ABSTRACT
Although chimeric antigen receptor (CAR) T cell therapies have demonstrated promising clinical outcomes, durable remissions remain limited. To extend the efficacy of CAR T cells, we develop a CAR enhancer (CAR-E), comprising a CAR T cell antigen fused to an immunomodulatory molecule. Here we demonstrate this strategy using B cell maturation antigen (BCMA) CAR T cells for the treatment of multiple myeloma, with a CAR-E consisting of the BCMA fused to a low-affinity interleukin 2 (IL-2). This selectively induces IL-2 signaling in CAR T cells upon antigen-CAR binding, enhancing T cell activation and antitumor activity while reducing IL-2-associated toxicities. We show that the BCMA CAR-E selectively binds CAR T cells and increases CAR T cell proliferation, clearance of tumor cells and development of memory CAR T cells. The memory cells retain the ability to re-expand upon restimulation, effectively controlling tumor growth upon rechallenge. Mechanistic studies reveal the involvement of both CAR and IL-2 receptor endodomains in the CAR-E mechanism of action. The CAR-E approach avoids the need for specific engineering and enables CAR T cell therapy with lower cell doses.
ABSTRACT
INTRODUCTION AND HYPOTHESIS: The objective was to investigate the incidence and risk factors of postoperative de novo stress urinary incontinence (SUI) in stress-continent women following minimally invasive sacrocolpopexy without an anti-incontinence procedure. METHODS: We completed a multicenter, retrospective cohort study of women undergoing laparoscopic sacrocolpopexy without concurrent anti-incontinence procedures from October 2006 through January 2021. RESULTS: Of the 169 women who underwent minimally invasive sacrocolpopexy, 17.1% (n=30) developed de novo SUI, and 7.1% eventually underwent a midurethral sling placement. On logistic regression, BMI, preoperative urinary urgency, and history of transvaginal mesh repair were found to be significantly associated with and predictive of de novo SUI. When the concordance index (C-index) was calculated with the model published by Jelovsek et al. for women who developed de novo SUI within 12 months of the prolapse surgery, the current de novo SUI calculator was able to discriminate de novo SUI outcome (C-index = 0.71). CONCLUSIONS: The incidence of de novo SUI after minimally invasive sacrocolpopexy without anti-incontinence procedure correlates directly with higher BMI, preoperative urinary urgency, and transvaginal mesh history for POP. Preoperative counseling for minimally invasive sacrocolpopexy should include discussing the risk of de novo SUI and preoperative factors that may increase this risk.
Subject(s)
Pelvic Organ Prolapse , Suburethral Slings , Urinary Incontinence, Stress , Female , Humans , Pelvic Organ Prolapse/surgery , Pelvic Organ Prolapse/complications , Urinary Incontinence, Stress/epidemiology , Urinary Incontinence, Stress/etiology , Urinary Incontinence, Stress/surgery , Incidence , Retrospective Studies , Suburethral Slings/adverse effects , Postoperative Complications/epidemiology , Postoperative Complications/etiologyABSTRACT
Fibrosis is a hallmark of aging tissues which often leads to altered architecture and function. The ovary is the first organ to show overt signs of aging, including increased fibrosis in the ovarian stroma. How this fibrosis affects ovarian biomechanics and the underlying mechanisms are unknown. Using instrumental indentation, we demonstrated a quantitative increase in ovarian stiffness, as evidenced by an increase in Young's modulus, when comparing ovaries from reproductively young (6-12 weeks) and old (14-17 months) mice. This ovarian stiffness was dependent on collagen because ex vivo enzyme-mediated collagen depletion in ovaries from reproductively old mice restored their collagen content and biomechanical properties to those of young controls. In addition to collagen, we also investigated the role of hyaluronan (HA) in regulating ovarian stiffness. HA is an extracellular matrix glycosaminoglycan that maintains tissue homeostasis, and its loss can change the biomechanical properties of tissues. The total HA content in the ovarian stroma decreased with age, and this was associated with increased hyaluronidase (Hyal1) and decreased hyaluronan synthase (Has3) expression. These gene expression differences were not accompanied by changes in ovarian HA molecular mass distribution. Furthermore, ovaries from mice deficient in HAS3 were stiffer compared to age-matched WT mice. Our results demonstrate that the ovary becomes stiffer with age and that both collagen and HA matrices are contributing mechanisms regulating ovarian biomechanics. Importantly, the age-associated increase in collagen and decrease in HA are conserved in the human ovary and may impact follicle development and oocyte quality.
