Jolan Harsfalvi

Seroreactivity to microbial components in Crohn's disease is associated with ileal involvement, noninflammatory disease behavior and NOD2/CARD15 genotype, but not with risk for surgery in a Hungarian cohort of IBD patients

Background: Antibodies directed against Saccharomyces cerevisiae (ASCA), perinuclear components of neutrophils (pANCA), and porin protein C of Escherichia coli (anti‐OmpC) are reported to be associated with disease phenotype and may be of diagnostic importance in inflammatory bowel disease (IBD). Since limited data are available from Eastern Europe, we assessed the above antibodies in Hungarian IBD patients. Methods: In all, 653 well‐characterized, unrelated consecutive IBD patients (Crohns disease [CD]: 558, m/f: 263/295, duration: 8.1 ± 10.7 years; ulcerative colitis [UC]: 95, m/f: 44/51, duration: 8.9 ± 9.8 years) and 100 healthy subjects were investigated. Sera were assayed for anti‐Omp and ASCA by enzyme‐linked immunosorbent assay (ELISA) and ANCA by indirect immunofluorescence assay (IIF). TLR4 and NOD2/CARD15 variants were tested by polymerase chain reaction/restriction fragment length polymorphism (PCR‐RFLP). Detailed clinical phenotypes were determined by reviewing the medical charts. Results: Anti‐Omp, ASCA, and atypical pANCA antibodies were present in 31.2%, 59.3%, and 13.8% of CD, 24.2%, 13.7%, and 48.5% of UC patients, and in 20%, 16%, and 5.6% of controls, respectively. ASCA and anti‐Omp positivity were associated with increased risk for CD (odds ratio [OR]ASCA = 7.65, 95% confidence interval [CI]: 4.37–13.4; OROmp = 1.81, 95% CI: 1.08–3.05). In a logistic regression analysis, anti‐Omp and ASCA were independently associated with ileal and noninflammatory disease, but not with a risk for surgery or response to steroids or infliximab. A serology dosage effect was also observed. ASCA and anti‐Omp antibodies were associated with NOD2/CARD15, in addition to a gene dosage effect. No associations were found in UC. Conclusions: Serological markers were useful in the differentiation between CD and UC in an Eastern European IBD cohort. Reactivity to microbial components was associated with disease phenotype and NOD2/CARD15 genotype, further supporting the role of altered microbial sensing in the pathogenesis of CD.

Acute phase proteins in the diagnosis and prediction of cirrhosis associated bacterial infections

Bacterial infections are common cause of morbidity and mortality in patients with cirrhosis. The early diagnosis of these infections is rather difficult.

Factor XIII, clot structure, thrombosis.

Blood coagulation factor XIII (FXIII) is a tetrameric protein consisting of two catalytic A (FXIII-A) and two carrier/inhibitory B (FXIII-B) subunits. It is a zymogen, which becomes transformed into an active transglutaminase (FXIIIa) in the final phase of coagulation cascade by thrombin and Ca(2+). FXIII is essential for hemostasis, its deficiency results in severe bleeding diathesis. FXIIIa mechanically stabilizes fibrin by cross-linking its α-, and γ-chains. It also protects newly formed fibrin from fibrinolysis, primarily by cross-linking α(2)-plasmin inhibitor to fibrin. Beside the above prothrombotic effects, it is involved in limiting thrombus growth by down-regulating platelet adhesion to fibrin. Elevated FXIII level seems to be a gender-specific risk factor of both coronary artery disease and peripheral arterial disease, it represents an increased risk only in females. The association of FXIII level with the risk of ischemic stroke and venous thromboembolism was investigated only in a few studies from which no clear conclusion could be drawn. Among the FXIII subunit polymorphisms, concerning their effect on the risk of thrombotic diseases, only FXIII-A p.Val34Leu was investigated extensively. Meta-analyses of reported data suggest that this polymorphism provides a moderate protection against coronary artery disease and venous thromboembolism, but not against ischemic stroke. Gene-gene and gene-environmental interactions might modify its effect. Further studies are required to explore the effect of other FXIII subunit polymorphism on the risk of thrombotic diseases.

