Local Factor Production Research Articles

Distinct mucosal endotypes as initiators and drivers of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a potentially devastating autoimmune disease. The great majority of patients with RA are seropositive for anti-citrullinated protein antibodies (ACPAs), rheumatoid factors, or other autoantibodies. The onset of clinically apparent inflammatory arthritis meeting classification criteria (clinical RA) is preceded by ACPA seropositivity for an average of 3-5 years, a period that is designated as 'at-risk' of RA for ACPA-positive individuals who do not display signs of arthritis, or 'pre-RA' for individuals who are known to have progressed to developing clinical RA. Prior studies of individuals at-risk of RA have associated pulmonary mucosal inflammation with local production of ACPAs and rheumatoid factors, leading to development of the 'mucosal origins hypothesis'. Recent work now suggests the presence of multiple distinct mucosal site-specific mechanisms that drive RA evolution. Indicatively, subsets of individuals at-risk of RA and patients with RA harbour a faecal bacterial strain that has exhibited arthritogenic activity in animal models and that favours T helper 17 (TH17) cell responses in patients. Periodontal inflammation and oral microbiota have also been suggested to promote the development of arthritis through breaches in the mucosal barrier. Herein, we argue that mucosal sites and their associated microbial strains can contribute to RA evolution via distinct pathogenic mechanisms, which can be considered causal mucosal endotypes. Future therapies instituted for prevention in the at-risk period, or, perhaps, during clinical RA as therapeutics for active arthritis, will possibly have to address these individual mechanisms as part of precision medicine approaches.

HnRNP R promotes O-GlcNAcylation of eIF4G and facilitates axonal protein synthesis

Motoneurons critically depend on precise spatial and temporal control of translation for axon growth and the establishment and maintenance of neuromuscular connections. While defects in local translation have been implicated in the pathogenesis of motoneuron disorders, little is known about the mechanisms regulating axonal protein synthesis. Here, we report that motoneurons derived from Hnrnpr knockout mice show reduced axon growth accompanied by lowered synthesis of cytoskeletal and synaptic components in axons. Mutant mice display denervated neuromuscular junctions and impaired motor behavior. In axons, hnRNP R is a component of translation initiation complexes and, through interaction with O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (Ogt), modulates O-GlcNAcylation of eIF4G. Restoring axonal O-GlcNAc levels rescued local protein synthesis and axon growth defects of hnRNP R knockout motoneurons. Together, these findings demonstrate a function of hnRNP R in controlling the local production of key factors required for axon growth and formation of neuromuscular innervations.

Functional role of glial-derived neurotrophic factor in a mixed allergen murine model of asthma.

Our previous study showed that glial-derived neurotrophic factor (GDNF) expression is upregulated in asthmatic human lungs, and GDNF regulates calcium responses through its receptor GDNF family receptor α1 (GFRα1) and RET receptor in human airway smooth muscle (ASM) cells. In this study, we tested the hypothesis that airway GDNF contributes to airway hyperreactivity (AHR) and remodeling using a mixed allergen mouse model. Adult C57BL/6J mice were intranasally exposed to mixed allergens (ovalbumin, Aspergillus, Alternaria, house dust mite) over 4 wk with concurrent exposure to recombinant GDNF, or extracellular GDNF chelator GFRα1-Fc. Airway resistance and compliance to methacholine were assessed using FlexiVent. Lung expression of GDNF, GFRα1, RET, collagen, and fibronectin was examined by RT-PCR and histology staining. Allergen exposure increased GDNF expression in bronchial airways including ASM and epithelium. Laser capture microdissection of the ASM layer showed increased mRNA for GDNF, GFRα1, and RET in allergen-treated mice. Allergen exposure increased protein expression of GDNF and RET, but not GFRα1, in ASM. Intranasal administration of GDNF enhanced baseline responses to methacholine but did not consistently potentiate allergen effects. GDNF also induced airway thickening, and collagen deposition in bronchial airways. Chelation of GDNF by GFRα1-Fc attenuated allergen-induced AHR and particularly remodeling. These data suggest that locally produced GDNF, potentially derived from epithelium and/or ASM, contributes to AHR and remodeling relevant to asthma.NEW & NOTEWORTHY Local production of growth factors within the airway with autocrine/paracrine effects can promote features of asthma. Here, we show that glial-derived neurotrophic factor (GDNF) is a procontractile and proremodeling factor that contributes to allergen-induced airway hyperreactivity and tissue remodeling in a mouse model of asthma. Blocking GDNF signaling attenuates allergen-induced airway hyperreactivity and remodeling, suggesting a novel approach to alleviating structural and functional changes in the asthmatic airway.

