the sensitivity of MRD. Methods: We collected 546 BM from 511 B-ALL patients who detected in our lab from Feb.2024 to Jun. 2024. There were 300 male (58.7%) and 211 female (41.3%), with a median age of 13 years old (range,1-71 years). They were detected at three time points: post-induction (PI; day-35), post-consolidation (PC; day-78), and subsequent follow-up time-points (SFU). We created a novel 13-color single-tube FSFC panel as cTdT-FITC,CD10-PE,CD34-Percpcy5.5,CD19-Pecy7, CD20-Apccy7,cCD79a-APC,CD38-Percpeflour710,CD86-PE dazzle594,CD72-BV421,CD33-BV650,CD123-BV711,CD81-BV786, and CD45-V500. The prepared samples were detected on a three-laser 24 channel flow cytometer(Cytek Biosciences, USA) with a target of 1*106 events, and the data were analyzed with SpectroFlo software. Forward scatter (FSC)/side scatter (SSC) dot plots set the live cell gate, doublets were excluded on FSC-A/FSC-H, and synchronous gating was performed from the single live cells: CD45/SSC for background subsets, B cells were gated by CD19/SSC, cCD79a/SSC and CD72/SSC. By analyzing the developmental pattern of B-lineage cells in dot plots such as CD10/CD20, CD10/CD34, CD10/CD38, CD10/CD81, CD10/CD86, CD34/cTdT, CD38/CD72, CD19/cCD79a, CD33/CD72, CD123/CD34, CD19/CD45, cCD79a/ CD45, CD72/ CD45 and so on, we identified B-ALL MRD cells based on the combined DfN and LAIP strategy. To verify the accuracy of our 13c-FSFC method, 186 BM samples were analyzed in parallel to molecular monitoring. BCR-ABL1, TCF3-PBX1, ETV6-RUNX1, MLL-AF4, MLL-AF10, MLL-ELN, and UBTF-ATXN7L3 fusion genes, PAX5, ZNF384, MEF2D, and IgH rearrangements, MLL-PTD mutation, and IKZF1 deletion were detected by PCR. McNema's test was performed in SPSS Statistics version 27, and P < 0.05 was deemed to be statistically significant. Cohen's Kappa test was also performed in SPSS, Kappa≥0.75 indicates that the diagnostic results of two methods are in good consistency. Results: It was shown that CD19-directed therapies, such as blinatumomab and anti-CD19-CAR-T, have been receiving increasing attention in the treatment of B-ALL. In order to establish an alternate gating strategy, it is necessary to add cCD79a and CD72 into the panel since CD19 might downregulated. We found that out of 546 bone marrow samples, there were 49 MRD positive samples (8.97%) and 497 MRD negative samples (91.03%). The median tumor burden was 0.15% (range, 0.001%-89.77%). Among the 49 MRD positive samples, abnormalities in CD19 (1 bright , 3 loss) accounted for 8.61%, cCD79a (2 dim) accounted for 4.08%, CD72 (7 dim) accounted for 14.29%, CD34 (4 bright, 18 dim/-) accounted for 44.90%, CD10 (33 bright, 7 dim/-) accounted for 81.63%, CD20 (6 dim) accounted for 12.24%, CD38 (38 dim) accounted for 77.55%, CD81 (36 dim) accounted for 73.47%, cTdT (17 dim/-) accounted for 34.69%, CD123 (2 bright,5 dim) accounted for 20.00%, CD33 (6 positive) accounted for 12.24%, CD86 (9 positive, 8 dim) accounted for 34.69%. Besides CD19, cCD79a, CD72 and CD45, the contribution of these antibodies, in descending order, is CD10, CD38, CD81, CD34, cTdT, CD86, CD123, CD33, CD20. CD10 str/+ and CD38 dim/- and CD81 dim/+ are the most frequent aberrancies, we were able to identify 59.18% abnormal samples in the combination of CD10/CD38 (n=19) or CD10/CD81 (n=10) dot plots. Three MRD positive samples were found to have lost CD19 but were defined by cCD79a or CD72. Out of the186 samples analyzed by molecular method, MRD positive samples account for 8.60% (16/186), while the remaining 91.40% tested negative for MRD. When comparing the results of MRD using FSFC and PCR methods, 179 samples showed consistent analysis results: including 15 MRD positive samples and 164 MRD negative samples. There were seven samples with two inconsistent test results, and no statistical difference between the two methods by McNema's test (P=0.125). According to Cohen's Kappa test, the FSFC method has good consistency with PCR monitoring (Kappa=0.790). Conclusions: We have established a single-tube 13c-FSFC method for detecting MRD in B-ALL.

