Crosslinked Layered Surfaces of Heparin and Poly(L-Lysine) Enhance Mesenchymal Stromal Cell Behavior in the Presence of Soluble Interferon Gamma.

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for the treatment of immune-mediated diseases. Culturing hMSCs on tissue culture plastic reduces their therapeutic potential in part due to the lack of extracellular matrix components. The aim of this study is to evaluate multilayers of heparin and poly(L-lysine) (HEP/PLL) as a bioactive surface for hMSCs stimulated with soluble interferon gamma (IFN-γ). Multilayers were formed, via layer-by-layer assembly, with HEP as the final layer and supplemented with IFN-γ in the culture medium. Multilayer construction and chemistry were confirmed using Azure A staining, quartz crystal microbalance, and X-ray photoelectron spectroscopy. hMSCs adhesion, viability, and differentiation, were assessed. Results showed that (HEP/PLL) multilayer coatings were poorly adhesive for hMSCs. However, performing chemical crosslinking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide significantly enhanced hMSCs adhesion and viability. The immunosuppressive properties of hMSCs cultured on crosslinked (HEP/PLL) multilayers were confirmed by measuring indoleamine 2,3-dioxygenase activity. Lastly, hMSCs cultured on crosslinked (HEP/PLL) multilayers in the presence of soluble IFN- γ successfully differentiated towards the osteogenic and adipogenic lineages as confirmed by Alizarin red, and oil-red O staining, as well as alkaline phosphatase activity. This study suggests that crosslinked (HEP/PLL) films can modulate hMSCs response to soluble factors, which may improve hMSCs-based therapies aimed at treating several immune diseases.

Dual-Targeting Glycol Chitosan/Heparin-Decorated Polypyrrole Nanoparticle for Augmented Photothermal Thrombolytic Therapy.

Near-infrared (NIR)-light-modulated photothermal thrombolysis has been investigated to overcome the hemorrhage danger posed by clinical clot-busting substances. A long-standing issue in thrombosis fibrinolytics is the lack of lesion-specific therapy, which should not be ignored. Herein, a novel thrombolysis therapy using photothermal disintegration of a fibrin clot was explored through dual-targeting glycol chitosan/heparin-decorated polypyrrole nanoparticles (GCS-PPY-H NPs) to enhance thrombus delivery and thrombolytic therapeutic efficacy. GCS-PPY-H NPs can target acidic/P-selectin high-expression inflammatory endothelial cells/thrombus sites for initiating lesion-site-specific thrombolysis by hyperthermia using NIR irradiation. A significant fibrin clot-clearance rate was achieved with thrombolysis using dual-targeting/modality photothermal clot disintegration in vivo. The molecular level mechanisms of the developed nanoformulations and interface properties were determined using multiple surface specific analytical techniques, such as particle size distribution, zeta potential, electron microscopy, Fourier-transform infrared spectroscopy (FTIR), wavelength absorbance, photothermal, immunofluorescence, and histology. Owing to the augmented thrombus delivery of GCS-PPY-H NPs and swift treatment time, dual-targeting photothermal clot disintegration as a systematic treatment using GCS-PPY-H NPs can be effectively applied in thrombolysis. This novel approach possesses a promising future for thrombolytic treatment.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

Mast Cell Regulation and Irritable Bowel Syndrome: Effects of Food Components with Potential Nutraceutical Use.

Mast cells are key actors in inflammatory reactions. Upon activation, they release histamine, heparin and nerve growth factor, among many other mediators that modulate immune response and neuron sensitization. One important feature of mast cells is that their population is usually increased in animal models and biopsies from patients with irritable bowel syndrome (IBS). Therefore, mast cells and mast cell mediators are regarded as key components in IBS pathophysiology. IBS is a common functional gastrointestinal disorder affecting the quality of life of up to 20% of the population worldwide. It is characterized by abdominal pain and altered bowel habits, with heterogeneous phenotypes ranging from constipation to diarrhea, with a mixed subtype and even an unclassified form. Nutrient intake is one of the triggering factors of IBS. In this respect, certain components of the daily food, such as fatty acids, amino acids or plant-derived substances like flavonoids, have been described to modulate mast cells' activity. In this review, we will focus on the effect of these molecules, either stimulatory or inhibitory, on mast cell degranulation, looking for a nutraceutical capable of decreasing IBS symptoms.

Amphiphilic multi-charged cyclodextrins and vitamin K co-assembly as a synergistic coagulant.

