Morphological study of TNPO3 and SRSF1 interaction during myogenesis by combining confocal, structured illumination and electron microscopy analysis.

Transportin3 (TNPO3) shuttles the SR proteins from the cytoplasm to the nucleus. The SR family includes essential splicing factors, such as SRSF1, that influence alternative splicing, controlling protein diversity in muscle and satellite cell differentiation. Given the importance of alternative splicing in the myogenic process and in the maintenance of healthy muscle, alterations in the splicing mechanism might contribute to the development of muscle disorders. Combining confocal, structured illumination and electron microscopy, we investigated the expression of TNPO3 and SRSF1 during myogenesis, looking at nuclear and cytoplasmic compartments. We investigated TNPO3 and its interaction with SRSF1 and we observed that SRSF1 remained mainly localized in the nucleus, while TNPO3 decreased in the cytoplasm and was strongly clustered in the nuclei of differentiated myotubes. In conclusion, combining different imaging techniques led us to describe the behavior of TNPO3 and SRSF1 during myogenesis, showing that their dynamics follow the myogenic process and could influence the proteomic network necessary during myogenesis. The combination of different high-, super- and ultra-resolution imaging techniques led us to describe the behavior of TNPO3 and its interaction with SRSF1, looking at nuclear and cytoplasmic compartments. These observations represent a first step in understanding the role of TNPO3 and SRFSF1 in complex mechanisms, such as myogenesis.

Targeting Wnt/ß-catenin and PI3K/Akt/mTOR pathways in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological disorder that results from the clonal transformation of T-cell precursors. Phosphatidylinositol 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) and canonical Wnt/ß-catenin signaling pathways play a crucial role in T-cell development and in self-renewal of healthy and leukemic stem cells. Notably, ß-catenin is a transcriptional regulator of several genes involved in cancer cell proliferation and survival. In this way, aberrations of components belonging to the aforementioned networks contribute to T-ALL pathogenesis. For this reason, inhibition of both pathways could represent an innovative strategy in this hematological malignancy. Here, we show that combined targeting of Wnt/ß-catenin pathway through ICG-001, a CBP/ß-catenin transcription inhibitor, and of the PI3K/Akt/mTOR axis through ZSTK-474, a PI3K inhibitor, downregulated proliferation, survival, and clonogenic activity of T-ALL cells. ICG-001 and ZSTK-474 displayed cytotoxic effects, and, when combined together, induced a significant increase in apoptotic cells. This induction of apoptosis was associated with the downregulation of Wnt/ß-catenin and PI3K/Akt/mTOR pathways. All these findings were confirmed under hypoxic conditions that mimic the bone marrow niche where leukemic stem cells are believed to reside. Taken together, our findings highlight potentially promising treatment consisting of cotargeting Wnt/ß-catenin and PI3K/Akt/mTOR pathways in T-ALL settings.

Functionalized Keratin as Nanotechnology-Based Drug Delivery System for the Pharmacological Treatment of Osteosarcoma.

Osteosarcoma therapy might be moving toward nanotechnology-based drug delivery systems to reduce the cytotoxicity of antineoplastic drugs and improve their pharmacokinetics. In this paper, we present, for the first time, an extensive chemical and in vitro characterization of dual-loaded photo- and chemo-active keratin nanoparticles as a novel drug delivery system to treat osteosarcoma. The nanoparticles are prepared from high molecular weight and hydrosoluble keratin, suitably functionalized with the photosensitizer Chlorin-e6 (Ce6) and then loaded with the chemotherapeutic drug Paclitaxel (PTX). This multi-modal PTX-Ce6@Ker nanoformulation is prepared by both drug-induced aggregation and desolvation methods, and a comprehensive physicochemical characterization is performed. PTX-Ce6@Ker efficacy is tested on osteosarcoma tumor cell lines, including chemo-resistant cells, using 2D and 3D model systems. The single and combined contributions of PTX and Ce6 is evaluated, and results show that PTX retains its activity while being vehiculated through keratin. Moreover, PTX and Ce6 act in an additive manner, demonstrating that the combination of the cytostatic blockage of PTX and the oxidative damage of ROS upon light irradiation have a far superior effect compared to singularly administered PTX or Ce6. Our findings provide the proof of principle for the development of a novel, nanotechnology-based drug delivery system for the treatment of osteosarcoma.

