Low Molecular Weight Dextran Sulfate Research Articles

Gut Protective Effect from Newly Isolated Bacteria as Probiotics against Dextran Sulfate Sodium and Carrageenan-Induced Ulcerative Colitis.

Gut microbiome dysbiosis might be linked to certain diseases such as inflammatory bowel diseases (IBDs), which are categorized by vigorous inflammation of the gastrointestinal tract. Several studies have shown the favorable anti-inflammatory effect of certain probiotics in IBD therapy. In the present investigation, the possible gut protective effects of commensal bacteria were examined in an IBD model mouse that was cost-effectively induced with low molecular weight dextran sulfate sodium (DSS) and kappa carrageenan. Our conclusions show that certain probiotic supplementation could result in the attenuation of the disease condition in the IBD mouse, suggesting a favorable therapeutic capability for considerably improving symptoms of gut inflammation with an impact on the IBD therapy. However, the molecular mechanisms require further investigation.

Optimization of Heparin Monitoring with Anti-FXa Assays and the Impact of Dextran Sulfate for Measuring All Drug Activity.

Heparins, unfractionated or low molecular weight, are permanently in the spotlight of both clinical indications and laboratory monitoring. An accurate drug dosage is necessary for an efficient and safe therapy. The one-stage kinetic anti-FXa assays are the most widely and universally used with full automation for large series, without needing exogenous antithrombin. The WHO International Standards are available for UFH and LMWH, but external quality assessment surveys still report a high inter-assay variability. This heterogeneity results from the following: assay formulation, designed without or with dextran sulfate to measure all heparin in blood circulation; calibrators for testing UFH or LMWH with the same curve; and automation parameters. In this study, various factors which impact heparin measurements are reviewed, and we share our experience to optimize assays for testing all heparin anticoagulant activities in plasma. Evidence is provided on the usefulness of low molecular weight dextran sulfate to completely mobilize all of the drug present in blood circulation. Other key factors concern the adjustment of assay conditions to obtain fully superimposable calibration curves for UFH and LMWH, calibrators’ formulations, and automation parameters. In this study, we illustrate the performances of different anti-FXa assays used for testing heparin on UFH or LMWH treated patients’ plasmas and obtained using citrate or CTAD anticoagulants. Comparable results are obtained only when the CTAD anticoagulant is used. Using citrate as an anticoagulant, UFH is underestimated in the absence of dextran sulfate. Heparin calibrators, adjustment of automation parameters, and data treatment contribute to other smaller differences.

ILB\xae resolves inflammatory scarring and promotes functional tissue repair

Fibrotic disease is a major cause of mortality worldwide, with fibrosis arising from prolonged inflammation and aberrant extracellular matrix dynamics. Compromised cellular and tissue repair processes following injury, infection, metabolic dysfunction, autoimmune conditions and vascular diseases leave tissues susceptible to unresolved inflammation, fibrogenesis, loss of function and scarring. There has been limited clinical success with therapies for inflammatory and fibrotic diseases such that there remains a large unmet therapeutic need to restore normal tissue homoeostasis without detrimental side effects. We investigated the effects of a newly formulated low molecular weight dextran sulfate (LMW-DS), termed ILB®, to resolve inflammation and activate matrix remodelling in rodent and human disease models. We demonstrated modulation of the expression of multiple pro-inflammatory cytokines and chemokines in vitro together with scar resolution and improved matrix remodelling in vivo. Of particular relevance, we demonstrated that ILB® acts, in part, by downregulating transforming growth factor (TGF)β signalling genes and by altering gene expression relating to extracellular matrix dynamics, leading to tissue remodelling, reduced fibrosis and functional tissue regeneration. These observations indicate the potential of ILB® to alleviate fibrotic diseases.

Low Molecular Weight Dextran Sulfate (ILB®) Administration Restores Brain Energy Metabolism Following Severe Traumatic Brain Injury in the Rat.

