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Find video protocols related to scientific articles indexed in Pubmed.
As little as needed: the extraordinary case of a mild recessive osteopetrosis owing to a novel splicing hypomorphic mutation in the TCIRG1 gene.
J. Bone Miner. Res.
PUBLISHED: 01-23-2014
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Mutations in the TCIRG1 gene, coding for a subunit of the osteoclast proton pump, are responsible for more than 50% of cases of human malignant autosomal recessive osteopetrosis (ARO), a rare inherited bone disease with increased bone density owing to a failure in bone resorption. A wide variety of mutations has been described, including missense, nonsense, small deletions/insertions, splice-site mutations, and large genomic deletions, all leading to a similar severe presentation. So far, to the best of our knowledge, no report of a mild phenotype owing to recessive TCIRG1 mutations is present neither in our series of more than 100 TCIRG1-dependent ARO patients nor in the literature. Here we describe an 8-year-old patient referred to us with a clinical diagnosis of ARO, based on radiological findings; of note, no neurological or hematological defects were present in this girl. Surprisingly, we identified a novel nucleotide change in intron 15 of the TCIRG1 gene at the homozygous state, leading to the production of multiple aberrant transcripts, but also, more importantly, of a limited amount of the normal transcript. Our results show that a low level of normal TCIRG1 protein can dampen the clinical presentation of TCIRG1-dependent ARO. On this basis, a small amount of protein might be sufficient to rescue, at least partially, the severe ARO phenotype, and this is particularly important when gene therapy approaches are considered. In addition, we would also recommend that the TCIRG1 gene be included in the molecular diagnosis of mild forms of human ARO.
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Severe combined immunodeficiency in Serbia and Montenegro between years 1986 and 2010: a single-center experience.
J. Clin. Immunol.
PUBLISHED: 01-20-2014
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Severe combined immunodeficiency (SCID), including the 'variant' Omenn syndrome (OS), represent a heterogeneous group of monogenic disorders characterized by defect in differentiation of T- and/or B lymphocytes and susceptibility to infections since birth. In the period of 25 years, between January 1986 and December 2010, a total of 21 patients (15 SCID, 6 OS) were diagnosed in Mother & Child Health Institute of Serbia, a tertiary-care teaching University hospital and a national referral center for patients affected with primary immunodeficiency (PID). The diagnoses were based on anamnestic data, clinical findings, and immunological and genetic analysis. The median age at the onset of the first infection was the 2nd month of life. Seven (33 %) patients had positive family history for SCID. Out of five male infants with T-B+NK- SCID phenotype, mutation analysis revealed interleukin-2 (common) gamma-chain receptor (IL2RG) mutations in 3 with positive X-linked family history, and Janus-kinase (JAK)-3 gene defects in the other two. Six patients had T-B-NK+ SCID phenotype and further 6 features of OS, 11 of which had recombinase-activating gene (RAG1or RAG2) and 1 Artemis gene mutations. One child with T+B+NK+ SCID phenotype as well had proven RAG mutation. One child each with T-B+NK+ SCID phenotype, CD8 lymphopenia and unknown phenotype remained without known underlying genetic defect. Of the eight patients who underwent hematopoetic stem cell transplant (HSCT) 5 survived, the other 13 died between 2 days and 12 months after diagnosis was made. Early diagnosis of SCID, before onset of severe infections, offers possibility for HSCT and cure. Education of primary-care pediatricians, in particular including awareness of the risk of using live vaccines and non-irradiated blood products, should improve prognosis of SCID in our setting.
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Sphingosine-1-phosphate receptors control B-cell migration through signaling components associated with primary immunodeficiencies, chronic lymphocytic leukemia, and multiple sclerosis.
J. Allergy Clin. Immunol.
PUBLISHED: 01-07-2014
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Five different G protein-coupled sphingosine-1-phosphate (S1P) receptors (S1P1-S1P5) regulate a variety of physiologic and pathophysiologic processes, including lymphocyte circulation, multiple sclerosis (MS), and cancer. Although B-lymphocyte circulation plays an important role in these processes and is essential for normal immune responses, little is known about S1P receptors in human B cells.
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Osteopetrosis mimicking juvenile myelomonocytic leukemia.
Pediatr Int
PUBLISHED: 01-06-2014
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A 5-month-old boy developed splenomegaly, anemia, thrombocytopenia with elevated white cells, monocytosis and immature granulocytes in the peripheral blood. Bone marrow showed dysplasia without blastosis. Increased colony-forming unit-granulocyte-macrophage was found in the peripheral blood, mimicking granulocyte-macrophage colony-stimulating factor hypersensitivity. These findings fulfilled the diagnosis criteria for juvenile myelomonocytic leukemia (JMML), but no mutations in the CBL, NRAS, KRAS, or PTPN11 genes were detected. In addition to these findings severe hypogammaglobulinemia and elevated alkaline phosphatase were present. Bone X-ray showed dense and radiopaque bones with a bone-in-bone appearance characteristic of infantile malignant osteopetrosis (IMO). Genetic mutation in T-cell, immune regulator 1 (TCIRG1) was identified, confirming the diagnosis of IMO. Careful differential diagnosis including osteopetrosis, is therefore recommended in patients with clinical features and hematologic findings consistent with JMML.
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Rag defects and thymic stroma: lessons from animal models.
Front Immunol
PUBLISHED: 01-01-2014
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Thymocytes and thymic epithelial cells (TECs) cross-talk is essential to support T cell development and preserve thymic architecture and maturation of TECs and Foxp3(+) natural regulatory T cells. Accordingly, disruption of thymic lymphostromal cross-talk may have major implications on the thymic mechanisms that govern T cell tolerance. Several genetic defects have been described in humans that affect early stages of T cell development [leading to severe combined immune deficiency (SCID)] or late stages in thymocyte maturation (resulting in combined immunodeficiency). Hypomorphic mutations in SCID-causing genes may allow for generation of a limited pool of T lymphocytes with a restricted repertoire. These conditions are often associated with infiltration of peripheral tissues by activated T cells and immune dysregulation, as best exemplified by Omenn syndrome (OS). In this review, we will discuss our recent findings on abnormalities of thymic microenvironment in OS with a special focus of defective maturation of TECs, altered distribution of thymic dendritic cells and impairment of deletional and non-deletional mechanisms of central tolerance. Here, taking advantage of mouse models of OS and atypical SCID, we will discuss how modifications in stromal compartment impact and shape lymphocyte differentiation, and vice versa how inefficient T cell signaling results in defective stromal maturation. These findings are instrumental to understand the extent to which novel therapeutic strategies should act on thymic stroma to achieve full immune reconstitution.
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Development of central nervous system autoimmunity is impaired in the absence of Wiskott-Aldrich syndrome protein.