Поиск биомаркеров диабетической ретинопатии и ее осложнений в стекловидном теле и внутриглазной жидкости

Today diabetes mellitus (DM) is one of the leading medical and social problems. Its complications lead to formidable comorbidities that significantly affect the lives of patients. At the beginning of 2021, there were more than 537 million ¹ people with DM in the world and this number is increasing every year. Diabetic retinopathy (DR) is one of the most severe complications of diabetes affecting the organ of vision. The formation of moorings on the retina, the formation of traction syndrome, hemorrhagic complications, retinal detachment and hemophthalmia, as well as neovascular glaucoma (NG) is a manifestation of the terminal stage of proliferative DR. NG is considered one of the most severe terminal forms of refractory glaucoma, which is steadily progressing, leading to disability and impaired social adaptation [1]. The main method of treating diabetic complications in the posterior segment of the eye is vitrectomy, and in the event of NG, implantation of the Ahmed valve due to the ineffectiveness of conservative therapy. At the same time, one should not forget about multiple complications both during the operation, and in the early and late postoperative periods. To date the pathogenesis of DR development remains unexplored. Apparently, it is based on hyperglycemia, chronic diffuse chorioretinal hypoxia and ischemia of the corresponding structures and tissues. These unfavorable factors trigger a pathogenetic cascade of reactions that cause the release of various angiogenic factors and cytokines. Many researchers have shown the role of the immune response in the development of proliferative DR. This was the reason for the study of the local production of various cytokines and growth factors in patients with DM, proliferative DR and NG. Keywords: diabetes mellitus, diabetic retinopathy, neovascular glaucoma, biomarker, cytokines

RECOVERING AND STANDARDIZING OF THE ORIGINAL GENOTHERAPEUTIC CONSTRUCTION FOR STIMULATION OF NERVOUS TISSUE REGENERATION

Traumatic nerve injury is one of the most common cause of permanent disability. The problem of nerve regeneration is based on the slow growth of nerve fibers and short-term production of neutrophic factors (NGF, BDNF, NT-3, GDNF, etc.), which stimulate the survival of damaged neurons and growth of nerve fibers. In most cases, the intrinsic regenerative potential is insufficient to restore innervation, which requires the replacement therapy with neurotrophins. One of the most promising therapeutic approaches for regenerative medicine is gene therapy that allows to prolong the local production of neurotrophic factors. Previously, we created a bicistronic gene therapy construct pNCure (a plasmid for the treatment of nerves), based on the modified pVax1 plasmid construct approved by the FDA (U.S. Food and Drug Administration), and encoding the cDNA sequences of brain-derived neurotrophic factor (BDNF) and urokinase-type plasminogen activator (uPA). It revealed a prominent therapeutic activity in a model of traumatic nerve injury in mice. Gene therapy products is a particular class of drugs that need particular approaches to production, purification and standartization. There is practically no information on the purification and standardization of gene therapy drugs in Russian, and this is the topic of our manuscript. Here, we have elucidat-ed the issues of plasmid production in E. coli cells with subsequent multistage purification, as well as the issues of evaluation the identity and quantity of the plasmid (using PCR and UV spectrometry methods), and determination of pH and quantity of related and secondary impurities within the sub-stance. The results of the study have demonstrated that the proposed approach to plasmid production, purification and standartization allows to obtain the desired high-quality gene therapy drug that meets all the requirements of the XIV State Pharmacopia.

Bone marrow stromal cells are sensitive to discrete surface alterations in build and post-build modifications of bioinspired Ti6Al4V 3D-printed in vitro testing constructs.

