Genotyping of coeliac-specific human leucocyte antigen in children with type 1 diabetes: does this screening method make sense?

OBJECTIVES: Due to a high linkage disequilibrium of diabetes and coeliac-specific human leucocyte antigen (HLA) genotypes, the prevalence of coeliac disease (CD) in children and adolescents with diabetes mellitus type 1 (T1D) is much higher than in the general population. Recently, the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) revised new screening guidelines in which genotyping for coeliac-specific HLA alleles is recommended for high-risk patients as patients with T1D. The aim of our study was to investigate the frequency and distribution of coeliac-specific HLA genotypes in paediatric patients with T1D. STUDY DESIGN: HLA genotyping was performed on paediatric patients with T1D, recruited at the Medical University Hospital of Innsbruck and Graz. The test was done by PCR. Statistical analysis was performed with IBM-SPSS V.20. RESULTS: In 121 paediatric patients with T1D (52% male), mean age 13.3 (SD 3.9) years, mean age at diabetes diagnosis 7.4 (SD 3.8) and mean diabetes duration of 5.9 (SD 3.3) years, HLA genotyping was conducted. Ninety-two per cent showed positive HLA DQ2 and/or HLA DQ8 genotypes. Thirty-four per cent carried HLA DQ2, 33% were HLA DQ2+DQ8 positive and 25% of the patients showed positive results for HLA DQ8 alone. Only 8% had no coeliac-specific HLA markers. Four (3%) patients were diagnosed with CD. CONCLUSIONS: The majority of paediatric patients with T1D has positive coeliac-specific HLA genotypes DQ2 and/or DQ8. Therefore, genotyping for coeliac-specific HLA alleles as a first-line test in patients with T1D as recommended in the ESPGHAN guidelines does not seem reasonable. Screening for coeliac-specific antibodies needs to be performed on a regular basis for patients with T1D.

Predicting Transfusion Requirements During Extracorporeal Membrane Oxygenation.

OBJECTIVE: Patients requiring extracorporeal membrane oxygenation (ECMO) have a well-known bleeding risk and the potential for experiencing possibly fatal thromboembolic complications. Risk factors and predictors of transfusion requirements during ECMO support remain uncertain. The authors hypothesized that compromised organ function immediately before ECMO support will influence transfusion requirements. DESIGN: A prospective observational study. SETTING: A tertiary, single-institutional university hospital. PARTICIPANTS: The study included 40 adult patients requiring ECMO for intractable cardiac and respiratory failure between July 2010 and December 2012. Blood samples were taken before initiation of ECMO (baseline), after 24 and 48 hours on ECMO, and 24 hours after termination of ECMO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Independent of veno-arterial or veno-venous support, 26% of patients required≥2 packed red blood cells per day (PRBC/d) and 74% of patients required<2 PRBC/d during ECMO. Requirements of≥2 PRBC/d during ECMO support were associated with higher creatinine levels and lower prothrombin times (PT, %) at baseline and with impaired platelet function after 24 hours on ECMO. Platelet function, activated by thrombin receptor-activating peptide stimulation, decreased by 30% to 40% over time on ECMO. Receiver operating characteristic curve analysis showed cut-off values for creatinine of 1.49 mg/dL (sensitivity 70%, specificity 70%; area under the curve [AUC] 0.76, 95% confidence interval [CI] 0.58-0.94), for PT of 48% (sensitivity 80%, specificity 59%; AUC 0.69, 95% CI 0.50-0.87), and for thrombin receptor-activating peptide (TRAP) 32 U (sensitivity 90%, specificity 68%; AUC 0.76, 95% CI 0.59-0.93). CONCLUSIONS: The results of this study demonstrated that increased creatinine levels and lower PT before ECMO and secondary impaired platelet function significantly increased transfusion requirement.

Extracorporeal membrane oxygenation induces short-term loss of high-molecular-weight von Willebrand factor multimers.

