Higher Levels Of Glycosaminoglycans Research Articles

Some pathogenetic relationships in gastroesophageal reflux disease in children

Objective. To study the relationship of the upper gastrointestinal tract acidity index and collagen metabolites and proteoglycans in children with GERD. Materials and methods. 62 children with GERD were observed. The control group included 32 children. All children underwent esophagogastroduodenoscopy with parietal endoscopic pH-metry. The content of free hydroxyproline (FHOP), peptide-bonded hydroxyproline (PSHOP), glycosaminoglycans (GAG), as well as a-mannosidase activity in gastric juice were studied. Results. The hyperacidity of the pH in the esophagus, the gastric “lake” and the body of the stomach in children with GERD was revealed. A relationship between hyperacidity and the breakdown of collagen in the mucous membrane of the gastroesophageal area demonstrated by high levels of GAG, FHOP, and PSНOP a-mannosidase in the gastric juice has been shown. Conclusions. In children with GERD, the dynamic balance of aggressive-protective factors in the gastroduodenal area is disturbed. As a result of it, degradation of collagen in the mucous and submucous layer of the esophagus and stomach is observed.

Facial skin ageing: Key concepts and overview of processes.

IntroductionThe face is a cosmetically sensitive region where the process of ageing is most clearly manifested. With increased focus on anti‐ageing and longevity, more anti‐senescent treatments are being proposed despite limited evidence. This study outlines the pathways and mechanisms underpinning the biological process of ageing in the face.MethodsComprehensive searches of MEDLINE, EMBASE, Cochrane Library and CINAHL from inception to 2020. Inclusion criteria included all empirical human research studies specific to facial ageing features, written in the English language.ResultsA total of 65 papers met inclusion criteria for analysis. Pathways were subdivided into intrinsic and extrinsic senescence mechanisms. Intrinsic pathways included genetics, generation of reactive oxygen species and hormonal changes. Extrinsic pathways included photoageing and damage to skin layers. The combined intrinsic and extrinsic pathway alterations result in wrinkles, higher laxity, slackness and thinning of the skin. Skin functions such as barrier immune function, wound healing, thermoregulation and sensory function are also impaired.ConclusionThe ageing process is unique to the individual and depends on the interplay between an individual's genetics and external environmental factors. Through understanding the molecular and cellular mechanisms, an appreciation of the consequent structural and functional changes can be achieved. Based on this knowledge, further research can focus on how to slow or impede the ageing process and identify specific targets to develop and evolve new treatment strategies.

Mucopolysaccharidoses diagnosis in the era of enzyme replacement therapy in Egypt

BackgroundUndegraded glycosaminoglycans (GAGs) induced by deficiency of enzymes are the primary cause of mucopolyscchardoses. Mucopolysacchardoses (MPS) are a group of rare lysosomal storage diseases (LSD). The quantification of a specific enzymatic activity is needed for accurate diagnosis. The objectives of this work were: first, to continue the study of mucopolysacchardoses disease in Egypt after the start of using the enzyme replacement therapy (ERT). Second, to define the commonest types among our population after 18 years experience with the disease. Third, to compare the different MPS types’ distribution, diagnosed after the start of the ERT, to identify the impact of using ERT on the number and type of diagnosed patients. MethodUrinary GAGs were measured for all referred cases followed by two-dimensional electrophoretic separation for cases with high levels of GAGs; the specific enzyme activity was assayed for each type depending on the abnormal electrophoretic pattern obtained. Clinically suspected cases of Morquio syndrome were directly subjected to measuring the specific enzyme. ResultsOut of 1448 suspected cases, 622 (42.9%) MPS patients were diagnosed revealing the following distribution: MPS I (172, 27.7%), MPS II (57, 9.1%), MPS III [(177, 28.5%: 134 type B and 43 types A, C or D)], MPS IVA (124, 19.9%), MPS VI (90, 14.5%) and MPS VII (2, 0.3%). MPS III was the most commonly diagnosed type followed by MPS I and MPS IVA. MPS IVA represented the most common type receiving treatment, followed by MPS I, MPS II and MPS VI. ConclusionThe presence of treatment encouraged the affected families and physicians to seek diagnosis. MPS III was the commonest type among our studied group after 7 years of diagnosis, while MPS IVA was the commonest type receiving treatment.

