GAG Levels Research Articles

Evaluate the Clinical and Laboratory Manifestations of Children With Type 6 of Mucopolysaccharidosis Before and After Enzyme Therapy: A Quasi-Experimental One Group Study

The purpose of this study is to evaluate the clinical and laboratory manifestations of children with mucopolysaccharidosis type 6 before and after enzyme therapy. In this quasi-experimental study, 8 patients with MPS-6 referred to the pediatric endocrinology department of Imam Reza Hospital in Mashhad were followed up for 12 months. The level of urinary glycosaminoglycan was measured to check the response to the treatment. The range of motion of the shoulder and elbow joints was evaluated using a goniometer, and abdominal ultrasound was performed to check the size of the liver and spleen in the midclavicular line. The 6-minute walking test and the 3-minute stair climbing test were performed for the patients at the mentioned times. The height and weight of the patients were also measured, and echocardiography was performed. Then patients underwent weekly enzyme treatment. One of the patients (seventh patient) was excluded from the study. Patients were treated with enzyme from the beginning of the study. The patients were evaluated at 12 months later. Statistical analysis showed that changes in urinary GAG level, height, weight, changes in 6-minute walk and range of motion (extension and flexion) of the shoulder were significant (P<0.05). Changes in liver and spleen size compared to height, climbing stairs, corneal opacity and heart changes after 12 months of enzyme therapy were not significant. It is suggested that even though this method of treatment is not definitive, that can be continued to improve the current condition of the patient.

Astragaloside IV Protects Against IL-1β-Induced Chondrocyte Damage via Activating Autophagy.

Osteoarthritis (OA) is a chronic inflammatory condition that affects the articular cartilage. Astragaloside IV (AS-IV) constitutes the primary active component of the Chinese herbal medicine Huangqi (Radix Astragali Mongolici). AS-IV demonstrates anti-inflammatory and anti-apoptotic attributes, exhibiting therapeutic potential across various inflammatory and apoptosis-related disorders. Nevertheless, its pharmaceutical effects in OA are yet to be fully defined. This study aimed to investigate the protective impact of AS-IV on rat chondrocytes treated with IL-1β and ascertain whether autophagy plays a role in this effect. Chondrocytes were isolated and cultivated from the knee joints of neonatal SD mice. The study included the blank control group, the model group, and the AS-IV concentration gradient group (50, 100, 200 μmol/L) to intervene with chondrocytes. The MTT assay was employed to assess cell viability at varying culture periods, enabling the determination of suitable concentration and duration. Subsequently, chondrocytes were treated with the optimal AS-IV concentration and divided into three groups: the model group replicated IL-1β-induced inflammatory chondrocyte injury, the AS-IV group received a co-culture of AS-IV and IL-1β, and a blank control group was established. Changes in cell morphology and structure were observed using ghost pen cyclic peptide staining. ELISA was used to measure TNF-α and GAG levels in cell supernatants. RT-qPCR assessed p62 and LC3 mRNA expression, while Western Blot evaluated p62 and LC3Ⅱ/Ⅰ protein expression. AS-IV promoted chondrocyte proliferation and concurrently inhibited cell apoptosis. An optimal AS-IV dose of 200 μmol/L and a suitable reaction time of 48 h were identified. Ghost pen cyclic peptide staining indicated that the model group's cytoskeleton exhibited fusiform changes with reduced immunofluorescence intensity, as opposed to the blank control group. The AS-IV group displayed more polygonal cytoskeletal morphology with increased immunofluorescence intensity. AS-IV reduced TNF-α levels and elevated GAG levels in the culture supernatant. Additionally, AS-IV lowered p62 mRNA and protein expression while increasing LC3 mRNA expression in cultured chondrocytes. Our findings suggest that AS-IV mitigates inflammatory chondrocyte injury, safeguarding chondrocytes through a potential autophagy suppression mechanism. These results imply that AS-IV could offer preventive advantages for OA.

Identification of key pathways and mRNAs in interstitial cystitis/bladder pain syndrome treatment with quercetin through bioinformatics analysis of mRNA-sequence data.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition with painful bladder. At present, the pathogenesis of IC/BPS is still unknown. Quercetin (QCT) is a kind of natural flavonoid with wide sources and multiple biological activities. The purpose of this study was to explore the effects of QCT on mRNA expression and related regulatory signal pathways in IC model rats. LL-37 was used to induce the IC/BPS model rats. 20 mg/kg QCT was injected intraperitoneally into IC/BPS rats. ELISA, HE, Masson and TB staining were used to evaluate the level of inflammation and pathology. The concentration of QCT in rats was detected by HPLC. The mRNA sequencing was used to detect the differentially expressed (DE) mRNA in each group. The over-expression experiment of Lpl was carried out in IC/BPS model rats. QCT treatment significantly decreased the level of MPO, IL-1β, IL-6 and TNF-α induced by LL-37 in rats, and alleviated bladder injury and mast cell degranulation. There were significant differences in mRNA sequencing data between groups, and the hub gene Lpl were screened by Cytohubba. The expression of Lpl was downregulated in IC/BPS rats. QCT intervention promoted Lpl expression. Overexpression of Lpl reduced the bladder injury induced by LL-37, increased GAG level and decreased the expression of MPO, IL-1β, IL-6 and TNF-α. In this study, we provided the DE mRNA in IC/BPS rats treated with QCT, the signaling pathways for DE enrichment, screened out the hub genes, and revealed that Lpl overexpression alleviated IC/BPS model rats.

