Miet Peeters

Clustering of increased small intestinal permeability in families with Crohn's disease

BACKGROUND & AIMS Small intestinal permeability is increased in a proportion of patients with Crohns disease (CD) and a subset of their healthy relatives. A primary permeability defect was postulated in the pathogenesis of the disease. The aim of this study was to identify a possible genetic pattern in the distribution of CD and/or abnormal permeability. METHODS Differential urinary excretion of lactulose and mannitol (L/ M) in complete CD families was determined. Controls included healthy families and families with ulcerative colitis. Pedigrees were used to compare the distribution of CD and/or increased permeability. RESULTS The L/M was significantly increased in patients with CD. Seventeen of 67 first-degree relatives (25%) had a ratio greater than the upper limit (P95 = 0.0170). Permeability results of CD families showed a highly significant familial aggregation. The lack of a genetic pattern in relation with CD and occurrence of disturbed permeability especially within generation, points toward a shared environmental factor. Five of 14 healthy spouses (36%) of patients with CD had also an increased permeability, and prevalence of increased permeability was not higher in families with known familial occurrence (P = 0.85). CONCLUSIONS This large family study confirms an increased permeability in a subset of healthy relatives of patients with CD. However, the absence of a typical family pattern and the high prevalence in spouses is in favor of a common nongenetic factor or a subclinical disease manifestation.

Development of a Genotype 325–Specific proCPU/TAFI ELISA

Objective—A Thr/Ile polymorphism at position 325 in the coding region of proCPU has been reported. Immunological assays, fully characterized (including genotype dependency), are required for the quantitation of proCPU levels. Methods and Results—We have generated a panel of monoclonal antibodies against human, plasma-derived proCPU. Two combinations exhibiting distinct reactivities were selected for measurement of proCPU in plasma. T12D11/T28G6-HRP yielded values of 10.1±3.1 &mgr;g/mL (mean±SD, n=86; normal donors), and T32F6/T9G12-HRP yielded values of 5.4±3.0 &mgr;g/mL. Grouping according to the 325 genotype demonstrated that T12D11/T28G6-HRP was independent to this polymorphism whereas T32F6/T9G12-HRP revealed a complete lack of reactivity with the Ile/Ile genotype (ie, 0.0±0.0, 4.2±1.7, and 7.3±2.9 &mgr;g/mL for the Ile/Ile, Ile/Thr, and Thr/Thr isoforms, respectively). Commercially available antigen assays appeared to be partially dependent on the 325 genotype (eg, 44±8.9% and 100±30% for the Ile/Ile and Thr/Thr isoforms, respectively). Conclusions—Our data demonstrate that great care should be taken when evaluating proCPU antigen values as a putative causative agent or as a diagnostic risk marker for cardiovascular events.

Anticipation in familial Crohn’s disease

Background—Offspring with a family history of Crohn’s disease have an earlier age of onset than their parents. This might be due to genetic anticipation, characterised by earlier and/or more severe disease in subsequent generations. Aims—To investigate the possibility of genetic anticipation in affected parent-child pairs with Crohn’s disease from France and Belgium. Patients and methods—In a cohort of 160 multiply affected families with Crohn’s disease, 57 parent-first affected child pairs were detected. Clinical characteristics (age at diagnosis, disease extent, and type) of both parents and children were registered and compared. Results—Children were younger than their parents at diagnosis in 48/57 (84%) pairs. The median age at diagnosis was 16 years younger in children than in parents (p<0.0001). However, the difference was related to the age at diagnosis in the parents and was not present in 12 parent-child pairs with an early age at diagnosis for the parents. In most cases, disease extent and type were not considered more severe in children than in parents. Parental sex affected neither age at diagnosis nor extent and type of disease in children. Conclusion—Patients in the second affected generation acquire their disease at an earlier time in life in some but not all familial cases of Crohn’s disease. Several explanations including genetic anticipation and environmental factors might explain this phenomenon.

