Granulocyte colony-stimulating factor (GCSF) is currently the most widely used cytokine for stem cell mobilization. There are few studies suggesting GCSF administration may induce activation of both coagulation and endothelial cells that could favor the developing of thrombotic events. We report a 58-year-old female with vasculitis and renal impairment. She was found to have an underlying monoclonal gammopathy of unknown significance (MGUS). The monoclonal protein was felt to play a role in her underlying renal disease and peripheral neuropathy. She was considered a candidate for peripheral blood stem cell transplantation to manage the monoclonal protein. During stem cell mobilization with GCSF, she developed worsening of anemia; thrombocytopenia and worsening of renal function. She was diagnosed with thrombotic microangiopathy (TMA) which was successfully treated with therapeutic plasma exchange and rituximab. It is possible that GCSF may have directly (activating endothelial cells) or indirectly (activation of underlying autoimmune disorder) contributed to TMA in this patient.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18857号 / 医博第3968号 / 新制||医||1008(附属図書館) / 31808 / 京都大学大学院医学研究科医学専攻 / (主査)教授 前川 平, 教授 江藤 浩之, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Abstract Background: Deficiency of Von Willebrand Factor Cleaving Protease (VWFCP) has been implicated as the cause of Thrombotic Thrombocytopenic Purpura (TTP). TTP is a lifethreatening disease characterised by microangiopathic thrombosis due to accumulation of Ultralarge Von Willebrand Factor (ULVWF) multimers. The clinical features of TTP include microangiopathic haemolysis and thrombocytopenia. TTP is being seen with increased frequency in the context of HIV. However, in the context of HIV infection, cytopenias are often multifactorial in nature and levels of VWFCP in HIV-related thrombocytopenia have not specifically been assessed. This study assessed VWFCP activity in the setting of patients with HIV and thrombocytopenia in the absence of TTP, in order to determine the utility of a VWFCP assay in the diagnosis of HIV-related TTP. Acquired VWFCP deficiency is generally assumed to be due to the presence of autoantibody inhibitors to the enzyme, but limited data are available regarding VWFCP activity in HIV positive TTP patients. There is also currently no assay available for measuring VWFCP activity in our laboratory. Aim of Study: To establish a practical assay for VWFCP activity for routine use in our laboratory. The rapid collagen binding assay, based on the ELISA method of Rick, et al., 2002, was chosen. This was initially used to measure VWFCP activity in patients with HIV with and without thrombocytopenia (of any cause except TTP), in order to ascertain whether assessment of VWFCP activity is likely to be of value in facilitating early diagnosis of HIV related TTP. The ELISA assay was performed to establish cut-off values for VWFCP in HIV negative controls and two HIV positive groups (HIV thrombocytopenia / low platelets and HIV normal platelets). Depending on the outcome of this, the assay could then be performed to assess VWFCP activity in HIV positive patients with TTP. Methods: The rapid collagen binding assay for VWFCP activity was established and optimised for routine use in our laboratory. The cut-off values for percentage Residual Collagen Binding Activity (RCBA) in both HIV negative and HIV positive groups were identified. The assay could then be used to assess VWFCP activity in 20 HIV positive patients with TTP at the time of presentation. In patients with reduced VWFCP activity, patient plasma was mixed with normal pool plasma in a 50:50 mix, to assess for the presence of inhibitors. Correlation of VWFCP activity, inhibitors and other laboratory and clinical parameters were performed. Results: The cut-off values for percentage RCBA in both HIV negative (<37.12%) and HIV positive (<51.51%) patients were established. The % RCBA for the HIV negative control group was statistically significantly different from the HIV positive group with normal platelets (p=0.0001) and from the HIV positive group with low platelets (p=0.0006). The cut-off value in the two HIV positive patient groups was higher than for HIV negative control patients, indicating mildly reduced VWFCP enzyme activity in HIV positive patients (regardless of the platelet count), in the absence of TTP. However, no significant difference in the cut-off value was noted between HIV positive patients with low platelet counts versus HIV positive patients with normal platelet counts (p=0.7783). The assay could therefore be used in HIV positive patients with TTP. VWFCP activity was assessed in twenty HIV positive patients with TTP. Two groups of HIV positive patients with TTP were identified based on VWFCP activity. Six patients (30%) had normal (one borderline) VWFCP activity (RCBA <51.51%), while the remaining 14 patients had severely reduced VWFCP levels (RCBA >90%). Of the patients with reduced VWFCP activity, only 5 patients had a detectable inhibitor, while an inhibitor was not detected in the remaining 8 patients. Conclusion: The rapid collagen binding ELISA assay is a cost effective semi-quantitative assay for the assessment of VWFCP activity. VWFCP activity in HIV positive patients appears to be slightly lower, however is not related to the platelet count. This suggests a slight baseline deficiency of VWFCP in the setting of HIV. The baseline VWFCP cut-off value in HIV allowed assessment of HIV positive patients with TTP. The results suggest heterogeneity of VWFCP activity in HIV-related TTP. A negative result (normal VWFCP activity) does not exclude TTP in patients with HIV-related TTP and other pathogenic factors may therefore be involved.