Overexpression of lncRNA LINC00665 inhibits the proliferation and chondroblast differentiation of bone marrow mesenchymal stem cells by targeting miR-214-3p

BackgroundOsteoarthritis is a chronic disease mainly involving the damage of articular cartilage and the whole articular tissue, which is the main cause of disability in the elderly. To explore more effective treatment measures, this study analyzed the regulatory role and molecular mechanism of lncRNA LINC00665 (LINC00665) in the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), providing a valuable theoretical basis for the pathogenesis and patient treatment of osteoarthritis.MethodsOsteoarthritis tissues and healthy tissues were obtained from 52 patients with osteoarthritis and 34 amputated patients without osteoarthritis, and the levels of LINC00665 and miR-214-3p were assessed by RT-qPCR. BMSCs were cultured and induced chondrogenic differentiation. The proliferation ability of BMSCs was detected by CCK-8 method, and the apoptosis level of BMSCs was evaluated by flow cytometry. The content of proteoglycan-glycosaminoglycan (GAG) in cartilage matrix was determined by Alcian blue staining. In addition, the binding relationship between LINC00665 and miR-214-3p was verified by luciferase reporter assay, and the molecular mechanism was further analyzed.ResultsIn osteoarthritis tissues, LINC00665 was elevated and miR-214-3p was down-regulated. With the chondrogenic differentiation of BMSCs, the level of GAG increased, and LINC00665 expression gradually decreased, while miR-214-3p level was on the contrary. After transfection of pcDNA3.1-LINC00665 in BMSCs, cell proliferation capacity was decreased, apoptosis rate was increased, and GAG content was reduced. Moreover, LINC00665 sponged miR-214-3p and negatively regulate its expression. Transfection of pcDNA3.1-LINC00665-miR-214-3p mimic changed the regulation of pcDNA3.1-LINC00665 on the viability and chondrogenic differentiation of BMSCs.ConclusionsOverexpression of lncRNA LINC00665 inhibited the proliferation and chondrogenic differentiation of BMSCs by targeting miR-214-3p. The LINC00665/miR-214-3p axis may improve joint damage and alleviate the progression of osteoarthritis.

Higher Expression of Human Endogenous Retrovirus-K was Observed in Peripheral B Lymphocytes of Leukemia and Lymphoma Patients.

Hematological malignant tumors (HMTs) are serious diseases that threaten human health and life with high mortality. Therefore, it is necessary to develop novel strategies for diagnosis and treatment. Human endogenous retroviruses (HERVs) have recently attracted increasing attention as potential targets for cancer diagnosis and therapy. In this study, we explored the association between HERV-K expression levels and HMTs development. Clinical data and peripheral blood samples were collected from 236 leukemia, 384 lymphoma patients, and 69 healthy controls. Quantitative polymerase chain reaction was used to detect the expression of HERV-K gag, pol, and env genes in peripheral blood mononuclear cells or different cell subpopulations. Differently expressed HERV-K genes were further tested by using deep sequencing method, and further analyzed with gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. B cell- and T cell-related cytokines in patients were also detected by enzyme-linked immunosorbent assay (ELISA). The results showed that the expression levels of the HERV-K gag, pol, and env genes in patients were significantly higher than in healthy controls. There was a correlation between the expression level of HERV-K and the clinicopathological parameters of leukemia patients. HERV-K expression was increased in the B lymphocytes of leukemia and lymphoma patients, but not in the T cells or neutrophils. The GO and KEGG analyses showed that abnormal expression of the HERV-K locus in patients affected immune regulation. The analysis of cytokines proved that the B cell-related cytokines, including interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon-gamma, were significantly decreased in patients, while the T cell-related cytokines, including IL-3, IL-12, and TNF-β, were not significantly changed. In conclusion, HERV-K genes might participate in the occurrence and development of leukemia and lymphoma, and might be biomarkers for the detection or evaluation of leukemia and lymphoma.