Modulation of TAFI function through different pathways – implications for the development of TAFI inhibitors

Summary.  Objective: To elucidate the mechanism and the binding regions of monoclonal antibodies (MA) that interfere with thrombin‐activatable fibrinolysis inhibitor (TAFI)/activated thrombin‐activatable fibrinolysis inhibitor (TAFIa) activity. Results: Of 42 MA, 19 interfere with the TAFI activation/TAFIa activity resulting in an inhibition of up to 92%. Characterization of the mechanism of inhibition revealed that 14 MA blocked the activation of TAFI by thrombin/thrombomodulin completely whereas five MA interfered directly with the enzymatic activity of TAFIa. Surprisingly, the former, except one, induced a significant reduction of clot lysis time whereas the latter did not. Affinity studies using a human/murine TAFI chimer revealed that the binding region of the 14 activation blocking MA is located between AA1 and AA67. MA that inhibit exclusively the activation of TAFI by thrombin/thrombomodulin bind to Gly66. A MA that inhibits the activation of TAFI by both thrombin/thrombomodulin and plasmin binds to Val41. The MA that interfere with the enzymatic activity bind to the TAFIa moiety. Conclusions: The current study reveals at least three different putative molecular targets in the search for pharmacologically active compounds to modulate TAFIa activity.

Evaluation of the profibrinolytic properties of an anti-TAFI monoclonal antibody in a mouse thromboembolism model.

The enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. Activated thrombin activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis and is an attractive target to develop profibrinolytic drugs. TAFI can be activated by thrombin, thrombin/thrombomodulin, or plasmin, but the in vivo physiologic TAFI activator(s) are unknown. Here, we generated and characterized MA-TCK26D6, a monoclonal antibody raised against human TAFI, and examined its profibrinolytic properties in vitro and in vivo. In vitro, MA-TCK26D6 showed a strong profibrinolytic effect caused by inhibition of the plasmin-mediated TAFI activation. In vivo, MA-TCK26D6 significantly decreased fibrin deposition in the lungs of thromboembolism-induced mice. Moreover, in the presence of MA-TCK26D6, plasmin-α(2)-antiplasmin complexes in plasma of thromboembolism-induced mice were significantly increased compared with a control antibody, indicative of an acceleration of fibrinolysis through MA-TCK26D6. In this study, we show that plasmin is an important TAFI activator that hampers in vitro clot lysis. Furthermore, this is the first report on an anti-TAFI monoclonal antibody that demonstrates a strong profibrinolytic effect in a mouse thromboembolism model.

Development of ELISAs Measuring the Extent of TAFI Activation

Objectives—To date, quantitation of TAFI antigen levels has been mainly focused on “total” antigen levels and has been shown to yield ambiguous results because of the existence of different isoforms and various degrees of activation. Our objective was to develop assays that allow measuring the extent of TAFI activation. Methods and Results—A variety of enzyme-linked immunosorbent assays (ELISAs) were evaluated for their preferential reactivity toward TAFI before and after activation, and toward the recombinantly expressed activation peptide. Three ELISAs with distinct reactivities were selected: recognizing either exclusively nonactivated TAFI, the released activation peptide, or exclusively TAFIa (activated TAFI). Evaluation of TAFI activation during clot lysis revealed that decreases of TAFI levels are associated with increases of the released activation peptide and TAFIa levels. In addition, antigenic measurement of TAFIa parallels activity measured by chromogenic assay. Analyzing plasma samples revealed that subjects with hyperlipidemia had significantly higher plasma levels of both the activation peptide (109.2 versus 95.5; P<0.001) and TAFIa (112.1 versus 103.3; P=0.03), and not of TAFI antigen (92.5 versus 87.9; P=0.07) (results in % of plasma pooled from normolipidemic subjects). Conclusion—ELISAs that allow to measure the extent of TAFI activation were developed. These ELISAs constitute more sensitive markers in studies on the relationship between TAFI and cardiovascular diseases.

Development of a universal anti-adalimumab antibody standard for interlaboratory harmonization.