PLoS ONE
PUBLISHED: 01-01-2014
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Wiskott-Aldrich Syndrome protein (WASP) is a key regulator of the actin cytoskeleton in hematopoietic cells. Defective expression of WASP leads to multiple abnormalities in different hematopoietic cells. Despite severe impairment of T cell function, WAS patients exhibit a high prevalence of autoimmune disorders. We attempted to induce EAE, an animal model of organ-specific autoimmunity affecting the CNS that mimics human MS, in Was(-/-) mice. We describe here that Was(-/-) mice are markedly resistant against EAE, showing lower incidence and milder score, reduced CNS inflammation and demyelination as compared to WT mice. Microglia was only poorly activated in Was(-/-) mice. Antigen-induced T-cell proliferation, Th-1 and -17 cytokine production and integrin-dependent adhesion were increased in Was(-/-) mice. However, adoptive transfer of MOG-activated T cells from Was(-/-) mice in WT mice failed to induce EAE. Was(-/-) mice were resistant against EAE also when induced by adoptive transfer of MOG-activated T cells from WT mice. Was(+/-) heterozygous mice developed an intermediate clinical phenotype between WT and Was(-/-) mice, and they displayed a mixed population of WASP-positive and -negative T cells in the periphery but not in their CNS parenchyma, where the large majority of inflammatory cells expressed WASP. In conclusion, in absence of WASP, T-cell responses against a CNS autoantigen are increased, but the ability of autoreactive T cells to induce CNS autoimmunity is impaired, most probably because of an inefficient T-cell transmigration into the CNS and defective CNS resident microglial function.
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Hypomorphic mutation in the RAG2 gene affects dendritic cell distribution and migration.
J. Leukoc. Biol.
PUBLISHED: 09-19-2013
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OS is a severe combined immunodeficiency characterized by erythrodermia and protracted diarrhea as a result of infiltration of oligoclonal-activated T cells, caused by hypomorphic mutations in RAGs. The RAG2(R229Q) mouse model fully recapitulates the clinical OS phenotype. We evaluated whether T and B cell defects, together with the abnormal lymphoid structure, could affect DC homeostasis and function. High density of LCs was observed in skin biopsies of Omenn patients and in the derma of RAG2(R229Q) mice, correlating with the presence of erythrodermia. In vivo models of cutaneous skin painting and CHS demonstrated a decreased migration of RAG2(R229Q) DCs-in particular, LCs-into draining LNs. Interestingly, at steady state, RAG2(R229Q) mice showed a reduction in DC number in all hematopoietic organs except LNs. Analysis of the MHCII marker revealed a diminished expression also upon the LPS-driven inflammatory condition. Despite the decreased number of peripheral DCs, BM pre-cDCs were present in normal number compared with RAG2(+/+) controls, whereas pDCs and monocytes were reduced significantly. Overall, these results point to a secondary defect in the DC compartment, which contributes to clinical manifestations and autoimmunity in OS.
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Exome sequencing identifies CTSK mutations in patients originally diagnosed as intermediate osteopetrosis.
Bone
PUBLISHED: 09-10-2013
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Autosomal Recessive Osteopetrosis is a genetic disorder characterized by increased bone density due to lack of resorption by the osteoclasts. Genetic studies have widely unraveled the molecular basis of the most severe forms, while cases of intermediate severity are more difficult to characterize, probably because of a large heterogeneity. Here, we describe the use of exome sequencing in the molecular diagnosis of 2 siblings initially thought to be affected by "intermediate osteopetrosis", which identified a homozygous mutation in the CTSK gene. Prompted by this finding, we tested by Sanger sequencing 25 additional patients addressed to us for recessive osteopetrosis and found CTSK mutations in 4 of them. In retrospect, their clinical and radiographic features were found to be compatible with, but not typical for, Pycnodysostosis. We sought to identify modifier genes that might have played a role in the clinical manifestation of the disease in these patients, but our results were not informative. In conclusion, we underline the difficulties of differential diagnosis in some patients whose clinical appearance does not fit the classical malignant or benign picture and recommend that CTSK gene be included in the molecular diagnosis of high bone density conditions.
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Wiskott-Aldrich Syndrome protein deficiency perturbs the homeostasis of B-cell compartment in humans.
J. Autoimmun.
PUBLISHED: 08-09-2013
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Wiskott-Aldrich Syndrome protein (WASp) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the Wiskott-Aldrich Syndrome (WAS), a primary immunodeficiency with microthrombocytopenia, eczema and a higher susceptibility to develop tumors. Autoimmune manifestations, frequently observed in WAS patients, are associated with an increased risk of mortality and still represent an unsolved aspect of the disease. B cells play a crucial role both in immune competence and self-tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity. We performed a phenotypical and molecular analysis of central and peripheral B-cell compartments in WAS pediatric patients. We found a decreased proportion of immature B cells in the bone marrow correlating with an increased presence of transitional B cells in the periphery. These results could be explained by the defective migratory response of WAS B cells to SDF-1?, essential for the retention of immature B cells in the BM. In the periphery, we observed an unusual expansion of CD21(low) B-cell population and increased plasma BAFF levels that may contribute to the high susceptibility to develop autoimmune manifestations in WAS patients. WAS memory B cells were characterized by a reduced in vivo proliferation, decreased somatic hypermutation and preferential usage of IGHV4-34, an immunoglobulin gene commonly found in autoreactive B cells. In conclusion, our findings demonstrate that WASp-deficiency perturbs B-cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in WAS patients.
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Osteopetrosis: genetics, treatment and new insights into osteoclast function.
Nat Rev Endocrinol
PUBLISHED: 07-23-2013
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Osteopetrosis is a genetic condition of increased bone mass, which is caused by defects in osteoclast formation and function. Both autosomal recessive and autosomal dominant forms exist, but this Review focuses on autosomal recessive osteopetrosis (ARO), also known as malignant infantile osteopetrosis. The genetic basis of this disease is now largely uncovered: mutations in TCIRG1, CLCN7, OSTM1, SNX10 and PLEKHM1 lead to osteoclast-rich ARO (in which osteoclasts are abundant but have severely impaired resorptive function), whereas mutations in TNFSF11 and TNFRSF11A lead to osteoclast-poor ARO. In osteoclast-rich ARO, impaired endosomal and lysosomal vesicle trafficking results in defective osteoclast ruffled-border formation and, hence, the inability to resorb bone and mineralized cartilage. ARO presents soon after birth and can be fatal if left untreated. However, the disease is heterogeneous in clinical presentation and often misdiagnosed. This article describes the genetics of ARO and discusses the diagnostic role of next-generation sequencing methods. The management of affected patients, including guidelines for the indication of haematopoietic stem cell transplantation (which can provide a cure for many types of ARO), are outlined. Finally, novel treatments, including preclinical data on in utero stem cell treatment, RANKL replacement therapy and denosumab therapy for hypercalcaemia are also discussed.
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Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.
Science
PUBLISHED: 07-11-2013
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Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.
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RANKL cytokine: from pioneer of the osteoimmunology era to cure for a rare disease.
Clin. Dev. Immunol.