Current standards in bone-facing implant fabrication by metal 3D (M3D) printing require post-manufacturing modifications to create distinct surface properties and create implant microenvironments that promote osseointegration. However, the biological consequences of build parameters and surface modifications are not well understood. This study evaluated the relative contributions of build parameters and post-manufacturing modification techniques to cell responses that impact osseointegration in vivo. Biomimetic testing constructs were created by using a M3D printer with standard titanium-aluminum-vanadium (Ti6Al4V) print parameters. These constructs were treated by either grit-blasting and acid-etching (GB + AE) or GB + AE followed by hot isostatic pressure (HIP) (GB + AE, HIP). Next, nine constructs were created by using a M3D printer with three build parameters: (1) standard, (2) increased hatch spacing, and (3) no infill, and additional contour trace. Each build type was further processed by either GB + AE, or HIP, or a combination of HIP treatment followed by GB + AE (GB + AE, HIP). Resulting constructs were assessed by SEM, micro-CT, optical profilometry, XPS, and mechanical compression. Cellular response was determined by culturing human bone marrow stromal cells (MSCs) for 7 days. Surface topography differed depending on processing method; HIP created micro-/nano-ridge like structures and GB + AE created micro-pits and nano-scale texture. Micro-CT showed decreases in closed pore number and closed porosity after HIP treatment in the third build parameter constructs. Compressive moduli were similar for all constructs. All constructs exhibited ability to differentiate MSCs into osteoblasts. MSCs responded best to micro-/nano-structures created by final post-processing by GB + AE, increasing OCN, OPG, VEGFA, latent TGFβ1, IL4, and IL10. Collectively these data demonstrate that M3D-printed constructs can be readily manufactured with distinct architectures based on the print parameters and post-build modifications. MSCs are sensitive to discrete surface topographical differences that may not show up in qualitative assessments of surface properties and respond by altering local factor production. These factors are vital for osseointegration after implant insertion, especially in patients with compromised bone qualities.

Commentary: Sport-specific Factors Impacting Solar Ultraviolet Exposure in Individuals Who Perform Outdoor Sport Activities

The epidemiologic and molecular links between ultraviolet radiation (UVR) exposure and subsequent development of both melanoma and nonmelanoma skin cancers have been well elucidated. This relationship is explained by the tendency of UVR to form DNA-damaging reactive oxygen species, increase production of local growth factors, and impair cutaneous immune function in the skin.

Nano-hydroxyapatite coated TiO2 nanotubes on Ti-19Zr-10Nb-1Fe alloy promotes osteogenesis in vitro

Titanium and titanium alloys have broad applications in orthopedic implants due to their excellent mechanical properties and biocompatibility. The biological activity of the metallic implants can be improved by implementing a nano-hydroxyapatite (nano-HA) coating, while it is still challenging to synthesize uniform and stable nano-HA on the metallic materials. The characterization results confirmed that the nanotube array with a diameter of 87 ± 21 nm and a length of 8.1 ± 1.3 μm is achieved by using double anodic oxidation, and then microsphere-like nano-HA crystals are formed on the TiO2 nanotube arrays. Through X-ray diffraction (XRD) and Fourier Transform Infrared Spectrometer (FT-IR) analysis, it is determined that the chemical composition of the coating is hydroxyapatite. in vitro cell experiments, compared to the TZNF metal surface, the TZNF-NTs/HA is beneficial to the proliferation and adhesion of osteoblasts, and the activity of ALP was 6.93 ± 0.47 DEA unit and the content of OCN was 7.04 ± 0.51 ng/L. In terms of the expression of osteogenic gene information as osterix, osteopontin, and osteonectin, the mRNA levels of TZNF-NTs/HA were 2.6-fold, 1.6-fold, and 4.3-fold higher than that of TZNF samples, respectively, at 14 days. The results suggested that the introduction of nano-HA improves osteoblast differentiation and local factor production, as well as indicates the potential for improved implant osseointegration.

Dexamethasone as a ropivacaine adjuvant to pre-emptive incision-site infiltration analgesia in pediatric craniotomy patients: A prospective, multicenter, randomized, double-blind, controlled trial.