Increased plasma von Willebrand factor antigen levels but normal von Willebrand factor cleaving protease (ADAMTS13) activity in preeclampsia

The activity of ADAMTS13, the von Willebrand factor (VWF) cleaving protease is low in several conditions, including HELLP (haemolysis, elevated liver enzymes, and low platelet count) syndrome. As HELLP syndrome develops in most cases on the basis of preeclampsia, our aim was to determine whether plasma ADAMTS13 activity is decreased in preeclampsia. Sixty-seven preeclamptic patients, 70 healthy pregnant women and 59 healthy non-pregnant women were involved in this case-control study. Plasma ADAMTS13 activity was determined with the FRETS-VWF73 assay, while VWF antigen (VWF:Ag) levels with an enzyme-linked immunosorbent assay. The multimeric pattern of VWF was analyzed by SDS-agarose gel electrophoresis. There was no significant difference in plasma ADAMTS13 activity between the preeclamptic and the healthy pregnant and non-pregnant groups (median [25-75 percentile]: 98.8 [76.5-112.8] %, 96.3 [85.6-116.2] % and 91.6 [78.5-104.4] %, respectively; p > 0.05). However, plasma VWF:Ag levels were significantly higher in preeclamptic patients than in healthy pregnant and non-pregnant women (187.1 [145.6-243.1] % versus 129.3 [105.1-182.8] % and 70.0 [60.2-87.3] %, respectively; p < 0.001). The multimeric pattern of VWF was normal in each group. Primiparas had lower plasma ADAMTS13 activity than multi-paras (92.6 [75.8-110.6] % versus 104.2 [92.1-120.8] %; p = 0.011). No other relationship was found between clinical characteristics, laboratory parameters and plasma ADAMTS13 activity in either study group. In conclusion, plasma ADAMTS13 activity is normal in preeclampsia despite the increased VWF:Ag levels. However, further studies are needed to determine whether a decrease in plasma ADAMTS13 activity could predispose preeclamptic patients to develop HELLP syndrome.

Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross‐linking enzyme, which forms isopeptide bonds between protein‐linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine‐donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine‐donor substrate. Twenty‐six Gln‐containing sequences from the second and third biopanning rounds were susceptible for TG2‐mediated incorporation of 5‐(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage‐selected sequences, and the N‐terminal glutamine‐rich domain of SWI1/SNF1‐related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry‐based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q6, Q8, and Q22 are modified by TG2. Kinetic parameters of SnQ1 transamidation (KMapp = 250 μM, kcat = 18.3 sec−1, and kcat/KMapp = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full‐length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.

4‐Thio‐deoxyuridylate‐modified thrombin aptamer and its inhibitory effect on fibrin clot formation, platelet aggregation and thrombus growth on subendothelial matrix

Summary.  Background: The consensus thrombin aptamer C15‐mer is a single‐stranded DNA of 15 nucleotides [d(GGTTGGTGTGGTTGG)] that was identified by the selection of thrombin‐binding molecules from a large combinatorial library of oligonucleotides. It is capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite 1. As has been shown in our earlier studies, the 4‐thio‐deoxyuridylate (s4dU)‐containing oligonucleotides have high affinity for a number of proteins, due to the reduced hydrophilic character of the modified oligonucleotide. Methods: Three different analogs of the original thrombin‐inhibiting sequence, in which some of the thymidylate residues were replaced by 4‐thio‐deoxyuridylates, were synthesized. The inhibitory effect of modified aptamers was tested on thrombin‐catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin‐induced platelet aggregation/secretion, and the formation of thrombus on coverslips coated with human collagen type III, thrombin‐treated fibrinogen or subendothelial matrix of human microvascular endothelial cells. Results: As compared with the C15‐mer, the analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15‐mer) showed a 2‐fold increased inhibition of thrombin‐catalyzed fibrin clot formation, fibrinopeptide A release, platelet aggregation and secretion in human plasma and thrombus formation on thrombin‐treated fibrinogen surfaces under flow conditions. Concerning the inhibition of thrombin‐induced fibrin formation from purified fibrinogen and activation of washed platelets, UC15‐mer was 3‐fold and twelve‐fold more effective than C15‐mer, respectively. Conclusion: The replacement of four thymidylate residues in C15‐mer by 4‐thio‐deoxyuridylate resulted in a new thrombin aptamer with increased anticoagulant and antithrombotic properties.

Interaction between tissue transglutaminase and phospholipid vesicles

A specific interaction between purified liver transglutaminase and small unilamellar phospholipid vesicles at the lipid phase transition have been revealed. The enzyme‐induced perturbation of the bilayer is sufficient for phase transition release of encapsulated carboxyfluorescein from the vesicles. The size of the enzyme—phospholipid recombinants depends upon the protein—phospholipid ratio as shown on Sepharose 4B elution profile. The activity of transglutaminase inserted into the bilayer is greatly reduced. The interaction does not occur when the phospholipid vesicle are in the solid or liquid phase and it requires the structural integrity of the enzyme.