PUBLISHED: 03-29-2013
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Since its identification, the RANKL cytokine has been demonstrated to play a crucial role in bone homeostasis and lymphoid tissue organization. Genetic defects impairing its function lead to a peculiar form of autosomal recessive osteopetrosis (ARO), a rare genetic bone disease presenting early in life and characterized by increased bone density due to failure in bone resorption by the osteoclasts. Hematopoietic stem cell transplantation (HSCT) is the only option for the majority of patients affected by this life-threatening disease. However, the RANKL-dependent ARO does not gain any benefit from this approach, because the genetic defect is not intrinsic to the hematopoietic osteoclast lineage but rather to the mesenchymal one. Of note, we recently provided proof of concept of the efficacy of a pharmacological RANKL-based therapy to cure this form of the disease. Here we provide an overview of the diverse roles of RANKL in the bone and immune systems and review the clinical features of RANKL-deficient ARO patients and the results of our preclinical studies. We emphasize that these patients present a continuous worsening of the disease in the absence of a cure and strongly wish that the therapy we propose will be further developed.
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Lentiviral gene transfer of TCIRG1 into peripheral blood CD34(+) cells restores osteoclast function in infantile malignant osteopetrosis.
Bone
PUBLISHED: 03-25-2013
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Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by non-functional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to restore the resorptive function of IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA. CD34(+) cells from peripheral blood of five IMO patients and from normal cord blood were transduced with lentiviral vectors expressing TCIRG1 and GFP under a SFFV promoter, expanded in culture and differentiated on bone slices to mature osteoclasts. qPCR analysis and western blot revealed increased mRNA and protein levels of TCIRG1, comparable to controls. Vector corrected IMO osteoclasts generated increased release of Ca(2+) and bone degradation product CTX-I into the media as well as increased formation of resorption pits in the bone slices, while non-corrected IMO osteoclasts failed to resorb bone. Resorption was approximately 70-80% of that of osteoclasts generated from cord blood. Furthermore, transduced CD34(+) cells successfully engrafted in NSG-mice. In conclusion we provide the first evidence of lentiviral-mediated correction of a human genetic disease affecting the osteoclastic lineage.
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Wiskott-Aldrich syndrome protein-mediated actin dynamics control type-I interferon production in plasmacytoid dendritic cells.
J. Exp. Med.
PUBLISHED: 01-21-2013
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Mutations in Wiskott-Aldrich syndrome (WAS) protein (WASp), a regulator of actin dynamics in hematopoietic cells, cause WAS, an X-linked primary immunodeficiency characterized by recurrent infections and a marked predisposition to develop autoimmune disorders. The mechanisms that link actin alterations to the autoimmune phenotype are still poorly understood. We show that chronic activation of plasmacytoid dendritic cells (pDCs) and elevated type-I interferon (IFN) levels play a role in WAS autoimmunity. WAS patients display increased expression of type-I IFN genes and their inducible targets, alteration in pDCs numbers, and hyperresponsiveness to TLR9. Importantly, ablating IFN-I signaling in WASp null mice rescued chronic activation of conventional DCs, splenomegaly, and colitis. Using WASp-deficient mice, we demonstrated that WASp null pDCs are intrinsically more responsive to multimeric agonist of TLR9 and constitutively secrete type-I IFN but become progressively tolerant to further stimulation. By acute silencing of WASp and actin inhibitors, we show that WASp-mediated actin polymerization controls intracellular trafficking and compartmentalization of TLR9 ligands in pDCs restraining exaggerated activation of the TLR9-IFN-? pathway. Together, these data highlight the role of actin dynamics in pDC innate functions and imply the pDC-IFN-? axis as a player in the onset of autoimmune phenomena in WAS disease.
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Recombination-activating gene 1 (Rag1)-deficient mice with severe combined immunodeficiency treated with lentiviral gene therapy demonstrate autoimmune Omenn-like syndrome.
J. Allergy Clin. Immunol.
PUBLISHED: 01-03-2013
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Recombination-activating gene 1 (RAG1) deficiency results in severe combined immunodeficiency (SCID) caused by a complete lack of T and B lymphocytes. If untreated, patients succumb to recurrent infections.
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SNX10 mutations define a subgroup of human autosomal recessive osteopetrosis with variable clinical severity.
J. Bone Miner. Res.
PUBLISHED: 01-03-2013
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Human Autosomal Recessive Osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. In 2000, we found that mutations in the TCIRG1 gene encoding for a subunit of the proton pump (V-ATPase) are responsible for more than one-half of ARO cases. Since then, five additional genes have been demonstrated to be involved in the pathogenesis of the disease, leaving approximately 25% of cases that could not be associated with a genotype. Very recently, a mutation in the sorting nexin 10 (SNX10) gene, whose product is suggested to interact with the proton pump, has been found in 3 consanguineous families of Palestinian origin, thus adding a new candidate gene in patients not previously classified. Here we report the identification of 9 novel mutations in this gene in 14 ARO patients from 12 unrelated families of different geographic origin. Interestingly, we define the molecular defect in three cases of "Västerbottenian osteopetrosis," named for the Swedish Province where a higher incidence of the disease has been reported. In our cohort of more than 310 patients from all over the world, SNX10-dependent ARO constitutes 4% of the cases, with a frequency comparable to the receptor activator of NF-?B ligand (RANKL), receptor activator of NF-?B (RANK) and osteopetrosis-associated transmembrane protein 1 (OSTM1)-dependent subsets. Although the clinical presentation is relatively variable in severity, bone seems to be the only affected tissue and the defect can be almost completely rescued by hematopoietic stem cell transplantation (HSCT). These results confirm the involvement of the SNX10 gene in human ARO and identify a new subset with a relatively favorable prognosis as compared to TCIRG1-dependent cases. Further analyses will help to better understand the role of SNX10 in osteoclast physiology and verify whether this protein might be considered a new target for selective antiresorptive therapies.
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Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID.
Blood
PUBLISHED: 12-19-2011
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Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.
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Omenn syndrome does not live by V(D)J recombination alone.
Curr Opin Allergy Clin Immunol
PUBLISHED: 10-18-2011
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During the past decade, easy access to sequence analyses has allowed us to increase our understanding of the pathogenesis of severe combined immunodeficiencies. Here, we describe the expanding clinical and immunological spectrum associated with Omenn syndrome phenotype. In particular, we review the cellular and molecular mechanisms involved in the pathophysiology of classical Omenn syndrome due to the recombination activating gene (RAG) defects and of a new subgroup of Omenn-like disorders.
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Identification of the first deletion in the LRP5 gene in a patient with autosomal dominant osteopetrosis type I.
Bone
PUBLISHED: 04-04-2011
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In the last decade, the low-density lipoprotein receptor-related protein 5 (LRP5) gene, coding for a coreceptor in the canonical Wnt signalling pathway, has been shown to play an important role in regulating bone mass and to be involved in the pathogenesis of several bone disorders. Here we describe a patient who presented with a clinical picture of Autosomal Dominant Osteopetrosis type I (ADO I), in whom we could identify the first deletion in the LRP5 gene causing increased bone mass. This mutation caused the in-frame deletion of two amino acids in the fourth blade of the first propeller of the protein, namely the highly conserved glycine at position 171 and the following glutamate residue. In vitro studies suggested that the pathogenic effect of this novel mutation could be due to a decreased inhibition of Wnt signalling by the antagonistic proteins sclerostin and Dickkopf-1, encoded respectively by the SOST and DKK1 genes, in the presence of mutated LRP5. Our results highlight an increasing molecular heterogeneity in LRP5-related bone diseases.