Dexamethasone added to incision-site infiltration has been routinely used to reduce pain after tonsillectomy in children. However, this has not been studied in pediatric craniotomy patients yet. We hypothesized that incision-site infiltration with a combination of ropivacaine and dexamethasone might provide superior analgesia to ropivacaine alone in pediatric craniotomy patients. In this multicenter, double-blind, randomized, controlled trial, children aged 2-12years, scheduled for craniotomy, were prospectively enrolled at two study centers, from September 2, 2019, to July 5, 2020. Eighty children were randomly assigned (1:1) to either ropivacaine plus dexamethasone group who received pre-emptive incision-site infiltration with 0.2% ropivacaine plus 0.025% dexamethasone, or ropivacaine group who received 0.2% ropivacaine alone. Primary outcome was the modified Children's Hospital of Eastern Ontario Pain Scale (mCHEOPS) at 24h postoperatively. Primary analysis was performed using the modified intention-to-treat principle. Pre-emptive incision-site infiltration with ropivacaine plus dexamethasone had a reduced pain score of 2.0, compared with the pain score of 2.9 in the ropivacaine group, at 24h postoperatively (mean difference -0.9, 95% confidence interval [CI], -1.7 to -0.2; p=.019). Estimated median of the time of first rescue analgesic demand was 24h in the ropivacaine plus dexamethasone group and 8.5h in the ropivacaine group [hazard ratio 0.43, 95% CI 0.24 to 0.08; Log-rank p=.0025]. No adverse events related to incision-site infiltration with dexamethasone were observed in this study. Dexamethsone reduces the local production of pro-inflammatory factors after tissue damage and as a ropivacaine adjuvant for incision-site infiltration reduced the pain scores by 31% at 24 h postoperatively. The results were similar to several prior studies on to tonsillectomy patients. However, this changes on pain scores might has limited clinical significance. The addition of dexamethasone to ropivacaine for preoperative incision-site infiltration has better postoperative analgesic effect than ropivacaine alone in pediatric craniotomy patients.

Review of the immune mechanisms of preeclampsia and the potential of immune modulating therapy

Successful pregnancy relies on maternal immunologic tolerance mechanisms limit maladaptive immune responses against the semi-allogeneic fetus and placenta and support fetal growth. Preeclampsia is a common disorder of pregnancy that affects 4–10% of pregnancies and is a leading cause of maternal and neonatal morbidity and mortality. Preeclampsia clinically manifests as maternal hypertension, proteinuria, and progressive multi-organ injury likely triggered by hypoxic injury to the placenta, resulting in local and systemic anti-angiogenic and inflammatory factor production. Despite the steady rising rates of preeclampsia in the United States, effective treatment options are limited to delivery, which improves maternal status often at the cost of prematurity in the newborn. Preeclampsia also increases the lifelong risk of cardiovascular disease for both mother and infant. Thus, identifying new therapeutic targets is a high priority area to improve maternal, fetal, and infant health outcomes. Immune abnormalities in the placenta and in the maternal circulation have been reported to precede the clinical onset of disease. In particular, excessive systemic and placental complement activation and impaired adaptive T cell tolerance with Th1/Th2/Th17/Treg imbalance has been reported in humans and in animal models of preeclampsia. In this review, we focus on the evidence for the immune origins of preeclampsia, discuss the promise of immune modulating therapy for prevention or treatment, and highlight key areas for future research.

Immune response in glioma's microenvironment.

ObjectivesGlioma is the most common tumor of the central nervous system. In this review, we outline the immunobiological factors that interact with glioma cells and tumor microenvironment (TME), providing more potential targets for clinical inhibition of glioma development and more directions for glioma treatment.ContentRecent studies have shown that glioma cells secrete a variety of immune regulatory factors and interact with immune cells such as microglial cells, peripheral macrophages, myeloid-derived suppressor cells (MDSCs), and T lymphocytes in the TME. In particular, microglia plays a key role in promoting glioma growth. Infiltrating immune cells induce local production of cytokines, chemokines and growth factors. Further leads to immune escape of malignant gliomas.Summary and OutlookThe complex interaction of tumor cells with the TME has largely contributed to tumor heterogeneity and poor prognosis. We review the immunobiological factors, immune cells and current immunotherapy of gliomas, provide experimental evidence for future research and treatment of gliomas.

Features of local and systemic production of pro-inflammatory, chemoattractant mediators and vascular growth factors in high-risk corneal transplants.