High Levels of Glycosaminoglycans in the Urines of Children with Attention-Deficit/Hyperactivity Disorder (ADHD).

Attention-deficit/hyperactivity disorder (ADHD) is a common neurobehavioral/neurodevelopmental disorder. Some early studies indicated that increased intake of added sugars might have a role in ADHD. In the present study, we tested this possibility by evaluating the urinary excretion of oligosaccharides and glycosaminoglycans (GAGs) in ADHD and control subjects. Forty ADHD subjects matched with 34 controls were enrolled in the study. The subjects underwent a standardized dietary regimen. The urine levels of oligosaccharides and GAGs were quantified biochemically, and their covariance and association were evaluated statistically. Fructose (21/40, 52.5%), maltose (26/40, 65%), galactose (30/40, 75%), and lactose (38/40, 95%) excretions were frequently found in the urine of ADHD subjects (p < 0.05), an excretion which does not occur normally. Furthermore, these subjects showed a pathologic tGAG (glycosaminoglycan) excretion (40/40, 100%). The present study supportsthe thesis that carbohydrate metabolism differs in ADHD subjects compared with control subjects.

Matrix composition in 3-D collagenous bioscaffolds modulates the survival and angiogenic phenotype of human chronic wound dermal fibroblasts

There is a substantial need for new strategies to stimulate cutaneous tissue repair in the treatment of chronic wounds. To address this challenge, our team is developing modular biomaterials termed “bead foams”, comprised of porous beads synthesized exclusively of extracellular matrix (ECM) and assembled into a cohesive three-dimensional (3-D) network. In the current study, bead foams were fabricated from human decellularized adipose tissue (DAT) or commercially-sourced bovine tendon collagen (COL) to explore the effects of ECM composition on human wound edge dermal fibroblasts (weDF) sourced from chronic wound tissues. The DAT and COL bead foams were shown to be structurally similar, but compositionally distinct, containing different levels of glycosaminoglycan content and collagen types IV, V, and VI. In vitro testing under conditions simulating stresses within the chronic wound microenvironment indicated that weDF survival and angiogenic marker expression were significantly enhanced in the DAT bead foams as compared to the COL bead foams. These findings were corroborated through in vivo assessment in a subcutaneous athymic mouse model. Taken together, the results demonstrate that weDF survival and paracrine function can be modulated by the matrix source applied in the design of ECM-derived scaffolds and that the DAT bead foams hold promise as cell-instructive biological wound dressings. Statement of SignificanceBiological wound dressings derived from the extracellular matrix (ECM) can be designed to promote the establishment of a more permissive microenvironment for healing in the treatment of chronic wounds. In the current work, we developed modular biomaterials comprised of fused networks of porous ECM-derived beads fabricated from human decellularized adipose tissue (DAT) or commercially-available bovine collagen. The bioscaffolds were designed to be structurally similar to provide a platform for investigating the effects of ECM composition on human dermal fibroblasts isolated from chronic wounds. Testing in in vitro and in vivo models demonstrated that cell survival and pro-angiogenic function were enhanced in the adipose-derived bioscaffolds, which contained higher levels of glycosaminoglycans and collagen types IV, V, and VI. Our findings support that the complex matrix composition within DAT can induce a more pro-regenerative cellular response for applications in wound healing.

