Surface-Functionalized Microgels as Artificial Antigen-Presenting Cells to Regulate Expansion of T Cells.

Artificial antigen-presenting cells (aAPCs) are currently used to manufacture T cells for adoptive therapy in cancer treatment, but a readily tunable and modular system could enable both rapid T cell expansion and control over T cell phenotype. Here, we show that microgels with tailored surface biochemical properties can serve as aAPCs to mediate T cell activation and expansion. Surface functionalization of microgels was achieved via layer-by-layer coating using oppositely charged polymers, forming a thin but dense polymer layer on the surface. This facile and versatile approach is compatible with a variety of coating polymers and allows efficient and flexible surface-specific conjugation of defined peptides or proteins. We demonstrate that tethering appropriate stimulatory ligands on the microgel surface efficiently activates T cells for polyclonal and antigen-specific expansion. The expansion, phenotype and functional outcome of primary mouse and human T cells can be regulated by modulating the concentration, ratio and distribution of stimulatory ligands presented on microgel surfaces as well as the stiffness and viscoelasticity of the microgels. This article is protected by copyright. All rights reserved.

Reversible Mechanical Contraception and Endometriosis Treatment Using Stimuli-Responsive Hydrogels.

Female sterilization via fallopian tube ligation is a common procedure; However, after the operation, over 10% of women seek re-fertilization, which is frequently unsuccessful. In addition, there is evidence that fallopian tubes contribute to the spread of endometriotic tissue as they serve as channels for proinflammatory media entering the abdominal cavity via retrograde menstruation. Here, stimuli-degradable hydrogel implants are presented for the functional, biocompatible, and reversible occlusion of fallopian tubes. The hydrogel implants, designed with customized swelling properties, mechanically occlude fallopian tubes in a high-performance manner with burst pressures reaching 255-558 mmHg, exceeding normal abdominal pressures (95 mmHg). Their damage-free removal can be achieved within 30 min using near-visible UV light or a glutathione solution, employing a method akin to standard fallopian tube perfusion diagnostics. Ultrasound-guided implant placement is demonstrated using a clinical hysteroscope in a human-scale uterus model and biocompatibility in a porcine in vivo model. Importantly, the prevention of live sperm as well as endometrial cell passage through blocked fallopian tubes is demonstrated. Overall, a multifunctional system is presented that constitutes a possible means of on-demand, reversible contraception along with the first-ever mechanical approach to abdominal endometriosis prevention and treatment.

ADP-Ribosylation Factor-Interacting Protein 2 Acts as a Novel Regulator of Mitophagy and Autophagy in Podocytes in Diabetic Nephropathy.

(1) Background: Differentiated podocytes are particularly vulnerable to oxidative stress and cellular waste products. The disease-related loss of postmitotic podocytes is a direct indicator of renal disease progression and aging. Podocytes use highly specific regulated networks of autophagy and endocytosis that counteract the increasing number of damaged protein aggregates and help maintain cellular homeostasis. Here, we demonstrate that ARFIP2 is a regulator of autophagy and mitophagy in podocytes both in vitro and in vivo. (2) Methods: In a recent molecular regulatory network analysis of mouse glomeruli, we identified ADP-ribosylation factor-interacting protein 2 (Arfip2), a cytoskeletal regulator and cofactor of ATG9-mediated autophagosome formation, to be differentially expressed with age. We generated an Arfip2-deficient immortalized podocyte cell line using the CRISPR/Cas technique to investigate the significance of Arfip2 for renal homeostasis in vitro. For the in vivo analyses of Arfip2 deficiency, we used a mouse model of Streptozotozin-induced type I diabetes and investigated physiological data and (patho)histological (ultra)structural modifications. (3) Results: ARFIP2 deficiency in immortalized human podocytes impedes autophagy. Beyond this, ARFIP2 deficiency in human podocytes interferes with ATG9A trafficking and the PINK1-Parkin pathway, leading to the compromised fission of mitochondria and short-term increase in mitochondrial respiration and induction of mitophagy. In diabetic mice, Arfip2 deficiency deteriorates autophagy and leads to foot process effacement, histopathological changes, and early albuminuria. (4) Conclusions: In summary, we show that ARFIP2 is a novel regulator of autophagy and mitochondrial homeostasis in podocytes by facilitating ATG9A trafficking during PINK1/Parkin-regulated mitophagy.