BACKGROUND: High-molecular-weight (HMW) von Willebrand factor (vWF) multimers are crucial for primary hemostasis. Increased shear stress from ventricular assist devices can provoke premature degradation of HMW vWF multimers. Whether similar loss of vWF multimers occurs during extracorporeal membrane oxygenation (ECMO) is not clear. METHODS: We conducted a prospective observational study in a clinical cohort of patients who required ECMO for intractable cardiac and/or respiratory failure. The primary end point was the quantity and quality of HMW vWF multimer bands before, during, and after ECMO support. To investigate further changes in primary hemostasis, we also measured vWF antigen activity (vWF:Ag), vWF ristocetin cofactor activity (vWF:RCo), and factor VIII in 38 patients who required ECMO support before initiation of ECMO (baseline), after 24 and 48 hours on ECMO, and 24 hours after termination of ECMO therapy. RESULTS: Compared with baseline, vWF:Ag and vWF:RCo decreased after 24 hours of ECMO (mean ± SD, vWF:Ag, 307% ± 152% to 261% ± 138%, P = 0.002; vWF:RCo 282% ± 145% to 157% ± 103%, P < 0.0001) and remained lower during ongoing support (vWF:Ag 265% ± 128%, P = 0.025; vWF:RCo 163% ± 94%, P < 0.0001). After termination of ECMO, vWF:Ag was greater than baseline (359% ± 131%, P = 0.004) and vWF:RCo was similar to baseline levels (338% ± 142%, P = 0.046). Compared with baseline, the calculated vWF:RCo/vWF:Ag ratio decreased after 24 hours on support (0.96 ± 0.23 to 0.61 ± 0.17, P ≤ 0.0001) and remained lower during 48 hours on ECMO (0.63 ± 0.18, P ≤ 0.0001). After termination of ECMO support (0.94 ± 0.19, P = 0.437), values rapidly returned to baseline. The number of HMW vWF multimers (n) decreased from baseline after 24 hours on ECMO (21 ± 1.4 to 14 ± 1.8, P ≤ 0.0001) and after 48 hours on ECMO (15 ± 2.1, P ≤ 0.0001). Twenty-four hours after termination of ECMO support, HMW vWF multimeric pattern had returned to baseline values (21 ± 1.8, P = 0.551). CONCLUSIONS: Loss of HMW vWF multimer bands occurred in patients undergoing ECMO support and resolved after the termination of ECMO. Although not detectable with coagulation screening tests, a vWF:RCo/vWF:Ag ratio <0.7 during ECMO was highly indicative for loss of HMW vWF multimers. Our findings may at least in part explain increased bleeding tendency during ECMO therapy. Administration of vWF concentrates may support restoration of primary hemostasis in patients with relevant bleeding during ECMO support.

Anti-red blood cell antibodies, free light chains, and antiphospholipid antibodies in intravenous immunoglobulin preparations.

BACKGROUND: Anti-red blood cell antibodies, free light chains (FLC) and prothrombotic proteins (PTP) may co-elute with intact immunoglobulin (IgG), and may be the cause of adverse reactions to intravenous immunoglobulin preparations (IVIG). OBJECTIVES: To investigate the presence of residual amounts of these components in IVIG and their effects on ABO blood group agglutination. METHODS: Iso-agglutinin anti-A and anti-B activity was determined with a direct hemagglutination assay of red blood cell (RBC) suspensions from 1% of 46 blood donors together with the serial dilutions of five IVIG (IV1, IV2, IV3, IV4, IV5). Anti-A1 monoclonal antibody was used to confirm reactivity with the A1-reference RBC. The selected IVIG were diluted to a final concentration of 25 mg/ml in 0.15 M NaCI and 0.01 M phosphate-buffered saline, pH 7.4, with or without a further twofold dilution in a low ionic strength solution. RESULTS: A variation up to fivefold in the titer strength of anti-A/B activity was observed between the IVIG preparations. A2-type RBC required higher IVIG inputs when tested in 0.15 M NaCl. The differences in FLC kappa and lambda concentrations were as high as > 400 mg/L among the various IVIG. Only IV1 had a significantly high level of antiphospholipid IgG antibodies (18 U/ml). We demonstrated the presence of anti-RBC antibodies, FLC and PTP in IVIG preparations. CONCLUSIONS: Our findings provide clear evidence that IVIG may harbor pathophysiological substrates with a potential risk for adverse effects such as iatrogenic hemolysis, FLC-associated disorders, and thromboembolism.

Interference of the new oral anticoagulant dabigatran with frequently used coagulation tests.