Human White Adipocytes Convert Into "Rainbow" Adipocytes In Vitro.

White adipocytes are plastic cells able to reversibly transdifferentiate into brown adipocytes and into epithelial glandular cells under physiologic stimuli in vivo. These plastic properties could be used in future for regenerative medicine, but are incompletely explored in their details. Here, we focused on plastic properties of human mature adipocytes (MA) combining gene expression profile through microarray analysis with morphologic data obtained by electron and time lapse microscopy. Primary MA showed the classic morphology and gene expression profile of functional mature adipocytes. Notably, despite their committed status, MA expressed high levels of reprogramming genes. MA from ceiling cultures underwent transdifferentiation toward fibroblast-like cells with a well-differentiated morphology and maintaining stem cell gene signatures. The main morphologic aspect of the transdifferentiation process was the secretion of large lipid droplets and the development of organelles necessary for exocrine secretion further supported the liposecretion process. Of note, electron microscope findings suggesting liposecretion phenomena were found also in explants of human fat and rarely in vivo in fat biopsies from obese patients. In conclusion, both MA and post-liposecretion adipocytes show a well-differentiated phenotype with stem cell properties in line with the extraordinary plasticity of adipocytes in vivo. J. Cell. Physiol. 232: 2887-2899, 2017. © 2016 Wiley Periodicals, Inc.

Effect of a one-step self-etch adhesive on endogenous dentin matrix metalloproteinases.

Degradation of the hybrid layer created in dentin by dentin adhesives is caused by enzyme activities present within the dentin matrix that destroy unprotected collagen fibrils. The aim of the present study was to evaluate the effect of a one-step self-etch adhesive system on dentinal matrix metalloproteinases 2 and 4 (MMP-2 and MMP-9, respectively) using in situ zymography and an enzymatic activity assay. The null hypothesis tested was that there are no differences in the activities of dentinal MMPs before and after treatment with a one-step adhesive system. The MMP-2 and MMP-9 activities in dentin treated with the one-step adhesive, Adper Easy Bond, were quantified using an enzymatic activity assay system. The MMP activities within the hybrid layer created by the one-step adhesive tested were also evaluated using in situ zymography. The enzymatic assay revealed an increase in MMP-2 and MMP-9 activities after treatment with adhesive. In situ zymography indicated that gelatinolytic activity is present within the hybrid layer created with the one-step self-etch adhesive. The host-derived gelatinases were localized within the hybrid layer and remained active after the bonding procedure. It is concluded that the one-step self-etch adhesive investigated activates endogenous MMP-2 and MMP-9 with the dentin matrix, which may cause collagen degradation over time.

Effect of mechanical strain on the collagen VI pericellular matrix in anterior cruciate ligament fibroblasts.

Cell-extracellular matrix interaction plays a major role in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. Collagen VI is a widely expressed non-fibrillar collagen, which regulates tissues homeostasis. The objective of the present investigation was to extend our understanding of the role of collagen VI in human ACL. This study shows that collagen VI is associated both in vivo and in vitro to the cell membrane of knee ACL fibroblasts, contributing to the constitution of a microfibrillar pericellular matrix. In cultured cells the localization of collagen VI at the cell surface correlated with the expression of NG2 proteoglycan, a major collagen VI receptor. The treatment of ACL fibroblasts with anti-NG2 antibody abolished the localization of collagen VI indicating that collagen VI pericellular matrix organization in ACL fibroblasts is mainly mediated by NG2 proteoglycan. In vitro mechanical strain injury dramatically reduced the NG2 proteoglycan protein level, impaired the association of collagen VI to the cell surface, and promoted cell cycle withdrawal. Our data suggest that the injury-induced alteration of specific cell-ECM interactions may lead to a defective fibroblast self-renewal and contribute to the poor regenerative ability of ACL fibroblasts.

Desmin and Plectin Recruitment to the Nucleus and Nuclei Orientation Are Lost in Emery-Dreifuss Muscular Dystrophy Myoblasts Subjected to Mechanical Stimulation.