Subject(s)
Collagen/metabolism , Extracellular Matrix/metabolism , Hyaluronan Synthases/metabolism , Ovary/physiopathology , Adult , Aging , Animals , Female , Humans , MiceABSTRACT
The ovarian stroma, the microenvironment in which female gametes grow and mature, becomes inflamed and fibrotic with age. Hyaluronan is a major component of the ovarian extracellular matrix (ECM), and in other aging tissues, accumulation of low molecular weight (LMW) hyaluronan fragments can drive inflammation. Thus, we hypothesized that LMW hyaluronan fragments contribute to female reproductive aging by stimulating an inflammatory response in the ovarian stroma and impairing gamete quality. To test this hypothesis, isolated mouse ovarian stromal cells or secondary stage ovarian follicles were treated with physiologically relevant (10 or 100 µg/mL) concentrations of 200 kDa LMW hyaluronan. In ovarian stromal cells, acute LMW hyaluronan exposure, at both doses, resulted in the secretion of a predominantly type 2 (Th2) inflammatory cytokine profile as revealed by a cytokine antibody array of conditioned media. Additional qPCR analyses of ovarian stromal cells demonstrated a notable up-regulation of the eotaxin receptor Ccr3 and activation of genes involved in eosinophil recruitment through the IL5-CCR3 signaling pathway. These findings were consistent with an age-dependent increase in ovarian stromal expression of Ccl11, a major CCR3 ligand. When ovarian follicles were cultured in 10 or 100 µg/mL LMW hyaluronan for 12 days, gametes with compromised morphology and impaired meiotic competence were produced. In the 100 µg/mL condition, LMW hyaluronan induced premature meiotic resumption, ultimately leading to in vitro aging of the resulting eggs. Further, follicles cultured in this LMW hyaluronan concentration produced significantly less estradiol, suggesting compromised granulosa cell function. Taken together, these data demonstrate that bioactive LMW hyaluronan fragments may contribute to reproductive aging by driving an inflammatory stromal milieu, potentially through eosinophils, and by directly compromising gamete quality through impaired granulosa cell function.
Subject(s)
Germ Cells/metabolism , Hyaluronic Acid/metabolism , Inflammation/metabolism , Ovary/metabolism , Stromal Cells/metabolism , Aging/metabolism , Animals , Extracellular Matrix/metabolism , Female , Granulosa Cells/metabolism , Hyaluronan Receptors/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular WeightABSTRACT
Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. The spatial-temporal localization of HA can be visualized in situ using biotinylated HA binding proteins (HABPs). This assay is sensitive to fixation conditions, and there are currently no best practices for HA detection. Thus, the goal of this study was to optimize fixation conditions for visualizing HA in the ovary, kidney, and liver through analysis of six commonly used fixatives for HA detection: Bouin's Solution, Carnoy's Solution, Ethanol-Formalin-Glacial Acetic Acid (EFG), Histochoice, Modified Davidson's Solution, and 10% Neutral Buffered Formalin. Organs were harvested from CB6F1 mice and fixed with one of the identified fixatives. Fixed organs were sectioned, and the HABP assay was performed on sections in parallel. Hematoxylin and eosin staining was also performed to visualize tissue architecture. HABP signal localization and intensity varied between fixatives. EFG and Carnoy's Solution best preserved the HA signal intensity in the ovary and liver, showing HA localization in various sub-organ structures. In the kidney, only Modified Davidson's Solution was less than optimal. Our findings demonstrate that fixation can alter the ability to detect HA in tissue macro- and microstructures, as well as localization in a tissue-specific manner, in situ.