HIV-1 Restriction in Systemic Lupus Erythematosus Involves Iron Homeostasis

INTRODUCTION: Over the past two decades, there has been a growing emphasis on host restriction factors. However, a comprehensive understanding of their role in modulating HIV-1 infection in vivo requires further detailed investigation. Intracellular iron levels can affect HIV-1 replication by influencing expression of host cell viral restriction factors. Systemic Lupus Erythematosus (SLE) is characterized by upregulation of interferon response and the coexistence of SLE and HIV infection is rare. In lupus nephritis, deregulation of proteins involved in iron homeostasis have also been identified, with hepcidin suggested to play a role in disease severity . Hif1α, a protein regulated by hypoxia and under iron depletion, and reported to block HIV transcription, is upregulated in SLE. High levels of IL-10 and IL-16 which inhibits HIV infection are upregulated in SLE. APOBECs and oxysterols and Interferon Regulating Factors (IRFs) are also known to be upregulated in SLE. The current study is aimed to investigate ex vivo HIV-1 infection in peripheral blood mononuclear cells (PBMCs) derived from individuals SLE and identify factors that contributed to this inhibition. METHODS: We utilized replication deficient VSVg-pseudotype HIV-1 and replication competent HIV-IIIB to infect SLE PBMCs. Real-time PCR was conducted to determine the upregulation of genes involved in HIV-1 replication. RNA-seq of PBMCs were done to identify altered restriction factors. RESULTS: We investigated one round of HIV-1 infection in peripheral blood mononuclear cells (PBMCs) obtained from individuals with Systemic Lupus Erythematosus (SLE) and compared them to control PBMCs. We found that the HIV-1 infection in SLE-derived PBMCs was significantly reduced, as indicated by lower mRNA levels of Gag and Nef, two essential viral proteins. Our ongoing study, which focused on the inhibition of HIV-1 replication ex vivo in Sickle Cell Disease (SCD), revealed interesting findings. RNA sequencing of PBMCs from individuals with SCD showed an upregulation of APOBEC3A, APOBEC3B, and CH25H, which are known restriction factors against HIV-1. Given these intriguing results, we decided to analyze the RNA expression of these restriction factors in SLE-derived PBMCs. Surprisingly, we found a significant increase in the mRNA levels of APOBEC3A, APOBEC3B, and CH25H in SLE-derived PBMCs as well. This suggests that the enhanced expression of these restriction factors could be contributing to the reduced HIV-1 infection observed in SLE-derived PBMCs. Additionally, we explored the RNA expression of HIF-1α and TFR1, which are iron-regulated factors, and found that their mRNA levels were upregulated in SLE-derived PBMCs. Furthermore, we observed an upregulation of interferon regulatory factors, including IL-10, IFN-β, and IRF-7, which are involved in immune responses and antiviral defense mechanisms. Interestingly IRF-7 was upregulated in SCD PBMCs too. DISSUCION: The ongoing study in our lab on SCD and the study reported here are known for deregulated iron metabolism. Interestingly on one hand PBMCs derived from both inhibit HIV-1 and on other have some common restriction factors, iron regulated genes and IRF upregulated. Iron-regulated factors, such as TFR1, HIF-1α and ferroportin have been associated with antiviral effects. Modulating the expression or activity of these factors could influence HIV infection. CONCLUSIONs: Taken together, our findings indicate that SLE-derived PBMCs exhibit a distinct gene expression profile that may confer protection against HIV-1 infection. The upregulation of APOBEC3A, APOBEC3B, CH25H, HIF-1α, TFR1, and interferon regulatory factors could collectively contribute to the observed reduction in HIV-1 replication in SLE-derived PBMCs. Further investigation is needed to fully understand the mechanisms underlying these observations and their implications for HIV-1 pathogenesis under altered intracellular iron levels influencing HIV-1 replication by modulating host cell factors. Acknowledgments This work was supported by NIH Research Grants 1R01HL125005, 5U54MD007597, P30AI087714 and P50AI150476. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Hsa-miR-181-5p inhibits human immunodeficiency virus type 1 replication by downregulating DDX3X expression

BackgroundHIV-1 infection affects expression profiles of microRNA. miR-181 is found negatively correlated with HIV-1 viral load. This study aimed to explain that miR-181 targets DDX3X, a host factor involved in HIV-1 nuclear export, thereby inhibiting HIV-1 replication. MethodsTo verify our hypothesis, first, the relationship between miR-181 expression, DDX3X expression, and HIV-1 viral load was analyzed. Second, miR-181 mimics were transfected into Jurkat cells infected with wild pNL4-3 strain or H9-IIIB cells with HIV-1 replication-competent for HIV-1 viral protein P24(Gag) detection. Besides the reporter gene plasmid containing the DDX3X mRNA sequence was transfected into 293T cells to demonstrate the targeting of miR-181 to the DDX3X mRNA. Finally, the spliced, unspliced, or incompletely spliced HIV-1 transcripts and HIV-1 Tat, Rev, and Gag mRNA were also detected after miR-181 transfection. ResultsOur result proved that miR-181 significantly reduced the HIV-1 viral protein Gag(P24) level and targeted DDX3X mRNA 3′-UTR, inhibiting the unspliced or incompletely spliced HIV-1 mRNA's nuclear export. ConclusionOur results confirmed that miR-181 is involved in HIV-1 viral replication in lymphocytes by downregulating DDX3X expression. The research provides a research basis for future HIV-1 antiviral research.

Anti-Aging and Depigmentation Effect of a Hyaluronic Acid Mechanically Stabilized Complex on Human Skin Explants.

Solar radiation and environmental pollutants are factors that cause changes in the skin that trigger skin aging. The objective of the study is to evaluate the rejuvenating effects of a complex formed by hyaluronic acid supplemented with vitamins, amino acids and oligopeptides in explants of human skin. For this, surplus skin samples have been obtained from donors that have been resected and cultivated on slides with membrane inserts. The complex was administered to some skin explants and the percentage of cells with low, medium and high levels of melanin was evaluated as an indicator of the degree of pigmentation. Other skin segments were irradiated with UVA/UVB, then the product was administered on several slides and the levels of collagen, elastin, sulfated GAG and MMP1 were evaluated. The results show that the administration of the complex significantly reduces the percentage of skin cells with a high melanin content by 16%, and that in skin irradiated with UVA/UVB, there is a decrease in the content of collagen, elastin and sulfate GAGs, and the complex reverses this reduction without changing MMP1 levels. This suggests that the compound has anti-aging and depigmentation effects on the skin, giving a skin rejuvenation appearance.

