A Bivalent Aptamer-Based DNA Agonist for EGFR Signaling Effectively Alleviates Ulcerative Colitis In Vivo.

While epidermal growth factor (EGF) shows promise in addressing the clinical manifestations of intestinal ulcerative diseases by activating the EGF receptor (EGFR)-mediated cell signaling, its clinical application is hampered by poor protein hydrolytic stability, low thermostability, and difficulty in modification. The development of a novel EGFR agonist for ulcerative colitis remains an urgent need, necessitating innovative solutions to overcome the limitations of current therapies via recombinant EGF protein. Herein, we introduce a novel DNA agonist for EGFR, Dimer-YL, which employs a bivalent aptamer to induce stable receptor dimerization, thereby activating the EGFR signaling and related cell behaviors. Dimer-YL has been demonstrated to recapitulate the EGF-promoted cellular behaviors, including proliferation and migration, as well as repair the damage of intercellular tight junctions. Furthermore, our findings demonstrate the potent therapeutic function of Dimer-YL in alleviating DSS-induced ulcerative colitis in vivo. Together, the present work has revealed Dimer-YL as an innovative DNA molecule for effective EGFR activation, offering promise for the development of EGFR-agonistic agents for therapeutic purposes.

Knowledge, Attitude, and Practice of Breast Cancer Patients Toward Lymphedema Complications: Cross-Sectional Study.

Breast cancer is commonly treated through surgical resection, but a common complication of the procedure is lymphedema of the upper limbs, which can significantly impact patients' daily life. This study aims to investigate the knowledge, attitude, and practice (KAP) of breast cancer patients with regard to lymphedema complications. This cross-sectional study was conducted by a self-administered questionnaire between August and October 2022 toward breast cancer patients in our Hospital of Traditional Chinese Medicine. A total of 529 breast cancer patients were enrolled, including 186 (35.16%) aged < 50 years old. Participants had moderate knowledge, attitudes, and practices with scores of 18.24 ± 3.145 (possible range: 0-30), 62.24 ± 10.260 (possible range: 17-85), and 63.27 ± 20.967 (possible range: 21-105), respectively. Multivariate logistic regression showed that high school/technical secondary school (OR = 1.880, 95% CI = 1.107-3.194, P = 0.019) and being retired (OR = 0.482, 95% CI = 0.245-0.947, P = 0.034) were independently associated with good knowledge. Knowledge (OR = 1.321, 95% CI = 1.222-1.428, P < 0.001) was independently associated with a good attitude. Furthermore, knowledge (OR = 1.262, 95% CI = 1.151-1.384, P < 0.001) and attitude (OR = 1.122, 95% CI = 1.085-1.160, P < 0.001) were independently associated with good practice. Breast cancer patients have moderate knowledge, attitudes, and practices regarding lymphedema complications. Effective education and self-management programs are needed to improve patients' KAP toward lymphedema.

Effects of different anesthesia methods on postoperative immune function in patients undergoing gastrointestinal tumor resection.

To investigate the effects of different anesthetic methods on postoperative immune function in patients undergoing gastrointestinal tumor resection. Ninety patients undergoing laparoscopic gastrointestinal tumor resection were divided into 3 groups. Patients in the GA group were anesthetized by total intravenous anesthesia. The GE group was anesthetized by general anesthesia combined with epidural anesthesia. The GN group was anesthetized by general anesthesia combined with bilateral Transversus Abdominis Plane block (TAP) and rectus sheath nerve blocks. General anesthesia is total intravenous anesthesia in all three groups. Blood samples were taken to test the changes of peripheral lymphocyte subtype analysis, and levels of plasma cortisol, epinephrine, norepinephrine. Also, the dosage of anesthetic drugs, recovery time, and visual analog scale (VAS) scores were recorded. Postoperative immune indexes, including CD4 count, CD8 count, B, and NK cells, in the GE group were significantly higher than those in NA and GA groups (P < 0.01). Perioperative stress indices, including epinephrine levels, norepinephrine level and aldosterone level, in the GE group were significantly lower than in the GA group and GN group (P < 0.01). The intraoperative/total sufentanil dosage and remifentanil dosage in the GE group were significantly lower than those in the GA and GN groups (P < 0.01). The VAS scores in the GE group were significantly better than those in GA and GN groups (P < 0.01). General anesthesia combined with epidural anesthesia attenuates the increase in inflammatory mediators. Its possible mechanisms include reducing perioperative stress response and reducing perioperative opioid use.