Glycol chitosan/oxidized hyaluronic acid hydrogels functionalized with cartilage extracellular matrix particles and incorporating BMSCs for cartilage repair

In this article, we fabricated a bioactive hydrogel composed of glycol chitosan (G-CS) and oxidized hyaluronic acid (OHA) via Schiff base reaction. Cartilage extracellular matrix (ECM) particles with different concentrations were used to functionalize G-CS/OHA (S1) hydrogel. The results demonstrated that S3 (G-CS/OHA/ECM 2% w/v) hydrogel exhibited the most suitable compression strength and provided the optimal environment for proliferation of bone marrow mesenchymal stem cells (BMSCs). To assess the chondroinductivity of ECM in vitro, we compared the chondrogenesis of BMSCs in S1 (G-CS/OHA) and S3 (G-CS/OHA/ECM 2% w/v) hydrogels. The results confirmed that the higher levels of glycosaminoglycans (GAGs) and collagen type II (COL II) were accumulated in S3 hydrogel. In vivo, cartilage defects of rats generated most mature tissue within BMSCs-laden S3 hydrogel (S3/BMSCs group). The tissues were more integrative and contained higher levels of COL II and GAGs compared to the other groups. All these results suggested that the G-CS/OHA hydrogel functionalized with ECM particles is a good candidate biomaterial to be applied in cartilage tissue engineering.

Differences in elements between intact and disrupted human ligamenta capitum femorum.

To compare the element compositions between intact (i.e., intact throughout its length) and disrupted (i.e., ligament no longer attached to the attachment) ligaments, the contents of elements in the human ligamenta capitum femorum (LCF) were analyzed by inductively coupled plasma-atomic emission spectrometry. Histological and immunohistological assessments were also performed in both groups. The subjects were 8 men and 32 women. Trace element analyses showed that the sulfur and iron contents were significantly greater in the intact group than in the disrupted group, while the phosphorus and magnesium contents were significantly smaller in the intact group than in the disrupted group. The calcium and zinc contents were smaller in the intact group than in the disrupted group, with no significant differences. Histologically, there were fibrocartilage cells and extracellular matrix metachromasia in ligaments of the intact group. In contrast, fibrocartilage cells disappeared, and fat cells appeared instead of collagen fibrils in ligaments of the disrupted group. The LCFs of the intact group were immunohistologically positive for all components examined including collagens, glycosaminoglycans, and proteoglycans. The increase in sulfur suggested the presence of high glycosaminoglycan levels associated with fibrocartilaginous metaplasia in the ligament by compressive force. The reduction in iron may show a decreased number of blood vessels in the synovium after ligament disruption. The increases in phosphorus, magnesium, and calcium are indicative of degenerative changes including calcification and ossification. We conclude that differences in the contents of elements between intact and disrupted LCFs indicate degenerative alterations to the ligament structure after disruption.

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP- cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.

Age-related adaptation of bone-PDL-tooth complex: Rattus-Norvegicus as a model system.

Functional loads on an organ induce tissue adaptations by converting mechanical energy into chemical energy at a cell-level. The transducing capacity of cells alters physico-chemical properties of tissues, developing a positive feedback commonly recognized as the form-function relationship. In this study, organ and tissue adaptations were mapped in the bone-tooth complex by identifying and correlating biomolecular expressions to physico-chemical properties in rats from 1.5 to 15 months. However, future research using hard and soft chow over relevant age groups would decouple the function related effects from aging affects. Progressive curvature in the distal root with increased root resorption was observed using micro X-ray computed tomography. Resorption was correlated to the increased activity of multinucleated osteoclasts on the distal side of the molars until 6 months using tartrate resistant acid phosphatase (TRAP). Interestingly, mononucleated TRAP positive cells within PDL vasculature were observed in older rats. Higher levels of glycosaminoglycans were identified at PDL-bone and PDL-cementum entheses using alcian blue stain. Decreasing biochemical gradients from coronal to apical zones, specifically biomolecules that can induce osteogenic (biglycan) and fibrogenic (fibromodulin, decorin) phenotypes, and PDL-specific negative regulator of mineralization (asporin) were observed using immunohistochemistry. Heterogeneous distribution of Ca and P in alveolar bone, and relatively lower contents at the entheses, were observed using energy dispersive X-ray analysis. No correlation between age and microhardness of alveolar bone (0.7±0.1 to 0.9±0.2 GPa) and cementum (0.6±0.1 to 0.8±0.3 GPa) was observed using a microindenter. However, hardness of cementum and alveolar bone at any given age were significantly different (P<0.05). These observations should be taken into account as baseline parameters, during development (1.5 to 4 months), growth (4 to 10 months), followed by a senescent phase (10 to 15 months), from which deviations due to experimentally induced perturbations can be effectively investigated.