Evaluation of Archival HIV DNA in Brain and Lymphoid Tissues.

HIV reservoirs persist in anatomic compartments despite antiretroviral therapy (ART). Characterizing archival HIV DNA in the central nervous system (CNS) and other tissues is crucial to inform cure strategies. We evaluated paired autopsy brain-frontal cortex (FC), occipital cortex (OCC), and basal ganglia (BG)-and peripheral lymphoid tissues from 63 people with HIV. Participants passed away while virally suppressed on ART at the last visit and without evidence of CNS opportunistic disease. We quantified total HIV DNA in all participants and obtained full-length HIV-envelope (FL HIV-env) sequences from a subset of 14 participants. We detected HIV DNA (gag) in most brain (65.1%) and all lymphoid tissues. Lymphoid tissues had higher HIV DNA levels than the brain (P < 0.01). Levels of HIV gag between BG and FC were similar (P > 0.2), while OCC had the lowest levels (P = 0.01). Females had higher HIV DNA levels in tissues than males (gag, P = 0.03; 2-LTR, P = 0.05), suggesting possible sex-associated mechanisms for HIV reservoir persistence. Most FL HIV-env sequences (n = 143) were intact, while 42 were defective. Clonal sequences were found in 8 out of 14 participants, and 1 participant had clonal defective sequences in the brain and spleen, suggestive of cell migration. From 10 donors with paired brain and lymphoid sequences, we observed evidence of compartmentalized sequences in 2 donors. Our data further the idea that the brain is a site for archival HIV DNA during ART where compartmentalized provirus may occur in a subset of people. Future studies assessing FL HIV-provirus and replication competence are needed to further evaluate the HIV reservoirs in tissues. IMPORTANCE HIV infection of the brain is associated with adverse neuropsychiatric outcomes, despite efficient antiretroviral treatment. HIV may persist in reservoirs in the brain and other tissues, which can seed virus replication if treatment is interrupted, representing a major challenge to cure HIV. We evaluated reservoirs and genetic features in postmortem brain and lymphoid tissues from people with HIV who passed away during suppressed HIV replication. We found a differential distribution of HIV reservoirs across brain regions which was lower than that in lymphoid tissues. We observed that most HIV reservoirs in tissues had intact envelope sequences, suggesting they could potentially generate replicative viruses. We found that women had higher HIV reservoir levels in brain and lymphoid tissues than men, suggesting possible sex-based mechanisms of maintenance of HIV reservoirs in tissues, warranting further investigation. Characterizing the archival HIV DNA in tissues is important to inform future HIV cure strategies.

Non-invasive intravenous administration of AAV9 transducing iduronate sulfatase leads to global metabolic correction and prevention of neurologic deficits in a mouse model of Hunter syndrome

Hunter syndrome is a rare x-linked recessive genetic disorder that affects lysosomal metabolism due to deficiency of iduronate-2-sulfatase (IDS), with subsequent accumulation of glycosaminoglycans heparan and dermatan sulfates (GAG). Enzyme replacement therapy is the only FDA-approved remedy and is an expensive life-time treatment that alleviates some symptoms of the disease without neurocognitive benefit. We previously reported successful treatment in a mouse model of mucopolysaccharidosis type II (MPS II) using adeno-associated viral vector serotype 9 encoding human IDS (AAV9.hIDS) via intracerebroventricular injection. As a less invasive and more straightforward procedure, here we report intravenously administered AAV9.hIDS in a mouse model of MPS II. In animals administered 1.5 × 1012 vg of AAV9.hIDS at 2 months of age, we observed supraphysiological levels of IDS enzyme activity in the circulation (up to 9100-fold higher than wild-type), in the tested peripheral organs (up to 560-fold higher than wild-type), but only 4% to 50% of wild type levels in the CNS. GAG levels were normalized on both sides of the blood-brain-barrier (BBB) in most of tissues tested. Despite low levels of the IDS observed in the CNS, this treatment prevented neurocognitive decline as shown by testing in the Barnes maze and by fear conditioning. This study demonstrates that a single dose of IV-administered AAV9.hIDS may be an effective and non-invasive procedure to treat MPS II that benefits both sides of the BBB, with implications for potential use of IV-administered AAV9 for other neuronopathic lysosomal diseases.

Pulsed Electromagnetic Field Therapy and Direct Current Electric Field Modulation Promote the Migration of Fibroblast-like Synoviocytes to Accelerate Cartilage Repair In Vitro.