BACKGROUND: Dabigatran etexilate is a new oral anticoagulant for the therapy and prophylaxis of venous thromboembolism and stroke prevention in patients with atrial fibrillation. To investigate the extent of interactions of this new anticoagulant with frequently used coagulation assays, we completed a multicenter in vitro trial with Conformité Européenne(CE)-labeled dabigatran-spiked plasma samples. METHODS: Lyophilized plasma samples with dabigatran concentrations ranging from 0.00 to 0.48 µg/mL were sent to the coagulation laboratories of six major Austrian hospitals for evaluation. Coagulation assays were performed under routine conditions using standard reagents and analyzer. RESULTS: Dabigatran led to a dose-dependent prolongation of the clotting times in coagulometric tests and influenced the majority of the parameters measured. Statistically significant interference could be observed with the prothrombin time (PT), activated partial thromboplastin time (aPTT) and PT/aPTT-based assays (extrinsic/intrinsic factors, APC-resistance test) as well as lupus anticoagulant testing. Even non-clotting tests, such as the colorimetric factor XIII activity assay and to a minor extent the amidolytic antithrombin activity assay (via factor IIa) were affected. CONCLUSIONS: This multicenter trial confirms and also adds to existing data, demonstrating that laboratories should expect to observe strong interferences of coagulation tests with increasing concentrations of dabigatran. This finding might become particularly important in the elderly and in patients with renal impairment as well as patients whose blood is drawn at peak levels of dabigatran.

Electrospun small-diameter polyurethane vascular grafts: ingrowth and differentiation of vascular-specific host cells.

No small-diameter synthetic graft has yet shown comparable performance to autologous vessels. Synthetic conduits fail due to their inherent surface thrombogenicity and the development of intimal hyperplasia. In addressing these shortcomings, electrospinning offers an interesting alternative to other nanostructured, cardiovascular substitutes because of the close match of electrospun materials to the biomechanical and structural properties of native vessels. In this study, we investigated the in vivo behavior of electrospun, small-diameter conduits in a rat model. Vascular grafts composed of polyurethane were fabricated by electrospinning. Prostheses were implanted into the abdominal aorta in 40 rats for either 7 days, 4 weeks, 3 months, or 6 months. Retrieved specimens were evaluated by histology, immunohistochemical staining, confocal laser scanning microscopy, and scanning electron microscopy. At all time points, we found no evidence of foreign body reaction or graft degradation. The overall patency rate of the intravascular implants was 95%. Within 7 days, grafts revealed ingrowth of host cells. CD34+ cells increased significantly from 7 days up to 6 months of implantation (P < 0.05). Myofibroblasts and myocytes showed increasing cell numbers up to 3 months (P < 0.05). Ki67 staining indicated unaltered cell proliferation during the whole follow-up period. Besides biomechanical benefits, electrospun polyurethane grafts exhibit excellent biocompatibility in vivo. Cell immigration and differentiation seems to be promoted by the nanostructured artificial matrix.

Role of heme oxygenase-1 in human endothelial cells: lesson from the promoter allelic variants.

OBJECTIVE: Heme oxygenase-1 (HO-1) is an antioxidative, antiinflammatory, and cytoprotective enzyme that is induced in response to cellular stress. The HO-1 promoter contains a (GT)n microsatellite DNA, and the number of GT repeats can influence the occurrence of cardiovascular diseases. We elucidated the effect of this polymorphism on endothelial cells isolated from newborns of different genotypes. METHODS AND RESULTS: On the basis of HO-1 expression, we classified the HO-1 promoter alleles into 3 groups: short (S) (most active, GT < or = 23), medium (moderately active, GT=24 to 28), and long (least active, GT > or = 29). The presence of the S allele led to higher basal HO-1 expression and stronger induction in response to cobalt protoporphyrin, prostaglandin-J(2), hydrogen peroxide, and lipopolysaccharide. Cells carrying the S allele survived better under oxidative stress, a fact associated with the lower concentration of oxidized glutathione and more favorable oxidative status, as determined by measurement of the ratio of glutathione to oxidized glutathione. Moreover, they proliferated more efficiently in response to vascular endothelial growth factor A, although the vascular endothelial growth factor-induced migration and sprouting of capillaries were not influenced. Finally, the presence of the S allele was associated with lower production of some proinflammatory mediators, such as interleukin-1beta, interleukin-6, and soluble intercellular adhesion molecule-1. CONCLUSIONS: The (GT)n promoter polymorphism significantly modulates a cytoprotective, proangiogenic, and antiinflammatory function of HO-1 in human endothelium.