The Prognostic Effect of Immune Cell Infiltration Depends on Molecular Subtype in Endometrioid Ovarian Carcinomas.

PURPOSE: Endometrioid ovarian carcinoma (ENOC) is the second most-common type of ovarian carcinoma, comprising 10%-20% of cases. Recently, the study of ENOC has benefitted from comparisons to endometrial carcinomas including defining ENOC with four prognostic molecular subtypes. Each subtype suggests differential mechanisms of progression, although tumor-initiating events remain elusive. There is evidence that the ovarian microenvironment may be critical to early lesion establishment and progression. However, while immune infiltrates have been well studied in high-grade serous ovarian carcinoma, studies in ENOC are limited. EXPERIMENTAL DESIGN: We report on 210 ENOC, with clinical follow-up and molecular subtype annotation. Using multiplex IHC and immunofluorescence, we examine the prevalence of T-cell lineage, B-cell lineage, macrophages, and populations with programmed cell death protein 1 or programmed death-ligand 1 across subtypes of ENOC. RESULTS: Immune cell infiltrates in tumor epithelium and stroma showed higher densities in ENOC subtypes with known high mutation burden (POLEmut and MMRd). While molecular subtypes were prognostically significant, immune infiltrates were not (overall survival P > 0.2). Analysis by molecular subtype revealed that immune cell density was prognostically significant in only the no specific molecular profile (NSMP) subtype, where immune infiltrates lacking B cells (TILB minus) had inferior outcome (disease-specific survival: HR, 4.0; 95% confidence interval, 1.1-14.7; P < 0.05). Similar to endometrial carcinomas, molecular subtype stratification was generally superior to immune response in predicting outcomes. CONCLUSIONS: Subtype stratification is critical for better understanding of ENOC, in particular the distribution and prognostic significance of immune cell infiltrates. The role of B cells in the immune response within NSMP tumors warrants further study.

The magnetic field strength and the force distance dependency of the magnetically controlled growing rods used for early onset scoliosis.

Magnetically controlled growing rods (MCGR's) have revolutionized the treatment of early-onset scoliosis (EOS) because painless lengthenings can be done in the outpatient clinic without anesthesia. Untreated EOS leads to respiratory insufficiency and reduced life expectancy. However, MCGR's have inherent complications like non-functioning of the lengthening mechanism. We quantify an important failure mechanism and give advice on how to avoid this complication. The magnetic field strength was measured on new/explanted rods at different distances between the external remote controller and the MCGR and likewise in patients before/after distractions. The magnetic field strength of the internal actuator decayed fast with increasing distances and plateaued at 25-30 mm approximating zero. Two new and 12 explanted MCGRs was used for the lab measurements of the elicited force using a forcemeter. At a distance of 25 mm, the force was reduced to approximately 40% (ca. 100 N) compared to zero distance (ca. 250 N), most so for explanted rods. This is used to point out the importance of minimizing the implantation depth to ensure proper functionality of the rod lengthening in clinical use for EOS patients. A distance of 25 mm from skin to MCGR should be considered a relative contraindication to clinical use in EOS patients.

Inter- and intrarater reliability of measuring lengthening of magnetically controlled growing rods on digital radiographs.