Balancing and neutralizing heparin dosing after surgeries and hemodialysis treatment is of great importance in medical and clinical fields. In this study, a series of new amphiphilic multi-charged cyclodextrins (AMCD)s as anti-heparin coagulants were designed and synthesized. The AMCD assembly was capable of selective heparin binding through multivalent bonding and showed a better neutralizing effect towards both unfractionated heparin and low molecular weight heparin than protamine in plasma. Meanwhile, an AMCD and vitamin K (VK) co-assembly was prepared to realize heparin-responsive VK release and provide a novel VK deficiency treatment for hemodialysis patients. This AMCD-VK co-assembly for heparin neutralization & vitamin K supplementation synergistic coagulation represents a promising candidate as a clinical anti-heparin coagulant.

A magnetically modified black phosphorus nanosheet-based heparin delivery platform for preventing DVT accurately.

A new heparin targeting delivery platform was developed based on iron oxide (Fe3O4) nanoparticles and polyethyleneimine (PEI) functionalized black phosphorus nanosheets (BP NSs). Both in and ex vivo studies suggested that this drug delivery platform (PEI/Fe3O4@BP NSs) possessed high heparin loading capacity (≈450%), accurate magnetic enrichment capacity, and good biocompatibility. With the aid of near-infrared (NIR) laser irradiation, this BP NS based delivery platform could further enhance the photothermal thrombolysis effect. Most importantly, the experiments in vivo confirmed that the proposed PEI/Fe3O4@BP NSs could considerably prolong the effective drug concentration duration of heparin. By which means, accurate, long-acting, and effective thromboprophylaxis could be accomplished with limited drug dosage, which could radically reduce the perniciousness of drug overdose.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening.

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer's disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

Cell salvage using the continuous autotransfusion device CATSmart - an observational bicenter technical evaluation.

BACKGROUND: The use of cell salvage and autologous blood transfusion has become an important method of blood conservation. So far, there are no clinical data about the performance of the continuous autotransfusion device CATSmart. METHODS: In total, 74 patients undergoing either cardiac or orthopedic surgery were included in this prospective, bicenter and observational technical evaluation to validate red cell separation process and washout quality of CATSmart. The target of red cell separation process was defined as a hematocrit value in the packed red cell unit of 55-75% and of washout quality of 80-100% removal ratio. RESULTS: Hematocrit values measured by CATSmart and laboratory analysis were 78.5% [71.3%; 84.0%] and 73.7% [67.5%; 75.5%], respectively. Removal ratios for platelets 94.7% [88.2%; 96.7%], free hemoglobin 89.3% [85.2%; 94.9%], albumin 97.9% [96.6%; 98.5%], heparin 99.9% [99.9%; 100.0%], and potassium 92.5% [90.8%; 95.0%] were within the target range while removal of white blood cells was slightly worse 72.4% [57.9%; 87.3%]. CONCLUSION: The new autotransfusion device enables sufficient red cell separation and washout quality.

Secretion of Tau via an Unconventional Non-vesicular Mechanism.

Tauopathies are characterized by cerebral accumulation of Tau protein aggregates that appear to spread throughout the brain via a cell-to-cell transmission process that includes secretion and uptake of pathological Tau, followed by templated misfolding of normal Tau in recipient cells. Here, we show that phosphorylated, oligomeric Tau clusters at the plasma membrane in N2A cells and is secreted in vesicle-free form in an unconventional process sensitive to changes in membrane properties, particularly cholesterol and sphingomyelin content. Cell surface heparan sulfate proteoglycans support Tau secretion, possibly by facilitating its release after membrane penetration. Notably, secretion of endogenous Tau from primary cortical neurons is mediated, at least partially, by a similar mechanism. We suggest that Tau is released from cells by an unconventional secretory mechanism that involves its phosphorylation and oligomerization and that membrane interaction may help Tau to acquire properties that allow its escape from cells directly through the plasma membrane.

Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding.

BACKGROUND: Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. METHODS: Human IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. RESULTS: Secreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. CONCLUSION: The biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. GENERAL SIGNIFICANCE: These data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis.

New insight into chondroitin and heparosan-like capsular polysaccharide synthesis by profiling of the nucleotide sugar precursors.

