Fanconi anemia and genome stability : the role of FANCM

RÉSUMÉ: Le génome de toute cellule est susceptible d'être attaqué par des agents endogènes et exogènes. Afin de préserver l'intégrité génomique, les cellules ont développé des multitudes de mécanismes. La réplication de l'ADN, une étape importante durant le cycle cellulaire, constitue un stress et présente un danger important pour l'intégrité du génome. L'anémie de Fanconi est une maladie héréditaire rare dont les protéines impliquées semblent jouer un rôle crucial dans la réponse au stress réplicatif. La maladie est associée à une instabilité chromosomique ainsi qu'à une forte probabilité de développer des cancers. Les cellules des patients souffrant de l'anémie de Fanconi sont sensibles à des agents interférant avec la réplication de l'ADN, et plus particulièrement àdes agents qui fient les deux brins d'ADN d'une manière covalente. L'anémie de Fanconi est une maladie génétiquement hétérogène. Treize protéines ont pu être identifiées. Elles semblent figurer dans une même voie de signalisation qui est aussi connue sous le nom de « FA/BRCA pathway », car un des gènes est identique au gène BRCA2 (breast cancer susceptibility gene 2). Huit protéines forment un complexe nucléaire dont l'intégrité est nécessaire à la monoubiquitination de deux autres protéines, FANCD2 et FANCI, en réponse à un stress réplicatif. A ce jour, la fonction moléculaire des protéines du « FA/BRCA pathway »reste encore mal décrite. Au début de mon travail de thèse, nous avons donc décidé de purifier les protéines du complexe nucléaire et d'étudier leurs propriétés biochimiques. Nous avons tout d'abord étudié les cinq protéines connues à l'époque qui sont FANCA, FANCC, FANCE, FANCF et FANCG. Par la suite, nous avons étendu notre étude à des protéines découvertes plus récemment, FANCL, FANCM et FAAP24, en concentrant finalement notre travail sur la caractérisation de FANCM. FANCM, contrairement aux autres protéines du complexe, est constituée de deux domaines conservés suggérant un rôle important dans le métabolisme de l'ADN. Il s'agit d'un domaine « DEAH box hélicase »situé dans la partie N-terminale et d'un domaine « ERCC4 nuclease »situé dans la partie C-terminale de la protéine. Dans cette étude, nous avons purifié avec succès la protéine FANCM entière à partir d'un système hétérologue. Nous montrons que FANCM s'attache de manière spécifique à des jonctions de Holliday et des fourches de réplication. De plus, nous démontrons que FANCM peut déplacer le point de jonction de ces structures via son domaine hélicase de manière dépendante de l'ATP. FANCM est aussi capable de dissocier de grands intermédiaires de la recombinaison, via la migration de jonctions de Holliday à travers une région d'homologie de 2.6 kb. Tous ces résultats suggèrent que FANCM peut s'attacher spécifiquement à des fourches de réplication et à des jonctions de Holliday in vitro et que son domaine hélicase est associé à une activité migratoire efficace. Nous pensons que FANCM peut avoir un rôle direct sur les intermédiaires de réplication. Ceci est en accord avec l'idée que les protéines de l'anémie de Fanconi coordonnent la réparation de l'ADN au niveau des fourches de réplication arrêtées. Nos résultats donnent une première indication quant au rôle de FANCM dans la cellule et peuvent contribuer à élucider la fonction de cette voie de signalisation peu comprise jusqu'à présent. SUMMARY: The genome of every cell is subject to a constant offence by endogenous and exogenous agents. Not surprisingly; cells have evolved a multitude of mechanisms which aim at preserving genomic integrity. A key step during the life cycle of a cell, DNA replication itself, constitutes a special danger to the integrity of the genome. The proteins defective in the rare hereditary disease Fanconi anemia (FA) are suspected to play a crucial role in the cellular response to DNA replication stress. The disease is associated with chromosomal instability and pronounced cancer susceptibility. Cells from Fanconi anemia patients are sensitive to a variety of agents which interfere with DNA replication, DNA interstrand cross-linking agents being particularly threatening to their survival. Fanconi anemia is a genetically heterogeneous disease with 13 different proteins identified, which seem to work together in a common pathway. Since one of the FA genes is identical to the breast cancer susceptibility gene BRCA2, it is also referred to as the FA/BRCA pathway. Eight proteins form a nuclear complex, whose integriry is required for the monoubiquitination of two other FA proteins, FANCD2 and FANCI, in response to DNA replication stress. Despite intensive research, the function of the FA/BRCA pathway at a molecular level has remained largely elusive so far. At the beginning of my thesis, we therefore decided to purify the proteins of the FA core complex and to investigate their biochemical properties. We started with the five proteins which were known at that time, FANCA, FANCC, FANCE, FANCF, and FACG. Later on, we extended our studies to the newly discovered proteins FANCL, FANCM, and FAAP24, and eventually focused our work on the characterisation of FANCM. In contrast to the other core complex proteins, FANCM contains two conserved domains, which point to a role in DNA metabolism: an N-terminal DEAH box helicase domain and a C-terminal ERCC4 nuclease domain. In this study, we have successfully purified full-length FANCM from a recombinant source. We show that purified FANCM binds to branched DNA molecules, such as Holliday junctions and replication forks, with high specificity and affinity. In addition, we demonstrate that FANCM can translocate the junction point of branched DNA molecules due to its helicase domain in an ATPase-dependent manner. FANCM can even dissociate large recombination intermediates, via branch migration of Holliday junctions through a 2.6 kb region of homology. Taken together, our data suggest that FANCM can specifically bind to replication forks and Holliday junctions in vitro, and that its DEAH box helicase domain is associated with a potent branch migration activity. We propose that FANCM might have a direct role in the processing of DNA replication intermediates. This is consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks. Our findings provide a first hint as to the context in which FANCM might play a role in the cell. We are optimistic that they might be key to further elucidate the function of a pathway which is far from being understood.

Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations.

The cause of many autoimmune and inflammatory diseases is unresolved, although dysregulated production of tumor necrosis factor (TNF) family members appears to be important in many cases. BAFF, a new member of the TNF family, binds to B cells and costimulates their growth in vitro. Mice transgenic for BAFF have vastly increased numbers of mature B and effector T cells, and develop autoimmune-like manifestations such as the presence of high levels of rheumatoid factors, circulating immune complexes, anti-DNA autoantibodies, and immunoglobulin deposition in the kidneys. This phenotype is reminiscent of certain human autoimmune disorders and suggests that dysregulation of BAFF expression may be a critical element in the chain of events leading to autoimmunity.

T-dependent B cell activation and differentiation in Lat Y136F mice : polyclonal response leading to autoimmune disease

SUMMARY LatY136F knock-in mice harbor a point mutation in tyrosine 136 of the linker for activation of T cells (LAT), and show accumulation of TH2 effector cells leading to IgG1 and IgE hypergammaglobulinemia. The observed polyclonal. B cell activation was not a direct effect of the mutation on B cells since in the absence of T cells mutant B cells did not show an activated phenotype. After adoptive transfer of LAT mutant T cells into wild type (WT) Tcell-deficient recipients, recipient B cells became activated. We show in vivo and in vitro that the LatY136F mutation promotes Tcell-dependent B cell activation leading to germinal center, memory and plasma cell formation even in the absence of MHC class II. This effect was, however, dependant on CD40 and CD80/CD86. All the plasma and memory B cell populations found in physiological T cell-dependent B cell responses were found. Characterization of the abundant plasmablasts observed in. secondary lymphoid organs of LatY136F mice revealed the presence of a previously uncharacterized CD93expressing subpopulation, whose existence was confirmed in WT mice after immunization. In LatY136F mice, B cell activation was polyclonal and not antigen-driven, since the increase in serum IgG1 and IgE concentrations involved antibodies and autoantibodies with different specificities equally. Although the non-complement-fixing IgG1 and IgE were the only isotypes significantly increased in LatY136F serum, we observed early onset of systemic autoimmunity with nephritis showing IgE autoantibody deposits and severe proteinuria. These results show that TH2 cells developing in LatY136F mice can trigger polyclonal B cell activation and thereby lead to systemic autoimmune disease. RESUME Les souris présentent une mutation ponctuelle au niveau de la tyrosine 136 de l'adaptateur requis pour l'activation des cellules T (LAT) et développent, de ce fait, une accumulation de cellules T effectrices de type TH2 ainsi qu'une hypergammaglobulémie des isotypes IgG1 et IgE. Dans ce modèle murin, l'activation des cellules B et la production d'anticorps qui y est associée ne sont pas dues à un effet direct de la mutation. Nous avons mis en évidence que l'interaction physique entre cellules T activées et cellules B est indispensable au développement de ce phenotype. D'un point de vue moléculaire, cette interaction ne requiert pas l'intervention des complexes majeurs d'histocompatibilité de classe II, garant de la spécificité d'une réponse immunitaire. Cependant, les molécules de costimulation CD40 et CD80/CD86 sont indispensables à une réponse complète des cellules B. Les souris LatY136F développent