PURPOSE: The aim of this study was to determine inter- and intraobserver reliability of delta rod extension, and total rod length measured on digital x-rays in patients with early onset scoliosis (EOS) treated with magnetically controlled growing rods (MCGR). For the last decade, patients with EOS have been treated with MCGR. Replacement of MCGR relies heavily on the measurement done at every lengthening session. Only a few studies have looked at inter- and intraobserver reliability of rod lengthening, and none have used the delta extension before. METHODS: 202 radiographs presented in random order were rated and measured twice with at least a 14-day interval and differing order of the radiographs. The measuring was done at both rods. All x-rays came from 15 patients diagnosed with EOS and treated with MCGR from 2009 until 2019. The total extension length and the delta extension (the difference in total extension length between two lengthening in succession) were measured, and the intraclass correlation coefficient (ICC) calculated for both measurements RESULTS: Intrarater ICC scores varied from moderate to good, but non-significantly. Interrater reliability increased significantly from moderate (ICC 0.72 [0.68; 0.76] and 0.73 [0.69; 0.77] to excellent (ICC 0.91 [0.88; 0.93] and 0.97 [0.96: 0.98]), when examining delta extension every sixth instead of every second month. CONCLUSION: Measuring rod lengthening on x-rays can be done every 6 months, with an ample reliability. The ICC's for the delta extension with 2-3 months interval were only moderately precise, compared to the near perfect ICC's for the total extension length.

Pro-cachectic factors link experimental and human chronic kidney disease to skeletal muscle wasting programs.

Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified increased production and elevated blood levels of soluble pro-cachectic factors, including activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single-cell sequencing data identified the expression of activin A in specific kidney cell populations of fibroblasts and cells of the juxtaglomerular apparatus. We propose that persistent and increased kidney production of pro-cachectic factors, combined with a lack of kidney clearance, facilitates a vicious kidney/muscle signaling cycle, leading to exacerbated blood accumulation and, thereby, skeletal muscle wasting. Systemic pharmacological blockade of activin A using soluble activin receptor type IIB ligand trap as well as muscle-specific adeno-associated virus-mediated downregulation of its receptor ACVR2A/B prevented muscle wasting in different mouse models of experimental CKD, suggesting that activin A is a key factor in CKD-induced cachexia. In summary, we uncovered a crosstalk between kidney and muscle and propose modulation of activin signaling as a potential therapeutic strategy for skeletal muscle wasting in CKD.

Magnetically controlled growing rods in early-onset scoliosis.

INTRODUCTION: Early-onset scoliosis (EOS) may result in disability and a reduced life expectancy. The aim of this study was to report the results of primary magnetically controlled growing rods (MCGR) in a consecutive group of patients with EOS diagnosed and operated at Aalborg University Hospital, Denmark, from 2009 and onwards and with at least two years of follow-up. METHODS: Data were extracted from the electronic patient records and the Picture Archiving and Communication System. All data were extracted by an unbiased observer. Demographics, any complication and the Cobb angles and maximal kyphosis angles preoperatively and post-operatively were recorded. Likewise, the total expansion of the MCGR and the increase in T1-T12 and T1-S1 heights were recorded. RESULTS: A total of 15 patients (three females) were followed for an average of 3.75 years. The Cobb angles were corrected on average by 68% and the maximal kyphosis angle by 45%. The thoracic height increased significantly with only two patients (still undergoing expansions) with a T1-S1 height below 22 cm. Four complications were recorded (one deep infection and three non-functioning rods), all resulting in rod exchange. The complication rate was 27% or 0.07 per patient per year. CONCLUSIONS: The MCGR may reduce the deformity and support thoracic and pulmonary growth without any need for repeated surgeries. The number of complications in the present series was low compared with the literature with an average of 0.07 complications per year per patient or a total complication rate of 27%. FUNDING: none. TRIAL REGISTRATION: not relevant.

The MyoRobot technology discloses a premature biomechanical decay of skeletal muscle fiber bundles derived from R349P desminopathy mice.