Electrospun polyurethane vascular grafts: in vitro mechanical behavior and endothelial adhesion molecule expression.

Engineered small diameter vascular grafts must closely match mechanical characteristics of native vessels and exhibit stimulus-responsive bioactivity. In this study, mechanical homogeneity of electrospun small diameter polyurethane grafts as well as spontaneous attachment, proliferation, and adhesion molecule expression of endothelial cells (EC) in their presence was studied in vitro. Axial and circumferential tensile strengths were measured and found to be twofold higher in the circumferential direction. EC attachment was easily achieved without precoating the fiber matrix. Stimulation of EC with interleukin-1beta (IL-1beta) led to a statistically significant upregulation of the adhesion molecules E-Selectin, ICAM-1, and VCAM-1. Quantification of adhesion molecule expression by means of energy-dispersive X-ray microanalysis revealed no differences in the stimulatory responses of EC cultured on electrospun polyurethane when compared with cells grown on tissue culture-treated cover slips. Summarizing, highly uniform small diameter polyurethane grafts were fabricated and shown to allow spontaneous EC attachment. The synthetic graft surface neither impaired the endothelial response toward IL-1beta stimulation nor did it adversely affect the regulation of expression of endothelial adhesion molecules.

Scanning electron microscopy and energy-dispersive x-ray microanalysis: a valuable tool for studying cell surface antigen expression on tissue-engineered scaffolds.

The influence of an acellular porcine matrix on proinflammatory activation of endothelial cells (EC) during normoxia and hypoxia was investigated by a newly established semi-quantitative electron microscopic procedure. As a model, three adhesion molecules (E-selectin, ICAM-1, and VCAM-1) were localized by silver-enhanced immunogold staining and energy dispersive X-ray microanalysis after normoxic or hypoxic pretreatment of the cells and subsequent stimulation with IL-1beta. Morphology of EC grown on porcine matrix or coverslips was recorded simultaneously using secondary electron imaging. EC appeared tightly attached to the underlying surfaces with their typical cobblestone-like morphology. Statistically significant upregulations upon stimulation with IL-1beta were observed in both groups for all three adhesion molecules. Hypoxic pretreatment of the specimens with subsequent reoxygenation neither induced morphological changes nor caused an upregulation of adhesion molecule expression in cells grown on acellular porcine tissue. Unexpectedly, in cells seeded onto the acellular matrix, IL-1beta failed to upregulate ICAM-1 expression after a short period of hypoxia. The surface expression of VCAM-1 was also significantly lower even under normoxic conditions, which might indicate the development of functional impairment of cells in contact with acellular porcine tissue. The method presented in this study has proven valuable for the determination of antigen expression on scaffold materials in parallel with the characterization of surface morphology.

15d-PGJ2 upregulates synthesis of IL-8 in endothelial cells through induction of oxidative stress.

15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) is a cyclopentenone prostaglandin regarded as antiinflammatory mediator, which can act through peroxisome proliferator-activated receptor-gamma (PPARgamma) or through G protein-coupled surface receptors. It has been demonstrated that 15d-PGJ(2) potently increases the generation of interleukin-8 (IL-8) in human microvascular endothelial cells (HMEC-1s); however, the mechanism of this induction is not known. The aim of the study was to find the pathway involved in 15d-PGJ(2)-mediated IL-8 stimulation. Our data confirmed that the effect of 15d-PGJ(2) is independent of PPARgamma. For the first time, we excluded the activation of G proteins and the contribution of G protein-coupled surface receptors in endothelial cells treated with 15d-PGJ(2). Instead, we demonstrated that stimulation of IL-8 involved induction of oxidative stress, activation of p38 kinases, and increase in stability of IL-8 mRNA. Upregulation of IL-8 promoter, although measurable, seemed to play a less-pronounced role. Additionally, our results indicate the involvement of cAMP elevation and may suggest a role for ATF2 transcription factor. Concomitant induction of heme oxygenase-1 in HMEC-1s did not influence the synthesis of IL-8. In summary, we showed that 15d-PGJ(2), acting through oxidative stress, may exert proinflammatory effects. The upregulation of IL-8 is mostly associated with p38-mediated stabilization of mRNA.