In muscle cells subjected to mechanical stimulation, LINC complex and cytoskeletal proteins are basic to preserve cellular architecture and maintain nuclei orientation and positioning. In this context, the role of lamin A/C remains mostly elusive. This study demonstrates that in human myoblasts subjected to mechanical stretching, lamin A/C recruits desmin and plectin to the nuclear periphery, allowing a proper spatial orientation of the nuclei. Interestingly, in Emery-Dreifuss Muscular Dystrophy (EDMD2) myoblasts exposed to mechanical stretching, the recruitment of desmin and plectin to the nucleus and nuclear orientation were impaired, suggesting that a functional lamin A/C is crucial for the response to mechanical strain. While describing a new mechanism of action headed by lamin A/C, these findings show a structural alteration that could be involved in the onset of the muscle defects observed in muscular laminopathies.

Collagen VI Deficiency Impairs Tendon Fibroblasts Mechanoresponse in Ullrich Congenital Muscular Dystrophy.

The pericellular matrix (PCM) is a specialized extracellular matrix that surrounds cells. Interactions with the PCM enable the cells to sense and respond to mechanical signals, triggering a proper adaptive response. Collagen VI is a component of muscle and tendon PCM. Mutations in collagen VI genes cause a distinctive group of inherited skeletal muscle diseases, and Ullrich congenital muscular dystrophy (UCMD) is the most severe form. In addition to muscle weakness, UCMD patients show structural and functional changes of the tendon PCM. In this study, we investigated whether PCM alterations due to collagen VI mutations affect the response of tendon fibroblasts to mechanical stimulation. By taking advantage of human tendon cultures obtained from unaffected donors and from UCMD patients, we analyzed the morphological and functional properties of cellular mechanosensors. We found that the length of the primary cilia of UCMD cells was longer than that of controls. Unlike controls, in UCMD cells, both cilia prevalence and length were not recovered after mechanical stimulation. Accordingly, under the same experimental conditions, the activation of the Hedgehog signaling pathway, which is related to cilia activity, was impaired in UCMD cells. Finally, UCMD tendon cells exposed to mechanical stimuli showed altered focal adhesions, as well as impaired activation of Akt, ERK1/2, p38MAPK, and mechanoresponsive genes downstream of YAP. By exploring the response to mechanical stimulation, for the first time, our findings uncover novel unreported mechanistic aspects of the physiopathology of UCMD-derived tendon fibroblasts and point at a role for collagen VI in the modulation of mechanotransduction in tendons.

Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

Ewing sarcoma (EWS) is the second most common pediatric bone tumor. The EWS tumor microenvironment is largely recognized as immune-cold, with macrophages being the most abundant immune cells and their presence associated with worse patient prognosis. Expression of CD99 is a hallmark of EWS cells, and its targeting induces inhibition of EWS tumor growth through a poorly understood mechanism. In this study, we analyzed CD99 expression and functions on macrophages and investigated whether the concomitant targeting of CD99 on both tumor and macrophages could explain the inhibitory effect of this approach against EWS. Targeting CD99 on EWS cells downregulated expression of the "don't eat-me" CD47 molecule but increased levels of the "eat-me" phosphatidyl serine and calreticulin molecules on the outer leaflet of the tumor cell membrane, triggering phagocytosis and digestion of EWS cells by macrophages. In addition, CD99 ligation induced reprogramming of undifferentiated M0 macrophages and M2-like macrophages toward the inflammatory M1-like phenotype. These events resulted in the inhibition of EWS tumor growth. Thus, this study reveals what we believe to be a previously unrecognized function of CD99, which engenders a virtuous circle that delivers intrinsic cell death signals to EWS cells, favors tumor cell phagocytosis by macrophages, and promotes the expression of various molecules and cytokines, which are pro-inflammatory and usually associated with tumor regression. This raises the possibility that CD99 may be involved in boosting the antitumor activity of macrophages.

Replicative senescence and high glucose induce the accrual of self-derived cytosolic nucleic acids in human endothelial cells.