d'importantes populations de cellules B des centres germinatifs, de cellules B mémoires ainsi que de cellules sécrétant des anticorps, qui présentent les mêmes caractéristiques que lors d'une réponse immunitaire à un antigène classique. En observant plus précisément les plasmablastes présents dans les ganglions des souris LatY13sF, nous avons détecté une sous-population exprimant CD93; l'expression de ce marqueur par les cellules B n'a jamais été mise en évidence durant une réponse immunitaire. Cependant, notre étude a permis de confirmer sa présence, dans les ganglions de souris de type sauvage, lors d'immunisation avec différents antigènes. Nous avons montré que l'activation des cellules B des souris LatY136F est polyclonale et n'est pas dirigée par un antigène; les taux d'autoanticorps augmentent de manière proportionnelle à ceux des anticorps totaux. Bien que les IgG1 et les IgE ne soient pas des isotypes connus pour leurs propriétés pathogéniques, nous avons observé le développement d'une autoimmunité systémique caractérisée par une néphrite impliquant des dépôts d'autoanticorps du type IgE ainsi que par une sévère proteinurée.

Protective effect of intravitreal injection of vasoactive intestinal peptide-loaded liposomes on experimental autoimmune uveoretinitis.

PURPOSE: The aim of this study was to investigate the effect of a single intravitreal (i.v.t.) injection of vasoactive intestinal peptide (VIP) loaded in rhodamine-conjugated liposomes (VIP-Rh-Lip) on experimental autoimmune uveoretinitis (EAU). METHODS: An i.v.t. injection of VIP-Rh-Lip, saline, VIP, or empty-(E)-Rh-Lip was performed simultaneously, either 6 or 12 days after footpad immunization with retinal S-antigen in Lewis rats. Clinical and histologic scores were determined. Immunohistochemistry and cytokine quantification by multiplex enzyme-linked immunosorbent assay were performed in ocular tissues. Systemic immune response was determined at day 20 postimmunization by measuring proliferation and cytokine secretion of cells from inguinal lymph nodes (ILNs) draining the immunization site, specific delayed-type hypersensitivity (DTH), and the serum concentration of cytokines. Ocular and systemic biodistribution of VIP-Rh-Lip was studied in normal and EAU rats by immunofluorescence. RESULTS: The i.v.t. injection of VIP-Rh-Lip performed during the afferent, but not the efferent, phase of the disease reduced clinical EAU and protected against retinal damage. No effect was observed after saline, E-Rh-Lip, or VIP injection. VIP-Rh-Lip and VIP were detected in intraocular macrophages and in lymphoid organs. In VIP-Rh-Lip-treated eyes, macrophages expressed transforming growth factor-beta2, low levels of major histocompatibility complex class II, and nitric oxide synthase-2. T-cells showed activated caspase-3 with the preservation of photoreceptors. Intraocular levels of interleukin (IL)-2, interferon-gamma (IFN-gamma), IL-17, IL-4, GRO/KC, and CCL5 were reduced with increased IL-13. At the systemic level, treatment reduced retinal soluble autoantigen lymphocyte proliferation, decreased IL-2, and increased IL-10 in ILN cells, and diminished specific DTH and serum concentration of IL-12 and IFN-gamma. CONCLUSIONS: An i.v.t. injection of VIP-Rh-Lip, performed during the afferent stage of immune response, reduced EAU pathology through the immunomodulation of intraocular macrophages and deviant stimulation of T-cells in ILN. Thus, the encapsulation of VIP within liposomes appears as an effective strategy to deliver VIP into the eye and is an efficient means of the prevention of EAU severity.

