Elevated ectodomain of type 23 collagen is a novel biomarker of the intestinal epithelium to monitor disease activity in ulcerative colitis and Crohn's disease.

BACKGROUND: Impaired intestinal epithelial barrier is highly affected in inflammatory bowel disease. Transmembrane collagens connecting the epithelial cells to the extracellular matrix have an important role in epithelial cell homeostasis. Thus, we sought to determine whether the transmembrane type 23 collagen could serve as a surrogate marker for disease activity in patients with Crohn's disease and ulcerative colitis. METHODS: We developed an enzyme-linked immunosorbent assay to detect the ectodomain of type 23 collagen (PRO-C23) in serum, followed by evaluation of its levels in both acute and chronic dextran sulphate sodium colitis models in rats and human inflammatory bowel disease cohorts. Serum from 44 Crohn's disease and 29 ulcerative colitis patients with active and inactive disease was included. RESULTS: In the acute and chronic dextran sulphate sodium-induced rat colitis model, the PRO-C23 serum levels were significantly increased after colitis and returned to normal levels after disease remission. Serum levels of PRO-C23 were elevated in Crohn's disease (p < 0.05) and ulcerative colitis (p < 0.001) patients with active disease compared to healthy donors. PRO-C23 differentiated healthy donors from ulcerative colitis (area under the curve [AUC]: 0.81, p = 0.0009) and Crohn's disease (AUC: 0.70, p = 0.0124). PRO-C23 differentiated ulcerative colitis patients with active disease from those in remission (AUC: 0.75, p = 0.0219) and Crohn's disease patients with active disease from those in remission (AUC: 0.68, p = 0.05). CONCLUSION: PRO-C23 was elevated in rats with active colitis, and inflammatory bowel disease patients with active disease. Therefore, PRO-C23 may be used as a surrogate marker for monitoring disease activity in ulcerative colitis and Crohn's disease.

Prolonging the circulatory retention of SPIONs using dextran sulfate: in vivo tracking achieved by functionalisation with near-infrared dyes.

The rapid reticuloendothelial system (RES) mediated clearance of superparamagnetic iron oxide nanoparticles (SPIONs) from circulation is considered a major limitation of their clinical utility. We aimed to address this by using dextran sulfate 500 (DSO4 500), a Kupffer cell blocking agent, to prolong SPIONs circulatory time. Blood concentrations of SPIONs are difficult to quantify due to the presence of haemoglobin. We therefore developed methods to functionalise SPIONs with near-infrared (NIR) dyes in order to trace their biodistribution. Two SPIONs were investigated: Nanomag®-D-spio-NH(2) and Ferucarbotran. Nanomag®-D-spio-NH(2) was functionalised using NHS (N-hydroxysuccinimide) ester NIR dye and Ferucarbotran was labelled using periodate oxidation followed by reductive amination or a combination of EDC (ethyl(dimethylaminopropyl) carbodiimide )/NHS and click chemistries. Stability after conjugation was confirmed by dynamic light scattering (DLS), superconducting quantum interference device (SQUID) and transmission electron microscopy (TEM). In vivo experiments with the functionalised SPIONs showed a significant improvement in SPIONs blood concentrations in mice pre-treated with dextran sulfate sodium salt 500 (DSO4 500).

Zinc-dependent contact system activation induces vascular leakage and hypotension in rodents.

Contact to polyanions induces autoactivation of the serine protease factor XII that triggers the kallikrei-kinin system. Recent studies indicate that polysaccharide-induced autoactivation of factor XII has a role in allergy-related vascular leakage, and angioedema. Here, we characterize in vivo effects of the synthetic polysaccharide dextran sulfate in human plasma and in rodent models. Minute amounts of high-molecular-weight dextran sulfate-initiated factor XII-autoactivation and triggered formation of the inflammatory mediator bradykinin via plasma kallikrein-mediated cleavage of high-molecular-weight kininogen. High-molecular-weight kininogen fragments, containing the HKH20 sequence in domain D5H, blocked dextran sulfate-initiated bradykinin-generation by depleting plasma Zn2+ ions. Topical application of high molecular weight dextran sulfate increased leakage in murine skin microvessels, in a bradykinin-dependent manner. Intravital laser scanning microscopy showed a greater than two-fold elevated and accelerated fluid extravasation in C1 esterase inhibitor deficient mice that lack the major inhibitor of factor XII, compared to wild-type controls. Intra-arterial infusion of dextran sulfate induced a rapid transient drop in arterial blood pressure in rats and preinjection of kinin B2 receptor antagonists or HKH20 peptide blunted dextran sulfate-triggered hypotensive reactions. The data characterize dextran sulfate as a potent in vivo activator of factor XII with implications for bradykinin-mediated vascular permeability and blood pressure control.

