Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, ...we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPβ. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.
Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA ...silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.
Summary Objective To compare the tensile biomechanical properties of age-matched adult human knee articular cartilage exhibiting distinct stages of degenerative or osteoarthritic deterioration and to ...determine the relationships between tensile properties and biochemical and structural properties hypothesized to underlie functional biomechanical deterioration. Methods Age-matched articular cartilage samples, obtained from the lateral and medial femoral condyles (LFC and MFC), exhibited (1) minimal fibrillation, characteristic of normal aging (NLA), (2) overt fibrillation associated with degeneration (DGN), or (3) overt fibrillation associated with osteoarthritis (OA). DGN samples were from knees that exhibited degeneration but not osteophytes while OA samples were from fragments removed during total knee arthroplasty. Cartilage samples were analyzed for tensile properties, cell and matrix composition, and histopathological structure. Results Differences in tensile, compositional and surface structural properties were indicative of distinct stages of cartilage degeneration, early (OA) advanced (DGN) and late (OA) with early degenerative changes in NLA samples being more advanced in the MFC than the LFC, including higher surface fibrillation, lower intrinsic fluorescence, and lower mechanical integrity. The transition from early to advanced degeneration involved a diminution in mechanical function, surface integrity, and intrinsic fluorescence. The transition from advanced to late degeneration involved an increase in cartilage water content, an increase in degraded collagen, and loss of collagen. Conclusions These results provide evidence of coordinated mechanical dysfunction, collagen network remodeling, and surface fibrillation. Even in the cartilage of knees exhibiting overt fibrillation but not extensive erosions characteristic of clinical osteoarthritis, most features of advanced cartilage degeneration were present.
Summary Continuous passive motion (CPM) is currently a part of patient rehabilitation regimens after a variety of orthopedic surgical procedures. While CPM can enhance the joint healing process, the ...direct effects of CPM on cartilage metabolism remain unknown. Recent in vivo and in vitro observations suggest that mechanical stimuli can regulate articular cartilage metabolism of proteoglycan 4 (PRG4), a putative lubricating and chondroprotective molecule found in synovial fluid and at the articular cartilage surface. Objectives (1) Determine the topographical variation in intrinsic cartilage PRG4 secretion. (2) Apply a CPM device to whole joints in bioreactors and assess effects of CPM on PRG4 biosynthesis. Methods A bioreactor was developed to apply CPM to bovine stifle joints in vitro . Effects of 24 h of CPM on PRG4 biosynthesis were determined. Results PRG4 secretion rate varied markedly over the joint surface. Rehabilitative joint motion applied in the form of CPM regulated PRG4 biosynthesis, in a manner dependent on the duty cycle of cartilage sliding against opposing tissues. Specifically, in certain regions of the femoral condyle that were continuously or intermittently sliding against meniscus and tibial cartilage during CPM, chondrocyte PRG4 synthesis was higher with CPM than without. Conclusions Rehabilitative joint motion, applied in the form of CPM, stimulates chondrocyte PRG4 metabolism. The stimulation of PRG4 synthesis is one mechanism by which CPM may benefit cartilage and joint health in post-operative rehabilitation.
Activation of the RET receptor tyrosine kinase by glial‐derived neurotrophic factor family members is dependent on a family of coreceptors, GFRα1–4. GFRα3 preferentially binds the newest member of ...the glial‐derived neurotrophic factor family of ligands, artemin. The major site of GFRα3 expression is in the dorsal root ganglion; however, the class of sensory neurons that expresses GFRα3 has not been reported previously. Using immunohistochemical methods, we show that the majority of dorsal root ganglion cells that express GFRα3 also express vanilloid receptor type 1, peripherin, RET, trkA and calcitonin gene‐related peptide. In addition, a significant subpopulation of GFRα3‐expressing cells also binds the lectin IB4. We demonstrate that GFRα3 artemin neurons are immunopositive for markers expected of nociceptors and include a subset of neurons distinct from the GDNF‐responsive population. Our results indicate artemin may exert selective effects on pain sensation.