Traumatic brain injury (TBI) is the leading cause of death and disability in people less than 40 years of age in Western countries. Currently, there are no satisfying pharmacological treatments for TBI patients. In this study, we subjected rats to severe TBI (sTBI), testing the effects of a single subcutaneous administration, 30 min post-impact, of a new low molecular weight dextran sulfate, named ILB®, at three different dose levels (1, 5, and 15 mg/kg body weight). A group of control sham-operated animals and one of untreated sTBI rats were used for comparison (each group n = 12). On day 2 or 7 post-sTBI animals were sacrificed and the simultaneous HPLC analysis of energy metabolites, N-acetylaspartate (NAA), oxidized and reduced nicotinic coenzymes, water-soluble antioxidants, and biomarkers of oxidative/nitrosative stress was carried out on deproteinized cerebral homogenates. Compared to untreated sTBI rats, ILB® improved energy metabolism by increasing ATP, ATP/ adenosine diphosphate ratio (ATP/ADP ratio), and triphosphate nucleosides, dose-dependently increased NAA concentrations, protected nicotinic coenzyme levels and their oxidized over reduced ratios, prevented depletion of ascorbate and reduced glutathione (GSH), and decreased oxidative (malondialdehyde formation) and nitrosative stress (nitrite + nitrate production). Although needing further experiments, these data provide the first evidence that a single post-injury injection of a new low molecular weight dextran sulfate (ILB®) has beneficial effects on sTBI metabolic damages. Due to the absence of adverse effects in humans, ILB® represents a promising therapeutic agent for the treatment of sTBI patients.

Control of IBMIR Induced by Fresh and Cryopreserved Hepatocytes by Low Molecular Weight Dextran Sulfate versus Heparin

Rapid destruction of hepatocytes after hepatocyte transplantation has hampered the application of this procedure clinically. The instant blood-mediated inflammatory reaction (IBMIR) is a plausible underlying cause for this cell loss. The present study was designed to evaluate the capacity of low molecular weight dextran sulfate (LMW-DS) to control these initial reactions from the innate immune system. Fresh and cryopreserved hepatocytes were tested in an in vitro whole-blood model using ABO-compatible blood. The ability to elicit IBMIR and the capacity of LMW-DS (100 μg/ml) to attenuate the degree of activation of the cascade systems were monitored. The effect was also compared to conventional anticoagulant therapy using unfractionated heparin (1 IU/ml). Both fresh and freeze–thawed hepatocytes elicited IBMIR to the same extent. LMW-DS reduced the platelet loss and maintained the cell counts at the same degree as unfractionated heparin, but controlled the coagulation and complement systems significantly more efficiently than heparin. LMW-DS also attenuated the IBMIR elicited by freeze–thawed cells. Therefore, LMW-DS inhibits the cascade systems and maintains the cell counts in blood triggered by both fresh and cryopreserved hepatocytes in direct contact with ABO-matched blood. LMW-DS at a previously used and clinically applicable concentration (100 μg/ml) inhibits IBMIR in vitro and is therefore a potential IBMIR inhibitor in hepatocyte transplantation.

A biomimetic approach to active self-microencapsulation of proteins in PLGA

A biomimetic approach to organic solvent-free microencapsulation of proteins based on the self-healing capacity of poly (dl)-lactic-co-glycolic acid (PLGA) microspheres containing glycosaminoglycan-like biopolymers (BPs), was examined. To screen BPs, aqueous solutions of BP [high molecular weight dextran sulfate (HDS), low molecular weight dextran sulfate (LDS), chondroitin sulfate (CS), heparin (HP), hyaluronic acid (HA), chitosan (CH)] and model protein lysozyme (LYZ) were combined in different molar and mass ratios, at 37°C and pH7. The BP-PLGA microspheres (20–63μm) were prepared by a double water–oil–water emulsion method with a range of BP content, and trehalose and MgCO3 to control microclimate pH and to create percolating pores for protein. Biomimetic active self-encapsulation (ASE) of proteins [LYZ, vascular endothelial growth factor165 (VEGF) and fibroblast growth factor (FgF-20)] was accomplished by incubating blank BP-PLGA microspheres in low concentration protein solutions at ~24°C, for 48h. Pore closure was induced at 42.5°C under mild agitation for 42h. Formulation parameters of BP-PLGA microspheres and loading conditions were studied to optimize protein loading and subsequent release. LDS and HP were found to bind >95% LYZ at BP:LYZ>0.125 w/w, whereas HDS and CS bound >80% LYZ at BP:LYZ of 0.25–1 and <0.33, respectively. HA-PLGA microspheres were found to be not ideal for obtaining high protein loading (>2% w/w of LYZ). Sulfated BP-PLGA microspheres were capable of loading LYZ (~2–7% w/w), VEGF (~4% w/w), and FgF-20 (~2% w/w) with high efficiency. Protein loading was found to be dependent on the loading solution concentration, with higher protein loading obtained at higher loading solution concentration within the range investigated. Loading also increased with content of sulfated BP in microspheres. Release kinetics of proteins was evaluated in-vitro with complete release media replacement. Rate and extent of release were found to depend upon volume of release (with non-sink conditions observed <5ml release volume for ~18mg loaded BP-PLGA microspheres), ionic strength of release media and loading solution concentration. HDS-PLGA formulations were identified as having ideal loading and release characteristics. These optimal microspheres released ~73–80% of the encapsulated LYZ over 60days, with >90% of protein being enzymatically active. Nearly 72% of immunoreactive VEGF was similarly released over 42days, without significant losses in heparin binding affinity in the release medium.