Purpose: to study the composition and content of proinflammatory cytokines, chemoattractant mediators, vascular growth factors in blood serum (SB) and lacrimal fluid (LF) in patients with KPHR. Materials and methods. A total of 106 patients with postinflammatory corneal spots and turbid transplant were examined. The control group consisted of 20 practically healthy donors. Cytokines: IL-2, IL-18, MCP-1 / CCL2, MIP-1β / CCL4, RANTES / CCL5, Eotaxin / CCL11, IP-10 / CXCL10, SDF-1α / CXCL12, VEGF-A, VEGF-D, PIGF-1, HGF / SF. Results. The study of the levels of systemic production of immunomodiators in the groups of patients revealed significant changes in the concentrations of MCP-1 / CCL2, RANTES / CCL5, Eotaxin / CCL11, IP-10 / CXCL10, SDF-1α / CXCL12, as well as angiogenic factors VEGF-A and HGF / SF compared to controls. In the group of primary KPHR in the presence of vascularization, an increase in the production of IL-18, RANTES / CCL5, Eotaxin / CCL11 was observed. 1 / CCL2, VEGF-A, PIGF-1 and HGF / SF (p<0.05). Graft vascularization in repeated (2 and higher) corneal transplants (group II) was associated with significant increases in SC IL-2, PIGF-1, and HGF/SF (p<0.05). Analysis of cytokine content in the LF of patients with repeated KPHR and vascularized graft showed a statistically significant increase in IP-10 / CXCL10, MIP-1β / CCL4, SDF-1α / CXCL12, and VEGF-A. A retrospective analysis of the content of cytokines in the SB, depending on the outcomes of keratoplasty, showed a significant increase in the concentration of 7 out of 11 studied cytokines in patients of the KPHR group with various complications in the postoperative period, including those with a developed graft rejection reaction. Conclusions. It has been shown that the formation of vascularized corneal spots is associated with a significant increase in systemic production of IL-18, an increase in the concentration of chemokines with angiogenic activity RANTES / CCL5 and Eotaxin / CCL11 in the blood of patients with KPHR. During vascularization of the graft with repeated KPHR in the SB and LF, a unidirectional increase in the levels of IL-2, MCP-1 / CCL2, growth of VEGF-A and HGF / SF is observed. A factor in the development of graft rejection is the initially high concentrations in the SB of patients with KPHR IL-2, IL-18, chemokines RANTES / CCL5, MIP1β / CCL4, VEGF-A, PLGF, HGF / SF (p<0.05), indicating about the activation of pathological angiogenesis against the background of inflammation and an activated adaptive immune response.

Growth factors produced by bone marrow stromal cells on nanoroughened titanium-aluminum-vanadium surfaces program distal MSCs into osteoblasts via BMP2 signaling.

Statement of Clinical Significance: There remains the need to develop materials and surfaces that can increase the rate of implant osseointegration. Though osteoanabolic agents, like bone morphogenetic protein (BMP), can provide signaling for osteogenesis, the appropriate design of implants can also produce an innate cellular response that may reduce or eliminate the need to use additional agents to stimulate bone formation. Studies show that titanium implant surfaces that mimic the physical properties of osteoclast resorption pits regulate cellular responses of bone marrow stromal cells (MSCs) by altering cell morphology, transcriptomes, and local factor production to increase their differentiation into osteoblasts without osteogenic media supplements required for differentiation of MSCs on tissue culture polystyrene (TCPS). The goal of this study was to determine how cells in contact with biomimetic implant surfaces regulate the microenvironment around these surfaces in vitro. Two different approaches were used. First, unidirectional signaling was assessed by treating human MSCs grown on TCPS with conditioned media from MSC cultures grown on Ti6Al4V biomimetic surfaces. In the second set of studies, bidirectional signaling was assessed by coculturing MSCs grown on mesh inserts that were placed into culture wells in which MSCs were grown on the biomimetic Ti6Al4V substrates. The results show that biomimetic Ti6Al4V surface properties induce MSCs to produce factors within 7 days of culture that stimulate MSCs not in contact with the surface to exhibit an osteoblast phenotype via endogenous BMP2 acting in a paracrine signaling manner.

Recent advances in polymeric biomaterials-based gene delivery for cartilage repair

Untreated articular cartilage damage normally results in osteoarthritis and even disability that affects millions of people. However, both the existing surgical treatment and tissue engineering approaches are unable to regenerate the original structures of articular cartilage durably, and new strategies for integrative cartilage repair are needed. Gene therapy provides local production of therapeutic factors, especially guided by biomaterials can minimize the diffusion and loss of the genes or gene complexes, achieve accurate spatiotemporally release of gene products, thus provideing long-term treatment for cartilage repair. The widespread application of gene therapy requires the development of safe and effective gene delivery vectors and supportive gene-activated matrices. Among them, polymeric biomaterials are particularly attractive due to their tunable physiochemical properties, as well as excellent adaptive performance. This paper reviews the recent advances in polymeric biomaterial-guided gene delivery for cartilage repair, with an emphasis on the important role of polymeric biomaterials in delivery systems.