Mutations in the Des gene coding for the muscle-specific intermediate filament protein desmin lead to myopathies and cardiomyopathies. We previously generated a R349P desmin knock-in mouse strain as a patient-mimicking model for the corresponding most frequent human desmin mutation R350P. Since nothing is known about the age-dependent changes in the biomechanics of affected muscles, we investigated the passive and active biomechanics of small fiber bundles from young (17-23 wks), adult (25-45 wks) and aged (>60 wks) heterozygous and homozygous R349P desmin knock-in mice in comparison to wild-type littermates. We used a novel automated biomechatronics platform, the MyoRobot, to perform coherent quantitative recordings of passive (resting length-tension curves, visco-elasticity) and active (caffeine-induced force transients, pCa-force, 'slack-tests') parameters to determine age-dependent effects of the R349P desmin mutation in slow-twitch soleus and fast-twitch extensor digitorum longus small fiber bundles. We demonstrate that active force properties are not affected by this mutation while passive steady-state elasticity is vastly altered in R349P desmin fiber bundles compatible with a pre-aged phenotype exhibiting stiffer muscle preparations. Visco-elasticity on the other hand, was not altered. Our study represents the first systematic age-related characterization of small muscle fiber bundle preparation biomechanics in conjunction with inherited desminopathy.

P2Y2R Signaling Is Involved in the Onset of Glomerulonephritis.

Endogenously released adenosine-5'-triphosphate (ATP) is a key regulator of physiological function and inflammatory responses in the kidney. Genetic or pharmacological inhibition of purinergic receptors has been linked to attenuation of inflammatory disorders and hence constitutes promising new avenues for halting and reverting inflammatory renal diseases. However, the involvement of purinergic receptors in glomerulonephritis (GN) has only been incompletely mapped. Here, we demonstrate that induction of GN in an experimental antibody-mediated GN model results in a significant increase of urinary ATP-levels and an upregulation of P2Y2R expression in resident kidney cells as well as infiltrating leukocytes pointing toward a possible role of the ATP/P2Y2R-axis in glomerular disease initiation. In agreement, decreasing extracellular ATP-levels or inhibition of P2R during induction of antibody-mediated GN leads to a reduction in all cardinal features of GN such as proteinuria, glomerulosclerosis, and renal failure. The specific involvement of P2Y2R could be further substantiated by demonstrating the protective effect of the lack of P2Y2R in antibody-mediated GN. To systematically differentiate between the function of P2Y2R on resident renal cells versus infiltrating leukocytes, we performed bone marrow-chimera experiments revealing that P2Y2R on hematopoietic cells is the main driver of the ATP/P2Y2R-mediated disease progression in antibody-mediated GN. Thus, these data unravel an important pro-inflammatory role for P2Y2R in the pathogenesis of GN.

Genetic and pharmacological inhibition of microRNA-92a maintains podocyte cell cycle quiescence and limits crescentic glomerulonephritis.

Crescentic rapidly progressive glomerulonephritis (RPGN) represents the most aggressive form of acquired glomerular disease. While most therapeutic approaches involve potentially toxic immunosuppressive strategies, the pathophysiology remains incompletely understood. Podocytes are glomerular epithelial cells that are normally growth-arrested because of the expression of cyclin-dependent kinase (CDK) inhibitors. An exception is in RPGN where podocytes undergo a deregulation of their differentiated phenotype and proliferate. Here we demonstrate that microRNA-92a (miR-92a) is enriched in podocytes of patients and mice with RPGN. The CDK inhibitor p57Kip2 is a major target of miR-92a that constitutively safeguards podocyte cell cycle quiescence. Podocyte-specific deletion of miR-92a in mice de-repressed the expression of p57Kip2 and prevented glomerular injury in RPGN. Administration of an anti-miR-92a after disease initiation prevented albuminuria and kidney failure, indicating miR-92a inhibition as a potential therapeutic strategy for RPGN. We demonstrate that miRNA induction in epithelial cells can break glomerular tolerance to immune injury.

Space matters: meristem expansion triggers corona formation in Passiflora.