Dextran sulfate included in factor Xa assay reagent overestimates heparin activity in patients after heparin reversal by protamine.

A lack of correlation between activated partial thromboplastin time (aPTT), thrombin time (TT) and anti-factor Xa (AXa) activity was observed in patients after cardiac surgery with cardiopulmonary bypass (CBP). Indeed, AXa activity measured by the chromogenic assay, Coamatic Heparin, was higher than expected with regard to results obtained in coagulation assays. To account for this discrepancy, another AXa chromogenic assay was tested. First, AXa activity was measured with two chromogenic assays (Coamatic Heparin and Rotachrom Heparin) in plasma samples of 25 patients undergoing cardiac surgery at two time points after heparin reversal by protamine. AXa activity was significantly higher when measured with Coamatic Heparin than with Rotachrom Heparin in samples collected just after protamine infusion (p<0.01). Next, since Coamatic( Heparin contains dextran sulfate (DXS) to reduce the influence of heparin antagonists such as platelet factor 4 (PF4), whereas Rotachrom Heparin does not, we hypothesized that the dextran sulfate contained in the reagent might explain this discrepancy. We therefore performed in vitro studies consisting in neutralizing unfractionated heparin (UFH) with protamine and measuring AXa activity with the two chromogenic assays. An AXa activity was still measurable with Coamatic Heparin after neutralization, thus strongly suggesting that dextran sulfate dissociates protamine/heparin complexes. We conclude that Coamatic Heparin assays should be avoided when measuring AXa activity in plasma samples immediately after protamine infusion, as inaccurate results may lead to inadequate management of heparin reversal.

The potentiation of human C1-inhibitor by dextran sulphate is transient in vivo: studies in a rat model.

C1-inhibitor (C1-Inh) is an important regulator of inflammatory reactions because it is a potent inhibitor of the contact and complement system. C1-Inh application in inflammatory disease is, however, restricted because of the high doses required. The glycosaminoglycan-like molecule dextran sulphate (DXS) enhances C1-Inh function in vitro. Hence, we investigated whether co-administration with dextran sulphate reduces the amount of C1-Inh required, through enhancement in vivo. C1-Inh potentiation was measured in a newly developed C1s-inactivation assay that is based on activation of C4 by purified C1s. Activated C4 in rat plasma was quantified with a newly developed ELISA. Human C1-Inh (2.5 microM) inhibited C1s in rat plasma 55-fold faster in the presence of dextran sulphate (15 kDa, 5 microM). To study the stability of the complex in vivo, rats were given a mixture of C1-Inh (10 mg/kg) and dextran sulphate (3 mg/kg). C1-Inh activity during 5 h was analyzed ex vivo with the C1s inactivation assay. The noncovalent C1-Inh-dextran sulphate complex resulted in a transient enhancement of the inhibitory capacity of C1-Inh, lasting for 60-90 min. Dextran sulphate did not affect plasma clearance of C1-Inh. We conclude that the enhanced inhibitory capacity of C1-Inh complexed to dextran sulphate is transient in vivo. Hence, co-administration of these compounds seems a feasible approach to achieve short-term inhibition of complement in vivo.

Correlation of transarterial transport of various dextrans with their physicochemical properties.