Effects of dextran sulfates on the acute infection and growth stages of Toxoplasma gondii

Toxoplasma gondii is one of the most prevalent parasites, causing toxoplasmosis in various warm-blooded animals, including humans. Because of the broad range of hosts susceptible to T. gondii, it had been postulated that a universal component of the host cell surface, such as glycosaminoglycans (GAGs), may act as a receptor for T. gondii infection. Carruthers et al. (Infect Immun 68:4005-4011, 2000) showed that soluble GAGs have also been shown to disrupt parasite binding to human fibroblasts. Therefore, we investigated the inhibitory effect of GAGs and their analogue dextran sulfate (DS) on T. gondii infection. For up to 24h of incubation after inoculation of T. gondii, the inhibitory effect of GAGs on T. gondii infection and growth inside the host cell was weak. In contrast, DS markedly inhibited T. gondii infection. Moreover, low molecular weight DS particularly slowed the growth of T. gondii inside host cells. DS10 (dextran sulfate MW 10kDa) was the most effective agent in these in vitro experiments and was therefore tested for its inhibitory effects in animal experiments; infection inhibition by DS10 was confirmed under these in vivo conditions. In this report, we showed that DSs, especially DS10, have the potential of a new type of drug for toxoplasmosis.

Effect of low molecular weight dextran sulfate on tourniquet-induced limb ischemia/reperfusion injury and distant lung damage

Effect of low molecular weight dextran sulfate on tourniquet-induced limb ischemia/reperfusion injury and distant lung damage

Low Molecular Weight Dextran Sulfate Binds to Human Myoblasts and Improves their Survival after Transplantation in Mice

Myoblast transplantation represents a promising therapeutic strategy in the treatment of several genetic muscular disorders including Duchenne muscular dystrophy. Nevertheless, such an approach is impaired by the rapid death, limited migration, and rejection of transplanted myoblasts by the host. Low molecular weight dextran sulfate (DXS), a sulfated polysaccharide, has been reported to act as a cytoprotectant for various cell types. Therefore, we investigated whether DXS could act as a "myoblastprotectant" either in vitro or in vivo after transplantation in immunodeficient mice. In vitro, DXS bound human myoblasts in a dose-dependent manner and significantly inhibited staurosporine-mediated apoptosis and necrosis. DXS pretreatment also protected human myoblasts from natural killer cell-mediated cytotoxicity. When human myoblasts engineered to express the renilla luciferase transgene were transplanted in immunodeficient mice, bioluminescence imaging analysis revealed that the proportion of surviving myoblasts 1 and 3 days after transplantation was two times higher when cells were preincubated with DXS compared to control (77.9 ± 10.1% vs. 39.4 ± 4.9%, p = 0.0009 and 38.1 ± 8.5% vs. 15.1 ± 3.4%, p = 0.01, respectively). Taken together, we provide evidence that DXS acts as a myoblast protectant in vitro and is able in vivo to prevent the early death of transplanted myoblasts.