Low Carotid Calcium Score Is Associated With Higher Levels of Glycosaminoglycans, Tumor Necrosis Factor-Alpha, and Parathyroid Hormone in Human Carotid Plaques

Computed tomography (CT) is used to study coronary artery plaques, but little is known about its potential to characterize plaque composition. This study assesses the relation between carotid calcium score (CCS) by CT and plaque composition, namely extracellular matrix, inflammatory mediators, and calcium metabolites. Thirty patients with significant carotid stenosis underwent preoperative CT. CCS was quantified by Agaston calcium score. Plaque components were studied histologically and biochemically (collagen, elastin, and glycosaminoglycans). Fraktalkine, interferon-γ, interleukin-10, interleukin-12 p70, interleukin-1β, interleukin-6, monocyte chemoattractant protein-1, platelet-derived growth factor-AB/BB, RANTES and tumor necrosis factor-α, and parathyroid hormone were measured using Luminex technology. Plaques with CCS ≥400 had more calcium (P=0.012), less glycosaminoglycan (P=0.002), tumor necrosis factor-α (P=0.013), and parathyroid hormone (P=0.028) than those with CCS <400. CCS correlated with plaque content of calcium (r=0.62; P<0.001) and inversely with glycosaminoglycan (r=-0.49; P=0.006) and tumor necrosis factor-α (r=-0.56; P=0.001). Human carotid plaques with high CCS are richer in calcium and have lower amounts of glycosaminoglycan, parathyroid hormone, and tumor necrosis factor-α, which is one of the main proinflammatory cytokines involved in atherosclerosis. This suggests that CCS not only reflects the degree of calcification, but also other important biological components relevant for stability such as inflammation.

Chondrogenesis on BMP‐6 loaded chitosan scaffolds in stationary and dynamic cultures

We originally investigated the suitability of chitosan scaffolds loaded with bone morphogenetic protein 6 (BMP-6) in both stationary and dynamic conditions for cartilage tissue engineering. In the first part of the present study, ATDC5 murine chondrogenic cells were seeded in chitosan and BMP-6 loaded chitosan scaffolds and cultured for 28 days under static conditions. In the following part, we examined the influence of dynamic cultivation conditions over BMP-6 loaded chitosan scaffolds by using rotating bioreactor with perfusion (RCMW). Tissue engineered constructs were characterized by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) assay, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and biochemical assays for glycosaminoglycans (GAG) deoxyribonucleic acid (DNA) and collagen Type II quantification. At the end of 4 weeks static incubation period high levels of GAG (21.22 mg/g dry weight), DNA amounts (1.37 mg/g dry weight) and collagen Type II amounts (1.94 microg/g dry weight) were achieved for BMP-6 loaded chitosan scaffolds compared to chitosan scaffolds. However, the results obtained from morphological observations suggested hypertrophic differentiation of ATDC5 cells in the presence of BMP-6 under stationary conditions. The influence of mechanical stimulation appeared significantly with differentiated cells, cultured under dynamic conditions, showing the effect of retaining their phenotypes without hypertrophy.

Glycosaminoglycans as Anticoagulants in Mucopolysaccharidosis Type I (MPS I).