The aim of our research was to explore the character of autophagy related 12 (ATG12) in the development of hepatocellular carcinoma (HCC). A total of 145 HCC tissues as well as paired adjacent normal tissues were collected, then immunohistochemistry was conducted to access the expression of ATG12. HCC cells were transfected with pcDNA ATG12 or si-ATG12 to overexpress ATG12 or downregulate ATG12. The vitality of HCC cells was accessed using CCK-8 assay, and the ability of invasion of them was tested through Transwell assay. The apoptotic rate of HCC cells was calculated by flow cytometry. The expression of ATG12 was lower in HCC tissues than that in normal tissues, and HCC patients with high ATG12 level survived longer. Overexpressed of ATG12 restrained vitality and invasion of HCC cells, while elevated apoptotic rate of HCC cells. Silence of ATG12 expression yielded opposite results to overexpression of ATG12 in HCC cells. In conclusion, ATG12 is low expressed in HCC, which attenuated the growth and invasion of HCC, while induced the apoptosis of HCC cells. Current research suggested that ATG12 might be a potential target for the diagnosis and treatment of HCC.

Background Mechano-growth factor (MGF), which is a growth factor produced specifically in response to mechanical stimuli, with potential of tissue repair and regeneration. Our previous research has shown that MGF plays a crucial role in repair of damaged periodontal ligaments by promoting differentiation of periodontal ligament stem cells (PDLSCs). However, the molecular mechanism is not fully understood. This study aimed to investigated the regulatory effect of MGF on differentiation of PDLSCs and its molecular mechanism. Methods Initially, we investigated how MGF impacts cell growth and differentiation, and the relationship with the activation of Fyn-p-YAPY357 and LATS1-p-YAPS127. Then, inhibitors were used to interfere Fyn phosphorylation to verify the role of Fyn-p-YAP Y357 signal after MGF stimulation; moreover, siRNA was used to downregulate YAP expression to clarify the function of YAP in PDLSCs proliferation and differentiation. Finally, after C3 was used to inhibit the RhoA expression, we explored the role of RhoA in the Fyn-p-YAP Y357 signaling pathway in PDLSCs proliferation and differentiation. Results Our study revealed that MGF plays a regulatory role in promoting PDLSCs proliferation and fibrogenic differentiation by inducing Fyn-YAPY357 phosphorylation but not LATS1-YAP S127 phosphorylation. Moreover, the results indicated that Fyn could not activate YAP directly but rather activated YAP through RhoA in response to MGF stimulation. Conclusion The research findings indicated that the Fyn-RhoA-p-YAPY357 pathway is significant in facilitating the proliferation and fibrogenic differentiation of PDLSCs by MGF. Providing new ideas for the study of MGF in promoting periodontal regenerative repair.

Objective:To investigate the significance and value of peripheral blood lymphocyte subset characteristics in patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT) for distinguishing cytomegalovirus (CMV) infection.Methods:This retrospective study gathered data from allo-HSCT patients treated at Beijing Ludaopei Hospital between May 2021 and January 2023. This study included 50 patients in the CMV infection group and 47 patients in the non-CMV infection group. Flow cytometry was used to detect peripheral blood lymphocyte subsets 1 month after the allo-HSCT. The absolute lymphocyte count and their subsets were analyzed to assess their predictive significance in the occurrence of CMV infection. Mamn-whiney U test and χ 2 test were used. Indicators with statistical significance in univariate analysis were further subjected to binary logistic regression analysis and receiver operating characteristic (ROC) curves construction. The generation of survival curves for different outcomes was conducted using the Kaplan-Meier method, and the Log-rank test was applied. A two-sided P-value of<0.05 was considered statistically significant. Results:1. Among patients in the CMV infection group, 22% (11/50) were diagnosed with CMV viremia combined with CMV disease. The maximum CMV viral load in peripheral whole blood in these patients was 1 600 (1 400, 5 800) copies/ml, which was significantly higher than that in patients without confirmed CMV disease 970 (670, 2 300) copies/ml, with a statistically significant difference ( Z=-2.281, P=0.029). 2. The overall survival (OS) at the follow-up endpoint was 80% (40/50) in the CMV infection group and 87.2% (41/47) in the non-CMV infection group. There was no statistically significant difference in OS between the two groups at the follow-up endpoint ( χ2=0.231, P=0.630). 3. In the binary logistic regression model, the number of CD34 cells transfused into the patient, the percentage of CD3+CD38+T cells in CD3+T cells, and the percentage of CD4+CD38+T cells in CD4+lymphocytes were identified as important independent risk factors for infection. The ROC curve analysis demonstrated that the area under the curve (AUC) for these independent risk factors of CMV infection was 0.914 (>0.7, P<0.001), with a decision value of 0.702, a sensitivity of 0.857, and a specificity of 0.844. Conclusion:Followingallo-HSCT, the immune subsets in the CMV infection group exhibited a diminished proportion of T cells and CD4+T cells in comparison to the non-CMV infection group. In terms of T cell differentiation, there was an increase in TEM cells and a decrease in Naive T cells. Additionally, the expression level of the activation marker CD38 on T cells exhibited a high degree of predictive value for the occurrence of CMV infection. Understanding the immune characteristics of post-transplant CMV-infected patients is beneficial for the analysis of their immune reconstitution status, providing valuable insights for clinicians in the diagnosis and treatment of CMV infection after transplantation.