Analysis of the role of helminth TGF-β mimic in tissue repair

Abstract Intestinal parasites express excretory/secretory (ES) molecules, which modulate the type-2 immune response, including anti-inflammatory and tissue repair mechanisms. TGF-β mimic (TGM), an ES molecule secreted by H. polygyrus (Hp), binds TGF-β receptors yet lacks structural homology to TGF-β and exhibits distinct receptor interactions. Studies suggest TGM generates higher expression of immunoregulatory Foxp3+ Tregs and decreased fibrotic markers in vitro compared to TGF-β. Host TGF-β also influences wound healing. In cutaneous wounds, effective tissue repair requires rapid suppression of inflammation and local production of wound healing factors. We investigated whether TGM modulates wound healing using an in vivo wound biopsy model during which TGM is injected beneath a Tegaderm® layer to mimic cutaneous wound treatment. We showed that TGM accelerates wound healing when applied to the wound surface at multiple doses and modulates the associated inflammatory response. LC-MS/MS analysis of TGM-treated wound exudate revealed increases in proteins mediating leukocyte chemotaxis and proliferation, collagen deposition and fibrotic regulation. There was also enhanced immune cell infiltration of alternatively activated CD206+F4/80+ macrophages and epithelial cells concurrent with accelerated wound closure. These macrophages are CD301b+ and are essential for effective wound healing and potentially regulating fibrosis. Our findings indicate that TGM can enhance skin wound healing through enhanced production of proteins facilitating wound repair and through recruitment of specific macrophage subsets. This study will investigate TGM as a potential novel and effective therapeutic option for enhanced wound healing.

Pivotal Role of STAT3 in Shaping Glioblastoma Immune Microenvironment.

Glioblastoma belongs to the most malignant intracranial tumors characterized by indispensable growth and aggressiveness that highly associates with dismal prognosis and therapy resistance. Tumor heterogeneity that often challenges therapeutic schemes is largely attributed to the complex interaction of neoplastic cells with tumor microenvironment (TME). Soluble immunoregulatory molecules secreted by glioma cells attract astrocytes, circulating stem cells and a range of immune cells to TME, inducing a local production of cytokines, chemokines and growth factors that reprogram immune cells to inflammatory phenotypes and manipulate host’s immune response in favor of cancer growth and metastasis. Accumulating evidence indicates that these tolerogenic properties are highly regulated by the constitutive and persistent activation of the oncogenic signal transducer and activator of transcription 3 (STAT3) protein, which impairs anti-tumor immunity and enhances tumor progression. Herein, we discuss current experimental and clinical evidence that highlights the pivotal role of STAT3 in glioma tumorigenesis and particularly in shaping tumor immune microenvironment in an effort to justify the high need of selective targeting for glioma immunotherapy.

Insights in the immunobiology of glioblastoma.

Glioblastoma, a grade IV astrocytoma, is considered as the most malignant intracranial tumor, characterized by poor prognosis and therapy resistance. Tumor heterogeneity that often leads to distinct functional phenotypes contributes to glioblastoma (GB) indispensable growth and aggressiveness. The complex interaction of neoplastic cells with tumor microenvironment (TME) along with the presence of cancer stem-like cells (CSCs) largely confers to extrinsic and intrinsic GB heterogeneity. Recent data indicate that glioma cells secrete a variety of soluble immunoregulatory factors to attract different cell types to TME including astrocytes, endothelial cells, circulating stem cells, and a range of immune cells. These further induce a local production of cytokines, chemokines, and growth factors which upon crosstalk with extracellular matrix (ECM) components reprogram immune cells to inflammatory or anti-inflammatory phenotypes and manipulate host's immune response in favor of cancer growth and metastasis. Herein, we provide an overview of the immunobiologic factors that orchestrate the complex network of glioma cells and TME interactions in an effort to identify potential therapeutic targets for GB malignancy. Current therapeutic schemes and advances in targeting GB-TME crosstalk are further discussed. KEY MESSAGES: • Intrinsic and extrinsic tumor heterogeneity affects GB growth and aggressiveness. • GB cells secrete growth factors and chemoattractants to recruit immune cells to TME. • GAMs are a critical cell type in promoting GB growth. • GAMs change from pro-inflammatory, anti-tumor M1 phenotype to pro-tumorigenic M2. • Novel therapeutic agents target the crosstalk of neoplastic cells with TME.