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Lentiviral-mediated gene therapy leads to improvement of B-cell functionality in a murine model of Wiskott-Aldrich syndrome.
J. Allergy Clin. Immunol.
PUBLISHED: 03-22-2011
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Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, infections, autoimmunity, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical donors is curative, but it is not available to all patients. We have developed a gene therapy (GT) approach for WAS by using a lentiviral vector encoding for human WAS promoter/cDNA (w1.6W) and demonstrated its preclinical efficacy and safety.
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Severe combined immunodeficiency in Greek children over a 20-year period: rarity of ?c-chain deficiency (X-linked) type.
J. Clin. Immunol.
PUBLISHED: 03-05-2011
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Severe combined immunodeficiencies (SCID) are a heterogeneous group of genetic disorders characterized by a blockade or impairment of both cellular and humoral immunity. Several epidemiological studies in different geographic areas have shown that the most common type of SCID affecting almost half of these patients is the X-linked common ?-chain (?(c)) deficiency. The objective of the study was to document the incidence and types of SCID in our area. We conducted a retrospective analysis of patients who were diagnosed with SCID in the major immunology center in Greece for a 20-year period. During the study period, 30 children from 27 unrelated families with final diagnosis of SCID were identified. The incidence of SCID in Greece is estimated at 1.7 cases per 100,000 live births. Out of 30 children, 19 were boys (63.3%) and 26 (86.7%) had Greek maternal origin. Lymphocyte immunophenotypes that were identified were T(-)B(-)NK(+) in 12 (40%) children, T(-)B(+)NK(-) in six (20%), T(-)B(+)NK(+) in three (10%), T(-)B(-)NK(-) in two (6.7%) and T(+)B(+/-)NK(+) in seven (23.4%) (among them, four [13.4%] females with Omenns syndrome). Molecular diagnosis was available for 12 children: ?(c) (2) with non Greek maternal origin, Jak3 (2), Rag1 (2), Artemis (3), ADA deficiency (2), PNP deficiency (1). Out of the 26 children of Greek maternal origin diagnosed with SCID representing 23 distinct families, only two (8.7%) had lymphocyte immunophenotype compatible with ?(c)-chain gene mutation (no molecular testing or enough DNA was available for them at the time of diagnosis). Findings of the present study suggest that, for unknown reasons, mutations of the ?(c) chain of several cytokine receptors have a rare occurrence in our area.
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Noninvasive assessment of breast cancer risk using time-resolved diffuse optical spectroscopy.
J Biomed Opt
PUBLISHED: 08-09-2010
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Breast density is a recognized strong and independent risk factor for breast cancer. We propose the use of time-resolved transmittance spectroscopy to estimate breast tissue density and potentially provide even more direct information on breast cancer risk. Time-resolved optical mammography at seven wavelengths (635 to 1060 nm) is performed on 49 subjects. Average information on breast tissue of each subject is obtained on oxy- and deoxyhemoglobin, water, lipids, and collagen content, as well as scattering amplitude and power. All parameters, except for blood volume and oxygenation, correlate with mammographic breast density, even if not to the same extent. A synthetic optical index proves to be quite effective in separating different breast density categories. Finally, the estimate of collagen content as a more direct means for the assessment of breast cancer risk is discussed.
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Homeostatic expansion of autoreactive immunoglobulin-secreting cells in the Rag2 mouse model of Omenn syndrome.
J. Exp. Med.
PUBLISHED: 06-14-2010
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Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2(R229Q) knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2(R229Q) knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.
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A new familial sclerosing bone dysplasia.
J. Bone Miner. Res.
PUBLISHED: 04-28-2010
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Osteoscleroses are a heterogeneous group of bone remodeling disorders characterized by an increase in bone density. Here we report on a consanguineous Lebanese family in which two sisters, aged 39 and 36 years, exhibit a severe genu varum, a square-face appearance, high forehead, slight proptosis of the eyes, symmetric enlargement of the jaw, protruding chin, and short stature. Bone X-rays showed the presence of hyperostosis of the cranial base and vault with increased density of the orbits, hyperostosis of the bones, thickening of the cortices, diaphyseal modeling defects, cortical thickening of the medullary cavity, mild enlargement of the medullary cavity of the short long bones, short femoral necks, increased width of the ribs, and narrow interpedicular distances of the lower lumbar spine. Osteodensitometry showed values 200% to 300% above values for age. A cervical MRI revealed the presence of a diffuse osteosclerosis with calcification of the posterior vertebral ligament and a narrow canal between C2 and T2. Blood test results were unremarkable. Serum osteocalcin levels were in the normal range, whereas high values of serum C-telopeptide were noted. A bone biopsy showed only the presence of compact bone and did not allow for histomorphometric analysis. Molecular studies excluded genes known to be involved in sclerosing bone dysplasias as the cause of this condition. In vitro analysis of osteoclast function indicated that contrary to most cases of autosomal recessive osteopetrosis, osteoclasts both formed and resorbed but exhibited a small decrease in resorptive activity compared with osteoclasts generated from normal control individuals. Differential diagnoses are discussed, and the possibility that this may be a novel clinical entity is raised.
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Revertant T lymphocytes in a patient with Wiskott-Aldrich syndrome: analysis of function and distribution in lymphoid organs.
J. Allergy Clin. Immunol.
PUBLISHED: 02-18-2010
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The Wiskott-Aldrich syndrome (WAS) is a rare genetic disease characterized by thrombocytopenia, immunodeficiency, autoimmunity, and hematologic malignancies. Secondary mutations leading to re-expression of WAS protein (WASP) are relatively frequent in patients with WAS.
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Defect of regulatory T cells in patients with Omenn syndrome.
J. Allergy Clin. Immunol.
PUBLISHED: 01-30-2010
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Omenn syndrome (OS) is an autosomal-recessive disorder characterized by severe immunodeficiency and T-cell-mediated autoimmunity. The disease is caused by hypomorphic mutations in recombination-activating genes that hamper the process of Variable (V) Diversity (D) Joining (J) recombination, leading to the generation of autoreactive T cells. We have previously shown that in OS the expression of autoimmune regulator, a key factor governing central tolerance, is markedly reduced.
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Human peripheral lymphoid tissues contain autoimmune regulator-expressing dendritic cells.
Am. J. Pathol.