Lymphocyte activation and correlation with IMPDH activity under therapy with mycophenolate mofetil.

BACKGROUND: In an attempt to monitor the pharmacodynamics of mycophenolate mofetil (MMF) we investigated the association of inosine monophosphate dehydrogenase (IMPDH) activity in peripheral blood mononuclear cells with the expression of lymphocyte activation markers in stable cardiac transplant recipients treated with MMF. METHODS: Twenty-four study patients were switched from azathioprine to MMF 7.2+/-4.1 years after heart transplantation. RESULTS: While the MPA trough level remained unchanged, the mean activity of IMPDH declined from 890 to 462 pmol/10(6)PBMC/h three months after onset of MMF therapy, was almost completely inhibited at six months and partially restored to 160 pmol/10(6)PBMC/h 12 months after switch to MMF (p< .0001). We detected also significant changes in a number of activated lymphocyte subsets: CD4+/25+, CD8+/38+, CD19+/69+, CD3+/16+/56+, natural killer (NK) cells, and monocytes. Moreover, the IMPDH activity profile correlated positively with the number of CD8+/38+ T cells (correlation coefficient (CC) +0.53), and inversely with NK cells (CC -0.52) and CD19+/69+ cells (CC -0.61). CONCLUSIONS: We revealed a close association of IMPDH baseline activity in mononuclear cells with the expression of lymphocyte activation markers in stable heart transplant patients after introduction of MMF therapy. This supports the assumption of a rather immunomodulatory than immunosuppressive effect of MMF.

Decellularized, xenogeneic small-diameter arteries: transition from a muscular to an elastic phenotype in vivo.

Reports regarding the biocompatibility of xenogeneic, decellularized bioprosthetic implants differ between bioinertness and complete graft degradation. We investigated heparin-crosslinked and nonheparinized, xenogeneic vascular substitutes in a rat model. Porcine arteries (15 x 1.5 mm) were decellularized by multistep detergent and enzymatic techniques, which were followed by heparin-crosslinking in 50% of the implants. Prostheses were implanted into the abdominal aorta of 76 rats for 1 day and up to 6 months. Retrieved specimens were evaluated by histology, immunohistochemistry, laser scanning, and scanning electron microscopy. Graft patency did not differ between groups (97.3%). Heparinized grafts showed a statistically significant lower rate of aneurysm formation (p = 0.04 %). Implants revealed infiltration with granulocytes and macrophages up to 3 months. Recellularization with endothelial cells and myofibroblasts was detectable within 1 month. After 6 months elastin biosynthesis and complete graft remodeling toward an elastic vessel was evident. These results indicate that temporary inflammation does not interfere with long-term vascular remodeling.

IgG deposition and activation of the classical complement pathway involvement in the activation of human granulocytes by decellularized porcine heart valve tissue.

Decellularization treatment of heart valves has been thought to eliminate tissue immunogenicity. Early failure of tissue-engineered xenogeneic heart valves was seen in children and has been a major drawback in this promising field of research. This study was designed to characterize the effects of acellular porcine heart valve tissue on immune activation in vitro. Incubation of decellularized porcine tissue with human plasma led to adsorption of IgG, activation of the classical complement pathway and adhesion of activated polymorphonuclear leukocytes (PMN). This inflammatory response was strongly inhibited by proteins extracted from native porcine tissue which might indicate that inhibitors of PMN activation present in the extracellular matrix (ECM) are lost during the decellularization process.

Beck and back: a paradigm change in coronary sinus interventions--pulsatile stretch on intact coronary venous endothelium.

