Dehydroepiandrosterone sulfate in the assessment of the hypothalamic-pituitary-adrenal axis

The role of dehydroepiandrosterone-sulfate (DHEA-S) in assessing the integrity of the hypothalamic-pituitary-adrenal (HPA) axis in patients with suspected insufficiency is uncertain.

Genetic polymorphisms of manganese-superoxide dismutase and glutathione-S-transferase in chronic alcoholic pancreatitis

Chronic alcohol consumption is a major risk factor for the development of chronic pancreatitis. However, chronic pancreatitis occurs only in a minority of heavy drinkers. This variability may be due to yet unidentified genetic factors. Several enzymes involved in the degradation of reactive oxidants and xenobiotics, such as glutathione-S-transferase P1 (GSTP1) and manganese-superoxide dismutase (MnSOD) reveal functional polymorphisms that affect the antioxidative capacity and may therefore modulate the development of chronic pancreatitis and long-term complications like endocrine and exocrine pancreatic insufficiency. Two functional polymorphisms of the MnSOD and the GSTP1 gene were assessed by polymerase chain reaction and restriction fragment length polymorphism in 165 patients with chronic alcoholic pancreatitis, 140 alcoholics without evidence of pancreatic disease and 160 healthy control subjects. The distribution of GSTP1 and MnSOD genotypes were in Hardy-Weinberg equilibrium in the total cohort. Genotype and allele frequencies for both genes were not statistically different between the three groups. Although genotype MnSOD Ala/Val was seemingly associated with the presence of exocrine pancreatic insufficiency, this subgroup was too small and the association statistically underpowered. None of the tested genotypes affected the development of endocrine pancreatic insufficiency. Polymorphisms of MnSOD and GSTP1 are not associated with chronic alcoholic pancreatitis. The present data emphasize the need for stringently designed candidate gene association studies with well-characterized cases and controls and sufficient statistical power to exclude chance observations.

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.

Addition of dextran sulfate to blood cardioplegia attenuates reperfusion injury in a porcine model of cardiopulmonary bypass

OBJECTIVE: Contact of blood with artificial surfaces and air as well as ischemia/reperfusion injury to the heart and lungs mediate systemic and local inflammation during cardiopulmonary bypass (CPB). Activation of complement and coagulation cascades leads to and accompanies endothelial cell damage. Therefore, endothelial-targeted cytoprotection with the complement inhibitor and endothelial protectant dextran sulfate (DXS, MW 5000) may attenuate CBP-associated myocardial and pulmonary injury. METHODS: Eighteen pigs (DXS, n=10; phosphate buffered saline [PBS], n=8) underwent standard cardiopulmonary bypass. After aortic cross-clamping, cardiac arrest was initiated with modified Buckberg blood cardioplegia (BCP), repeated after 30 and 60 min with BCP containing either DXS (300 mg/10 ml, equivalent to 5mg/kg) or 10 ml of PBS. Following 30 min reperfusion, pigs were weaned from CPB. During 2h of observation, cardiac function was monitored by echocardiography and invasive pressure measurements. Inflammatory and coagulation markers were assessed regularly. Animals were then sacrificed and heart and lungs analyzed. RESULTS: DXS significantly reduced CK-MB levels (43.4+/-14.8 ng/ml PBS, 35.9+/-11.1 ng/ml DXS, p=0.042) and significantly diminished cytokine release: TNFalpha (1507.6+/-269.2 pg/ml PBS, 222.1+/-125.6 pg/ml DXS, p=0.0071), IL1beta (1081.8+/-203.0 pg/ml PBS, 110.7+/-79.4 pg/ml DXS, p=0.0071), IL-6 (173.0+/-91.5 pg/ml PBS, 40.8+/-19.4 pg/ml DXS, p=0.002) and IL-8 (304.6+/-81.3 pg/ml PBS, 25.4+/-14.2 pg/ml DXS, p=0.0071). Tissue endothelin-1 levels were significantly reduced (6.29+/-1.90 pg/100mg PBS, 3.55+/-1.15 pg/100mg DXS p=0.030) as well as thrombin-anti-thrombin formation (20.7+/-1.0 microg/ml PBS, 12.8+/-4.1 microg/ml DXS, p=0.043). Also DXS reduced cardiac and pulmonary complement deposition, neutrophil infiltration, hemorrhage and pulmonary edema (measured as lung water content, 81+/-3% vs 78+/-3%, p=0.047), indicative of attenuated myocardial and pulmonary CPB-injury. Diastolic left ventricular function (measured as dp/dt(min)), pulmonary artery pressure (21+/-3 mmHg PBS, 19+/-3 mmHg DXS, p=0.002) and right ventricular pressure (21+/-1 mmHg PBS, 19+/-3 mmHg DXS p=0.021) were significantly improved with the use of DXS. CONCLUSIONS: Addition of DXS to the BCP solution ameliorates post-CPB injury and to a certain extent improves cardiopulmonary function. Endothelial protection in addition to myocyte protection may improve post-CPB outcome and recovery.