MPS IH (Hurler syndrome) is caused by severe mutations in a-L-iduronidase gene, leading to accumulation of complex sugars, termed glycosaminoglycans (GAG), in tissues. The disorder is lethal unless treated with hematopoietic stem cell transplantation (HSCT). When compared to other recipients of HSCT, MPS I patients are well-known to have an increased incidence of bleeding complications in peri-transplant period. To investigate whether this bleeding propensity is related to the enzyme deficiency and GAG accumulation we evaluated five MPS IH patients (2 females and 3 males, mean age: 1.1 years) prior to any therapy. We observed that three of the five patients had an elevated activated partial thromboplastin time (aPTT, normal range 22–35 seconds). As no heparin was used in any of these patients, we reasoned that the accumulation of heparan sulfate, a hallmark of MPS IH, may serve as a heparin-like anticoagulant. Should this assumption be correct, higher levels of GAG would be expected in the three patients with the elevated aPTT. This, in fact, was what we have observed (Figure), suggesting that dose - effect relationship exists between whole body GAG load (assessed as milligrams of urinary GAG normalized to grams of creatinine; x-axis) and level of anticoagulation (aPTT in seconds; y-axis). [Display omitted] In this context, it is important to note that the patient with the second highest value of urinary GAG and aPTT experienced pulmonary bleeding on day 12 after HSCT requiring intubation and ventilatory support for 25 days. We hypothesized that endogenous GAG heparan sulfate exerts its antithrombotic effect through antithrombin III-mediated inhibition of factor Xa, as in clinically used low molecular weight heparinoid, danaparoid. To test this hypothesis, we have assessed anti-Xa levels in mice with a-L-iduronidase deficiency, an animal model of MPS IH. As expected, the anti-Xa level were not significantly different in wild type C57Bl/6 mice (N=3) and heterozygous mice (N=6): 0.11±0.005 IU/cc plasma versus 0.10±0.004 IU/cc plasma (p=0.2). Anti-Xa values of MPSI mutant mice (N=14), however, were significantly elevated when compared to the age-matched sex-matched wild type controls (0.19±0.05 IU/cc plasma versus 0.11±0.005 IU/cc plasma; p<0.001). In addition, we have observed that GAG anticoagulant activity was neutralized by addition of the heparinase enzyme (MPS I, N=3, 0.16±0.02 IU/cc plasma versus MPS I + heparinase, N =3, 0.10±0.002 IU/cc plasma; p<0.01). In conclusion, the current study links GAG produced in excess as a result of a-L-iduronidase deficiency and a hemostatic defect in MPS I mice and humans. The implications of the current study are that an impaired hemostasis may exist in untreated patients with MPS IH, and that MPS IH patients with high urinary GAG are likely to experience bleeding complications. This information may be useful in identifying MPS IH patients at higher risk for bleeding, especially in the peri-transplant period, when GAG levels have not yet normalized and additional, HSCT-related factors (e.g., thrombocytopenia and hepatic dysfunction affecting clotting factors) contribute to the high risk of bleeding.

Glycosaminoglycans as Anticoagulants in Mucopolysaccharidosis Type I (MPS I).