Local vascular drug delivery provides elevated concentrations of drug in the target tissue while minimizing systemic side effects. To better characterize local pharmacokinetics we examined the arterial transport of locally applied dextran and dextran derivatives in vivo. Using a two-compartment pharmacokinetic model to correct the measured transmural flux of these compounds for systemic redistribution and elimination as delivered from a photopolymerizable hydrogel surrounding rat carotid arteries, we found that the diffusivities and the transendothelial permeabilities were strongly dependent on molecular weight and charge. For neutral dextrans, the effective diffusive resistance in the media increased with molecular weight approximately 4.1-fold between the molecular weights of 10 and 282 kDa. Similarly, endothelial resistance increased 28-fold over the same molecular weight range. The effective medial diffusive resistance was unaffected by cationic charge as such molecules moved identically to neutral compounds, but increased approximately 40% when dextrans were negatively charged. Transendothelial resistance was 20-fold lower for the cationic dextrans, and 11-fold higher for the anionic dextrans, when both were compared to neutral counterparts. These results suggest that, while low molecular weight drugs will rapidly traverse the arterial wall with the endothelium posing a minimal barrier, the reverse is true for high molecular weight agents. With these data, the deposition and distribution of locally released vasotherapeutic compounds might be predicted based upon chemical properties, such as molecular weight and charge.

Serum amyloid A and P protein levels are lowered by dextran sulfate cellulose low-density lipoprotein apheresis.

The present study describes the short-term effect of dextran sulfate cellulose (DSC) low-density lipoprotein (LDL) apheresis using a plasma separator equipped with a polysulfone (PS) membrane filter (PS/DSC-LDL apheresis) on the serum amyloid A (SAA) and P (SAP) protein levels during treatment in a patient with familial hypercholesterolemia (type IIa, heterozygote). PS/DSC-LDL apheresis markedly lowered both the SAA (reduction percentage, 84.1+/-8.2%) and SAP (91.4+/-5%) levels, which returned to their respective initial levels within 4 days. Experimentally, the levels of both proteins also decreased on passage through the DSC minicolumn without a PS membrane, indicating that the DSC resin had an affinity to both proteins. These results suggest that PS/DSC-LDL apheresis may be advantageous for amyloid protein accumulating disorders, including amyloidosis and atherosclerosis.

Circulating hyaluronan, chondroitin sulphate and dextran sulphate bind to a liver receptor that does not recognize heparin.

Chondroitin sulphate, injected intravenously into rats and given prior to intravenous 125I-labelled hyaluronan with a mean Mw of about 400 kDa, was shown to inhibit the rapid receptor-mediated uptake of hyaluronan by the liver. The labelled hyaluronan that remained in the circulation was shown, by size exclusion chromatography of serum and urine, to be rapidly degraded down to fragments of lower Mw and filtered out into the urine and tissues. When the uptake of 125I-hyaluronan was inhibited by unlabelled hyaluronan, only very low degradation and urinary excretion were found. Liver uptake could also be inhibited by dextran sulphate but not by heparin. Unlabelled hyaluronan could inhibit the liver uptake of labelled chondroitin sulphate but not labelled heparin. Unlabelled chondroitin sulphate and dextran sulphate inhibited cell association of labelled hyaluronan to liver endothelial cells in culture more effectively than unlabelled hyaluronan. Our data show that the liver hyaluronan receptors also recognize and effectively bind chondroitin sulphate and dextran sulphate but not heparin and that a hyaluronan-specific saturable degradative mechanism exists in the circulation. Such a mechanism could explain why hyaluronan in the general circulation has a much lower Mw than the hyaluronan in lymph. The results also indicate that increased hyaluronan levels in serum, and increased urinary excretion of hyaluronan, may be secondary to increased outflow of chondroitin sulphate from the tissues during some pathological conditions.

Determination of polyanionic macromolecules by size-exclusion chromatography.

This report presents an extension of a method developed for determination of dextran sulfate in rat serum. The drug is a negatively charged polysaccharide with a molecular mass of 8000. It is fractionated by molecular size and separated from serum components by high-performance size-exclusion chromatography. Sensitive detection is achieved by the post-column complexation of the analyte with 1,9-dimethylmethylene blue (DMMB). A metachromatic complex is formed; the absorbance maximum of the complex is shifted from that of the free dye. Various glycosaminoglycans and other macromolecular polyanions interact with DMMB. Several can be determined using the chromatographic conditions developed for dextran sulfate. The method provides a simple procedure for quantitation of these compounds. Compared to spectrophotometric assays, less sample preparation is required, selectivity is enhanced, and molecular mass information is provided. With modification of eluent composition, dye concentration, and detection wavelength, the method can be validated for determination of additional compounds.

Size-exclusion chromatographic determination of dextran sulfate in rat serum.