Activated B cells suppress T-cell function through metabolic competition.

BACKGROUND: B cells play a pivotal role in regulating the immune response. The induction of B cell-mediated immunosuppressive function requires B cell activating signals. However, the mechanisms by which activated B cells mediate T-cell suppression are not fully understood. METHODS: We investigated the potential contribution of metabolic activity of activated B cells to T-cell suppression by performing in vitro experiments and by analyzing clinical samples using mass cytometry and single-cell RNA sequencing. RESULTS: Here we show that following activation, B cells acquire an immunoregulatory phenotype and promote T-cell suppression by metabolic competition. Activated B cells induced hypoxia in T cells in a cell-cell contact dependent manner by consuming more oxygen via an increase in their oxidative phosphorylation (OXPHOS). Moreover, activated B cells deprived T cells of glucose and produced lactic acid through their high glycolytic activity. Activated B cells thus inhibited the mammalian target of rapamycin pathway in T cells, resulting in suppression of T-cell cytokine production and proliferation. Finally, we confirmed the presence of tumor-associated B cells with high glycolytic and OXPHOS activities in patients with melanoma, associated with poor response to immune checkpoint blockade therapy. CONCLUSIONS: We have revealed for the first time the immunomodulatory effects of the metabolic activity of activated B cells and their possible role in suppressing antitumor T-cell responses. These findings add novel insights into immunometabolism and have important implications for cancer immunotherapy.

Targeting the αv integrin/TGF-ß axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-ß activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-ß signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-ß axis as a potentially useful therapeutic target in GBM.

Metabolic Reprogramming of GMP Grade Cord Tissue Derived Mesenchymal Stem Cells Enhances Their Suppressive Potential in GVHD.

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.

Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

Large-scale GMP-compliant CRISPR-Cas9-mediated deletion of the glucocorticoid receptor in multivirus-specific T cells.

Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)-grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.

The effect of CYP2D6 *10 polymorphism on adjuvant tamoxifen in Asian breast cancer patients: a meta-analysis.

OBJECTIVE: To evaluate the effect of CYP2D6 *10 polymorphism (C 100C>T, rs1065852) on clinical outcomes of female Asian breast cancer patients with tamoxifen adjuvant treatment. METHODS: Meta-analysis of retrospective cohort studies published in July 2017 was performed. Fifteen studies with 1,794 Asian breast cancer patients were included, using strict eligibility requirements. Associations of disease-free survival (DFS), overall survival (OS) and recurrence rate after tamoxifen intake, with CYP2D6 *10 polymorphism were investigated through random effects models. RESULTS: CYP2D6 *10 polymorphism was found to have effect on DFS and recurrence rate in various comparison models, but not on overall survival in the female Asian breast cancer patients. CONCLUSION: In conclusion, our meta-analysis suggests that significant association of *10/*10 (TT) genotype with poorer DFS and recurrence exists in female Asian breast cancer patients with tamoxifen 20 mg/day adjuvant treatment. In the future, large and well-designed studies are required to illustrate the interactions of CYP2D6 genetic variants, including *10 polymorphism and tamoxifen response on female breast cancer patients.

Non-fucosylated CB CD34+ cells represent a good target for enforced fucosylation to improve engraftment following cord blood transplantation.

BACKGROUND AIMS: Despite ethnic diversity and ready availability of cryopreserved, human leukocyte antigen-typed cord blood (CB), delayed engraftment remains a significant hurdle to successful CB transplantation. Suboptimal homing of CB hematopoietic stem and progenitor cells (HSPCs) to the hematopoietic microenvironment (HM) is thought to be responsible and due to low levels of HSPC fucosylation. Fucosylation (decoration with sialyl-LewisX) may improve HSPC homing to HM by increasing the strength of HSPC/E-selectin interactions, where E-selectin is constitutively expressed by HM microvasculature. Enforced fucosylation of CB HSPCs using fucosyltransferases, increases the rate and magnitude of engraftment in xenogeneic transplant models. However, it is unclear whether endogenously fucosylated and non-fucosylated CB HSPC are qualitatively identical or whether endogenous fucosylation marks a qualitative difference between CB HSPC. If qualitatively identical, non-fucosylated CB HSPCs represent a good target for enforced fucosylation with improved engraftment conferred on an increased number of otherwise qualitatively identical HSPC. If qualitatively different, then conferring engraftment upon a majority, possibly lower "quality," non-fucosylated HSPCs by enforced fucosylation might inadvertently compromise engraftment. METHODS: Functional (xenogeneic engraftment, colony-forming unit and selectin-binding assays) and phenotypic analyses of fluorescence-activated cell sorting-isolated, endogenously fucosylated and non-fucosylated CB CD34+ cells were performed. RESULTS: Endogenous fucosylation of CB HSPCs exists as a continuum. Endogenously fucosylated HSPCs engrafted more efficiently in a xenogeneic transplantation model than non-fucosylated HSPCs. Outside of the differences in endogenous fucosylation, no other qualitative (functional and/or phenotypic) differences were identified. DISCUSSION: The majority of endogenously non-fucosylated CB HSPCs represent a good target for enforced fucosylation with the goal of improving engraftment following CB transplantation.