Haptoglobin polymorphisms are associated with crohn's disease, disease behavior, and extraintestinal manifestations in hungarian patients

Functional differences and association with inflammatory disorders were found relating to three major haptoglobin (Hp) phenotypes. Our aim was to investigate Hp polymorphisms in Hungarian patients with Crohn’s disease (CD). Four hundred sixty-eight CD patients and 384 healthy controls were examined. Hp phenotypes were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of the sera. The frequency of the Hp1 allele was significantly higher in CD (0.395; OR, 1.24; 95% CI, 1.02–1.52; P=0.03) compared to controls (0.345). In CD, Hp phenotype was associated with disease behavior (OR [Hp2‐1 vs other], 2.06; 95% CI, 1.29–3.28 for inflammatory behavior). Furthermore, an increased frequency of primary sclerosing cholangitis was observed in the Hp 2-2 compared to the Hp 1-1 phenotype (6.5% vs. 0.0%; P=0.039). We conclude that the Hp polymorphism is associated with CD, inflammatory disease behavior, and primary sclerosing cholangitis in Hungarian patients. Further studies are required to evaluate the significance of Hp polymorphisms in other populations from geographically diverse regions.

Enhanced release of IL-6 and IL-8 into tears in various anterior segment eye diseases

Objective: To determine the levels of interleukin 6 (IL-6) and interleukin 8 (IL-8/CXCL-8) in tears collected from the eyes of normal individuals and of patients with different irritative eye diseases, in order to acquire information on the immunological changes occurring during the early postoperative period following various forms of eye surgery, including penetrating keratoplasty (PKP). Methods: IL-6 and IL-8 levels were measured with the aid of human ultrasensitive ELISA kits in the non-stimulated tears of patients in the early postoperative period following PKP or cataract operation, and of patients with acute bacterial conjunctivitis or with a corneal foreign body. The IL-6 and IL-8 concentrations, the total amounts released in a given time and the rates of their release were calculated. Results: A significant increase in IL-6 release was observed in all patient groups compared with the normal controls (p ≤ 0.003). The IL-8 release levels were significantly higher in the tears of all patient groups (p ≤ 0.03), except for the cataract operation group, where the IL-8 release was not significantly higher (p = 0.053) than in the control samples. No significant differences in IL-6 or IL-8 release were observed when the various patient groups were compared with each other. Conclusion: The release of IL-6 and IL-8 into the tears is enhanced in various anterior segment eye diseases, and this may be used as an indicator of various inflammatory reactions in the early postoperative period.

Thrombotic and fibrinolytic alterations in the aseptic necrosis of femoral head

&NA; Recent reports seem to support the role of the thrombophilia and decreased fibrinolysis in the aetiopathogenesis of aseptic necrosis of bone. In the present study, haemostatic disturbances were analysed in adults (n = 49) and patients in childhood (Perthes disease) (n = 47) with aseptic necrosis of the femoral head. Fibrinolytic parameters (in vitro clot lysis, plasminogen, plasmatic plasminogen activator inhibitor‐1 activity, D‐dimer) along with lipoprotein (a) [Lp(a)] and fibrinogen were measured. von Willebrand factor, platelet activation and some thrombophilic factors (activated protein C resistance and factor V Leiden mutation, protein C, protein S activity) were also determined. Impaired fibrinolysis, an increased Lp(a) level along with slow clot lysis and increased platelet activation were found in adult cases. We detected five cases of factor V Leiden mutations (one heterozygotic and four homozygotic) among patients with Perthes disease. The clinical course of the heterozygous case was similar to the usual form of Perthes disease. The most severe form of Perthes disease has been observed in homozygous factor V Leiden mutation cases. The mutation of factor V Leiden per se probably does not induce the development of aseptic necrosis of bone tissue in childhood, but it does play a role in its acceleration. Homozygous factor V Leiden mutation definitely runs a more severe course. On the other hand, in adult cases, the disturbances of haemostasis, impaired fibrinolysis, elevated Lp(a) level, increased platelet activation and slight elevation of fibrinogen might have clinical relevance. Further studies should focus on proving the role of the haemostatic alterations in the pathogenesis of severe forms of aseptic bone necrosis. The use of antithrombotic drugs in order to slow the process of aseptic necrosis also has to be addressed in future surveys. Blood Coagul Fibrinolysis 14:243‐248