OBJECTIVES: Strategies to recover myocardium in therapeutically unresponsive patients are again under scrutiny, including techniques developed in the pioneering days of cardiothoracic surgery such as retroperfusion via the coronary sinus--the Beck procedure. An underestimated aspect of retroperfusion is the formation of new vessels. This early observation of neoangiogenesis may be an important mechanism in observed benefits. We hypothesized that periodic pressure elevation in coronary veins induces an analogy to shear stress angiogenic pulses by activating venous endothelium. Pulsatile stretch on venous endothelium can be achieved easily by a pressure-controlled intermittent balloon blockade of the coronary sinus outflow. METHODS: Three hours of myocardial ischemia was induced in 12 pigs. Pressure-controlled intermittent coronary sinus occlusion was applied in 6 animals 15 minutes after occlusion of the left anterior descending coronary artery. Postmortem myocardial specimens were taken, and heme oxygenase-1, vascular endothelial growth factor gene expression, and hypoxia-induced factor activity were measured. RESULTS: As compared with controls, treated animals released an angiogenic pulse by a 4-fold increase of heme oxygenase-1 gene expression in the infarct area (P < .001), together with a 2.5-fold enhanced transcription of vascular endothelial growth factor in the infarct (P < .006), border (P < .002), and remote (P < .02) areas, whereas hypoxia-induced factor activity was similar in both groups. A significant correlation (P < .01) of the achieved coronary sinus pressure elevation and gene expression was found. CONCLUSIONS: Mechanotransduction of pulsatile stretch on coronary venous endothelium by pressure-controlled intermittent coronary sinus occlusion induces heme oxygenase-1 and vascular endothelial growth factor gene expression, leaving the ischemic pathway of the hypoxia-induced factor activity unchanged. This cascade of molecular events closes the argument gap to historical reports of the Beck procedure on revascularization and myocardial salvage.

Activation of the purine salvage pathway in mononuclear cells of cardiac recipients treated with mycophenolate mofetil.

BACKGROUND: The objective of this study was to investigate purine nucleotide metabolism in peripheral blood mononuclear cells (PBMC) of cardiac transplant recipients switched from azathioprine to mycophenolate mofetil (MMF). METHODS: Concentrations of guanosine 5'triphosphate (GTP) and adenosine 5'triphosphate (ATP), the activities of inosine monophosphate dehydrogenase (IMPDH), guanine phosphoribosyltransferase (GPRT), and hypoxanthine phosphoribosyltransferase (HPRT) were determined in PBMC of 27 cardiac transplant recipients before switch to MMF and 3, 6, and 12 months thereafter. RESULTS: There was no difference in the activities of IMPDH and salvage pathway enzymes GPRT and HRPT as well as in intracellular GTP and ATP concentrations between the patients before switch to MMF and healthy controls. The GTP and ATP concentrations in PBMC of cardiac recipients did not change during the entire observation period. Although the MPA trough level remained similar, IMPDH activity declined from 897 to 316 pmol/10(6)PBMC/h 3 months after MMF onset, was almost completely inhibited after 6 months, and partially restored to 143 pmol/10(6)PBMC/h 12 months after switch to MMF. In contrast, GPRT activity increased after 3, 6, and 12 months of MMF therapy and HPRT activity 3 and 6 months after switch to MMF. CONCLUSIONS: We demonstrated for the first time an induction of salvage pathway enzyme activities in PBMC under MMF therapy. This probably accounts for the maintenance of intracellular purine nucleotide pools and prevents the GTP depletion.

Effects of 15d-PGJ(2) on VEGF-induced angiogenic activities and expression of VEGF receptors in endothelial cells.

15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) upregulates expression of vascular endothelial growth factor (VEGF), but may inhibit angiogenesis. We found that 15d-PGJ(2) (1-10muM) attenuated all VEGF-induced angiogenic activities in human umbilical vein endothelial cells (HUVEC). It blocked almost completely cell proliferation, potently reduced migration, assembly into tube-like network on matrigel, and growth of capillaries into collagen gel. 15d-PGJ(2) inhibited expression of VEGFR-1 and VEGFR-2 receptors both at mRNA and protein levels. This inhibition, however, was transient (observed after 6-12h, but not after 24h) and weak (20-30%), and could not fully explain inhibition of response to VEGF. Accordingly, proliferation was inhibited when 15d-PGJ(2) was added 24h after VEGF or in cells stimulated with basic fibroblast growth factor. Interestingly, 15d-PGJ(2) decreased activities of c-jun and c-myc in HUVEC and overexpression of c-myc attenuated its antiproliferative effects. This suggests that inhibition of this transcription factor by 15d-PGJ(2) contributes to decrease in angiogenic response.

Decellularization does not eliminate thrombogenicity and inflammatory stimulation in tissue-engineered porcine heart valves.