Le syndrome hémolytique et urémique atypique (SHUa) est une microangiopathie thrombotiquecomplément-dépendante dont l'atteinte majoritairement rénale reste, à ce jour, incomprise.L'objectif de ce travail était d'améliorer la compréhension de ce tropisme d’organe au moyen dedeux axes d'étude : i) la susceptibilité de l'endothélium glomérulaire à l'hémolyse, celle-ci étant à la fois la conséquence des microthromboses dans le SHUa et un amplificateur de la voie alterne du complément via l'hème libre, molécule issue des globules rouges lysés ; ii) le rôle potentiel du récepteur aux produits de glycation avancés ou RAGE. Ce récepteur membranaire aux fortes propriétés pro inflammatoires et pro thrombotiques a en effet été impliqué dans de nombreuses pathologies rénales, et sa liaison au C3a - anaphylatoxine libérée dans l'activation du complément - a été rapportée par une équipe.La première partie de ce travail a visé à expliquer la vulnérabilité de l'endothélium glomérulaire sous l'effet d'une hémolyse. Nous avons étudié plusieurs types de cellules endothéliales exposées +/- à l'hème, et mis au point un modèle murin traité par de l'hème. En conditions hémolytiques, plusieursfacteurs pouvant participer à cette susceptibilité endothéliale glomérulaire ont ainsi été mis en avant: i) une moindre liaison du facteur H, principal régulateur du complément, à sa surface ; ii) une faible expression de la thrombomoduline, protéine de la coagulation et régulatrice du complément ; iii) une faible expression de l'enzyme principale de dégradation de l'hème, l'hème-oxygénase 1. Ces deux derniers points étaient rattachés à une faible induction endothéliale glomérulaire de leurs facteurs de transcription, KFL2 et KLF4.La seconde partie de ce travail s'est concentrée sur le RAGE. N'ayant pas réussi à reproduire l'interaction RAGE/C3a, nous avons exploré l'hypothèse d'une liaison du RAGE à l'hème. En effet, le seul récepteur endothélial connu jusqu'à présent est le Toll Like Receptor 4 (TLR4), qui partage plusieurs ligands communs avec le RAGE (LPS - lipopolysaccharide, HMGB1 - high–mobility group box1). Nous avons découvert que le RAGE était un récepteur de l'hème, et identifié que le site de liaisonse trouvait sur le domaine V. A l'aide d'un modèle murin invalidé pour le RAGE et traité +/- par l'hème, nous avons mis en évidence que : i) l'invalidation de RAGE avait un effet protecteur en cas d’exposition à l'hème, marqué par une diminution de l'expression de gènes de l'inflammation (IL1β,TNFα) et du facteur tissulaire au niveau pulmonaire, organe exprimant le plus fortement RAGE ; ii)l'hème activait la phosphorylation des voies ERK1/2 et Akt via le RAGE.Par ces travaux, nous avons précisé les liens entre activation du complément, hémolyse et susceptibilité endothéliale glomérulaire dans le SHUa. Parallèlement, nous avons identifié le RAGE comme un nouveau récepteur à l'hème, dont la liaison à ce récepteur activerait différentes voies designalisation de l'inflammation. Le contrôle de l'hème et du RAGE pourrait ainsi constituer de nouvelles voies thérapeutiques dans le SHUa et les maladies hémolytiques. / The atypical haemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy of which the predominantly renal damage remains, to date, misunderstood. The aim of this work was to improve the understanding of this organ tropism by two axes of study: i) the susceptibility of the glomerular endothelium to hemolysis, which is the consequence of microthrombosis in aHUS, and also an complement pathway enhancer via free heme, hemoglobin-mediated hemolysis molecule, ii) the potential role of the receptor for advanced glycation end products, RAGE. Indeed, RAGE is described as an endothelial receptor with high proinflammatory and prothrombotic potential, involved in many kidney diseases; a team also reported that it was a receptor for the C3a molecule, anaphylatoxin released in complement activation.The first part of this work aimed to explain the vulnerability of the glomerular endothelium under the effect of hemolysis. We studied several types of endothelial cells exposed +/- to heme, and developed a murine model treated with heme. In hemolytic conditions, several factors that could participate in the endothelial glomerular susceptibility have been put forward: i) less binding of factor H, the main complement regulator, on its surface; ii) low expression of thrombomodulin, coagulation protein and complement regulator; iii) low expression of heme-oxygenase 1, the main heme degradation enzyme. These last two points were related to low induction, on glomerular endothelial cells, of transcription factors, KFL2 and KLF4.The second part of this work focused on RAGE. Having failed to reproduce the RAGE / C3a interaction, we explored the hypothesis of a linkage of RAGE to heme. Indeed, the only known endothelial receptor is Toll Like Receptor 4 (TLR4), which shares several common ligands (LPS - lipopolysaccharide, HMGB1 - high-mobility group box 1). We found that RAGE was a heme receptor, and identified that the binding site was on domain V. Using a mouse model knock out for RAGE and treated +/- with heme, we demonstrated that i) the invalidation of RAGE had a protective effect in case of exposure to heme, marked by a decrease in the expression of genes of inflammation (IL1β; TNFα; and tissue factor) at the pulmonary level, organ expressing most strongly RAGE, ii) the heme is an activator or the phosphorylation of ERK1 / 2 and Akt pathways via RAGE.Through this work, we have clarified the links between complement activation, hemolysis and glomerular endothelial susceptibility in aHUS. At the same time, we have identified RAGE as a new heme receptor, whose RAGE/heme bindind would activate different signaling pathways for inflammation. The control of heme and RAGE could constitute new therapeutic pathways in the aHUS, and hemolytic diseases.