Infrequent replication of parvovirus B19 and erythrovirus genotypes 2 and 3 among HIV-infected patients with chronic anemia.

We investigated the role that erythroviruses (parvovirus B19 and erythrovirus genotypes 2 and 3) play in the lives of immunosuppressed HIV-infected patients with chronic anemia. We screened the serum samples of 428 patients by specific ultrasensitive real-time polymerase chain reaction assay. Sixteen patients had circulating DNA, with no apparent clinical impact. Erythrovirus-associated anemia is an extremely rare event in HIV-infected patients.

Potential Role of TRAIL in the Management of Autoimmune Diabetes Mellitus.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease, due to the immune-mediated destruction of pancreatic β-cells, whose incidence has been steadily increasing during the last decades. Insulin replacement therapy can treat T1DM, which, however, is still associated with substantial morbidity and mortality. For this reason, great effort is being put into developing strategies that could eventually prevent and/or cure this disease. These strategies are mainly focused on blocking the immune system from attacking β-cells together with functional islet restoration either by regeneration or transplantation. Recent experimental evidences suggest that TNFrelated apoptosis-inducing ligand (TRAIL), which is an immune system modulator protein, could represent an interesting candidate for the cure for T1DM and/or its complications. Here we review the evidences on the potential role of TRAIL in the management of T1DM.

TL1A-DR3 interaction regulates Th17 cell function and Th17-mediated autoimmune disease.

T helper type 17 (Th17) cells play an important pathogenic function in autoimmune diseases; their regulation, however, is not well understood. We show that the expression of a tumor necrosis factor receptor family member, death receptor 3 (DR3; also known as TNFRSF25), is selectively elevated in Th17 cells, and that TL1A, its cognate ligand, can promote the proliferation of effector Th17 cells. To further investigate the role of the TL1A-DR3 pathway in Th17 regulation, we generated a TL1A-deficient mouse and found that TL1A(-/-) dendritic cells exhibited a reduced capacity in supporting Th17 differentiation and proliferation. Consistent with these data, TL1A(-/-) animals displayed decreased clinical severity in experimental autoimmune encephalomyelitis (EAE). Finally, we demonstrated that during EAE disease progression, TL1A was required for the optimal differentiation as well as effector function of Th17 cells. These observations thus establish an important role of the TL1A-DR3 pathway in promoting Th17 cell function and Th17-mediated autoimmune disease.

Persistent Candida albicans colonization and molecular mechanisms of azole resistance in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients.

Objectives: Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS-I) suffer from chronic candidosis caused mainly by Candida albicans, and repeated courses of azole antifungals have led to the development of resistance in the APECED patient population in Finland. The aim of our study was to address whether the patients are persistently colonized with the same or genetically closely related strains, whether epidemic strains are present and which molecular mechanisms account for azole resistance. Methods: Sets of C. albicans (n?=?19) isolates from nine APECED patients reported with decreased susceptibility to fluconazole isolated up to 9 years apart were included. The strains were typed by multilocus sequence typing. CDR1/2, MDR1 and ERG11 mRNA expression was analysed by northern blotting and Cdr1, Cdr2 and Mdr1 protein expression by western blotting, and TAC1 and ERG11 genes were sequenced. Results: All seven patients with multiple C. albicans isolates analysed were persistently colonized with the same or a genetically closely related strain for a mean of 5 years. All patients were colonized with different strains and no epidemic strains were found. The major molecular mechanisms behind the azole resistance were mutations in TAC1 contributing to overexpression of CDR1 and CDR2. Six new TAC1 mutations were found, one of which (N740S) is likely to be a gain-of-function mutation. Most isolates were found to have gained multiple TAC1 and ERG11 point mutations. Conclusions: Despite clinically successful treatment leading to relief of symptoms, colonization by C. albicans strains is persistent within APECED patients. Microevolution and point mutations occur within strains, leading to the development of azole-resistant isolates.