Hepatic encephalopathy (HE) represents a critical complications of end-stage liver disease, serving as an independent predictor of mortality among patients with cirrhosis. Despite effective treatment with rifaximin, some patients with HE still progress to recurrent episodes, posing a significant therapeutic challenge. Recurrent HE is defined as experiencing two or more episodes within a 6-month period. Previous research has suggested that FMT may emerge as a promising treatment for recurrent HE. However, there remains a critical need to explore the optimal dosage. This trial aims to abscess the efficacy and safety of two FMT dosages: 800 ml or 400 ml total bacterial count, including mortality and quality of life. This multicenter, prospective, randomized controlled trial will enroll 100 eligible patients from 31 hospitals in China. Participants will be randomly assigned in a 1:1 ratio to either the high-dose group (800 ml total bacterial count) or the low-dose group (400 ml total bacterial count). The primary objective is to assess the efficacy and safety of both dosages on outcomes at 24 and 48 weeks, including mortality and quality of life. If either or both dosages of FMT demonstrate safe and effective treatment of recurrent HE, leading to improve quality of life and survival at 24 and 48 weeks, this trial would address a significant gap in the management of recurrent HE, carrying innovative and clinically significant implications. NCT05669651 on ClinicalTrials.gov. Registered on 29 December 2022. CHiCTR2200067135 on China Registered Clinical Trial Registration Center. Registered on 27 December 2022.

This research investigated the effect and mechanism of trichosanthin(TCS) in inducing autophagy and apoptosis of HeLa cells in cervical cancer. Two-step chromatography was used to prepare TCS. MTT assay was used to detect the inhibition effect of TCS on the proliferation of HeLa cells. The formation of autophagic vesicles and apoptotic bodies in HeLa cells treated with TCS was observed under transmission electron microscopy, and green fluorescent protein(GFP)-microtubule-associated protein 1 light chain beta 3(LC3B) fusion protein location was observed under laser confocal microscopy. The expression of autophagy and apoptosis-related proteins was detected by Western blot. It was found that the prepared TCS had high purity and biological activity. TCS could inhibit the proliferation of HeLa cells significantly in a concentration-dependent and time-dependent manner. Obvious autophagic vesicles were observed after HeLa cells were treated with TCS for 12 h, and typical apoptotic bodies were formed after 48 h. Under laser confocal microscopy, LC3B protein was observed to shift from a diffuse distribution to a spotted aggregation distribution in the cytoplasm. Western blot results showed that the expression of autophagy-activating protein LC3BⅡ began to increase in HeLa cells after being treated with TCS for 12 h and increased with the increase in drug concentration. Apoptosis-related protein poly ADP-ribose polymerase(PARP) began to activate as cleaved PARP after being treated with TCS for 24 h. The combination of TCS and 3-methyladenine(3-MA) further promoted the activation of cleaved PARP. The results showed that TCS could significantly inhibit the growth of HeLa cells in cervical cancer. Moreover, TCS induced autophagy in HeLa cells earlier than apoptosis, and inhibiting autophagy could sensitize apoptosis of HeLa cells induced by TCS. This indicated that autophagy induced by TCS was a protective cell response, and TCS combined with autophagy inhibitors could enhance the anti-cervical cancer effect of TCS.