BACKGROUND AND AIM OF THE STUDY: In tissue engineering of heart valves using decellularized xenogenic valves, it has been suggested that cell elimination would result in a biologically inert matrix. The aim of this in-vitro investigation was to evaluate different decellularization methods in regard to the completeness of cell removal, inflammatory response, and thrombocyte activation. METHODS: Decellularized porcine Synergraft valves were compared with porcine pulmonary conduits decellularized with Triton X-100, sodium deoxycholate, Igepal CA-630 and ribonuclease. Completeness of decellularization was evaluated with staining for nuclei and alpha-Gal epitope. Decellularized heart valves with and without seeding with endothelial cells (ECs) were incubated with human platelet-rich plasma and stained for CD41 and PAC-1 to evaluate thrombocyte activation. Samples were processed for laser scanning microscopy (LSM) and scanning electron microscopy (SEM). Migration of human monocytic cells towards extracted valve proteins was tested. RESULTS: In contrast to the Synergraft, complete cell removal and elimination of the alpha-gal epitope was achieved with the new decellularization method. Numerous adherent and activated platelets were found on the decellularized matrix. This was inhibited by seeding with ECs. Even in completely cell-free valve tissue extracellular matrix proteins attracted human monocytic cells as in early inflammation, depending on whether porcine or human tissue was used. CONCLUSION: Important differences were found in the decellularization efficacy of treatment methods. However, even complete elimination of cells and their remnants did not result in a biologically inert matrix. The decellularized porcine heart valve matrix has the potential to attract inflammatory cells and to induce platelet activation. These findings suggest that it will be important to control the different inflammation-stimulating factors if porcine tissues are to be used successfully in tissue engineering.

Inosine 5'-monophosphate dehydrogenase inhibition by mycophenolic acid impairs maturation and function of dendritic cells.

BACKGROUND: The mechanism of action of mycophenolic acid (MPA) has been described as a blockade of inosine 5'-monophosphate dehydrogenase (IMPDH) and is thought to selectively influence T- and B-lymphocytes due to their strong dependency on guanine nucleotides synthesized via the de novo purine synthesis pathway. Recent evidence suggests MPA to affect antigen-presenting cells. METHODS: Using CD14+ derived human dendritic cells (DC) we have investigated the effects of MPA on differentiation, maturation and function and studied intracellular nucleotide content and IMPDH activity. RESULTS: GTP content and IMPDH activities of DC were strongly and dose-dependently decreased when MPA was present during the entire culture period or was added after the fifth (immature DC) or the seventh (mature DC) day of culture. Concurrent to low GTP levels, a dose-dependent reduction in the expression of CD80, CD86, CD40, CD54 and CD83 was seen which was accompanied by a decreased capacity of DC to stimulate T-cells. Our data for the first time shows a direct effect of MPA on the maturation and function of human CD14+ derived DC, indicates a role of IMPDH and a dependency on the de novo purine synthesis pathway.

Collection and storage of leukocyte depleted whole blood in autologous blood predeposit in elective surgery programs.

INTRODUCTION: The aim of this investigation was to provide evidence that leukocyte depleted whole blood meets the requirements for transfusion of the European Council and thus may be an alternative to leukocyte and plasma depleted packed red blood cells in autologous blood predeposit for patients undergoing elective surgery programs. MATERIAL AND METHODS: Standard units of 450mL blood were collected from 25 healthy male volunteers. Leukocyte depletion was done via inline filtration 4h after collection. Storage lesion was assessed by measuring the release of K(+), LDH, free hemoglobin, and lactate into the storage medium, as well as by the increase of hemolysis, the decrease of pH and consumption of glucose over a storage period of 35 days. As surrogate marker for red cell quality the intracellular concentrations of adenine nucleotides [ATP, ADP, AMP] were determined. RESULTS: The extent of storage lesion remained within the ranges of standard liquid storage conditions. Hemolysis was far below the threshold of 0.8% in all WB units at the end of their shelf life. Only minor changes of intracellular adenine nucleotide levels were measured indicating a preserved function of red blood cells in leukocyte depleted whole blood. At the end of shelf life 70%+/-18% of initial ATP levels were detected. CONCLUSION: Based on our data we propose that leukocyte depleted whole blood, stored for 35 days can be an option in the autologous blood supply as it meets the requirements for transfusion of the European Council.