Controlled Non‐Viral Gene Delivery in Cartilage and Bone Repair: Current Strategies and Future Directions

AbstractRecent advances in the approval and commercialization of gene therapeutics have fostered the return of gene therapy to center stage. But despite new optimism, no Food and Drug Administration approved product exists for the treatment of orthopedic disorders. Non‐viral gene delivery is a promising alternative to recombinant protein administration and viral gene transduction for orthopedic tissue engineering. When applied using appropriately designed systems, it enables temporal control of the overexpression of therapeutic genes, leading to local production of regulatory factors at physiologically relevant levels. Incorporating genetic material into 3D scaffold biomaterials, that is, gene activated scaffolds or hydrogels, presents a particular opportunity to utilize non‐viral gene therapy for in situ transfection of host cells and the regeneration of damaged tissues and organs. But controlled non‐viral gene delivery for musculoskeletal regeneration depends on a multifactorial design in which the choice of gene delivery method, therapeutic gene, and supportive biomaterial play a central role for the success of this strategy. This paper reviews the different modalities of non‐viral gene delivery used for the repair of bone and cartilage, and explores the current challenges and opportunities for the engineering of functional orthopedic tissues using gene activated scaffolds.

GMP-Compliant Human Fetal Skin Fibroblasts for Wound Healing

Background Among different kinds of cells involved in the wound healing process, fibroblasts play a pivotal role in the proliferation phase of this procedure wherein they induce the production of local growth factors and cytokines. Fetal fibroblasts with low immunogenic property and different wound healing process could be considered as a suitable alternative to neonatal foreskin cell based products in regenerative medicine and cell therapy. Cell therapy is an almost newly introduced method in the medical field and is also a challenging issue, which should assure the safety of the final product and the absence of viral or bacterial contamination with minimal immunogenic response. Therefore, based on current GMP (cGMP) principles, it is a matter of great importance to monitor raw materials’ quality and the conformity of all production procedures including cell culture, collection, and cryopreservation with validated standard operating procedures (SOPs). In the current study, we demonstrated the GMP-compatible and clinical-grade fetal fibroblast cells banking to be used for clinical applications.Methods All steps of isolation and culturing and cryopreservation of fetal fibroblast cells were performed under the sterile condition according to cGMP guidelines in the clean room. During the cell culture procedures, bacteriological investigation was performed in order to identify the probable contamination of the samples.Results Isolated fetal dermal fibroblasts were harvested and stored at early passages. The viability of fibroblasts was ≥ 98%, indicating successful establishment of fibroblast banking. Flow cytometry analysis has revealed the high purity of isolated and cultured fibroblastic population Karyotyping of fibroblasts at the 10th subculture shows a normal diploid male pattern.Conclusions The manufactured cells, according to the highest level of safety are considered as the most appropriate treatment option for cellular therapy. Based-on the current evidence, clinical-grade fetal fibroblasts throw new light on efficient and prompt healing of acute and chronic wounds of any etiology.

Proprotein convertase furin inhibits matrix metalloproteinase 13 in a TGF\u03b2-dependent manner and limits osteoarthritis in mice

Cartilage loss in osteoarthritis (OA) results from altered local production of growth factors and metalloproteases (MMPs). Furin, an enzyme involved in the protein maturation of MMPs, might regulate chondrocyte function. Here, we tested the effect of furin on chondrocyte catabolism and the development of OA. In primary chondrocytes, furin reduced the expression of MMP-13, which was reversed by treatment with the furin inhibitor α1-PDX. Furin also promoted the activation of Smad3 signaling, whereas activin receptor-like kinase 5 (ALK5) knockdown mitigated the effects of furin on MMP-13 expression. Mice underwent destabilization of the medial meniscus (DMM) to induce OA, then received furin (1 U/mice), α1-PDX (14 µg/mice) or vehicle. In mice with DMM, the OA score was lower with furin than vehicle treatment (6.42 ± 0.75 vs 9.16 ± 0.6, p < 0.01), and the number of MMP-13(+) chondrocytes was lower (4.96 ± 0.60% vs 20.96 ± 8.49%, p < 0.05). Moreover, furin prevented the increase in ALK1/ALK5 ratio in cartilage induced by OA. Conversely, α1-PDX had no effect on OA cartilage structure. These results support a protective role for furin in OA by maintaining ALK5 receptor levels and reducing MMP-13 expression. Therefore, furin might be a potential target mediating the development of OA.