PUBLISHED: 01-21-2010
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Autoimmune regulator (AIRE) modulates the expression of tissue-restricted antigens (TSAs) and promotes central tolerance in the thymus. However, few autoreactive T cells escape negative selection and reach the periphery, where peripheral tolerance is required to avoid autoimmunity. Murine lymph nodes (LNs) have been shown to contain "stromal" cells expressing AIRE and TSAs. Here we report the occurrence of AIRE-expressing cells in human peripheral lymphoid tissues, including LNs, tonsils, and gut-associated lymphoid tissue, with the exception of the spleen. Notably, AIRE+ cells are absent in fetal LNs and, in postnatal life, they are more numerous in abdominal than in superficial LNs, thus suggesting that their development in periphery may depend on instructive signals from microenvironment and antigen challenge. Extrathymic AIRE+ cells show a dendritic morphology, consistently express human leukocyte antigen-DR (HLADR) and fascin, and are largely positive for CD11c and S100 and for the dendritic cell-activation markers CD40, CD83, DC-LAMP/CD208, and CCR7. Lymphoid, myelomonocytic, mesenchymal, and epithelial cell lineage markers are negative. The HLADRhigh/AIRE+ cell fraction isolated from mesenteric LNs expressed TSAs (insulin, CYP17A1, and CYP21A2), as well as molecules associated with tolerogenic functions, such as interleukin-10 and indoleamine 2,3-dioxygenase. Data indicate that AIRE+ cells in human peripheral lymphoid tissues correspond to a subset of activated interdigitating dendritic cells expressing TSAs and the tolerogenic molecules indoleamine 2,3-dioxygenase and interleukin-10, suggestive of a potential tolerogenic function.
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Analysis of mutations from SCID and Omenn syndrome patients reveals the central role of the Rag2 PHD domain in regulating V(D)J recombination.
J. Clin. Invest.
PUBLISHED: 01-20-2010
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Rag2 plays an essential role in the generation of antigen receptors. Mutations that impair Rag2 function can lead to severe combined immunodeficiency (SCID), a condition characterized by complete absence of T and B cells, or Omenn syndrome (OS), a form of SCID characterized by the virtual absence of B cells and the presence of oligoclonal autoreactive T cells. Here, we present a comparative study of a panel of mutations that were identified in the noncanonical plant homeodomain (PHD) of Rag2 in patients with SCID or OS. We show that PHD mutant mouse Rag2 proteins that correspond to those found in these patients greatly impaired endogenous recombination of Ig gene segments in a Rag2-deficient pro-B cell line and that this correlated with decreased protein stability, impaired nuclear localization, and/or loss of the interaction between Rag2 and core histones. Our results demonstrate that point mutations in the PHD of Rag2 compromise the functionality of the entire protein, thus explaining why the phenotype of cells expressing PHD point mutants differs from those expressing core Rag2 protein that lacks the entire C-terminal region and is therefore devoid of the regulation imposed by the PHD. Together, our findings reveal the various deleterious effects of PHD Rag2 mutations and demonstrate the crucial role of this domain in regulating antigen receptor gene assembly. We believe these results reveal new mechanisms of immunodeficiency in SCID and OS.
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ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency.
Blood
PUBLISHED: 07-24-2009
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Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and childrens growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling.
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Plasma-induced graft-polymerization of polyethylene glycol acrylate on polypropylene films: chemical characterization and evaluation of the protein adsorption.
J Colloid Interface Sci
PUBLISHED: 06-30-2009
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This work deals with the optimization of argon plasma-induced graft-polymerization of polyethylene glycol acrylate (PEGA) on polypropylene (PP) films in order to obtain surfaces with a reduced protein adsorption for possible biomedical applications. To this end, we examined the protein adsorption on the treated and untreated surfaces. The graft-polymerization process consisted of four steps: (a) plasma pre-activation of the PP substrates; (b) immersion in a PEGA solution; (c) argon plasma-induced graft-polymerization; (d) washing and drying of the samples. The efficiency of these processes was evaluated in terms of the amount of grafted polymer, coverage uniformity and substrates wettability. The process was monitored by contact angle measurements, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray Photoelectron Spectroscopy (XPS) and atomic force microscopy (AFM) analyses. The stability of the obtained thin films was evaluated in water and in Phosphate Buffer Saline (PBS) at 37 degrees C. The adsorption of fibrinogen and green fluorescent protein (GFP)--taken as model proteins--on the differently prepared surfaces was evaluated through a fluorescence approach using laser scanning confocal microscopy with photon counting detection. After plasma treatments of short duration, the protein adsorption decreases by about 60-70% with respect to that of the untreated film, while long plasma exposure resulted in a higher protein adsorption, due to damaging of the grafted polymer.
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A single-center experience in 20 patients with infantile malignant osteopetrosis.
Am. J. Hematol.
PUBLISHED: 06-10-2009
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Infantile malignant osteopetrosis (IMO) includes various genetic disorders that affect osteoclast development and/or function. Genotype-phenotype correlation studies in IMO have been hampered by the rarity and heterogeneity of the disease and by the severity of the clinical course, which often leads to death early in life. We report on the clinical and molecular findings and treatment in 20 consecutive patients (11 males, nine females) with IMO, diagnosed at a single center in the period 1991-2008. Mean age at diagnosis was 3.9 months, and mean follow-up was 66.75 months. Mutations in TCIRG1, OSTM1, ClCN7, and TNFRSF11A genes were detected in nine, three, one, and one patients, respectively. Six patients remain genetically undefined. OSTM1 and ClCN7 mutations were associated with poor neurologic outcome. Among nine patients with TCIRG1 defects, six presented with hypogammaglobulinemia, and one showed primary pulmonary hypertension. Fourteen patients received hematopoietic cell transplantation; of these, nine are alive and eight of them have evidence of osteoclast function. These data may provide a basis for informed decisions regarding the care of patients with IMO.
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Early defects in human T-cell development severely affect distribution and maturation of thymic stromal cells: possible implications for the pathophysiology of Omenn syndrome.
Blood
PUBLISHED: 05-04-2009
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Thymocytes and thymic epithelial cell (TEC) cross-talk is crucial to preserve thymic architecture and function, including maturation of TECs and dendritic cells, and induction of mechanisms of central tolerance. We have analyzed thymic maturation and organization in 9 infants with various genetic defects leading to complete or partial block in T-cell development. Profound abnormalities of TEC differentiation (with lack of AIRE expression) and severe reduction of thymic dendritic cells were identified in patients with T-negative severe combined immunodeficiency, reticular dysgenesis, and Omenn syndrome. The latter also showed virtual absence of thymic Foxp3(+) T cells. In contrast, an IL2RG-R222C hypomorphic mutation permissive for T-cell development allowed for TEC maturation, AIRE expression, and Foxp3(+) T cells. Our data provide evidence that severe defects of thymopoiesis impinge on TEC homeostasis and may affect deletional and nondeletional mechanisms of central tolerance, thus favoring immune dysreactive manifestations, as in Omenn syndrome.
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Characterization of a novel Alu-Alu recombination-mediated genomic deletion in the TCIRG1 gene in five osteopetrotic patients.
J. Bone Miner. Res.
PUBLISHED: 04-25-2009
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Human malignant autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. Biallelic mutations in the TCIRG1 gene, encoding the a3 subunit of the vacuolar proton pump, are responsible for more than one half of ARO patients. However, a few patients with monoallelic mutations have been described, raising the possibility of a dominant-like TCIRG1-dependent osteopetrosis, of a digenic disease, or of peculiar mutations difficult to detect with standard methods. We describe here a novel genomic deletion in the TCIRG1 gene explaining why, in some patients, mutations in only one allele have previously been found. The analysis of a proband from a consanguineous Turkish family allowed us to define the deletion boundaries encompassing introns 10 and 13 and occurring within AluSx repeat sequences, suggesting Alu-mediated homologous recombination as a mechanism. An identical genomic deletion at the heterozygous level was found in four unrelated Italian families in whom only a single mutated allele has previously been found. TCIRG1 haplotype analysis in these five families suggests a possible common ancestral origin for this large deletion. In summary, we describe the identification of a novel genomic deletion in the TCIRG1 gene that is of clinical relevance, especially in prenatal diagnosis.