Articular cartilage injuries are a common source of joint pain and dysfunction. As articular cartilage is avascular, it exhibits a poor intrinsic healing capacity for self-repair. Clinically, osteochondral grafts are used to surgically restore the articular surface following injury. A significant challenge remains with the repair properties at the graft-host tissue interface as proper integration is critical toward restoring normal load distribution across the joint. A key to addressing poor tissue integration may involve optimizing mobilization of fibroblast-like synoviocytes (FLS) that exhibit chondrogenic potential and are derived from the adjacent synovium, the specialized connective tissue membrane that envelops the diarthrodial joint. Synovium-derived cells have been directly implicated in the native repair response of articular cartilage. Electrotherapeutics hold potential as low-cost, low-risk, non-invasive adjunctive therapies for promoting cartilage healing via cell-mediated repair. Pulsed electromagnetic fields (PEMFs) and applied direct current (DC) electric fields (EFs) via galvanotaxis are two potential therapeutic strategies to promote cartilage repair by stimulating the migration of FLS within a wound or defect site. PEMF chambers were calibrated to recapitulate clinical standards (1.5 ± 0.2 mT, 75 Hz, 1.3 ms duration). PEMF stimulation promoted bovine FLS migration using a 2D in vitro scratch assay to assess the rate of wound closure following cruciform injury. Galvanotaxis DC EF stimulation assisted FLS migration within a collagen hydrogel matrix in order to promote cartilage repair. A novel tissue-scale bioreactor capable of applying DC EFs in sterile culture conditions to 3D constructs was designed in order to track the increased recruitment of synovial repair cells via galvanotaxis from intact bovine synovium explants to the site of a cartilage wound injury. PEMF stimulation further modulated FLS migration into the bovine cartilage defect region. Biochemical composition, histological analysis, and gene expression revealed elevated GAG and collagen levels following PEMF treatment, indicative of its pro-anabolic effect. Together, PEMF and galvanotaxis DC EF modulation are electrotherapeutic strategies with complementary repair properties. Both procedures may enable direct migration or selective homing of target cells to defect sites, thus augmenting natural repair processes for improving cartilage repair and healing.

Endogenous Thrombin Generation Potential in Patients with Pulmonary Embolism and Its Relevance to Thrombo-Inflammatory Biomarkers

Background: Pulmonary embolism (PE) is a catastrophic outcome in patients with venous thrombosis and third leading cardiovascular cause of death after myocardial infraction and stroke, estimates around 100,000 deaths annually.Inflammation, hemostatic dysregulation including fibrinolytic imbalance, endothelial compromise, and generation of thrombotic mediators, along with cellular defects and hemodynamic derangement contribute to the pathogenesis of PE.Endogenous thrombin generation contributes to the overall pathology of PE. The purpose of this study is to investigate endogenous thrombin potential (ETP) and the circulating biomarkers of fibrinolytic, inflammatory, and endothelial dysfunction in PE samples. Methods: Citrated blood samples from 400 patients with confirmed PE were collected at Loyola University Medical Center and Gottlieb Memorial Hospital under an IRB approved protocol. Normal human plasma (NHP) comprised of 25 male and 25 female samples were obtained from a commercial source. Thrombin generation studies were carried out using a commercially available a kinetic system. Endogenous GAG's contents were measured by fluorogenic quenching method. Thrombin generation parameters such as peak thrombin, lag time and area under the curve (AUC) were compiled. Plasma samples were retrospectively analyzed for biomarkers, including D-Dimer, PAI-1, tPA, TAFIa, vWF, CRP, IL6, TFPI and VEGF using commercially available sandwich ELISA methods. Results were compiled as mean ± SD and analyzed for significance and correlation by using GraphPad Prism software. Result: On a cumulative basis, PE patients showed a wide variation in thrombin generation parameters (Table 1). Biomarker analysis showed elevated levels of D-Dimer (37.05-fold), CRP (35.34-folds), IL6 (21.62-folds), tPA (4.69-folds), PAI-1a (3.95-folds), VEGF (2.38-folds), TFPI (2.17-folds), GAG's (1.92-folds), vWF (1.89-folds), and TAFIa (1.21-folds). Peak thrombin levels exhibited varying degree of positive and negative correlations with various biomarkers as illustrated in figure 1. Thrombin levels showed significant negative correlations with tPA, endogenous GAG's & TFPI, while positively correlated with VEGF. D-dimer showed positive correlations with CRP & IL6. PAI-1 demonstrated positive correlations with tPA, IL6, and endogenous GAG's. tPA showed negative correlation with TAFI, while positive correlations with endogenous GAG's, and TFPI. vWF showed positive correlations CRP, IL6, and endogenous GAG's. CRP showed positive correlations with IL6. Endogenous GAG's showed positive correlations with TFPI while TFPI showed negative correlation with VEGF. Conclusion: PE patients exhibit a wide variation in endogenous thrombin generation parameters. Overall, peak thrombin and ETP were decreased, whereas the lag time was increased. The increase in thrombo-inflammatory markers is suggestive of upregulation of thrombogenesis, and inflammatory processes in these patients. Thus, the profiling of thrombo-inflammatory biomarkers along with thrombin generation profile may provide additional information on the pathogenesis and severity of PE. However, biomarkers of hemostatic activation, vascular dysfunction and inflammation were increased. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Activities of (Poly)phenolic Antioxidants and Other Natural Autophagy Modulators in the Treatment of Sanfilippo Disease: Remarkable Efficacy of Resveratrol in Cellular and Animal Models