Low Molecular Weight Dextran Sulfate Binds to Human Myoblasts and Improves their Survival after Transplantation in Mice

Introduction: Myoblasts are skeletal muscle progenitors cells responsible for skeletal muscle growth and regeneration. As such and because myoblasts faces the complex challenges of clinical application, myoblast transplantation appears to be a promising approach for treatment of extensive skeletal muscle destruction occurring in severe myopathies such as Duchenne muscular dystrophy. Nevertheless, even in autologous combination, such approaches are impaired by the rapid death, limited migration and rejection of transplanted myoblasts by the host. Low molecular weight dextran sulfate (DXS), a sulfated polysaccharide, has been reported to act as a cytoprotectant in vitro and in vivo. These beneficial effects of DXS in modulating innate immunity make this substance a possible candidate to improve the success of myoblast transplantation. Methods: Clonal cultures of human myoblasts were prepared from human muscle biopsies obtained during corrective orthopedic surgery. Myoblasts were 100% positives for the myogenic markers CD56 and desmin as assessed by flow cytometry. For in vivo experiments, human myoblasts were transduced with a lentiviral vector encoding the renilla luciferase bioluminescence marker gene (Rluc). Non-invasive bioluminescence imaging was used to quantify survival of engrafted human myoblasts after transplantation in immunodeficient mice. All research with human samples and mice was performed in compliance with the institutional guidelines, national as well as international guidelines. Results: In vitro, DXS binds dose dependently to human myoblasts and significantly inhibits staurosporine mediated apoptosis and necrosis. DXS pretreatment also protects human myoblasts from natural killer cell-mediated cytotoxicity in a dose dependent manner. At a multiplicity of infection of 1, 80% of the cells expressed the renilla luciferase transgene after lentiviral infection. Rluc transduced myoblasts kept their myogenic capacity to proliferate and to differentiate in vitro as assessed by fusion indexes. In vivo, we observed a two-fold improvement in survival of human myoblasts that were pretreated with DXS (10 mg/ml) as compared to untreated cells, at 1 day and at 3 days after transplantation (77.9 ± 10.1% vs 39.4 ± 4.9%; p = 0.0009 and 38.1 ± 8.5% vs 15.1 ± 3.4%; p = 0.01, respectively). No significant difference was observed at 7 days (8.0 ± 2.1 % vs 3.6 ± 0.7%; p = 0.06) and at 14 days post injection with values that were indistinguishable from background levels. Conclusion: Taken together, we provide evidence that DXS act as a myoblast protectant in vitro and in vivo, able locally to prevent early cell death. This protective effect of DXS was efficient up to 72h after pretreatment. DXS might represent a useful therapeutic reagent to improve the success of myoblast cell transplantation. More experiments are needed to evaluate if combination of DXS pretreatment of human myoblasts with systemic DXS administration of the recipient may be envisaged to enhance myoblast transplantation success.

Protection of Human Islets from Instant Blood-Mediated Inflammatory Reaction by NF-κB Blockade

Introduction: Instant blood-mediated inflammatory reaction (IBMIR) is known to cause the loss of a significant mass of transplanted islets. Major characteristics of IBMIR include activation of complement and coagulation cascades and infiltration of innate immune cells into the islets. We provide evidence using an in vitro model that blockade of transcription factor NF-κB results in significant inhibition of this inflammatory reaction. Methods: Purified human islets were mixed with heparinized autologous or ABO-matched allogeneic blood in an in vitro tube model. The in vitro model was validated by measurement of pO2, pH and glucose in the blood+islet mix which showed that the mixing of islets and blood retained normal physiological conditions up to 6 h. Low-molecular weight dextran sulfate (LMWDS) alone and in combination with Withaferin A (WA)or resveratrol (known NF-kB inhibitors) were added to the blood-islet mix. Islet viability was measured using Hoechst 33341/propidium iodide staining followed by image analysis. Plasma samples and islet clots were retrieved at regular time points to analyze cytokines and cellular infiltration respectively. Panel of pro-inflammatory cytokines including IFN-γ, IL-6, and IL-8 were measured using Luminex assay. Results: Islet viability increased by 26% over control in the presence of LMWDS; however, addition of WA or resveratrol significantly improved islet viability over control (107-112%; p< 0.02). Exposure of purified islets to both autologous and allogeneic blood increased production of inflammatory cytokines with significant release at 6 hour. Mixing of allogeneic blood invoked a stronger cytokine response (>10 fold) when compared to autologous combination. Presence of WA or resveratrol significantly inhibited release of IFN-γ (4.6±0.1 vs. 2.0±1.0 pg/mL; p< 0.04), IL-6 (6444±196 vs. 712±127 pg/mL; p< 0.001), IL-8 (6368±309 vs. 4257±84 pg/mL; p< 0.005) in allogeneic combination. Immunohistochemistry of islets showed significant reduction in infiltration of neutrophils in both allogeneic (8.2±2.9 vs. 0.5±0.7 cells/islet; p< 0.005) and autologous samples (8.1±2.7 vs. 1.7±0.8 cells/islet; p< 0.001). Conclusion: The results showed that combination of LMWDS and NF-κB blockers offered remarkable protection of islets from IBMIR during the initial phase of the exposure of islets to blood. In summary, control of NF-kB activation could be a key strategy to minimize damage to both autologous and allogeneic islet transplants due to IBMIR.