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Recent advances in understanding the pathophysiology of Wiskott-Aldrich syndrome.
Blood
PUBLISHED: 04-07-2009
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Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency caused by mutations in the gene encoding for WASP, a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in WASP result in a wide spectrum of clinical manifestations ranging from the relatively mild X-linked thrombocytopenia to the classic full-blown WAS phenotype characterized by thrombocytopenia, immunodeficiency, eczema, and high susceptibility to developing tumors and autoimmune manifestations. The life expectancy of patients affected by severe WAS is reduced, unless they are successfully cured by bone marrow transplantation from related identical or matched unrelated donors. Because many patients lack a compatible bone marrow donor, the administration of WAS gene-corrected autologous hematopoietic stem cells could represent an alternative therapeutic approach. In the present review, we focus on recent progress in understanding the molecular and cellular mechanisms contributing to the pathophysiology of WAS. Although molecular and cellular studies have extensively analyzed the mechanisms leading to defects in T, B, and dendritic cells, the basis of autoimmunity and thrombocytopenia still remains poorly understood. A full understanding of these mechanisms is still needed to further implement new therapeutic strategies for this peculiar immunodeficiency.
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Impaired gastric acidification negatively affects calcium homeostasis and bone mass.
Nat. Med.
PUBLISHED: 04-06-2009
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Activation of osteoclasts and their acidification-dependent resorption of bone is thought to maintain proper serum calcium levels. Here we show that osteoclast dysfunction alone does not generally affect calcium homeostasis. Indeed, mice deficient in Src, encoding a tyrosine kinase critical for osteoclast activity, show signs of osteopetrosis, but without hypocalcemia or defects in bone mineralization. Mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells, have the expected defects in gastric acidification but also secondary hyperparathyroidism and osteoporosis and modest hypocalcemia. These results suggest that alterations in calcium homeostasis can be driven by defects in gastric acidification, especially given that calcium gluconate supplementation fully rescues the phenotype of the Cckbr-mutant mice. Finally, mice deficient in Tcirg1, encoding a subunit of the vacuolar proton pump specifically expressed in both osteoclasts and parietal cells, show hypocalcemia and osteopetrorickets. Although neither Src- nor Cckbr-deficient mice have this latter phenotype, the combined deficiency of both genes results in osteopetrorickets. Thus, we find that osteopetrosis and osteopetrorickets are distinct phenotypes, depending on the site or sites of defective acidification.
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Ethidium bromide as a vital probe of mitochondrial DNA in carcinoma cells.
Eur. J. Cancer
PUBLISHED: 03-27-2009
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The interaction of ethidium bromide (EB) with mitochondria in human breast and lung carcinoma cells was investigated under living conditions, employing a laser scanning confocal fluorescence microscopy (LSCFM) with a photon counting detection to reduce drastically the laser power excitation and the fluorescent probe concentration. In sensitive and multidrug-resistant MCF-7 cell lines, which are important model systems for the study of mitochondria in tumour cells, two distinct populations of mitochondria were observed, each characterised by a different EB fluorescence, membrane potential, cellular localisation and morphology. By image analysis, these peripheral mitochondria showed a peculiar morphology, consisting of punctuate organelles (0.8mum in size) organised in rosette-like assemblies. Unexpectedly, an intense EB fluorescence was observed in these mitochondria, indicating a high accessibility to EB of their mtDNA, which is likely to be in an active replicative or transcriptional state. These results might, therefore, suggest an active biogenesis and metabolism of the peripheral mitochondria that could be a consequence of the increased energetic needs of the cells, after their tumour transformation. Indeed, the pool of peripheral mitochondria, as well as their peculiar morphology and spatial organisation, was found to be a characteristic feature of all the carcinoma cells examined here, but not of their non-transformed parental MCF10A cells.
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The Wiskott-Aldrich syndrome protein is required for iNKT cell maturation and function.
J. Exp. Med.
PUBLISHED: 03-23-2009
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The Wiskott-Aldrich syndrome (WAS) protein (WASp) is a regulator of actin cytoskeleton in hematopoietic cells. Mutations of the WASp gene cause WAS. Although WASp is involved in various immune cell functions, its role in invariant natural killer T (iNKT) cells has never been investigated. Defects of iNKT cells could indeed contribute to several WAS features, such as recurrent infections and high tumor incidence. We found a profound reduction of circulating iNKT cells in WAS patients, directly correlating with the severity of clinical phenotype. To better characterize iNKT cell defect in the absence of WASp, we analyzed was(-/-) mice. iNKT cell numbers were significantly reduced in the thymus and periphery of was(-/-) mice as compared with wild-type controls. Moreover analysis of was(-/-) iNKT cell maturation revealed a complete arrest at the CD44(+) NK1.1(-) intermediate stage. Notably, generation of BM chimeras demonstrated a was(-/-) iNKT cell-autonomous developmental defect. was(-/-) iNKT cells were also functionally impaired, as suggested by the reduced secretion of interleukin 4 and interferon gamma upon in vivo activation. Altogether, these results demonstrate the relevance of WASp in integrating signals critical for development and functional differentiation of iNKT cells and suggest that defects in these cells may play a role in WAS pathology.
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Evidence for long-term efficacy and safety of gene therapy for Wiskott-Aldrich syndrome in preclinical models.
Mol. Ther.
PUBLISHED: 03-03-2009
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Wiskott-Aldrich Syndrome (WAS) is a life-threatening X-linked disease characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancies. Gene therapy could represent a therapeutic option for patients lacking a suitable bone marrow (BM) donor. In this study, we analyzed the long-term outcome of WAS gene therapy mediated by a clinically compatible lentiviral vector (LV) in a large cohort of was(null) mice. We demonstrated stable and full donor engraftment and Wiskott-Aldrich Syndrome protein (WASP) expression in various hematopoietic lineages, up to 12 months after gene therapy. Importantly, we observed a selective advantage for T and B lymphocytes expressing transgenic WASP. T-cell receptor (TCR)-driven T-cell activation, as well as B-cells ability to migrate in response to CXCL13, was fully restored. Safety was evaluated throughout the long-term follow-up of primary and secondary recipients of WAS gene therapy. WAS gene therapy did not affect the lifespan of treated animals. Both hematopoietic and nonhematopoietic tumors arose, but we excluded the association with gene therapy in all cases. Demonstration of long-term efficacy and safety of WAS gene therapy mediated by a clinically applicable LV is a key step toward the implementation of a gene therapy clinical trial for WAS.
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Infantile malignant, autosomal recessive osteopetrosis: the rich and the poor.
Calcif. Tissue Int.
