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Kinetics of IDO1 expression during LPS-induced DC maturation in vitro.

IDO1 gene expression in common human malignancies.

Supplemental References

Kinetics of LPS-induced DC maturation.

Characterization and specificity of anti-IDO1 antibodies by Western blot and immunohistochemistry.

Proliferation of T cells stimulated with allogeneic dendritic cells expressing IDO1.

Immunohistochemistry of a normal lung FFPE section with the anti-IDO1 antibody 4.16H1.

Clinical characteristics of the patients analyzed for IDO1 expression in tumor-draining lymph nodes.

Published series of IDO1 expression assessed by immunohistochemistry in specific tumor types.

In vitro characterization of IDO1-expressing DCs.

Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tumoral resistance to immune rejection. In humans, constitutive expression of IDO1 has been observed in several tumor types. However, a comprehensive analysis of its expression in normal and tumor tissues is still required to anticipate the risks and potential benefits of IDO1 inhibitors. Using a newly validated monoclonal antibody to human IDO1, we performed an extensive immunohistochemical analysis of IDO1 expression in normal and tumor tissues. In normal tissues, IDO1 was expressed by endothelial cells in the placenta and lung and by epithelial cells in the female genital tract. In lymphoid tissues, IDO1 was expressed in mature dendritic cells with a phenotype (CD83+, DC-LAMP+, langerin−, CD123−, CD163−) distinct from plasmacytoid dendritic cells. Importantly, IDO1-expressing dendritic cells were not enriched in tumor-draining lymph nodes, in contrast with previously reported findings. IDO1-expressing cells were observed in a large fraction (382/624, 61%) of human tumors. They comprised tumor cells, endothelial cells, and stromal cells in proportions that varied depending on the tumor type. Tumors showing the highest proportions of IDO1-immunolabeled samples were carcinomas of the cervix, followed by endometrium, bladder, kidney, and lung. This hierarchy of IDO1 expression was confirmed by gene expression data mined from The Cancer Genome Atlas database. Expression of IDO1 may be used to select tumors likely to benefit from targeted therapy with IDO1 inhibitors.

Objective. Endometrial carcinoma (EC) is the sixth most common cancer in women and therapies are limited for advanced and recurrent disease. Patient-derived tumor xenograft (PDTX) models are becoming popular tools in translational research because of their histological and genetic similarity to the original tumors and the ability to predict therapeutic response to treatments. Here, we established and characterized a panel of 24 EC PDTX models which includes the major histological and genetic subtypes observed in patients. Methods. Fresh tumor tissues collected from primary, metastatic and recurrent type I and type II EC patients were engrafted in immunocompromised mice. Histology, vimentin, and cytokeratin expression were evaluated, together with Microsatellite instability (MSI), mutation profiling by Whole Exome Sequencing and copy number profiling by Whole Genome Low Coverage Sequencing. The efficacy of both PI3K and MEK inhibitors was evaluated in a model of endometrioid carcinoma harboring PTEN, PIK3CA and KRAS mutations. Results. We observed good similarity between primary tumors and the corresponding xenografts, at histological and genetic level. Among the engrafted endometrioid models, we found a significant enrichment of MSI and POLE mutated tumors, compared to non-engrafted samples. Combination treatment with NVP-EZ235 and AZD6244 showed the possibility to stabilize the tumor growth in one model originated from a patient who already received several lines of chemotherapy. Conclusion. The established EC PDTX models, resembling the original human tumors, promise to be useful for preclinical evaluation of novel combination and targeted therapies in specific EC subgroups. (C) 2015 Elsevier Inc. All rights reserved

Non-autonomous thyroid nodules are common in the general population with a proportion found to be cancerous. A current challenge in the field is to be able to distinguish benign adenoma (FA) from preoperatively malignant thyroid follicular carcinoma (FTC), which are very similar both histologically and genetically. One controversial issue, which is currently not understood, is whether both tumor types represent different molecular entities or rather a biological continuum. To gain a better insight into FA and FTC tumorigenesis, we defined their molecular profiles by mRNA and miRNA microarray. Expression data were analyzed, validated by qRT-PCR and compared with previously published data sets. The majority of deregulated mRNAs were common between FA and FTC and were downregulated, however FTC showed additional deregulated mRNA. Both types of tumors share deregulated pathways, molecular functions and biological processes. The additional deregulations in FTC include the lipid transport process that may be involved in tumor progression. The strongest candidate genes which may be able to discriminate follicular adenomas and carcinomas, CRABP1, FABP4 and HMGA2, were validated in independent samples by qRT-PCR and immunohistochemistry. However, they were not able to adequately classify FA or FTC, supporting the notion of continuous evolving tumors, whereby FA and FTC appear to show quantitative rather than qualitative changes. Conversely, miRNA expression profiles showed few dysregulations in FTC, and even fewer in FA, suggesting that miRNA play a minor, if any, role in tumor progression., SCOPUS: ar.j, info:eu-repo/semantics/published

Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tumoral resistance to immune rejection. In humans, constitutive expression of IDO1 has been observed in several tumor types. However, a comprehensive analysis of its expression in normal and tumor tissues is still required to anticipate the risks and potential benefits of IDO1 inhibitors. Using a newly validated monoclonal antibody to human IDO1, we performed an extensive immunohistochemical analysis of IDO1 expression in normal and tumor tissues. In normal tissues, IDO1 was expressed by endothelial cells in the placenta and lung and by epithelial cells in the female genital tract. In lymphoid tissues, IDO1 was expressed in mature dendritic cells with a phenotype (CD83+, DC-LAMP+, langerin−, CD123−, CD163−) distinct from plasmacytoid dendritic cells. Importantly, IDO1-expressing dendritic cells were not enriched in tumor-draining lymph nodes, in contrast with previously reported findings. IDO1-expressing cells were observed in a large fraction (382/624, 61%) of human tumors. They comprised tumor cells, endothelial cells, and stromal cells in proportions that varied depending on the tumor type. Tumors showing the highest proportions of IDO1-immunolabeled samples were carcinomas of the cervix, followed by endometrium, bladder, kidney, and lung. This hierarchy of IDO1 expression was confirmed by gene expression data mined from The Cancer Genome Atlas database. Expression of IDO1 may be used to select tumors likely to benefit from targeted therapy with IDO1 inhibitors.