Escherichia coli K4 and K5 capsular polysaccharides (K4 and K5 CPSs) have been used as starting material for the biotechnological production of chondroitin sulfate (CS) and heparin (HP) respectively. The CPS covers the outer cell wall but in late exponential or stationary growth phase it is released in the surrounding medium. The released CPS concentration was used, so far, as the only marker to connect the strain production ability to the different cultivation conditions employed. Determining also the intracellular UDP-sugar precursor concentration variations, during the bacterial growth, and correlating it with the total CPS production (as sum of the inner and the released ones), could help to better understand the chain biosynthetic mechanism and its bottlenecks. In the present study, for the first time, a new capillary electrophoresis method was set up to simultaneously analyse the UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) and the inner CPS portion, extracted at the same time from the bacterial biomasses; separation was performed at 18°C and 18 kV with a borate-based buffer and detection at 200 nm. The E. coli K4 and K5 UDP-sugar pools were profiled, for the first time, at different time points of shake flask growths on a glycerol-containing medium and on the same medium supplemented with the monosaccharide precursors of the CPSs: their concentrations varied from 0.25 to 11 µM·gcdw-1, according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced.

Conformational activation of antithrombin by heparin involves an altered exosite interaction with protease.

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr(253)/His(319) residues were mutated. Mutation of factor Xa Arg(143), Lys(148), and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg(150) for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.

In silico discovery of a compound with nanomolar affinity to antithrombin causing partial activation and increased heparin affinity.

The medical and socioeconomic relevance of thromboembolic disorders promotes an ongoing effort to develop new anticoagulants. Heparin is widely used as activator of antithrombin but incurs side effects. We screened a large database in silico to find alternative molecules and predicted d-myo-inositol 3,4,5,6-tetrakisphosphate (TMI) to strongly interact with antithrombin. Isothermal titration calorimetry confirmed a TMI affinity of 45 nM, higher than the heparin affinity (273 nM). Functional studies, fluorescence analysis, and citrullination experiments revealed that TMI induced a partial activation of antithrombin that facilitated the interaction with heparin and low affinity heparins. TMI improved antithrombin inhibitory function of plasma from homozygous patients with antithrombin deficiency with a heparin binding defect and also in a model with endothelial cells. Our in silico screen identified a new, non-polysaccharide scaffold able to interact with the heparin binding domain of antithrombin. The functional consequences of this interaction were experimentally characterized and suggest potential anticoagulant therapeutic applications.

Gekko-sulfated glycopeptide inhibits tumor angiogenesis by targeting basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a therapeutic target of anti-angiogenesis. Here, we report that a novel sulfated glycopeptide derived from Gekko swinhonis Guenther (GSPP), an anticancer drug in traditional Chinese medicine, inhibits tumor angiogenesis by targeting bFGF. GSPP significantly decreased the production of bFGF in hepatoma cells by suppressing early growth response-1. GSPP inhibited the release of bFGF from extracellular matrix by blocking heparanase enzymatic activity. Moreover, GSPP competitively inhibited bFGF binding to heparin/heparan sulfate via direct binding to bFGF. Importantly, GSPP abrogated the bFGF-stimulated proliferation and migration of endothelial cells, whereas it had no inhibitory effect on endothelial cells in the absence of bFGF. Further study revealed that GSPP prevented bFGF-induced neovascularization and inhibited tumor angiogenesis and tumor growth in a xenograft mouse model. These results demonstrate that GSPP inhibits tumor angiogenesis by blocking bFGF production, release from the extracellular matrix, and binding to its low affinity receptor, heparin/heparan sulfate.

TSG-6 protein, a negative regulator of inflammatory arthritis, forms a ternary complex with murine mast cell tryptases and heparin.

TSG-6 (TNF-α-stimulated gene/protein 6), a hyaluronan (HA)-binding protein, has been implicated in the negative regulation of inflammatory tissue destruction. However, little is known about the tissue/cell-specific expression of TSG-6 in inflammatory processes, due to the lack of appropriate reagents for the detection of this protein in vivo. Here, we report on the development of a highly sensitive detection system and its use in cartilage proteoglycan (aggrecan)-induced arthritis, an autoimmune murine model of rheumatoid arthritis. We found significant correlation between serum concentrations of TSG-6 and arthritis severity throughout the disease process, making TSG-6 a better biomarker of inflammation than any of the other arthritis-related cytokines measured in this study. TSG-6 was present in arthritic joint tissue extracts together with the heavy chains of inter-α-inhibitor (IαI). Whereas TSG-6 was broadly detectable in arthritic synovial tissue, the highest level of TSG-6 was co-localized with tryptases in the heparin-containing secretory granules of mast cells. In vitro, TSG-6 formed complexes with the tryptases murine mast cell protease-6 and -7 via either heparin or HA. In vivo TSG-6-tryptase association could also be detected in arthritic joint extracts by co-immunoprecipitation. TSG-6 has been reported to suppress inflammatory tissue destruction by enhancing the serine protease-inhibitory activity of IαI against plasmin. TSG-6 achieves this by transferring heavy chains from IαI to HA, thus liberating the active bikunin subunit of IαI. Because bikunin is also present in mast cell granules, we propose that TSG-6 can promote inhibition of tryptase activity via a mechanism similar to inhibition of plasmin.