PUBLISHED: 02-20-2009
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Human recessive osteopetrosis (ARO) represents a group of diseases in which, due to a defect in osteoclasts, bone resorption is prevented. The deficit could arise either from failure in osteoclast differentiation or from inability to perform resorption by mature, multinucleated, but nonfunctional cells. Historically, osteopetrosis due to both these mechanisms was found in spontaneous and artificially created mouse mutants, but the first five genes identified in human ARO (CA-II, TCIRG1, ClCN7, OSTM1, and PLEKHM1) were all involved in the effector function of mature osteoclasts, being linked to acidification of the cell/bone interface or to intracellular processing of the resorbed material. Differentiation defects in human ARO have only recently been described, following the identification of mutations in both RANKL and RANK, which define a new form of osteoclast-poor ARO, as expected from biochemical, cellular, and animal studies. The molecular dissection of ARO has prognostic and therapeutic implications. RANKL-dependent patients, in particular, represent an interesting subset which could benefit from mesenchymal cell transplant and/or administration of soluble RANKL cytokine.
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Homozygous stop mutation in the SNX10 gene in a consanguineous Iraqi boy with osteopetrosis and corpus callosum hypoplasia.
Eur J Med Genet
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Recently a mutation in the SNX10 gene that belongs to the sorting nexin family was identified as a cause of a new subset of human autosomal recessive osteopetrosis. Here, we identified a novel homozygous mutation (c.46C > T, p.Arg16X) in SNX10, in an Iraqi boy from a consanguineous family with a history of infantile osteopetrosis. The proband exhibited macrocephaly, prominent forehead, proptosis of the eyes, strabismus, splenomegaly and joint hyperlaxity. Bone X-rays showed increased bone density, metaphyseal under-modelling, transverse alternating bands of greater and lesser density in tubular bones, anteriorly notched vertebral bodies and bone-in-bone appearance. Brain atrophy, external hydrocephalus and thin corpus callosum were noted at the brain MRI and CT scan. Blood test results revealed the presence of anaemia and leukopenia. Our findings confirm the role of SNX10 in autosomal recessive osteopetrosis and help to better define the core set of manifestations associated with this new pathological entity.
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Gene therapy for primary immunodeficiencies: Part 2.
Curr. Opin. Immunol.
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Gene therapy has become an attractive alternative therapeutic strategy to allogeneic transplant for primary immunodeficiencies (PIDs) owing to known genetic defects. Clinical trials using gammaretroviral vectors have demonstrated the proof of principle of gene therapy for Wiskott-Aldrich syndrome (WAS) and chronic granulomatous disease (CGD), but have also highlighted limitations of the technology. New strategies based on vectors that can achieve more robust correction with less risk of insertional mutagenesis are being developed. In this review we present the status of gene therapy for WAS and CGD, and discuss the emerging application of similar strategies to a broader range of PIDs, such as IPEX syndrome.
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The influence of plasma technology coupled to chemical grafting on the cell growth compliance of 3D hydroxyapatite scaffolds.
J Mater Sci Mater Med
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The development of advanced materials with biomimetic features in order to elicit desired biological responses and to guarantee tissue biocompatibility is recently gaining attention for tissue engineering applications. Bioceramics, such as hydroxyapatite-based biomaterials are now used in a number of different applications throughout the body, covering all areas of the skeleton, due to their biological and chemical similarity to the inorganic phases of bones. When bioactive sintered hydroxyapatite (HA) is desired, biomolecular modification of these materials is needed. In the present work, we investigated the influence of plasma surface modification coupled to chemical grafting on the cell growth compliance of HA 3D scaffolds.
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A homozygous contiguous gene deletion in chromosome 16p13.3 leads to autosomal recessive osteopetrosis in a Jordanian patient.
Calcif. Tissue Int.
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Human malignant autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. Mutations in the CLCN7 gene are responsible not only for a substantial portion of ARO patients but also for other forms of osteopetrosis characterized by different severity and inheritance. The lack of a clear genotype/phenotype correlation makes genetic counseling a tricky issue for CLCN7-dependent osteopetrosis. Here, we characterize the first homozygous interstitial deletion in 16p13.3, detected by array comparative genomic hybridization in an ARO patient of Jordanian origin. The deletion involved other genes besides CLCN7, while the proband displayed a classic ARO phenotype; however, her early death did not allow more extensive clinical investigations. The identification of this novel genomic deletion involving a large part of the CLCN7 gene is of clinical relevance, especially in prenatal diagnosis, and suggests the possibility that this kind of mutation has been underestimated so far. These data highlight the need for alternative approaches to genetic analysis also in other ARO-causative genes.
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Osteopetrosis rescue upon RANKL administration to Rankl(-/-) mice: a new therapy for human RANKL-dependent ARO.
J. Bone Miner. Res.
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In the last decades the molecular basis of monogenic diseases has been largely unraveled, although their treatment has often remained unsatisfactory. Autosomal recessive osteopetrosis (ARO) belongs to the small group of genetic diseases that are usually treated with hematopoietic stem cell transplantation (HSCT). However, this approach is not effective in the recently identified form carrying mutations in the receptor activator of NF-?B ligand (RANKL) gene. In this subset, therapy replacement approach based on RANKL delivery has a strong rationale. Here we demonstrate that the systematic administration of RANKL for 1 month to Rankl(-/-) mice, which closely resemble the human disease, significantly improves the bone phenotype and has beneficial effects on bone marrow, spleen and thymus; major adverse effects arise only when mice are clearly overtreated. Overall, we provide evidence that the pharmacological administration of RANKL represents the appropriate treatment option for RANKL-deficient ARO patients, to be validated in a pilot clinical trial.
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Autoimmunity in wiskott-Aldrich syndrome: an unsolved enigma.
Front Immunol
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Wiskott-Aldrich Syndrome (WAS) is a severe X-linked Primary Immunodeficiency that affects 1-10 out of 1 million male individuals. WAS is caused by mutations in the WAS Protein (WASP) expressing gene that leads to the absent or reduced expression of the protein. WASP is a cytoplasmic protein that regulates the formation of actin filaments in hematopoietic cells. WASP deficiency causes many immune cell defects both in humans and in the WAS murine model, the Was(-/-) mouse. Both cellular and humoral immune defects in WAS patients contribute to the onset of severe clinical manifestations, in particular microthrombocytopenia, eczema, recurrent infections, and a high susceptibility to develop autoimmunity and malignancies. Autoimmune diseases affect from 22 to 72% of WAS patients and the most common manifestation is autoimmune hemolytic anemia, followed by vasculitis, arthritis, neutropenia, inflammatory bowel disease, and IgA nephropathy. Many groups have widely explored immune cell functionality in WAS partially explaining how cellular defects may lead to pathology. However, the mechanisms underlying the occurrence of autoimmune manifestations have not been clearly described yet. In the present review, we report the most recent progresses in the study of immune cell function in WAS that have started to unveil the mechanisms contributing to autoimmune complications in WAS patients.
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Anti-CD3? mAb improves thymic architecture and prevents autoimmune manifestations in a mouse model of Omenn syndrome: therapeutic implications.