MPS IH (Hurler syndrome) is caused by severe mutations in a-L-iduronidase gene, leading to accumulation of complex sugars, termed glycosaminoglycans (GAG), in tissues. The disorder is lethal unless treated with hematopoietic stem cell transplantation (HSCT). When compared to other recipients of HSCT, MPS I patients are well-known to have an increased incidence of bleeding complications in peri-transplant period. To investigate whether this bleeding propensity is related to the enzyme deficiency and GAG accumulation we evaluated five MPS IH patients (2 females and 3 males, mean age: 1.1 years) prior to any therapy. We observed that three of the five patients had an elevated activated partial thromboplastin time (aPTT, normal range 22–35 seconds). As no heparin was used in any of these patients, we reasoned that the accumulation of heparan sulfate, a hallmark of MPS IH, may serve as a heparin-like anticoagulant. Should this assumption be correct, higher levels of GAG would be expected in the three patients with the elevated aPTT. This, in fact, was what we have observed (Figure), suggesting that dose - effect relationship exists between whole body GAG load (assessed as milligrams of urinary GAG normalized to grams of creatinine; x-axis) and level of anticoagulation (aPTT in seconds; y-axis). [Display omitted] In this context, it is important to note that the patient with the second highest value of urinary GAG and aPTT experienced pulmonary bleeding on day 12 after HSCT requiring intubation and ventilatory support for 25 days. We hypothesized that endogenous GAG heparan sulfate exerts its antithrombotic effect through antithrombin III-mediated inhibition of factor Xa, as in clinically used low molecular weight heparinoid, danaparoid. To test this hypothesis, we have assessed anti-Xa levels in mice with a-L-iduronidase deficiency, an animal model of MPS IH. As expected, the anti-Xa level were not significantly different in wild type C57Bl/6 mice (N=3) and heterozygous mice (N=6): 0.11±0.005 IU/cc plasma versus 0.10±0.004 IU/cc plasma (p=0.2). Anti-Xa values of MPSI mutant mice (N=14), however, were significantly elevated when compared to the age-matched sex-matched wild type controls (0.19±0.05 IU/cc plasma versus 0.11±0.005 IU/cc plasma; p<0.001). In addition, we have observed that GAG anticoagulant activity was neutralized by addition of the heparinase enzyme (MPS I, N=3, 0.16±0.02 IU/cc plasma versus MPS I + heparinase, N =3, 0.10±0.002 IU/cc plasma; p<0.01). In conclusion, the current study links GAG produced in excess as a result of a-L-iduronidase deficiency and a hemostatic defect in MPS I mice and humans. The implications of the current study are that an impaired hemostasis may exist in untreated patients with MPS IH, and that MPS IH patients with high urinary GAG are likely to experience bleeding complications. This information may be useful in identifying MPS IH patients at higher risk for bleeding, especially in the peri-transplant period, when GAG levels have not yet normalized and additional, HSCT-related factors (e.g., thrombocytopenia and hepatic dysfunction affecting clotting factors) contribute to the high risk of bleeding.

Comparative ultrastructural analysis of different regions of two digital flexor tendons of pigs

Tendons are parallel arrays of collagenous fibers which are specialized in resisting and transmitting tensile forces. In this work we examined the structure of the superficial digital flexor tendon (SDFT) and the deep digital flexor tendon (DDFT) of pigs, which are considered “wrap around” tendons and so receive compression and tension forces. In both tendons, fibrocartilaginous areas were observed in the regions subjected to compression plus frictional loading. Histological and ultrastructural analyses of the tensional region showed an extracellular matrix (ECM) rich in collagen bundles, that were all arranged in the same direction. Fibroblasts were seen closely associated with the collagen bundles. Chondrocyte-like cells and high levels of glycosaminoglycans (GAGs) were observed in the compressional regions. The collagen bundles in the compressional region were arranged in several directions and were associated with proteoglycans (PGs). The crimp pattern detected in the tensional region showed that the collagen fibrils were ordered aggregates which formed helical superstructures.

Purified human plasma glycosaminoglycans limit oxidative injury induced by iron plus ascorbate in skin fibroblast cultures

A number of studies in vivo and in vitro showed that high levels of glycosaminoglycans (GAGs) are found as a consequence of free radical damage. The GAG over production may represent an endogenous mechanism capable to limit oxidative damage. Based on these hypotheses, the aim of this study was to evaluate the antioxidant property of GAGs of human origin in fibroblast cultures. Purified human plasma GAGs were added to the fibroblast cultures in which oxidative stress was induced by the oxidizing system employing iron (Fe 2+) plus ascorbate. We assessed cell death, lactate dehydrogenase activity, membrane lipid peroxidation, DNA damage, protein oxidation, hydroxyl radical (OH ) generation and endogenous antioxidant depletion. The exposure of fibroblasts to FeSO 4 produced cell death and increased OH production. It also caused DNA strand breaks and protein oxidation as shown by the DNA fragment analysis and protein carbonyl content, respectively. In addition, FeSO 4 enhanced lactate dehydrogenase activity and lipid peroxidation while decreased antioxidant defences. Purified human GAGs, at three different doses, reduced cell death, limited DNA fragmentation and protein oxidation, decreased OH generation and lactate dehydrogenase activity, inhibited lipid peroxidation and improved endogenous antioxidant defences. These results further support the hypothesis that these molecules may function as antioxidants.