Recent literature shows that loss of replicative ability and acquisition of a proinflammatory secretory phenotype in senescent cells is coupled with the build-in of nucleic acids in the cytoplasm. Its implication in human age-related diseases is under scrutiny. In human endothelial cells (ECs), we assessed the accumulation of intracellular nucleic acids during in vitro replicative senescence and after exposure to high glucose concentrations, which mimic an in vivo condition of hyperglycemia. We showed that exposure to high glucose induces senescent-like features in ECs, including telomere shortening and proinflammatory cytokine release, coupled with the accrual in the cytoplasm of telomeres, double-stranded DNA and RNA (dsDNA, dsRNA), as well as RNA:DNA hybrid molecules. Senescent ECs showed an activation of the dsRNA sensors RIG-I and MDA5 and of the DNA sensor TLR9, which was not paralleled by the involvement of the canonical (cGAS) and non-canonical (IFI16) activation of the STING pathway. Under high glucose conditions, only a sustained activation of TLR9 was observed. Notably, senescent cells exhibit increased proinflammatory cytokine (IL-1ß, IL-6, IL-8) production without a detectable secretion of type I interferon (IFN), a phenomenon that can be explained, at least in part, by the accumulation of methyl-adenosine containing RNAs. At variance, exposure to exogenous nucleic acids enhances both IL-6 and IFN-ß1 expression in senescent cells. This study highlights the accrual of cytoplasmic nucleic acids as a marker of senescence-related endothelial dysfunction, that may play a role in dysmetabolic age-related diseases.

CAR+ extracellular vesicles predict ICANS in patients with B cell lymphomas treated with CD19-directed CAR T cells.

BACKGROUND: Predicting Immune-effector Cell Associated Neurotoxicity Syndrome (ICANS) in patients infused with Chimeric Antigen Receptor T cells (CAR-T) is still a conundrum. This complication, thought to be consequent to CAR-T cell activation, arises a few days after infusion, when circulating CAR-T cells are scarce and specific CAR-T cell-derived biomarkers are lacking. METHODS: Human CD19.CAR-T cells were generated to gain insight into CAR+ extracellular vesicle (CAR+EV) release upon target engagement. A prospective cohort of 100 B-cell lymphoma patients infused with approved CD19.CAR-T cell products (axi-cel, brexu-cel and tisa-cel) was assessed for plasma CAR+EVs as potential biomarkers of in vivo CD19.CAR-T cell activation and predictors of ICANS. Human induced pluripotent stem cells (iPSCs)-derived neural cells were used as a model for CAR+EV-induced neurotoxicity. RESULTS: In vitro, exosome-like CAR+EVs were released by CD19.CAR-T cells upon target engagement. In vivo, CAR+EVs were detectable as early as 1 hour in the plasma of patients. A concentration > 132.8 CAR+EVs/µl at hour +1 or > 224.5 CAR+EVs/µl at day +1 predicted ICANS in advance of 4 days, with a sensitivity up to 96.55% and a specificity up to 80.36%, outperforming other potential ICANS predictors. Enolase 2 (ENO2+) nanoparticles were released by iPSCs-derived neural cells upon CAR+EVs exposure and were increased in the plasma of ICANS patients. CONCLUSIONS: These results convey that plasma CAR+EVs are an immediate signal of CD19.CAR-T cell activation, are suitable predictors of neurotoxicity, and may be involved in ICANS pathogenesis. TRIAL REGISTRATION: NCT04892433, NCT05807789.

Targeting the Interplay of Independent Cellular Pathways and Immunity: A Challenge in Cancer Immunotherapy.

Immunotherapy is a cancer treatment that exploits the capacity of the body's immune system to prevent, control, and remove cancer. Immunotherapy has revolutionized cancer treatment and significantly improved patient outcomes for several tumor types. However, most patients have not benefited from such therapies yet. Within the field of cancer immunotherapy, an expansion of the combination strategy that targets independent cellular pathways that can work synergistically is predicted. Here, we review some consequences of tumor cell death and increased immune system engagement in the modulation of oxidative stress and ubiquitin ligase pathways. We also indicate combinations of cancer immunotherapies and immunomodulatory targets. Additionally, we discuss imaging techniques, which are crucial for monitoring tumor responses during treatment and the immunotherapy side effects. Finally, the major outstanding questions are also presented, and directions for future research are described.

Impaired synaptic plasticity in an animal model of autism exhibiting early hippocampal GABAergic-BDNF/TrkB signaling alterations.

In Neurodevelopmental Disorders, alterations of synaptic plasticity may trigger structural changes in neuronal circuits involved in cognitive functions. This hypothesis was tested in mice carrying the human R451C mutation of Nlgn3 gene (NLG3R451C KI), found in some families with autistic children. To this aim, the spike time dependent plasticity (STDP) protocol was applied to immature GABAergic Mossy Fibers (MF)-CA3 connections in hippocampal slices from NLG3R451C KI mice. These animals failed to exhibit STD-LTP, an effect that persisted in adulthood when these synapses became glutamatergic. Similar results were obtained in mice lacking the Nlgn3 gene (NLG3 KO mice), suggesting a loss of function. The loss of STD-LTP was associated with a premature shift of GABA from the depolarizing to the hyperpolarizing direction, a reduced BDNF availability and TrkB phosphorylation at potentiated synapses. These effects may constitute a general mechanism underlying cognitive deficits in those forms of Autism caused by synaptic dysfunctions.