Improved drug loading and antibacterial activity of minocycline-loaded PLGA nanoparticles prepared by solid/oil/water ion pairing method

BackgroundLow drug entrapment efficiency of hydrophilic drugs into poly(lactic-co-glycolic acid) (PLGA) nanoparticles is a major drawback. The objective of this work was to investigate different methods of producing PLGA nanoparticles containing minocycline, a drug suitable for periodontal infections.MethodsDifferent methods, such as single and double solvent evaporation emulsion, ion pairing, and nanoprecipitation were used to prepare both PLGA and PEGylated PLGA nanoparticles. The resulting nanoparticles were analyzed for their morphology, particle size and size distribution, drug loading and entrapment efficiency, thermal properties, and antibacterial activity.ResultsThe nanoparticles prepared in this study were spherical, with an average particle size of 85–424 nm. The entrapment efficiency of the nanoparticles prepared using different methods was as follows: solid/oil/water ion pairing (29.9%) > oil/oil (5.5%) > water/oil/water (4.7%) > modified oil/water (4.1%) > nano precipitation (0.8%). Addition of dextran sulfate as an ion pairing agent, acting as an ionic spacer between PEGylated PLGA and minocycline, decreased the water solubility of minocycline, hence increasing the drug entrapment efficiency. Entrapment efficiency was also increased when low molecular weight PLGA and high molecular weight dextran sulfate was used. Drug release studies performed in phosphate buffer at pH 7.4 indicated slow release of minocycline from 3 days to several weeks. On antibacterial analysis, the minimum inhibitory concentration and minimum bactericidal concentration of nanoparticles was at least two times lower than that of the free drug.ConclusionNovel minocycline-PEGylated PLGA nanoparticles prepared by the ion pairing method had the best drug loading and entrapment efficiency compared with other prepared nanoparticles. They also showed higher in vitro antibacterial activity than the free drug.

Heparin affects the interaction of kininogen on endothelial cells

In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Zn+2, HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PK binding to cell- or ECM-bound-HK and PK activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn2+. Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment.

Inhibition of direct and indirect TLR-mediated activation of human NK cells by low molecular weight dextran sulfate

NK cells express toll-like receptors (TLR) that recognize conserved pathogen or damage associated molecular patterns and play a fundamental role in innate immunity. Low molecular weight dextran sulfate (DXS), known to inhibit the complement system, has recently been reported by us to inhibit TLR4-induced maturation of human monocyte-derived dendritic cells (MoDC). In this study, we investigated the capability of DXS to interfere with human NK cell activation triggered directly by TLR2 agonists or indirectly by supernatants of TLR4-activated MoDC. Both TLR2 agonists and supernatants of TLR4-activated MoDC activated NK cells phenotypically, as demonstrated by the analysis of NK cell activation markers (CD56, CD25, CD69, NKp30, NKp44, NKp46, DNAM-1 and NKG2D), and functionally as shown by increased NK cell degranulation (CD107a surface expression) and IFN-γ secretion. DXS prevented the up-regulation of NK cell activation markers triggered by TLR2 ligands or supernatants of TLR4-activated MoDC and dose-dependently abrogated NK cell degranulation and IFN-γ secretion. In summary our results suggest that DXS may be a useful reagent to inhibit the direct and indirect TLR-mediated activation of NK cells.