Heparan sulfate/heparin promotes transthyretin fibrillization through selective binding to a basic motif in the protein.

Transthyretin (TTR) is a homotetrameric protein that transports thyroxine and retinol. Tetramer destabilization and misfolding of the released monomers result in TTR aggregation, leading to its deposition as amyloid primarily in the heart and peripheral nervous system. Over 100 mutations of TTR have been linked to familial forms of TTR amyloidosis. Considerable effort has been devoted to the study of TTR aggregation of these mutants, although the majority of TTR-related amyloidosis is represented by sporadic cases due to the aggregation and deposition of the otherwise stable wild-type (WT) protein. Heparan sulfate (HS) has been found as a pertinent component in a number of amyloid deposits, suggesting its participation in amyloidogenesis. This study aimed to investigate possible roles of HS in TTR aggregation. Examination of heart tissue from an elderly cardiomyopathic patient revealed substantial accumulation of HS associated with the TTR amyloid deposits. Studies demonstrated that heparin/HS promoted TTR fibrillization through selective interaction with a basic motif of TTR. The importance of HS for TTR fibrillization was illustrated in a cell model; TTR incubated with WT Chinese hamster ovary cells resulted in fibrillization of the protein, but not with HS-deficient cells (pgsD-677). The effect of heparin on TTR fibril formation was further demonstrated in a Drosophila model that overexpresses TTR. Heparin was colocalized with TTR deposits in the head of the flies reared on heparin-supplemented medium, whereas no heparin was detected in the nontreated flies. Heparin of low molecular weight (Klexane) did not demonstrate this effect.

Centrifugation protocols: tests to determine optimal lithium heparin and citrate plasma sample quality.

BACKGROUND: Currently, no clear guidelines exist for the most appropriate tests to determine sample quality from centrifugation protocols for plasma sample types with both lithium heparin in gel barrier tubes for biochemistry testing and citrate tubes for coagulation testing. METHODS: Blood was collected from 14 participants in four lithium heparin and one serum tube with gel barrier. The plasma tubes were centrifuged at four different centrifuge settings and analysed for potassium (K(+)), lactate dehydrogenase (LD), glucose and phosphorus (Pi) at zero time, poststorage at six hours at 21 °C and six days at 2-8°C. At the same time, three citrate tubes were collected and centrifuged at three different centrifuge settings and analysed immediately for prothrombin time/international normalized ratio, activated partial thromboplastin time, derived fibrinogen and surface-activated clotting time (SACT). RESULTS: The biochemistry analytes indicate plasma is less stable than serum. Plasma sample quality is higher with longer centrifugation time, and much higher g force. Blood cells present in the plasma lyse with time or are damaged when transferred in the reaction vessels, causing an increase in the K(+), LD and Pi above outlined limits. The cells remain active and consume glucose even in cold storage. The SACT is the only coagulation parameter that was affected by platelets >10 × 10(9)/L in the citrate plasma. CONCLUSION: In addition to the platelet count, a limited but sensitive number of assays (K(+), LD, glucose and Pi for biochemistry, and SACT for coagulation) can be used to determine appropriate centrifuge settings to consistently obtain the highest quality lithium heparin and citrate plasma samples. The findings will aid laboratories to balance the need to provide the most accurate results in the best turnaround time.

Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples.