Sanfilippo disease, caused by mutations in the genes encoding heparan sulfate (HS) (a glycosaminoglycan; GAG) degradation enzymes, is a mucopolysaccharidosis (MPS), which is also known as MPS type III, and is characterized by subtypes A, B, C, and D, depending on identity of the dysfunctional enzyme. The lack of activity or low residual activity of an HS-degrading enzyme leads to excess HS in the cells, impairing the functions of different types of cells, including neurons. The disease usually leads to serious psychomotor dysfunction and death before adulthood. In this work, we show that the use of molecules known as dietary (poly)phenolic antioxidants and other natural compounds known as autophagy activators (genistein, capsaicin, curcumin, resveratrol, trehalose, and calcitriol) leads to accelerated degradation of accumulated HS in the fibroblasts of all subtypes of MPS III. Both the cytotoxicity tests we performed and the available literature data indicated that the use of selected autophagy inducers was safe. Since it showed the highest effectivity in cellular models, resveratrol efficacy was tested in experiments with a mouse model of MPS IIIB. Urinary GAG levels were normalized in MPS IIIB mice treated with 50 mg/kg/day resveratrol for 12 weeks or longer. Behavioral tests indicated complete correction of hyperactivity and anxiety in these animals. Biochemical analyses indicated that administration of resveratrol caused autophagy stimulation through an mTOR-independent pathway in the brains and livers of the MPS IIIB mice. These results indicate the potential use of resveratrol (and possibly other autophagy stimulators) in the treatment of Sanfilippo disease.

Use of allogeneic freeze-dried conditioned serum for the prevention of degradation in cartilage exposed to IL-1ß

BackgroundAutologous conditioned serum (ACS) has been extensively used in the field of veterinary orthopaedics and sports medicine. Due to the autologous and blood-derived nature of this product, issues such as individual variability, need for storage at low temperatures and non-availability for immediate are frequently encountered for ACS use in the field. To address those issues, we proposed the evaluation of an off-the-shelf allogeneic freeze-dried version of conditioned serum in an in vitro model of osteoarthritis. In this study, we evaluated if origin (autologous and allogeneic) and preparation (frozen and freeze-dried) of conditioned serum could influence in its effect in an in vitro model.ResultsIL-1β stimulation in cartilage led to a significant increase in media GAG and decreased levels of GAG in cartilage explants at the termination of the experiment. No significant differences were noted in outcomes measured in the cartilage explants with respect to the main effects of treatment (frozen versus freeze-dried serum), autologous versus allogeneic preparations or based on serum concentration.ConclusionsThe study did not observe any substantial differences in the response of cartilage to allogeneic freeze-dried CS when compared to other independent parameters (autologous and frozen preparations). Further investigation using in vivo systems appears warranted.

Treatment Effects of Intra-Articular Allogenic Mesenchymal Stem Cell Secretome in an Equine Model of Joint Inflammation.

BackgroundAllogenic mesenchymal stem cell (MSC) secretome is a novel intra-articular therapeutic that has shown promise in in vitro and small animal models and warrants further investigation.ObjectivesTo investigate if intra-articular allogenic MSC-secretome has anti-inflammatory effects using an equine model of joint inflammation.Study DesignRandomized positively and negatively controlled experimental study.MethodIn phase 1, joint inflammation was induced bilaterally in radiocarpal joints of eight horses by injecting 0.25 ng lipopolysaccharide (LPS). After 2 h, the secretome of INFy and TNFα stimulated allogeneic equine MSCs was injected in one randomly assigned joint, while the contralateral joint was injected with medium (negative control). Clinical parameters (composite welfare scores, joint effusion, joint circumference) were recorded, and synovial fluid samples were analyzed for biomarkers (total protein, WBCC; eicosanoid mediators, CCL2; TNFα; MMP; GAGs; C2C; CPII) at fixed post-injection hours (PIH 0, 8, 24, 72, and 168 h). The effects of time and treatment on clinical and synovial fluid parameters and the presence of time-treatment interactions were evaluated. For phase 2, allogeneic MSC-secretome vs. allogeneic equine MSCs (positive control) was tested using a similar methodology.ResultsIn phase 1, the joint circumference was significantly (p < 0.05) lower in the MSC-secretome treated group compared to the medium control group at PIH 24, and significantly higher peak synovial GAG values were noted at PIH 24 (p < 0.001). In phase 2, no significant differences were noted between the treatment effects of MSC-secretome and MSCs.Main LimitationsThis study is a controlled experimental study and therefore cannot fully reflect natural joint disease. In phase 2, two therapeutics are directly compared and there is no negative control.ConclusionsIn this model of joint inflammation, intra-articular MSC-secretome injection had some clinical anti-inflammatory effects. An effect on cartilage metabolism, evident as a rise in GAG levels was also noted, although it is unclear whether this could be considered a beneficial or detrimental effect. When directly comparing MSC-secretome to MSCs in this model results were comparable, indicating that MSC-secretome could be a viable off-the-shelf alternative to MSC treatment.

Perturbing HIV-1 Ribosomal Frameshifting Frequency Reveals a cis Preference for Gag-Pol Incorporation into Assembling Virions.

HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding, while GP binds Gag to deliver the essential virion enzymes protease, reverse transcriptase, and integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (∼1:20), dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Here, we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched ∼3-fold in virions relative to cells, with viral infectivity being better maintained at subphysiological levels of GP than when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis (i.e., from the same gag-pol mRNA) than in trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans-delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taking these results together, we propose a "weighted Goldilocks" scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies. IMPORTANCE HIV-1 infectivity requires incorporation of the Gag-Pol (GP) precursor polyprotein into virions during the process of virus particle assembly. Mechanisms dictating GP incorporation into assembling virions are poorly defined, with GP levels in virions traditionally thought to solely reflect relative levels of Gag and GP expressed in cells, dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event that occurs in gag-pol mRNAs. Herein, we provide experimental support for a "weighted Goldilocks" scenario for GP incorporation, wherein the virus exploits both random and nonrandom mechanisms to buffer infectivity over a wide range of GP expression levels. These mechanistic data are relevant to ongoing efforts to develop antiviral strategies targeting PRF frequency and/or HIV-1 virion maturation.

Quantification of Bovine and Porcine Heparins Utilizing the Heparin Red Assay, Applications in the Study of Pharmacokinetics and Pharmacodynamics

Introduction Currently there is a major interest in developing bovine intestinal heparin for anticoagulant purposes. It is well known that bovine intestinal heparins exhibit a relatively lower potency than porcine intestinal heparins. The pharmacokinetic behavior of heparins has traditionally been based on antithrombin-dependent anti-Xa and anti-IIa activities. Heparin Red is a polycationic substance whose intrinsic fluorescence is quenched upon binding to heparin. As such, assays utilizing Heparin Red detect all heparin oligosaccharides in a sample: those that bind to AT and produce anti-Xa and anti-IIa activities, and those that do not. These non-AT binding heparin oligosaccharides can still impact coagulation through their interaction with heparin cofactor II, TFPI and platelets. Thus, the pharmacokinetic behavior of heparin (half-life, etc.) as determined using a Heparin Red assay may more closely reflect the clinical behavior of heparin (antithrombotic, bleeding) than pharmacodynamic measures where heparin levels are determined based only on AT-dependent activities (anti-Xa or anti-IIa). Methods Primates were administered PMH (Medefil, Glendale Heights, IL) or BMH (KinMaster, Passo Fundo, Brazil) at a dose of 100 anti-Xa U/kg IV. Blood samples were collected prior to and at 15, 30, 60 and 120 minutes post-heparin administration. Blood samples were centrifuged to produce platelet poor plasma which was aliquoted and stored frozen until analysis. Heparin levels were assessed using a chromogenic anti-Xa assay and a Heparin Red assay relative to product-specific calibration curves. Pharmacokinetic parameters were assessed using a non-compartmental model. Results Circulating functional drug levels based on anti-Xa activity were the same in PMH and BMH-treated primates. Peak levels of 1.45 ± 0.11 and 1.48 ± 0.08 U/ml were observed in PMH and BMH-treated primates, respectively. Using drug levels determined by anti-Xa assay, AUCs for bovine and porcine heparin treated animals were calculated to be 111.5 ± 11.0 and 108.8 ± 26.7 U*min*ml-1, respectively. By Heparin Red assay, peak circulating heparin levels were higher following BMH administration (10.7 ± 0.5 vs. 9.0 ± 0.2 mg/ml; t-test p<0.001) and the AUC for BMH-treated primates was approximately 22% larger than for PMH-treated primates (728.2 ± 35.3 vs. 594.9 ± 5.4 mg*min*ml-1; Mann-Whitney test p=0.029). Conclusions BMH, when administered at equivalent anti-Xa unit doses, produces comparable pharmacodynamic effects as PMH despite the presence of higher circulating GAG levels as measured by the Heparin Red assay. Measurement of BMH levels using the Heparin Red assay may be useful for studying its absolute pharmacokinetics and optimization of protamine sulfate dosage for its neutralization.

Rapamycin Ameliorates Age‐Associated Intervertebral Disc Degeneration in Male Marmosets

Introduction Age-associated intervertebral disc degeneration (IDD) is a major contributor to low back pain and physical disability in the US. Rapamycin has been shown in several animal models, including mammalian, to increase lifespan and decrease aging and age-associated pathologies, including cancer, cardiac and neurodegenerative diseases. Rapamycin is an FDA approved drug that inhibits mammalian target of rapamycin (mTOR) complex 1, which is a master regulator of cell growth and proliferation signals. It has not yet been determined if rapamycin treatment influences age-associated IDD. Hence, the goal of this study was to determine the effect of a one-year oral rapamycin treatment on IDD progression in old marmosets. Methods Old male marmosets were given rapamycin using a delivery system that microencapsulates rapamycin in a eudagrit coating and is mixed into yogurt for feeding via a syringe to accurately account for each daily dose. As control, old animals were given empty eudagrit capsules delivered the same way, for one year. Spine specimens were collected from young (<5 years old), old (> 10 years old) + vehicle, and old + rapamycin marmosets. Treatment was given for one year prior to sacrifice. Cellularity and histological structure were assessed using H&E staining. Disc matrix anabolism was assessed by aggrecan immunohistochemistry (IHC) and DMMB assay to quantify sulfated glycosaminoglycans. Disc matrix catabolism was assessed via WB for aggrecan fragments. Autophagy was measured via WB for established autophagy protein markers. Student's t-test was used for significance, n=3-4. Results Rapamycin treatment significantly decreased histological scoring for IDD characteristics compared to control old marmosets (Figure 1a-c.) Rapamycin treatment significantly increased older marmosets GAG levels and increased aggrecan IHC staining compared to control (Figure 2a, b). Similarly, rapamycin treatment decreased ADAMTS-mediated and MMP-mediated aggrecan fragmentation (Figure 2c, d). Rapamycin treatment did not significantly affect the autophagy markers LC3-II, Atg7, beclin-1, or Atg12-Atg5, but it did significantly decrease p62 expression. Discussion Overall, rapamycin treatment significantly improved disc catabolism, increasing aggrecan production, and histological score in old marmosets' discs. These results suggest that one-year daily rapamycin treatment delayed IDD progression in older male marmosets. Interestingly, autophagy related proteins were not significantly increased in older male marmosets treated with rapamycin, although rapamycin treatment has been shown to increase autophagy in other tissues, as it is a known inhibitor of mTOR. This implies either rapamycin given orally cannot diffuse into the disc, and systemic effects of rapamycin were observed in other assays, or autophagy is not regulated by rapamycin in the disc. This unique finding warrants further study. Significance This is the first study to investigate the in vivo effects of rapamycin treatment on age-associated IDD in a nonhuman primate model and suggests rapamycin might be a viable treatment to improve IDD characteristics in patients.