Low Molecular Weight Dextran Sulfate Is Well Tolerated in Humans and Increases Endogenous Expression of Islet Protective Hepatocyte Growth Factor

Low molecular weight dextran sulfate (LMW-DS) is a strong candidate to prevent early islet graft destruction caused by the instant blood-mediated inflammatory reaction. Pharmacokinetics, safety, tolerability, and the effect on endogenous release of various growth factors were studied in humans. Thirty healthy volunteers were given LMW-DS as a combined bolus and 20 min intravenous infusion followed by a continuous intravenous infusion for 5 hr to reach different predetermined target-activated partial thromboplastin times (APTT) in five study groups. Monitoring of APTT was used to estimate and control the plasma concentration. Safety, including hemostasis parameters, was evaluated before proceeding to a higher target APTT-level. Plasma was collected continuously during the infusion and was analyzed for additional safety markers and the presence of six different growth factors. Predetermined target APTT levels were reached and kept for 5 hr without extensive dose corrections. After the 5 hr 20 min of LMW-DS infusion, the subjects in the highest dose group (target APTT=150 s) were back at APTT-levels below 75 s within 60 min. Plasma levels of hepatocyte growth factor were increased 100-fold within 20 min of infusion start and persisted more than 8 hr in the two highest dose groups. At doses that maintain APTT at up to 150 s for 5 hrs 20 min, LMW-DS could be safely infused without affecting the platelet count or revealing other signs of increased bleeding risk. The observed endogenous release of islet protective hepatocyte growth factor could be an additional beneficial effect of LMW-DS during the first critical hours after transplantation.

The complement inhibitor low molecular weight dextran sulfate prevents TLR2-mediated activation of human natural killer cells

The complement inhibitor low molecular weight dextran sulfate prevents TLR2-mediated activation of human natural killer cells

The Complement Inhibitor Low Molecular Weight Dextran Sulfate Prevents TLR4-Induced Phenotypic and Functional Maturation of Human Dendritic Cells

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Dissecting the instant blood‐mediated inflammatory reaction in islet xenotransplantation

A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non-human primates. The detrimental instant blood-mediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss. This reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen. Low molecular weight dextran sulfate (LMW-DS) and the complement inhibitor Compstatin were used in a combination of in vitro and in vivo studies designed to dissect the xenogeneic IBMIR in a non-human primate model of pancreatic islet transplantation. Adult porcine islets (10,000 IEQs/kg) were transplanted intraportally into three pairs of cynomolgus monkeys that had been treated with LMW-DS or heparin (control), and the effects on the IBMIR were characterized. Porcine islets were also incubated in human blood plasma in vitro to assess complement inhibition by LMW-DS and Compstatin. Morphological scoring and immunohistochemical staining revealed that the severe islet destruction and macrophage, neutrophilic granulocyte, and T-cell infiltration observed in the control (heparin-treated) animals were abrogated in the LMW-DS-treated monkeys. Both coagulation and complement activation were significantly reduced in monkeys treated with LMW-DS, but IgM and complement fragments were still found on the islet surface. This residual complement activation could be inhibited by Compstatin in vitro. The xenogeneic IBMIR in this non-human primate model is characterized by an immediate binding of antibodies that triggers deleterious complement activation and a subsequent clotting reaction that leads to further complement activation. The effectiveness of LMW-DS (in vivo and in vitro) and Compstatin (in vitro) in inhibiting this IBMIR provides the basis for a protocol that can be used to abrogate the IBMIR in pig-human clinical islet transplantation.

Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C5

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.

Low molecular weight dextran sulfate site-specifically attenuates complement activation induced by ischemia/reperfusion and facilitates tolerance induction in allotransplantation

Low molecular weight dextran sulfate site-specifically attenuates complement activation induced by ischemia/reperfusion and facilitates tolerance induction in allotransplantation