No description available.

The first part of the thesis deals with the synthesis and biological evaluation of new platelets aggregation inhibitors, based on 12-HETE, 13-HODE and their analogues. In the second part we are interested in novel methodologies for the preparation of CF₂-containing molecules : First, a flexible strategy for the synthesis of gem-difluoro-bisarylic derivatives and heteroaromatic analogues was designed based on the easy synthesis and the reactivity of gem-difluoro propargylic intermediates, which by Diels-Alder cycloaddition and 1,3-dipolar cycloadditions afforded respectively the bisarylic and mixed arylic heteroarylic scaffolds. In addition, two small libraries were constructed around a bisarylic scaffold as representative examples. Second, we were interested in the synthesis of optically active functionalized molecules containing a gem-difluoro group, using asymmetric organocatalysis protocols. After preparation of the gem-difluoro enals, from their difluoropropargylic precursors, asymmetric organocalytic Diels-Alder cycloaddition and 1,4-conjugated additions were successfully performed.

Das Auftreten manifester transplantationsassoziierter thrombotischer Mikroangiopathie sowie das Auftreten leichterer, zumeist subklinischer mikroangiopathischer Veränderungen wurde in einem Kollektiv von Patienten untersucht, die zur Behandlung einer steroidrefraktären oder abhängigen akuten oder chronischen GvHD nach allogener Stammzelltransplantation eine Therapie mit extrakorporalen Photopheresen erhielten. Subklinische mikroangiopathische Schädigung wurde durch ein auf den Messwerten von Fragmentozyten, Thrombozyten sowie der LDH-Aktivität beruhendes Bewertungsschema festgestellt. Das Auftreten von ausgeprägten mikroangiopathischen Veränderungen (Schweregrad 2) ging mit einem erniedrigten Gesamtüberleben einher. Die Reduktion der Steroiddosis und die Verringerung der GvHD-Manifestationen wurden als Parameter für die Wirksamkeit der ECP analysiert. Ihre Effektivität wurde bestätigt. Trotz der nachgewiesenen Effektivität der ECP in der Kontrolle der GvHD zeigte sich eine Tendenz zur Zunahme mikroangiopathischer Veränderungen unter Therapie mit ECP, in Einzelfällen mit schwerwiegenden klinischen Konsequenzen.