Predicting Gag, Discomfort, and Laryngeal Visualization in Patients Undergoing Flexible Laryngoscopy with Stroboscopy.

To investigate potential associations between the Predictive Gagging Survey (PGS) with patient experience of gag and discomfort as well as provider perception of patient gag and level of laryngeal visualization during flexible laryngoscopy with stroboscopy (FL-S). A total of 53 adult patients undergoing FL-S were recruited for this prospective non-controlled study. PGS was completed before FL-S. Patients rated perceived level of gag and discomfort on a 10-point severity scale after FL-S. Additionally, providers completed a Gagging Severity Index (GSI) reflecting their impression of patient gag and level of laryngeal visualization following FL-S. Spearman rank correlation coefficients were used to assess associations. There was a positive association with PGS score and patient perception of gagging (0.34; P = .013) and patient perception of discomfort (0.38; P = .005). No significant association was found between PGS score and provider GSI (-0.12; P = .39) or level of laryngeal visualization (0.15; P = .29). A negative association was found between level of laryngeal visualization and patient perception of gagging (-0.34; P = .012) and discomfort (-0.44; P = .001). No significant differences were found between current and former smokers compared to never smokers for GSI or patient-perceived gag or discomfort. While not predictive of GSI or level of laryngeal visualization, the PGS was found to be a useful tool in predicting patient experience of gagging and discomfort during FL-S, further reinforcing the subjective experience of this procedure. Use of the PGS may be helpful in identifying specific candidates who may struggle with subjective discomfort or gagging during FL-S for future studies considering interventions to manage and meaningfully decrease discomfort. Having such an instrument is important given the low number of individuals who struggle with discomfort during the exam.

Treatment effects of intra-articular triamcinolone acetonide in an equine model of recurrent joint inflammation.

Intra-articular triamcinolone acetonide is a widely used treatment for joint inflammation despite limited scientific evidence of its efficacy. To investigate if intra-articular triamcinolone acetonide has sustained anti-inflammatory effects using an equine model of repeated joint inflammation. Randomised controlled experimental study. For three consecutive cycles 2weeks apart, inflammation was induced in both middle carpal joints of eight horses by injecting 0.25ng lipopolysaccharide (LPS). After the first LPS injection only, treatment with 12mg triamcinolone acetonide (TA) followed in one randomly assigned joint, while the contralateral joint was treated with sterile saline (control). Clinical parameters (composite welfare scores, joint effusion, joint circumference) were recorded and synovial fluid samples were analysed for various biomarkers (total protein, WBCC; PGE2 ; CCL2; TNFα; MMP; GAGs; C2C; CPII) at fixed timepoints (post injection hours 0, 8, 24, 72 and 168). The effects of time and treatment on clinical and synovial fluid parameters and the presence of time-treatment interactions were tested using a linear mixed model for repeated measures with horse as a random effect, and time and treatment as fixed effects. The TA treated joints showed significantly higher peak synovial GAG concentrations (Difference in means 283.1875µg/mL, 95% CI 179.8, 386.6, P<0.000), and PGE2 levels (Difference in means 77.8025pg/mL, 95% CI 21.2, 134.4, P<0.007) after the first inflammation induction. Significantly lower TP levels were seen with TA treatment after the second induction (Difference in means -7.5g/L, 95% CI -14.8, -0.20, P<0.04) . Significantly lower WBCC levels were noted with TA treatment after the first (Difference in means -23.7125×109 cells/L, 95% CI -46.7, -0.7, P<0.04) and second (Difference in means -35.95×109 cells/L, 95% CI -59.0, -12.9, P<0.002) inflammation inductions. Significantly lower general MMP activity was also seen with TA treatment after the second inflammation inductions (Difference in means -51.65 RFU/s, 95% CI -92.4, -10.9, P<0.01). This experimental study cannot fully reflect natural joint disease. In this model, intra-articular TA seems to have some anti-inflammatory activity (demonstrated by reductions in TP, WBCC and general MMP activity) up to 2weeks post treatment but not at 4weeks. This anti-inflammatory effect appeared to outlast a shorter-lived, potentially detrimental effect illustrated by increased synovial GAG and PGE2 levels after the first induction.