A model of mucopolysaccharidosis IIIB (Sanfilippo syndrome type IIIB): N‐acetyl‐α‐D‐glucosaminidase deficiency in Schipperke dogs

Mucopolysaccharidosis III (MPS III) is characterized by lysosomal accumulation of the glycosaminoglycan (GAG) heparan sulphate (HS). In humans, the disease manifests in early childhood, and is characterized by a progressive central neuropathy leading to death in the second decade. This disease has also been described in mice (MPS IIIA and IIIB), dogs (MPS IIIA), emus (MPS IIIB) and goats (MPS IIID). We now report on dogs with naturally occurring MPS IIIB, detailing the clinical signs, diagnosis, histopathology, tissue enzymology and substrate levels. Two 3-year-old Schipperke dogs were evaluated for tremors and episodes of stumbling. Examination of the animals found signs consistent with cerebellar disease including dysmetria, hind limb ataxia and a wide-based stance with truncal swaying. There were mildly dystrophic corneas and small peripheral foci of retinal degeneration. Magnetic resonance imaging of the brain and skeletal radiographs were normal. Intracytoplasmic granules were found in the white cells of peripheral blood and cerebral spinal fluid, and in myeloid lineages in bone marrow. Electrophoresis of urinary GAGs indicated the presence of HS, while assays of cultured fibroblasts found N-acetyl-alpha-D-glucosaminidase (Naglu) activity of between 4.3% and 9.2% of normal. Owing to neurological deterioration, both dogs were euthanized, and post-mortem examinations were performed. Biochemical studies of liver and kidney from both animals demonstrated profound deficiency of Naglu activity and abnormally high GAG levels. Pathology of the brain included severe cerebellar atrophy, Purkinje cell loss, and cytoplasmic vacuolation in neurons and perithelial cells throughout the central nervous system. Pedigree analyses and Naglu levels of family members supported an autosomal recessive mode of inheritance. Using an obligate heterozygote, a breeding colony has been established to aid in understanding the pathogenesis of MPS IIIB and testing of potential therapies.

Fetal tracheal augmentation with cartilage engineered from bone marrow-derived mesenchymal progenitor cells

Background/Purpose: The authors have described previously the use of engineered fetal cartilage in a large animal model of fetal tracheal repair. This study was aimed at comparing cartilage engineered from bone marrow-derived stromal cells (BMSC) to native and engineered cartilage, in this model. Methods: Ovine BMSC were expanded in vitro, seeded onto biodegradable scaffolds, and maintained in transforming growth factor beta 1 (TGF-β1)-supplemented medium for 3 months (group I). Identical scaffolds were seeded with fetal chondrocytes (group II). All constructs were analyzed in vitro, implanted into fetal tracheas, and harvested after birth for further analysis. Results: There were no differences in survival between the groups. All BMSC-based constructs exhibited chondrogenic differentiation. Matrix analyses in vitro showed that both groups had similar levels of glycosaminoglycans (GAG) and type II collagen (C-II), but lower levels of elastin when compared with native fetal cartilage. Yet, compared with group II, group I had higher levels of GAG, equal levels of C-II, and lower levels of elastin. However, remodeling resulted in no differences between the 2 groups in any of these variables in vivo. Conclusions: The bone marrow may be a useful cell source for cartilage engineering aimed at the surgical repair of severe congenital tracheal anomalies, such as tracheal atresia and agenesis, in utero.

Glycosaminoglycan binding properties of natural venezuelan equine encephalitis virus isolates.