T cells at the site of autoimmune inflammation show increased potential for trogocytosis

Les lymphocytes T CD4+ sont connus pour leur potentiel d'acquisition de fragments membraneux de cellules présentatrices d'antigène (CPA) dans un processus nommé trogocytose. Ce phénomène est observé lors de l'interaction entre le lymphocyte T CD4+ antigène-spécifique et la CPA lors de la présentation de l'antigène en question, et dépend donc de la spécificité du lymphocyte T CD4+. L'identification des lymphocytes T CD4+ sujets à la trogocytose en co-culture avec des CPA chargées d'un antigène connu permet d'enrichir des lymphocytes T antigène-spécifiques sans connaître leur spécificité exacte ou leur profil de production de cytokines. Dans cette étude, nous avons donc cherché à évaluer l'utilité de cette méthode dans l'identification de la spécificité des lymphocytes T effecteurs et régulateurs lors de l'inflammation auto-immune avec des spécificités souvent inconnues. La trogocytose a démontré son efficacité dans la détection de lymphocytes T réactifs à la protéine basique de myéline in vitro ainsi qu'ex vivo après immunisation. Cependant, le potentiel de la trogocytose à identifier des lymphocytes T régulateurs antigène-spécifiques est limité par le fait que les lymphocytes T régulateurs Foxp3+ montrent un taux élevé de manière constitutive de trogocytose comparé aux lymphocytes T Foxp3-, Un taux localement élevé de trogocytose lors d'un état inflammatoire (observé au niveau des lymphocytes T effecteurs et régulateurs isolés du système nerveux central enflammé) empêche l'utilisation de la trogocytose dans l'évaluation de la réactivité antigénique de cellules extraites d'un site inflammatoire. Nos résultats montrent la possibilité d'enrichir des lymphocytes T conventionnels antigène- réactifs en périphérie par détection au moyen de la trogocytose. Nous avons aussi montré les limitations de cette méthode dans sa capacité d'identifier des lymphocytes T effecteurs et régulateurs antigène- réactifs extraits de sites inflammatoires. Le potentiel de trogocytose élevé dans les sites d'inflammation soulève la question de la signification biologique de ce phénomène dans l'inflammation, dans la suppression médiée par les lymphocytes T régulateurs et dans le maintien de la tolérance immunologique dans des états de santé variables.

An unusual cause of high anion gap metabolic acidosis: pyroglutamic acidemia. A case report.

Pyroglutamic acidemia is an uncommon metabolic disorder, which is usually diagnosed at early ages. The mechanism of action is thought to be glutathione depletion, and its clinical manifestations consist of hemolytic anemia, mental retardation, ataxia, and chronic metabolic acidosis. However, an acquired form has been described in adult patients, who usually present with confusion, respiratory distress, and high anion gap metabolic acidosis (HAGMA). It is also associated with many conditions, including chronic acetaminophen consumption. A 68-year-old white male, with chronic acetaminophen use presented to our service on multiple occasions with severe HAGMA. The patient was admitted to the intensive care unit and required mechanical ventilation and aggressive supportive measures. After ruling out the most frequent etiologies for his acid-base disorder and considering the long history of Tylenol ingestion, his 5-oxiproline (pyroglutamic acid) levels were sent to diagnose pyroglutamic acidemia. Clinicians need to be aware of this cause for metabolic acidosis since it might be a more common metabolic disturbance in compromised patients than would be expected. Subjects with HAGMA that cannot be explained by common causes should be tested for the presence of 5-oxoproline. Discontinuation of the offending drug is therapeutic.