Adipose Stromal Cell Spheroids for Cartilage Repair: A Promising Tool for Unveiling the Critical Maturation Point.

Articular cartilage lacks intrinsic regenerative capabilities, and the current treatments fail to regenerate damaged tissue and lead only to temporary pain relief. These limitations have prompted the development of tissue engineering approaches, including 3D culture systems. Thanks to their regenerative properties and capacity to recapitulate embryonic processes, spheroids obtained from mesenchymal stromal cells are increasingly studied as building blocks to obtain functional tissues. The aim of this study was to investigate the capacity of adipose stromal cells to assemble in spheroids and differentiate toward chondrogenic lineage from the perspective of cartilage repair. Spheroids were generated by two different methods (3D chips vs. Ultra-Low Attachment plates), differentiated towards chondrogenic lineage, and their properties were investigated using molecular biology analyses, biophysical measurement of mass density, weight, and size of spheroids, and confocal imaging. Overall, spheroids showed the ability to differentiate by expressing specific cartilaginous markers that correlate with their mass density, defining a critical point at which they start to mature. Considering the spheroid generation method, this pilot study suggested that spheroids obtained with chips are a promising tool for the generation of cartilage organoids that could be used for preclinical/clinical approaches, including personalized therapy.

Aberrant MET activation impairs perinuclear actin cap organization with YAP1 cytosolic relocation.

Little is known about the signaling network responsible for the organization of the perinuclear actin cap, a recently identified structure holding unique roles in the regulation of nuclear shape and cell directionality. In cancer cells expressing a constitutively active MET, we show a rearrangement of the actin cap filaments, which crash into perinuclear patches associated with spherical nuclei, meandering cell motility and inactivation of the mechano-transducer YAP1. MET ablation is sufficient to reactivate YAP1 and restore the cap, leading to enhanced directionality and flattened nuclei. Consistently, the introduction of a hyperactive MET in normal epithelial cells, enhances nuclear height and alters the cap organization, as also confirmed by TEM analysis. Finally, the constitutively active YAP1 mutant YAP5SA is able to overcome the effects of oncogenic MET. Overall, our work describes a signaling axis empowering MET-mediated YAP1 dampening and actin cap misalignment, with implications for nuclear shape and cell motility.

Small Extracellular Vesicles from Inflamed Adipose Derived Stromal Cells Enhance the NF-κB-Dependent Inflammatory/Catabolic Environment of Osteoarthritis.

The last decade has seen exponentially growing efforts to exploit the effects of adipose derived stromal cells (ADSC) in the treatment of a wide range of chronic degenerative diseases, including osteoarthritis (OA), the most prevalent joint disorder. In the perspective of developing a cell-free advanced therapy medicinal product, a focus has been recently addressed to the ADSC secretome that lends itself to an allogeneic use and can be further dissected for the selective purification of small extracellular vesicles (sEVs). sEVs can act as "biological drug carriers" to transfer information that mirror the pathophysiology of the providing cells. This is important in the clinical perspective where many OA patients are also affected by the metabolic syndrome (MetS). ADSC from MetS OA patients are dysfunctional and "inflammatory" primed within the adipose tissue. To mimic this condition, we exposed ADSC to IL-1ß, and then we investigated the effects of the isolated sEVs on chondrocytes and synoviocytes, either cultured separately or in co-culture, to tease out the effects of these "IL-1ß primed sEVs" on gene and protein expression of major inflammatory and catabolic OA markers. In comparison with sEVs isolated from unstimulated ADSC, the IL-1ß primed sEVs were able to propagate NF-κB activation in bystander joint cells. The effects were more prominent on synoviocytes, possibly because of a higher expression of binding molecules such as CD44. These findings call upon a careful characterization of the "inflammatory fingerprint" of ADSC to avoid the transfer of an unwanted message as well as the development of in vitro "preconditioning" strategies able to rescue the antiinflammatory/anticatabolic potential of ADSC-derived sEVs.

The autocrine loop of ALK receptor and ALKAL2 ligand is an actionable target in consensus molecular subtype 1 colon cancer.

