Julia Driesen

Indoleamine 2,3-dioxygenase-expressing dendritic cells form suppurative granulomas following Listeria monocytogenes infection

Control of pathogens by formation of abscesses and granulomas is a major strategy of the innate immune system, especially when effector mechanisms of adaptive immunity are insufficient. We show in human listeriosis that DCs expressing indoleamine 2,3-dioxygenase (IDO), together with macrophages, are major cellular components of suppurative granulomas in vivo. Induction of IDO by DCs is a cell-autonomous response to Listeria monocytogenes infection and was also observed in other granulomatous infections with intracellular bacteria, such as Bartonella henselae. Reporting on our use of the clinically applied anti-TNF-alpha antibody infliximab, we further demonstrate in vitro that IDO induction is TNF-alpha dependent. Repression of IDO therefore might result in exacerbation of granulomatous diseases observed during anti-TNF-alpha therapy. These findings place IDO(+) DCs not only at the intersection of innate and adaptive immunity but also at the forefront of bacterial containment in granulomatous infections.

Prostaglandin E2 Impairs CD4+ T Cell Activation by Inhibition of lck: Implications in Hodgkin's Lymphoma

Many tumors, including Hodgkins lymphoma, are associated with decreased cellular immunity and elevated levels of prostaglandin E(2) (PGE(2)), a known inhibitor of CD4+ T cell activation, suggested to be involved in immune deviation in cancer. To address the molecular mechanisms tumor-derived PGE(2) might have on primary human CD4+ T cells, we used a whole genome-based transcriptional approach and show that PGE(2) severely limited changes of gene expression induced by signaling through the T cell receptor and CD28. This data suggests an interference of PGE(2) at an early step of T cell receptor signaling: indeed, PGE(2) stimulation of T cells leads to inactivation of lck and reduced phosphorylation of ZAP70. Antiapoptotic genes escaped PGE(2)-induced inhibition resulting in partial protection from apoptosis in response to irradiation or Fas-mediated signaling. As a functional consequence, PGE(2)-treated CD4+ T cells are arrested in the cell cycle associated with up-regulation of the cyclin/cyclin-dependent kinase inhibitor p27(kip1). Most importantly, CD4+ T cells in Hodgkins lymphoma show similar regulation of genes that were altered in vitro by PGE(2) in T cells from healthy individuals. These data strongly suggest that PGE(2) is an important factor leading to CD4+ T cell impairment observed in Hodgkins lymphoma.

Deficiencies of antithrombin, protein C and protein S – Practical experience in genetic analysis of a large patient cohort

Deficiencies of natural anticoagulant proteins including antithrombin (AT), protein C (PC) and protein S (PS) are important causes of inherited thrombophilia. This study aimed to report on the practical experience gained in performing genetic analyses of a large cohort of patients with AT, PC and PS deficiencies and to relate this knowledge to clinical application. We genotyped a large cohort of 709 unrelated patients with AT (231), PC (234) and PS (244) deficiencies referred to us by physicians throughout Germany. Mutations were detected by direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The highest mutation detection rate (MDR) was found for the SERPINC1 gene (83.5%), followed by the PROC (69%) and PROS1 (43%) genes. Even at AT activities close to the normal range (75%), the MDR was 70%. Contrastingly, for PC and PS deficiencies, the MDR dropped significantly and mildly lowered to subnormal values. At PS activities >55% for PS no mutations were detected. Mutation profiles of all three genes were similar with the highest prevalence for missense mutations (63-78%), followed by nonsense (7-11%), splice-site mutations (7-13%), small deletions (1-8%), small insertions/duplications (1-4%) and large deletions (3-6%). In conclusion, genetic testing is a useful diagnostic tool for diagnosing thrombophilia. Based on our data, genetic analysis for patients with AT deficiency is indicated for all subnormal activities. In contrast, genotyping is not advisable for PC activities >70% and for PS activities >55%.

CD25 as an immune regulatory molecule expressed on myeloid dendritic cells.

CD25 (alpha-chain of IL-2 receptor) on dendritic cells (DC) has been previously regarded as an activation marker. DC that concomitantly express surface CD25 and co-stimulatory molecules were considered to be fully mature. While both murine and human DC can express CD25, they do not express the beta-chain of the IL-2 receptor, which is indispensable for the execution of IL-2 signaling. The biological function of CD25 during the DC maturation therefore still remains undefined. In this review we focus on recent findings, describing CD25 expression and secretion by human myeloid regulatory DC. These DC co-express CD25 and the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) and inhibit T-cell function. CD25, expressed and secreted by such DC may capture IL-2 and thereby suppress T-cell proliferation, by this means providing an accessory mechanism of DC-mediated immune suppression. We also discuss the implication of DC-derived CD25 for human disease in both cancer and chronic infection.