Articular cartilage damage is a persistent and increasing problem with the aging population, and treatments to achieve biological repair or restoration remain a challenge. Cartilage tissue ...engineering approaches have been investigated for over 20 years, but have yet to achieve the consistency and effectiveness for widespread clinical use. One of the potential reasons for this is that the engineered tissues do not have or establish the normal zonal organization of cells and extracellular matrix that appears critical for normal tissue function. A number of approaches are being taken currently to engineer tissue that more closely mimics the organization of native articular cartilage. This review focuses on the zonal organization of native articular cartilage, strategies being used to develop such organization, the reorganization that occurs after culture or implantation, and future prospects for the tissue engineering of articular cartilage with biomimetic zones.
Clues from the epidemiology of schizophrenia, such as the increased risk in those born in winter/spring, have led to the hypothesis that prenatal vitamin D deficiency may increase the risk of later ...schizophrenia. We wish to explore this hypothesis in a large Danish case-control study (n = 2602). The concentration of 25 hydroxyvitamin D (25OHD) was assessed from neonatal dried blood samples. Incidence rate ratios (IRR) were calculated when examined for quintiles of 25OHD concentration. In addition, we examined statistical models that combined 25OHD concentration and the schizophrenia polygenic risk score (PRS) in a sample that combined the new sample with a previous study (total n = 3464; samples assayed and genotyped between 2008-2013). Compared to the reference (fourth) quintile, those in the lowest quintile (<20.4 nmol/L) had a significantly increased risk of schizophrenia (IRR = 1.44, 95%CI: 1.12-1.85). None of the other quintile comparisons were significantly different. There was no significant interaction between 25OHD and the PRS. Neonatal vitamin D deficiency was associated with an increased risk for schizophrenia in later life. These findings could have important public health implications related to the primary prevention of schizophrenia.
Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, ...we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPβ. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth. PUBLICATION ABSTRACT
Pancreatic beta cells synthesize and secrete the neurotransmitter γ-aminobutyric acid (GABA) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA ...release has classically been described as a secretory vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel (VRAC), functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from type 1 and type 2 diabetic patients, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.
BackgroundFresh osteochondral allograft transplantation is a well-established technique for the treatment of cartilage defects of the knee. It is believed that the basic paradigm of the technique is ...that the transplantation of viable chondrocytes maintains the articular cartilage matrix over time. Allograft tissue is typically transplanted up to forty-two days after the death of the donor, but it is unknown how the conditions and duration of storage affect the properties of fresh human osteochondral allografts. This study examined the quality of human allograft cartilage as a function of storage for a duration of one, seven, fourteen, and twenty-eight days. We hypothesized that chondrocyte viability, chondrocyte metabolic activity, and the biochemical and biomechanical properties of articular cartilage would remain unchanged after storage for twenty-eight days.MethodsSixty osteochondral plugs were harvested from ten fresh human femoral condyles within forty-eight hours after the death of the donor and were stored in culture medium at 4°C. At one, seven, fourteen, and twenty-eight days after harvest, the osteochondral plugs were analyzed for (1) viability and viable cell density by confocal microscopy, (2) proteoglycan synthesis by quantification of SO4 incorporation, (3) glycosaminoglycan content, (4) indentation stiffness, (5) compressive modulus and hydraulic permeability by static and dynamic compression testing, and (6) tensile modulus by equilibrium tensile testing.ResultsChondrocyte viability and viable cell density remained unchanged after storage for seven and fourteen days (p > 0.7) and then declined at twenty-eight days (p < 0.001). Proteoglycan synthesis remained unchanged at seven days (p > 0.1) and then declined at fourteen days (p < 0.01) and twenty-eight days (p < 0.001). No significant differences were detected in glycosaminoglycan content (p > 0.8), indentation stiffness (p > 0.4), compressive modulus (p > 0.05), permeability (p > 0.3), or equilibrium tensile modulus after storage for twenty-eight days (p > 0.9).ConclusionsThese data demonstrate that fresh human osteochondral allograft tissue stored for more than fourteen days undergoes significant decreases in chondrocyte viability, viable cell density, and metabolic activity, with preservation of glycosaminoglycan content and biomechanical properties. The cartilage matrix is preserved during storage for twenty-eight days, but the chondrocytes necessary to maintain the matrix after transplantation decreased over that time-period.Clinical RelevanceFresh osteochondral allografting is well established and increasingly used, but little information is available about the changes in the tissue that occur during storage over a period of time. Surgeons who perform fresh osteochondral allograft transplantation should understand the consequences of prolonged storage on the quality of the graft.
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