BACKGROUND: In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs). METHODS: In this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested cell lines, patient-derived organoids and mice with potent ALK inhibitors, already approved for clinical use. RESULTS: ALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D tumor spheroids. Consistently, ALK appeares constitutively phosphorylated in CMS1, and it signals mainly through the AKT axis. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Consequently, disruption of ALK axis mediates the pro-apoptotic action of CMS1 cell lines, both in 2D and 3D and enhanced cell-cell adhesion and e-cadherin organization. In agreement with all these findings, the ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, as a proof of concept, the efficacy of ALK inhibition was demonstrated in both patient-derived organoids and in tumor xenografts in vivo. CONCLUSIONS: Collectively, these findings suggest that ALK targeting may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.

Pre-transplant CD69+ extracellular vesicles are negatively correlated with active ATLG serum levels and associate with the onset of GVHD in allogeneic HSCT patients.

Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Rabbit anti-T lymphocyte globulin (ATLG) in addition to calcineurin inhibitors and antimetabolites is a suitable strategy to prevent GVHD in several transplant settings. Randomized studies already demonstrated its efficacy in terms of GVHD prevention, although the effect on relapse remains the major concern for a wider use. Tailoring of ATLG dose on host characteristics is expected to minimize its side effects (immunological reconstitution, relapse, and infections). Here, day -6 to day +15 pharmacokinetics of active ATLG serum level was first assayed in an explorative cohort of 23 patients by testing the ability of the polyclonal serum to bind antigens on human leukocytes. Significantly lower levels of serum active ATLG were found in the patients who developed GVHD (ATLG_AUCCD45: 241.52 ± 152.16 vs. 766.63 +/- 283.52 (µg*day)/ml, p = 1.46e-5). Consistent results were obtained when the ATLG binding capacity was assessed on CD3+ and CD3+/CD4+ T lymphocytes (ATLG_AUCCD3: 335.83 ± 208.15 vs. 903.54 ± 378.78 (µg*day)/ml, p = 1.92e-4; ATLG_AUCCD4: 317.75 ± 170.70 vs. 910.54 ± 353.35 (µg*day)/ml, p = 3.78e-5. Concomitantly, at pre-infusion time points, increased concentrations of CD69+ extracellular vesicles (EVs) were found in patients who developed GVHD (mean fold 9.01 ± 1.33; p = 2.12e-5). Consistent results were obtained in a validation cohort of 12 additional ATLG-treated HSCT patients. Serum CD69+ EVs were mainly represented in the nano (i.e. 100 nm in diameter) EV compartment and expressed the leukocyte marker CD45, the EV markers CD9 and CD63, and CD103, a marker of tissue-resident memory T cells. The latter are expected to set up a host pro-inflammatory cell compartment that can survive in the recipient for years after conditioning regimen and contribute to GVHD pathogenesis. In summary, high levels of CD69+ EVs are significantly correlated with an increased risk of GVHD, and they may be proposed as a tool to tailor ATLG dose for personalized GVHD prevention.

Matrix metalloproteinase 13 loss associated with impaired extracellular matrix remodeling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factors.

OBJECTIVE: To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. METHODS: MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and beta-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. RESULTS: Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, beta-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13-remodeled or -degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. CONCLUSION: MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.

Deletion of TrkB in adult progenitors alters newborn neuron integration into hippocampal circuits and increases anxiety-like behavior.

New neurons in the adult dentate gyrus are widely held to incorporate into hippocampal circuitry via a stereotypical sequence of morphological and physiological transitions, yet the molecular control over this process remains unclear. We studied the role of brain-derived neurotrophic factor (BDNF)/TrkB signaling in adult neurogenesis by deleting the full-length TrkB via Cre expression within adult progenitors in TrkB(lox/lox) mice. By 4 weeks after deletion, the growth of dendrites and spines is reduced in adult-born neurons demonstrating that TrkB is required to create the basic organization of synaptic connections. Later, when new neurons normally display facilitated synaptic plasticity and become preferentially recruited into functional networks, lack of TrkB results in impaired neurogenesis-dependent long-term potentiation and cell survival becomes compromised. Because of the specific lack of TrkB signaling in recently generated neurons a remarkably increased anxiety-like behavior was observed in mice carrying the mutation, emphasizing the contribution of adult neurogenesis in regulating mood-related behavior.