The widespread occurrence of heparin contaminated with oversulfated chrondroitin sulfate (OSCS) in 2008 initiated a comprehensive revision process of the Pharmacopoeial heparin monographs and stimulated research in analytical techniques for the quality control of heparin. Here, a set of 177 heparin samples from the market in 2008 as well as pure heparin sodium spiked with defined amounts of OSCS and DS were used to evaluate established and novel methods for the quality control of heparin. Besides (1)H nuclear magnetic resonance spectroscopy (NMR), the assessment included two further spectroscopic methods, i.e., attenuated total reflection-infrared spectroscopy (ATR-IR) and Raman spectroscopy, three coagulation assays, i.e., activated partial thromboplastin time (aPTT) performed with both sheep and human plasma and the prothrombin time (PT), and finally two novel purity assays, each consisting of an incubation step with heparinase I followed by either a fluorescence measurement (Inc-PolyH-assay) or by a chromogenic aXa-assay (Inc-aXa-assay). NMR was shown to allow not only sensitive detection, but also quantification of OSCS by using the peak-height method and a response factor determined by calibration. Chemometric evaluation of the NMR, ATR-IR, and Raman spectra by statistical classification techniques turned out to be best with NMR spectra concerning the detection of OSCS. The validity of the aPTT, the current EP assay, could be considerably improved by replacing the sheep plasma by human plasma. In this way, most of the contaminated heparin samples did not meet the novel potency limit of 180 IU/mg. However, also more than 50% of the uncontaminated samples had <180 IU/MG. In contrast to the aPTT, the PT specifically detects OSCS and other heparin mimetics (LOD 3%). About ten times more sensitive are both the Inc-PolyH-assay and the Inc-aXa-assay, two rapid and simple quantification assays for heparin mimetics. The determined OSCS contents of the heparin samples excellently correlated with those calculated from the NMR spectra. In conclusion, NMR proved to be the current spectroscopic method of choice. The two two-step-assays represent options to supplement NMR, especially as tests for the initial screening, since they detect any heparin mimetic without requiring special expertise for interpretation of the results.

Synthesis of heparan sulfate with cyclophilin B-binding properties is determined by cell type-specific expression of sulfotransferases.

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.

Vasoactive intestinal peptide (VIP) treatment of Parkinsonian rats increases thalamic gamma-aminobutyric acid (GABA) levels and alters the release of nerve growth factor (NGF) by mast cells.

The ventral anterior nucleus of the thalamus (VATh) gathers motor information from the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNpr) of the basal ganglia and projects directly to motor areas of cortex. GPi/SNpr send their tonically active gamma-aminobutyric acid (GABA)ergic outputs to VATh. The abnormal firing patterns of GABAergic neurons in GPi/SNpr lead to motor deficits. In Parkinson's disease, the spontaneous firing pattern of GPi/SNpr neurons is abnormal due to the degeneration of the nigrostriatal dopaminergic pathway. In a previous study, we found that systemically administered vasoactive intestinal peptide (VIP) was effective at reversing the motor deficits (but not the decline in striatal dopamine levels) in a rat model of Parkinson's disease (6-hydroxydopamine (6-OHDA) exposure). In addition to the beneficial effects on the motor response, VIP could also attenuate both neuronal cell death and the characteristic loss of the myelin sheath that is associated with 6-OHDA administration into the rat striatum. VIP was thought to preserve neurons by inducing native brain mast cells to adopt a nondegranulating phenotype that had the ability to secrete numerous neuroprotective substances, such as nerve growth factor (NGF) and heparin. In the present study, the effect of systemically administered VIP (25 ng/kg i.p.) was investigated on GABA levels of the VATh, dopamine/3,4-dihydroxyphenylacetic acid (DOPAC) levels in the corpus striatum, and the NGF, rat mast cell protease II (RMCPII), serotonin, and heparin content of brain mast cells in 6-OHDA-lesioned rats. Extracellular concentrations of GABA, dopamine, and DOPAC were measured by microdialysis and high-performance liquid chromatography. NGF, RMCPII, serotonin, and heparin levels were examined by immunohistochemical staining techniques. A total of 48 young adult Sprague-Dawley rats were used in the study, and these were assigned to one of six groups. Unilateral injection of 6-OHDA, 2 microl (6 mg/microl), was made into the right corpus striatum. VIP-treated animals received 25 ng/kg VIP i.p. at 2-day intervals for a period of 15 days. The present results demonstrated that VIP significantly increased the levels of GABA in the VATh that were reduced by 6-OHDA application and increased the number of NGF-immunoreactive mast cells but did not alter dopamine metabolism. Therefore, the protective effect of VIP on motor function is possibly related to the increased levels of GABA in the VATh, and its neuroprotective actions may be mediated by the release of NGF from brain mast cells.