Background: Therapeutic drug monitoring of adalimumab (ADM) has been introduced recently. When no detectable ADM serum concentrations can be found, the formation of antidrug antibodies (ADA) should be investigated. A variety of assays to measure the occurrence of ADA have been developed. Results are expressed as arbitrary units or as a titration value. The aim was to develop a monoclonal antibody (MA) that could serve as a universal calibrator to quantify the amount of ADA in ADM-treated patients. Methods: Hybridoma technology was used to generate a MA toward ADM. The functionality of the MA was tested in a bridging enzyme linked immunosorbent assay (ELISA) setup and in a cell-based assay. Sera from 25 anti–tumor necrosis factor naive patients with inflammatory bowel disease were used to determine the cutoff values. Sera from 9 ADM-treated patients with inflammatory bowel disease, with undetectable serum concentrations of ADM were used to quantify the ADA response. Results: In this study, MA-ADM6A10, an IgG1 that can be used as a calibrator in both an ELISA to quantify the amount of binding antibodies and in a cell-based assay to quantify the amount of neutralizing antibodies, was generated. Combining the results of both assays showed that the sera with high concentrations of anti-ADM binding antibodies also had the highest neutralizing capacity. Conclusions: The availability of a universal calibrator could facilitate the interlaboratory harmonization of antibody titers in patients who develop anti-adalimumab antibodies.

Generation of a stable activated thrombin activable fibrinolysis inhibitor variant.

Activated thrombin activable fibrinolysis inhibitor (TAFIa), generated upon activation of TAFI, exerts an antifibrinolytic effect. TAFIa is a thermolabile enzyme, inactivated through a conformational change. The objective of the current study was to generate a stable variant of human TAFIa. Using a site-directed as well as a random mutagenesis approach to generate a library of TAFI mutants, we identified two mutations that increase TAFIa stability, i.e. a Ser305 to Cys and a Thr329 to Ile mutation, respectively. Combining these mutations in TAFI-Ala147-Ile325, the most stable isoform of TAFIa (half-life of 9.4 ± 0.4 min), revealed a TAFIa half-life of 70 ± 3.1 min (i.e. an 11-fold increase versus 6.3 ± 0.3 min for TAFIa-Ala147-Thr325, the most frequently occurring isoform of TAFI in humans) at 37 °C. Moreover, clot lysis (induced by tissue plasminogen activator) experiments in which TAFI-Ala147-Cys305-Ile325-Ile329 was added to TAFI-depleted plasma revealed a 50% clot lysis time of 313 ± 77 min (i.e. a 3.0-fold increase versus 117 ± 10 min for TAFI-Ala147-Thr325). The availability of a more stable TAFIa variant will facilitate the search for inhibitors and allow further structural analysis to elucidate the mechanisms of the instability of TAFIa.

Announcing a TAFIa mutant with a 180‐fold increased half‐life and concomitantly a strongly increased antifibrinolytic potential

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Innovative thrombolytic strategy using a heterodimer diabody against TAFI and PAI-1 in mouse models of thrombosis and stroke

Circulating thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) are causal factors for thrombolytic failure. Therefore, we evaluated an antibody-engineered bispecific inhibitor against TAFI and PAI-1 (heterodimer diabody, Db-TCK26D6x33H1F7) in several mouse models of thrombosis and stroke. Prophylactic administration of the diabody (0.8 mg/kg) in a thromboplastin-induced model of thromboembolism led to decreased lung fibrin deposition. In a model of cerebral ischemia and reperfusion, diabody administration (0.8 mg/kg, 1 hour postocclusion) led to a mitigated cerebral injury with a 2.3-fold reduced lesion and improved functional outcomes. In a mouse model of thrombin-induced middle cerebral artery occlusion, the efficacy of the diabody was compared to the standard thrombolytic treatment with recombinant tissue-type plasminogen activator (tPA). Early administration of diabody (0.8 mg/kg) caused a twofold decrease in brain lesion size, whereas that of tPA (10 mg/kg) had a much smaller effect. Delayed administration of diabody or tPA had no effect on lesion size, whereas the combined administration of diabody with tPA caused a 1.7-fold decrease in lesion size. In contrast to tPA, the diabody did not increase accumulative bleeding. In conclusion, administration of a bispecific inhibitor against TAFI and PAI-1 results in a prominent profibrinolytic effect in mice without increased bleeding.