Equine-virulent, epidemic/epizootic strains of Venezuelan equine encephalitis (VEE) virus (VEEV) arise via mutation of progenitor enzootic strains that replicate poorly in equines. Sequencing studies have implicated positively charged amino acids on the surface of the E2 envelope glycoprotein in the acquisition of equine virulence and viremia potential, suggesting that changes in binding to cell surface glycosaminoglycans (GAGs) may mediate VEE emergence. Therefore, we evaluated the binding of natural enzootic and epizootic VEEV isolates to Chinese hamster ovary (CHO) cells expressing normal, high levels of GAGs as well as to mutant CHO cells lacking GAG expression. Binding to GAGs was not consistently associated with the epizootic phenotype, and cell culture passages resulted in increased GAG binding. The low levels of GAG binding exhibited by some low-passage, equine-virulent subtype IC VEEV strains indicate that the positive-charge E2 mutations implicated in VEE subtype IC emergence are not artifacts of laboratory passage and suggest that GAG binding does not play a major role in mediating VEE emergence. The increased GAG binding exhibited by VEEV strain CPA201 from the 1993 Mexican epizootic, when compared to that of closely related enzootic subtype IE strains, was shown to result from a Glu-to-Lys mutation at position 117 of the E2 envelope glycoprotein.

Envelope-dependent, cyclophilin-independent effects of glycosaminoglycans on human immunodeficiency virus type 1 attachment and infection.

Cell surface glycosaminoglycans (GAGs), in particular heparan sulfate (HS), have been proposed to mediate the attachment of human immunodeficiency virus type 1 (HIV-1) to target cells prior to virus entry, and both the viral gp120 envelope protein and virion-associated cyclophilin A (CypA) have been shown to directly interact with HS and its analogues. To determine the role of GAGs in HIV attachment and infection, we generated HIV-susceptible derivatives of CHO cell lines that either express high levels of GAGs (CHO-K1) or lack GAGs (pgsA745). Using a panel of HIV-1 envelopes, we found that cell surface GAG-mediated effects on virion attachment and infection vary in an envelope strain-dependent but coreceptor-independent manner. In fact, cell surface GAG-mediated enhancement of infection is confined to isolates that contain a highly positively charged V3-loop sequence, while infection by most strains is apparently inhibited by the presence of GAGs. Moreover, the enhancing and inhibitory effects of polycations and polyanions on HIV-1 infection are largely dependent on the presence of cell surface GAGs. These observations are consistent with a model in which GAGs influence in vitro HIV-1 infection primarily by modifying the charge characteristics of the target cell surface. Finally, the effects of GAGs on HIV-1 infection are observed to an equivalent extent whether CypA is present in or absent from virions. Overall, these data exclude a major role for GAGs in mediating the attachment of many HIV-1 strains to target cells via interactions with virion-associated gp120 or CypA.

Accumulation of Abnormal Amounts of Glycosaminoglycans in Murine Mucopolysaccharidosis Type VII Neural Progenitor Cells Does Not Alter the Growth Rate or Efficiency of Differentiation into Neurons

Mucopolysaccharidosis type VII (MPS VII) results from deficiencies in the gene encoding the lysosomal enzyme β-glucuronidase (GUSB). To study how the genetic and biochemical defects of MPS disease affect neural cell populations, neural progenitor cells (NPCs) were isolated from MPS VII mice and normal littermates. After growth in culture, approximately 90% of cells from both genotypes were nestin positive, a marker for NPCs, and lacked markers associated with lineage commitment. The mutant NPCs contained elevated levels of undegraded glycosaminoglycans (GAGs), the substrate for GUSB. Transduction with a retrovirus-vector expressing normal GUSB resulted in correction of the biochemical defects. Because of the demonstrated roles that GAGs and proteoglycans have in NPC biology and neural development, we tested whether the alterations in GAG metabolism affected MPS VII NPC properties regulated by GAG-containing molecules. MPS VII NPC cultures had growth rates in response to FGF-2 that were similar to normal cultures and the efficiency of differentiation into neurons was the same as with normal cells. Thus, even though isolated NPCs accumulate abnormally high levels of GAGs, these two key developmental properties were not altered when the cells were examined outside the milieu of the diseased brain.