Fucosylation with fucosyltransferase VI or fucosyltransferase VII improves cord blood engraftment.

BACKGROUND AIMS: Advantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo. METHODS: CD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment. RESULTS: Both FTVI and FTVII fucosylated CB CD34⁺ cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34⁺ cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes. CONCLUSIONS: Although FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34⁺ cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation.

Ex vivo graft purging and expansion of autologous blood progenitor cell products from patients with multiple myeloma.

Autologous peripheral blood progenitor cell (PBPC) transplantation is the treatment of choice for selected myeloma patients. However, tumor cells contaminating the apheresis product are a potential source of relapse. Here we report a sequential purging strategy targeting mature and immature clonogenic myeloma cell populations in the autograft. Thawed PBPC products of myeloma patients were treated with rituximab to kill CD138(-)20(+) B cells (highly clonogenic immature cells), and bortezomib to target CD138(+) cells (normal and differentiated myeloma plasma cells), followed by coculture with allogeneic mesenchymal stem cells (MSC) from normal donors. After 7 days of coculture, nonadherent cells were removed and cultured in the absence of MSC for an additional 7 days. Then, efficacy of purging (removal of CD138(-)20(+) and CD138(+) cells) was assessed by flow cytometry and PCR. We used our ex vivo purging strategy to treat frozen aphereses from 16 patients. CD138(+) and CD138(-)20(+)(19(+)) cells present in the initial products were depleted more than 3 and 4 logs, respectively based on 10(6) flow-acquisition events, and to levels below the limit of detection by PCR. In contrast, total nucleated cell (TNC), CD34(+) cell, and colony-forming cell numbers were increased by approximately 12 to 20, 8-, and 23-fold, respectively. Overall, ex vivo treatment of apheresis products with rituximab, bortezomib, and coculture with normal donor MSC depleted mature and immature myeloma cells from clinical aphereses while expanding the normal hematopoietic progenitor cell compartment.

Effect of toothpaste containing d-limonene on natural extrinsic smoking stain: a 4-week clinical trial.

PURPOSE: To determine whether natural smoking stain could be removed/inhibited effectively by a toothpaste containing 5% d-limonene. For comparison and contrast, the effects of d-limonene on tea stain were also assessed. METHODS: The design was a randomized controlled double-blind trial with parallel groups. Toothpastes were: A: positive control with perlite whitening formulation; B: A+5% d-limonene; C: D + 5% d-limonene; D: negative control. The extrinsic stains were measured using Lobene Stain Index. Following baseline examination, all subjects were randomly assigned to one of the four toothpaste groups and instructed to brush with the assigned products twice daily. Subjects returned to the clinic after 4-week brushing for stain removal assessment, then all extrinsic stains, plaque and supragingival calculus were removed and use of assigned products was continued for another 4 weeks, and the stain scores were repeated for inhibition assessment. RESULTS: A total of 408 subjects, 201 with smoking stains and 207 with tea stains, participated in the trial. 5% d-limonene combined with Perlite whitening formulation significantly reduced stain scores both for smoking stain removal and inhibition (P < 0.05). Furthermore, 5% d-limonene alone (in negative formulation) exhibited an additional advantage for smoking stain inhibition (P < 0.05), but the advantage was not found for long-standing smoking stain removal (P > 0.05). The additional advantage of 5% d-limonene was shown neither for removal nor for inhibition in the tea stain study (P > 0.05). All test products were well tolerated over the study period.