Infection of Myeloid Dendritic Cells with Listeria monocytogenes Leads to the Suppression of T Cell Function by Multiple Inhibitory Mechanisms

Myeloid dendritic cells (DC) and macrophages play an important role in pathogen sensing and antimicrobial defense. In this study we provide evidence that myeloid DC respond to infection with Listeria monocytogenes with simultaneous induction of multiple stimulatory and inhibitory molecules. However, the overall impact of infected DC during T cell encounter results in suppression of T cell activation, indicating that inhibitory pathways functionally predominate. Inhibitory activity of infected DC is effected mainly by IL-10 and cyclooxygenase 2-mediated mechanisms, with soluble CD25 acting as an IL-2 scavenger as well as by the products of tryptophan catabolism. These inhibitory pathways are strictly TNF-dependent. In addition to direct infection, DC bearing this regulatory phenotype can be induced in vitro by a combination of signals including TNF, TLR2, and prostaglandin receptor ligation and by supernatants derived from the infected cells. Both infection-associated DC and other in vitro-induced regulatory DC are characterized by increased resistance to infection and enhanced bactericidal activity. Furthermore, myeloid DC expressing multiple regulatory molecules are identified in vivo in granuloma during listeriosis and tuberculosis. Based on the in vivo findings and the study of in vitro models, we propose that in granulomatous infections regulatory DC may possess dual function evolved to protect the host from disseminating infection via inhibition of granuloma destruction by T cells and control of pathogen spreading.

Mutation distribution in the von Willebrand factor gene related to the different von Willebrand disease (VWD) types in a cohort of VWD patients

Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of the von Willebrand factor (VWF). VWD is classified into three types--type 1 (partial quantitative deficiencies), type 2 (qualitative defects) and type 3 (complete deficiency of VWF). In this study we explored genotype and phenotype characteristics of patients with VWD with the aim of dissecting the distribution of mutations in different types of VWD. One hundred fourteen patients belonging to 78 families diagnosed to have VWD were studied. Mutation analysis was performed by direct sequencing of the VWF . Large deletions were investigated by multiplex ligation-dependent probe amplification (MLPA) analysis. The impact of novel candidate missense mutations and potential splice site mutations was predicted by in silico assessments. We identified mutations in 66 index patients (IPs) (84.6%). Mutation detection rate was 68%, 94% and 94% for VWD type 1, 2 and 3, respectively. In total, 68 different putative mutations were detected comprising 37 missense mutations (54.4%), 10 small deletions (14.7%), two small insertions (2.9%), seven nonsense mutations (10.3%), five splice-site mutations (7.4%), six large deletions (8.8%) and one silent mutation (1.5%). Twenty-six of these mutations were novel. Furthermore, in type 1 and type 2 VWD, the majority of identified mutations (74% vs. 88.1%) were missense substitutions while mutations in type 3 VWD mostly caused null alleles (82%). Genotyping in VWD is a helpful tool to further elucidate the pathogenesis of VWD and to establish the relationship between genotype and phenotype.

Large deletions identified in patients with von Willebrand disease using multiple ligation-dependent probe amplification.

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In vitro characterization of recombinant factor VIII concentrates reveals significant differences in protein content, activity and thrombin activation profile

Recombinant factor VIII (rFVIII) concentrates differ due to cell lines, culture conditions, presence of the B domain and authorized potency assays. This study characterizes three commercially available rFVIII concentrates: a second‐generation full length (A), a third‐generation full length (B) and a third‐generation B domain‐deleted (BDD) product (C). rFVIII concentrates were characterized for FVIII activity (FVIII:C) by one‐stage clotting and chromogenic assays, FVIII antigen (FVIII:Ag), thrombin activation profile and FXa‐generation assay. The rFVIII concentrates exhibited significant differences with regard to FVIII:C, FVIII:Ag and thrombin activation profile. Product A had significantly greater FVIII:C and FVIII:Ag relative to the measured values of products B and C. In addition, product A demonstrated faster and more complete activation by thrombin than the two others. BDD product C had the slowest measured thrombin activation rate. Product A exhibited a greater in vitro FXa generation than products B and C. We found no differences in FXa generation among all three products when FXa generation was normalized for FVIII:Ag. The greater FVIII:C and FVIII:Ag values for product A compared with that for products B and C are due to application of different authorized potency assays (one‐stage assay for A vs. chromogenic assay for B and C). The variation in thrombin activation profiles may arise from differences in cell line‐dependent posttranslational modifications of the various recombinant proteins.