1 Univerzita Karlova v Praze 1. lékařská fakulta Některé aspekty patofyziologie plicní arteriální hypertenze a její výskyt v České republice Some aspects of pathophysiology of pulmonary arterial hypertension and its epidemiology in the Czech Republic MUDr. Pavel Jansa Praha 2011 2 Abstract Pulmonary arterial hypertension (PAH) is a group of diseases characterized by a progressive increase of resistance and pressure in pulmonary vascular bed. In all types of PAH the same four pathological processes are reported: vasoconstriction, inflammation, thrombosis and remodelling. The genetic background is essential for the development of PAH. We aimed to investigate the role of polymorphisms of endothelial nitric oxide synthase (eNOS) genes in PAH. We studied 142 PAH patients and 189 healthy subjects. We examined 3 polymorphisms of the eNOS gene, including the Glu298Asp polymorphism, 27-base pair (bp) variable numbers of tandem repeats (VNTR) and -786 T/C promoter gene polymorphism. Prevalence of 27-bp VNTR allele A was higher in patients with PAH compared with healthy controls. Patients with PAH associated with connective tissue diseases had higher prevalence of AA genotype compared with other PAH subgroups. The Glu298Asp polymorphism and -786 T/C polymorphism are not associated with PAH. Thrombotic arteriopathy is...

1 Univerzita Karlova v Praze 1. lékařská fakulta Některé aspekty patofyziologie plicní arteriální hypertenze a její výskyt v České republice Some aspects of pathophysiology of pulmonary arterial hypertension and its epidemiology in the Czech Republic MUDr. Pavel Jansa Praha 2011 2 Abstract Pulmonary arterial hypertension (PAH) is a group of diseases characterized by a progressive increase of resistance and pressure in pulmonary vascular bed. In all types of PAH the same four pathological processes are reported: vasoconstriction, inflammation, thrombosis and remodelling. The genetic background is essential for the development of PAH. We aimed to investigate the role of polymorphisms of endothelial nitric oxide synthase (eNOS) genes in PAH. We studied 142 PAH patients and 189 healthy subjects. We examined 3 polymorphisms of the eNOS gene, including the Glu298Asp polymorphism, 27-base pair (bp) variable numbers of tandem repeats (VNTR) and -786 T/C promoter gene polymorphism. Prevalence of 27-bp VNTR allele A was higher in patients with PAH compared with healthy controls. Patients with PAH associated with connective tissue diseases had higher prevalence of AA genotype compared with other PAH subgroups. The Glu298Asp polymorphism and -786 T/C polymorphism are not associated with PAH. Thrombotic arteriopathy is...

Dans la première partie nous décrivons la synthèse et l'évaluation biologique de nouveaux inhibiteurs de l'agrégation plaquettaire, composés dont la structure a été établie en partant du 12-HETE et du 13-HODE. Dans la seconde partie nous développons de nouvelles méthodologies pour la préparation de molécules contenant des motifs CF₂R. Tout d'abord une stratégie très flexible a été mise au point pour la préparation de composés gem-difluorobisaryliques et de leurs analogues hétéroaromatiques. Elle est basée sur l'emploi d'intermédiaires gem-difluoropropargyliques faciles d'accès. Par une séquence de Diels-alder-aromatisation on obtient les molécules cibles de la première série. Pour la seconde, des réactions de cycloaddition dipolaire 1,3 ont été utilisées. A partir de ces intermédiaires, des chimiothèques ciblées de molécules fluorées ont été préparées. Nous nous sommes intéressés ensuite à la synthèse de composés fluorés fonctionnalisés et chiraux à travers des réactions d'organocatalyse asymétrique. A partir d'énals gem-difluorés des réactions de Diels-Alder et des additions 1,4 asymétriques ont été réalisées avec succès. / The first part of the thesis deals with the synthesis and biological evaluation of new platelets aggregation inhibitors, based on 12-HETE, 13-HODE and their analogues. In the second part we are interested in novel methodologies for the preparation of CF₂-containing molecules : First, a flexible strategy for the synthesis of gem-difluoro-bisarylic derivatives and heteroaromatic analogues was designed based on the easy synthesis and the reactivity of gem-difluoro propargylic intermediates, which by Diels-Alder cycloaddition and 1,3-dipolar cycloadditions afforded respectively the bisarylic and mixed arylic heteroarylic scaffolds. In addition, two small libraries were constructed around a bisarylic scaffold as representative examples. Second, we were interested in the synthesis of optically active functionalized molecules containing a gem-difluoro group, using asymmetric organocatalysis protocols. After preparation of the gem-difluoro enals, from their difluoropropargylic precursors, asymmetric organocalytic Diels-Alder cycloaddition and 1,4-conjugated additions were successfully performed.