Determination of ethyl sulfate in human serum and urine by capillary zone electrophoresis

The use of capillary zone electrophoresis (CZE) with indirect absorbance detection for the analysis of ethyl sulfate (EtS) in serum and urine was investigated. EtS is a direct metabolite of ethanol employed as marker for recent alcohol consumption. Fused-silica capillaries of 60 cm total length were either coated with cetyltrimethylammonium bromide (CTAB, 50 microm I.D. capillary) or poly(diallyldimethylammonium chloride) (PDADMAC, 100 microm I.D. capillary) to allow CZE analyses to be performed with reversed polarity. At pH 2.2 with a maleic acid/phthalic acid background electrolyte, both approaches provided reliable EtS serum levels down to 0.2 mg L(-1) (1.6 microM) for the analysis of solid-phase extracts that were prepared after chloride precipitation. Analysis of urines diluted to a conductivity of 5 S m(-1) and analyzed in the two capillary formats resulted in limits of quantification (LOQs) of 2 and 1 mg L(-1), respectively. With urines adjusted to 10 S m(-1) via dilution or condensation, an LOQ of 0.6 mg L(-1) (4.8 microM) was obtained in the CTAB coated capillary whereas in the PDADMAC-coated capillary of equal length not all matrix components were resolved from EtS. The developed assays are robust and suitable to monitor EtS in samples of individuals who consumed as little as one standard drink of an alcoholic beverage containing about 14 g of ethanol.

Low molecular weight dextran sulfate as complement inhibitor and cytoprotectant in solid organ and islet transplantation

Complement is an essential part of the innate immune system and plays a crucial role in organ and islet transplantation. Its activation, triggered for example by ischemia/reperfusion (I/R), significantly influences graft survival, and blocking of complement by inhibitors has been shown to attenuate I/R injury. Another player of innate immunity are the dendritic cells (DC), which form an important link between innate and adaptive immunity. DC are relevant in the induction of an immune response as well as in the maintenance of tolerance. Modulation or inhibition of both components, complement and DC, may be crucial to improve the clinical outcome of solid organ as well as islet transplantation. Low molecular weight dextran sulfate (DXS), a well-known complement inhibitor, has been shown to prevent complement-mediated damage of the donor graft endothelium and is thus acting as an endothelial protectant. In this review we will discuss the evidence for this cytoprotective effect of DXS and also highlight recent data which show that DXS inhibits the maturation of human DC. Taken together the available data suggest that DXS may be a useful reagent to prevent the activation of innate immunity, both in solid organ and islet transplantation.

Dextran sulfate facilitates anti-CD4 mAb-induced long-term rat cardiac allograft survival after prolonged cold ischemia

Ischemia/reperfusion injury leads to activation of graft endothelial cells (EC), boosting antigraft immunity and impeding tolerance induction. We hypothesized that the complement inhibitor and EC-protectant dextran sulfate (DXS, MW 5000) facilitates long-term graft survival induced by non-depleting anti-CD4 mAb (RIB 5/2). Hearts from DA donor rats were heterotopically transplanted into Lewis recipients treated with RIB 5/2 (20 mg/kg, days-1,0,1,2,3; i.p.) with or without DXS (grafts perfused with 25 mg, recipients treated i.v. with 25 mg/kg on days 1,3 and 12.5 mg/kg on days 5,7,9,11,13,15). Cold graft ischemia time was 20 min or 12 h. Median survival time (MST) was comparable between RIB 5/2 and RIB 5/2+DXS-treated recipients in the 20-min group with >175-day graft survival. In the 12-h group RIB 5/2 only led to chronic rejection (MST = 49.5 days) with elevated alloantibody response, whereas RIB 5/2+DXS induced long-term survival (MST >100 days, p < 0.05) with upregulation of genes related to transplantation tolerance. Analysis of the 12-h group treated with RIB 5/2+DXS at 1-day posttransplantation revealed reduced EC activation, complement deposition and inflammatory cell infiltration. In summary, DXS attenuates I/R-induced acute graft injury and facilitates long-term survival in this clinically relevant transplant model.