Blood
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Omenn syndrome (OS) is an atypical primary immunodeficiency characterized by severe autoimmunity because of activated T cells infiltrating target organs. The impaired recombinase activity in OS severely affects expression of the pre-T-cell receptor complex in immature thymocytes, which is crucial for an efficient development of the thymic epithelial component. Anti-CD3? monoclonal antibody (mAb) treatment in RAG2(-/-) mice was previously shown to mimic pre-TCR signaling promoting thymic expansion. Here we show the effect of anti-CD3? mAb administration in the RAG2(R229Q) mouse model, which closely recapitulates human OS. These animals, in spite of the inability to induce the autoimmune regulator, displayed a significant amelioration in thymic epithelial compartment and an important reduction of peripheral T-cell activation and tissue infiltration. Furthermore, by injecting a high number of RAG2(R229Q) progenitors into RAG2(-/-) animals previously conditioned with anti-CD3? mAb, we detected autoimmune regulator expression together with the absence of peripheral immunopathology. These observations indicate that improving epithelial thymic function might ameliorate the detrimental behavior of the cell-autonomous RAG defect. Our data provide important therapeutic proof of concept for future clinical applications of anti-CD3? mAb treatment in severe combined immunodeficiency forms characterized by poor thymus function and autoimmunity.
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Correction of murine Rag2 severe combined immunodeficiency by lentiviral gene therapy using a codon-optimized RAG2 therapeutic transgene.
Mol. Ther.
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Recombination activating gene 2 (RAG2) deficiency results in severe combined immunodeficiency (SCID) with complete lack of T and B lymphocytes. Initial gammaretroviral gene therapy trials for other types of SCID proved effective, but also revealed the necessity of safe vector design. We report the development of lentiviral vectors with the spleen focus forming virus (SF) promoter driving codon-optimized human RAG2 (RAG2co), which improved phenotype amelioration compared to native RAG2 in Rag2(-/-) mice. With the RAG2co therapeutic transgene, T-cell receptor (TCR) and immunoglobulin repertoire, T-cell mitogen responses, plasma immunoglobulin levels and T-cell dependent and independent specific antibody responses were restored. However, the thymus double positive T-cell population remained subnormal, possibly due to the SF virus derived element being sensitive to methylation/silencing in the thymus, which was prevented by replacing the SF promoter by the previously reported silencing resistant element (ubiquitous chromatin opening element (UCOE)), and also improved B-cell reconstitution to eventually near normal levels. Weak cellular promoters were effective in T-cell reconstitution, but deficient in B-cell reconstitution. We conclude that immune functions are corrected in Rag2(-/-) mice by genetic modification of stem cells using the UCOE driven codon-optimized RAG2, providing a valid optional vector for clinical implementation.
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Ethidium bromide as a marker of mtDNA replication in living cells.
J Biomed Opt
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Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells.
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Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNA-PKcs.
J. Exp. Med.
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Artemis is an endonuclease that opens coding hairpin ends during V(D)J recombination and has critical roles in postirradiation cell survival. A direct role for the C-terminal region of Artemis in V(D)J recombination has not been defined, despite the presence of immunodeficiency and lymphoma development in patients with deletions in this region. Here, we report that the Artemis C-terminal region directly interacts with the DNA-binding domain of Ligase IV, a DNA Ligase which plays essential roles in DNA repair and V(D)J recombination. The Artemis-Ligase IV interaction is specific and occurs independently of the presence of DNA and DNA-protein kinase catalytic subunit (DNA-PKcs), another protein known to interact with the Artemis C-terminal region. Point mutations in Artemis that disrupt its interaction with Ligase IV or DNA-PKcs reduce V(D)J recombination, and Artemis mutations that affect interactions with Ligase IV and DNA-PKcs show additive detrimental effects on coding joint formation. Signal joint formation remains unaffected. Our data reveal that the C-terminal region of Artemis influences V(D)J recombination through its interaction with both Ligase IV and DNA-PKcs.
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Impaired osteoblastogenesis in a murine model of dominant osteogenesis imperfecta: a new target for osteogenesis imperfecta pharmacological therapy.
Stem Cells
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The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well-characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony-forming unit-adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony-forming unit-osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder.
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Preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vector for the treatment of Wiskott-Aldrich syndrome.
Mol. Ther.
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Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34(+) cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34(+) cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34(+) cells gene therapy for the treatment of WAS.
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Somatization in a primary care service for immigrants.
Ethn Health
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To study somatization in a large sample of immigrants attending a first visit to a primary care service. Differences in somatization among four large immigrant groups (Europeans, Asians, South Americans, and Africans) and 16 subgroups based on nationality were assessed.
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RANK-dependent autosomal recessive osteopetrosis: characterization of five new cases with novel mutations.
J. Bone Miner. Res.
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Autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder attributed to reduced bone resorption by osteoclasts. Most human AROs are classified as osteoclast rich, but recently two subsets of osteoclast-poor ARO have been recognized as caused by defects in either TNFSF11 or TNFRSF11A genes, coding the RANKL and RANK proteins, respectively. The RANKL/RANK axis drives osteoclast differentiation and also plays a role in the immune system. In fact, we have recently reported that mutations in the TNFRSF11A gene lead to osteoclast-poor osteopetrosis associated with hypogammaglobulinemia. Here we present the characterization of five additional unpublished patients from four unrelated families in which we found five novel mutations in the TNFRSF11A gene, including two missense and two nonsense mutations and a single-nucleotide insertion. Immunological investigation in three of them showed that the previously described defect in the B cell compartment was present only in some patients and that its severity seemed to increase with age and the progression of the disease. HSCT performed in all five patients almost completely cured the disease even when carried out in late infancy. Hypercalcemia was the most important posttransplant complication. Overall, our results further underline the heterogeneity of human ARO also deriving from the interplay between bone and the immune system, and highlight the prognostic and therapeutic implications of the molecular diagnosis.
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Wiskott-Aldrich syndrome protein deficiency in natural killer and dendritic cells affects antitumor immunity.
Eur. J. Immunol.
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Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency caused by reduced or absent expression of the WAS protein (WASP). WAS patients are affected by microthrombocytopenia, recurrent infections, eczema, autoimmune diseases and malignancies. Although immune deficiency has been proposed to play a role in tumor pathogenesis, there is little evidence on the correlation between immune cell defects and tumor susceptibility. Taking advantage of a tumor-prone model, we show that the lack of WASP induces early tumor onset because of defective immune surveillance. Consistently, the B16 melanoma model shows that tumor growth and the number of lung metastases are increased in the absence of WASP. We then investigated the in vivo contribution of Was(-/-) NK cells and DCs in controlling B16 melanoma development. We found fewer B16 metastases developed in the lungs of Was(-/-) mice that had received wild-type NK cells as compared with mice bearing Was(-/-) NK cells. Furthermore, we demonstrated that Was(-/-) DCs were less efficient in inducing NK-cell activation in vitro and in vivo. In summary, for the first time we demonstrate in in vivo models that WASP deficiency affects resistance to tumor and causes impairment in the antitumor capacity of NK cells and DCs. This article is protected by copyright. All rights reserved.
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