A sensitive and selective method for the determination of dextran sulfate in rat serum has been developed. The analysis is suitable for quantitation of the drug and for monitoring molecular mass changes occurring during biotransformation. Dextran sulfate is resolved from higher molecular mass serum components by high-performance aqueous size-exclusion chromatography. The method has been validated for the direct injection of serum. Sensitive detection is achieved by post-column reaction of the polyanionic drug with the dye 1,9-dimethylmethylene blue. Components of serum which inhibit complex formation are separated chromatographically from dextran sulfate. Absorbance of the metachromatic complex is monitored at 525 nm.

Adsorption profile of commercially available adsorbents: an in vitro evaluation.

Adsorbents from four commercially available devices, Protein A-Sepharose (Immunosorba Protein A-62,5; Excorim KB, Lund Sweden), Tryptophan-PVA (Immusorba TR-350; Asahi Medical Co., Tokyo, Japan), Phenylalanine-PVA (Immusorba PH-350; Asahi Medical Co., Tokyo, Japan), and Dextran sulfate (Liposorber LA-15; Kanegafuchi Chemical Co. Ltd, Osaka, Japan) were tested under optimal in vitro conditions to determine their adsorption capability for several plasma constituents which are usually the target of plasma therapy. The parameters of interest were: double stranded DNA-antibodies (anti-dsDNA), antiglomerular basement membrane antibodies (anti-GBM), anti-acetylcholin receptor antibodies (AChRAb), circulating immune complexes (CIC), rheumatoid factor (RF), IgA, IgG, IgM, IgE, C3c, C4, LDL-cholesterol, total cholesterol, erythropoietin (EPO) and beta 2-microglobulin (beta 2M). The IgG auto antibodies, CIC and RF can be removed by Protein A-Sepharose, Try-PVA and Phe-PVA. IgG is best adsorbed by Protein A-Sepharose, while IgE can be removed efficiently by Try-PVA. Dextran sulfate is without doubt the best adsorbent for LDL-cholesterol. All four adsorbents bind also complement components C3c and C4. No significant adsorption was found for EPO and beta 2M. The four devices exhibit a quite different adsorption profile which can be used as a guide for the optimal selection of an adsorption column in clinical apheresis.

Evidence from endothelium of gastric absorption of heparin and of dextran sulfates 8000.

Heparin, hydrogenated dextran sulfate 8000 (Usherdex 8), and dextran sulfate 8000 were administered to rats, and the total drug was separated and determined in endothelium and plasma. A large amount of each drug was recovered from endothelium 2.4 and 6 minutes after intravenous injection. This accounted for the drug missing from plasma. The drugs in water were placed in the stomach by catheter. All three drugs were recovered from the endothelium and identified unchanged by electrophoresis and specific staining. The amounts that were recovered at 2.4 and 6 minutes were equivalent to most of the drug administered. Thus heparin, Usherdex 8, and dextran sulfate 8000 enter the body immediately on oral administration. At longer time intervals after intravenous and oral administration, much of each drug was not demonstrable in endothelium by the method used. Some drug could be detected in endothelium after 4 hours. After oral administration, plasma levels of each drug were rarely more than 0.5% of the dose. Formalin-alcohol was applied to the jugular veins of anesthetized rats to produce a thrombus, (see Blake et al. J Clin Path 1959;12:118-22) and the drugs were immediately introduced into the stomach. Four hours later the injured veins were inspected for thrombi. Incidence of thrombotic plug was 80% in rats that received saline solution, 4% with Usherdex 8, 0% with dextran sulfate 8000, and 0% with heparin. Usherdex 8, dextran sulfate 8000, and heparin demonstrate low, moderate, and high in vitro anticoagulant activity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal polypeptide (VIP) stimulates fibrinolytic system in the rat.

We have studied the fibrinolytic effect of VIP in rats. Intravenous injection of VIP enhanced blood fibrinolytic activity in a dose-related manner. The euglobulin fraction obtained from intact rat plasma incubated with VIP did not produce an increase in fibrinolytic activity, while dextran sulfate (DS) and urokinase (UK) showed the activity. VIP solution placed on a plasminogen-rich fibrin plate did not show fibrinolysis. VIP had neither a plasminogen activator nor plasmin activity. VIP may release plasminogen activators into the blood.