BACKGROUND AND AIMS: Flower meristems differ from vegetative meristems in various aspects. One characteristic is the capacity for ongoing meristem expansion providing space for new structures. Here, corona formation in four species of Passiflora is investigated to understand the spatio-temporal conditions of its formation and to clarify homology of the corona elements. METHODS: One bird-pollinated species with a single-rowed tubular corona (Passiflora tulae) and three insect-pollinated species with three (P. standleyi Killip), four (P. foetida L. 'Sanctae Martae') and six (P. foetida L. var. hispida) ray-shaped corona rows are chosen as representative examples for the study. Flower development is documented by scanning electron microscopy. Meristem expansion is reconstructed by morphometric data and correlated with the sequential corona element formation. KEY RESULTS: In all species, corona formation starts late in ontogeny after all floral organs have been initiated. It is closely correlated with meristem expansion. The rows appear with increasing space in centripetal or convergent sequence. CONCLUSIONS: Based on the concept of fractionation, space induces primordia formation which is a self-regulating process filling the space completely. Correspondingly, the corona is interpreted as a structure of its own, originating from the receptacle. Considering the principle capacity of flower meristems to generate novel structures widens the view and allows new interpretations in combination with molecular, phylogenetic and morphogenetic data.

Combining regional expertise to form a bereavement support alliance.

Providing compassionate bereavement care for families experiencing perinatal loss is a standard of care in most healthcare organizations. In this article, we describe the development of The Alliance of Perinatal Bereavement Support Facilitators, begun over 25 years ago in Chicago by staff who identified the need to reach out to colleagues at other area institutions for advice and support in this work. This collaboration created a regional support network that has resulted in a long-lasting, active, sustainable organization of excellence focused on enhancing practice, education, and perinatal bereavement care. Alliance activities center around four main areas: education, networking/support, policy, and recognizing outstanding service to families. By continuing to draw upon the collective talent, wisdom, and expertise of its members, The Alliance still serves grieving families and provides mentoring for future interdisciplinary team members engaged in this work. The path taken to build this organization can be used by professionals in other specialties who are looking to create their own alliance infrastructure based on mutual benefit and interest.

N-wasp is required for stabilization of podocyte foot processes.

Alteration of cortical actin structures is the common final pathway leading to podocyte foot process effacement and proteinuria. The molecular mechanisms that safeguard podocyte foot process architecture and maintain the three-dimensional actin network remain elusive. Here, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which promotes actin nucleation, is required to stabilize podocyte foot processes. Mice lacking N-WASP specifically in podocytes were born with normal kidney function but developed significant proteinuria 3 weeks after birth, suggesting an important role for N-WASP in maintaining foot processes. In addition, inducing deletion of N-WASP in adult mice resulted in severe proteinuria and kidney failure. Electron microscopy showed an accumulation of electron-dense patches of actin and strikingly altered morphology of podocyte foot processes. Although basic actin-based processes such as cell migration were not affected, primary cultures of N-WASP-deficient podocytes revealed significant impairment of dynamic actin reorganization events, including the formation of circular dorsal ruffles. Taken together, our findings suggest that N-WASP-mediated actin nucleation of branched microfilament networks is specifically required for the maintenance of foot processes, presumably sustaining the mechanical resistance of the filtration barrier.

Molecular fingerprinting of the podocyte reveals novel gene and protein regulatory networks.

A thorough characterization of the transcriptome and proteome of endogenous podocytes has been hampered by low cell yields during isolation. Here we describe a double fluorescent reporter mouse model combined with an optimized bead perfusion protocol and efficient single cell dissociation to yield more than 500,000 podocytes per mouse allowing for global, unbiased downstream applications. Combining mRNA and miRNA transcriptional profiling with quantitative proteomic analyses revealed programs of highly specific gene regulation tightly controlling cytoskeleton, cell differentiation, endosomal transport, and peroxisome function in podocytes. Strikingly, the analyses further predict that these podocyte-specific gene regulatory networks are accompanied by alternative splicing of respective genes. Thus, our 'omics' approach will facilitate the discovery and integration of novel gene, protein, and organelle regulatory networks that deepen our systematic understanding of podocyte biology.