Insights into pathological mechanisms of missense mutations in C-terminal domains of von Willebrand factor causing qualitative or quantitative von Willebrand disease

The carboxyl-terminal domains of von Willebrand factor, D4-CK, are cysteine-rich implying that they are structurally important. In this study we characterized the impact of five cysteine missense mutations residing in D4-CK domains on the conformation and biosynthesis of von Willebrand factor. These variants were identified as heterozygous in type 1 (p.Cys2619Tyr and p.Cys2676Phe), type 2A (p.Cys2085Tyr and p.Cys2327Trp) and as compound heterozygous in type 3 (p.Cys2283Arg) von Willebrand disease. Transient expression of human cell lines with wild-type or mutant von Willebrand factor constructs was performed. The mutated and wild-type recombinant von Willebrand factors were quantitatively and qualitatively assessed and compared. Storage of von Willebrand factor in pseudo-Weibel-Palade bodies was studied with confocal microscopy. The structural impact of the mutations was analyzed by homology modeling. Homozygous expressions showed that these mutations caused defects in multimerization, elongation of pseudo-Weibel-Palade bodies and secretion of von Willebrand factor. Co-expressions of wild-type von Willebrand factor and p.Cys2085Tyr, p.Cys2327Trp and p.Cys2283Arg demonstrated defective multimer assembly, suggesting a new pathological mechanism for dominant type 2A von Willebrand disease due to mutations in D4 and B domains. Structural analysis revealed that mutations p.Cys2283Arg, p.Cys2619Tyr and p.Cys2676Phe disrupted intra-domain disulfide bonds, whereas p.Cys2327Trp might affect an inter-domain disulfide bond. The p.Cys2327Trp variant is distinguished from the other mutants by an electrophoretic mobility shift of the multimer bands. The results highlight the importance of cysteine residues within the carboxyl-terminal of von Willebrand factor on structural conformation of the protein and consequently multimerization, storage, and secretion of von Willebrand factor.

Transcatheter aortic valve implantation leads to a restoration of von Willebrand factor (VWF) abnormalities in patients with severe aortic stenosis – Incidence and relevance of clinical and subclinical VWF dysfunction in patients undergoing transfemoral TAVI

BACKGROUNDnIn this study, we sought to analyze the incidence and relevance of von Willebrand factor (VWF) abnormalities in patients undergoing transcatheter aortic valve implantation (TAVI), especially on perioperative bleeding. Furthermore, we hypothesized that, similar to aortic valve surgery, TAVI results in a restoration of VWF abnormalities.nnnMETHODS AND RESULTSnWe performed a prospective analysis of periinterventional VWF parameters in 74 patients (80±7years, female in 37.5%) undergoing transfemoral TAVI for severe symptomatic aortic valve stenosis. At baseline, VWF:Ag was 210±90IU/dl with a mean VWF activity of 166±106IU/dl; activity-to-antigen ratio was 0.85±0.45. Heydes syndrome (severe aortic stenosis plus GI bleeding from angiodyplasia) was observed in 2/74 (2.7%). Whereas preprocedural loss of high-molecular-weight (HMW) VWF multimers was found in thirty-six patients (48.6%), none of the patients fulfilled criteria for possible acquired VW syndrome. After TAVI, an increase of both VWF:Ag and activity compared to baseline was observed (p<0.01). In patients with HMW multimer loss, post-interventional recovery of multimers occurred in all cases. In the two patients with Heydes syndrome, a trend towards reduced VWF:Ag was seen, with loss of HMW multimers in one patient. Of interest, all patients suffering from periprocedural major bleeding (5/74; 6.8%) exhibited activity-to-antigen ratios <0.7, indicating subclinical VWF dysfunction.nnnCONCLUSIONnWhereas clinically relevant VWF dysfunction is rare, loss of HMW VWF multimers is common in TAVI patients. Similar to surgery, TAVI leads to a restoration of this loss. Furthermore, VWF parameters may be useful parameter to evaluate risk of periprocedural bleeding.