Locally targeted cytoprotection with dextran sulfate attenuates experimental porcine myocardial ischaemia/reperfusion injury

AIMS: Intravascular inflammatory events during ischaemia/reperfusion injury following coronary angioplasty alter and denudate the endothelium of its natural anticoagulant heparan sulfate proteoglycan (HSPG) layer, contributing to myocardial tissue damage. We propose that locally targeted cytoprotection of ischaemic myocardium with the glycosaminoglycan analogue dextran sulfate (DXS, MW 5000) may protect damaged tissue from reperfusion injury by functional restoration of HSPG. METHODS AND RESULTS: In a closed chest porcine model of acute myocardial ischaemia/reperfusion injury (60 min ischaemia, 120 min reperfusion), DXS was administered intracoronarily into the area at risk 5 min prior to reperfusion. Despite similar areas at risk in both groups (39+/-8% and 42+/-9% of left ventricular mass), DXS significantly decreased myocardial infarct size from 61+/-12% of the area at risk for vehicle controls to 39+/-14%. Cardioprotection correlated with reduced cardiac enzyme release creatine kinase (CK-MB, troponin-I). DXS abrogated myocardial complement deposition and substantially decreased vascular expression of pro-coagulant tissue factor in ischaemic myocardium. DXS binding, detected using fluorescein-labelled agent, localized to ischaemically damaged blood vessels/myocardium and correlated with reduced vascular staining of HSPG. CONCLUSION: The significant cardioprotection obtained through targeted cytoprotection of ischaemic tissue prior to reperfusion in this model of acute myocardial infarction suggests a possible role for the local modulation of vascular inflammation by glycosaminoglycan analogues as a novel therapy to reduce reperfusion injury.

Multimeric tyrosine sulfate acts as an endothelial cell protectant and prevents complement activation in xenotransplantation models

BACKGROUND: Activation of endothelial cells (EC) in xenotransplantation is mostly induced through binding of antibodies (Ab) and activation of the complement system. Activated EC lose their heparan sulfate proteoglycan (HSPG) layer and exhibit a procoagulant and pro-inflammatory cell surface. We have recently shown that the semi-synthetic proteoglycan analog dextran sulfate (DXS, MW 5000) blocks activation of the complement cascade and acts as an EC-protectant both in vitro and in vivo. However, DXS is a strong anticoagulant and systemic use of this substance in a clinical setting might therefore be compromised. It was the aim of this study to investigate a novel, fully synthetic EC-protectant with reduced inhibition of the coagulation system. METHOD: By screening with standard complement (CH50) and coagulation assays (activated partial thromboplastin time, aPTT), a conjugate of tyrosine sulfate to a polymer-backbone (sTyr-PAA) was identified as a candidate EC-protectant. The pathway-specificity of complement inhibition by sTyr-PAA was tested in hemolytic assays. To further characterize the substance, the effects of sTyr-PAA and DXS on complement deposition on pig cells were compared by flow cytometry and cytotoxicity assays. Using fluorescein-labeled sTyr-PAA (sTyr-PAA-Fluo), the binding of sTyr-PAA to cell surfaces was also investigated. RESULTS: Of all tested compounds, sTyr-PAA was the most effective substance in inhibiting all three pathways of complement activation. Its capacity to inhibit the coagulation cascade was significantly reduced as compared with DXS. sTyr-PAA also dose-dependently inhibited deposition of human complement on pig cells and this inhibition correlated with the binding of sTyr-PAA to the cells. Moreover, we were able to demonstrate that sTyr-PAA binds preferentially and dose-dependently to damaged EC. CONCLUSIONS: We could show that sTyr-PAA acts as an EC-protectant by binding to the cells and protecting them from complement-mediated damage. It has less effect on the coagulation system than DXS and may therefore have potential for in vivo application.