Rituximab displays therapeutic benefits in the treatment of patients with rheumatoid arthritis (RA) resistant to tumor necrosis factor (TNF) blockade. However, the precise role of B cells in the pathogenesis of RA is still unknown. We undertook this study to investigate the global molecular effects of rituximab in synovial biopsy samples obtained from anti-TNF-resistant RA patients before and after administration of the drug.Paired synovial biopsy samples were obtained from the affected knee of anti-TNF-resistant RA patients before (time 0) and 12 weeks after (time 12) initiation of rituximab therapy. Total RNA was extracted, labeled according to standard Affymetrix procedures, and hybridized on GeneChip HGU133 Plus 2.0 slides. Immunohistochemistry and quantitative real-time reverse transcriptase-polymerase chain reaction experiments were performed to confirm the differential expression of selected transcripts.According to Student's paired t-tests, 549 of 54,675 investigated probe sets were differentially expressed between time 0 and time 12. Pathway analysis revealed that genes down-regulated between time 0 and time 12 were significantly enriched in immunoglobulin genes and genes involved in chemotaxis, leukocyte activation, and immune responses (Gene Ontology annotations). In contrast, genes up-regulated between time 0 and time 12 were significantly enriched in transcripts involved in cell development (Gene Ontology annotation) and wound healing (Gene Set Enrichment Analysis). At baseline, higher synovial expression of immunoglobulin genes was associated with response to therapy.Rituximab displays unique effects on global gene expression profiles in the synovial tissue of RA patients. These observations open new perspectives in the understanding of the biologic effects of the drug and in the selection of patients likely to benefit from this therapy.

Contains fulltext : 98411.pdf (Publisher’s version ) (Closed access) OBJECTIVE: Rituximab has been shown to be successful in the treatment of rheumatoid arthritis (RA), and this unexpected finding indicates that B cells have an important role in this disease. The present study was undertaken to investigate the mechanism of action of rituximab in RA. METHODS: Twelve patients with active RA were treated with rituximab. Disease activity was evaluated using the 28-joint Disease Activity Score. Synovial biopsy samples obtained at baseline and 12 weeks after treatment initiation were analyzed by microarray, quantitative polymerase chain reaction, and immunohistochemistry. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and from 4 patients with X-linked agammaglobulinemia were stimulated with the Th17-inducing stimulus Candida albicans, and the response in the presence and absence of rituximab was examined. RESULTS: In RA patients, rituximab reduced expression of retinoic acid-related orphan receptor gammat and interleukin-22 (IL-22) and numbers of Th17-positive cells in synovial tissue, and this correlated with better clinical outcome. Rituximab did not affect tumor necrosis factor alpha (TNFalpha), Th1 cell, or Treg cell responses. Rituximab strongly reduced in vitro IL-17 and IL-22 production induced by C albicans. This effect was not observed in PBMCs from patients with X-linked agammaglobulinemia. CONCLUSION: Rituximab reduced the local Th17 response in RA patients, whereas it did not influence Th1 cell, Treg cell, or TNFalpha responses. The decreased Th17 response was associated with reduced inflammation and better clinical outcome. Moreover, inhibition of the Th17 response by rituximab was lost in the absence of B cells, providing evidence that the effects of rituximab are due to B cell depletion. These data demonstrate an unexpected role of B cells in the development of Th17 responses, which could possibly lead to B cell-based strategies for the treatment of Th17-related autoimmune diseases.

OBJECTIVE: Mutations in genes encoding succinate dehydrogenase and its anchoring subunits (SDH genes) are at the origin of hereditary head and neck paraganglioma (PGL) and a subset of apparently sporadic pheochromocytoma. METHODS: We describe a family including three patients harbouring bilateral head and neck PGL diagnosed before 25 years of age. Multiple hypervascular hepatic lesions were subsequently discovered in two of them. In both, liver biopsy confirmed the diagnosis of PGL. In addition, in one patient, MRI disclosed multiple target-like lesions of the spine, highly suggestive of metastatic PGL. Family history was compatible with autosomal dominant inheritance with possible maternal imprinting. RESULTS: Combined single-strand conformation polymorphism and heteroduplex analysis followed by sequencing did not show any mutation of the coding parts of SDHB, SDHC, SDHD, RET or VHL genes. Screening of copy number alterations and loss of heterozygosity in the three affected family members showed no deletion or amplification of the SDH, RET and VHL genes. Furthermore, succinate dehydrogenase activity measured in a liver PGL sample was not significantly decreased in the affected patient as compared with controls, underscoring the exclusion of the SDH genes. CONCLUSIONS: To our knowledge, this is the first reported family of hereditary head and neck PGL with metastatic dissemination in the liver and the spine. A large body of evidence supports the absence of mutations in SDH, RET and VHL genes, which suggests the existence of a yet unknown gene at the origin of this particular form of familial PGL.

Asymmetric positioning of internal organs is a characteristics of vertebrates. The normal left-right anatomic positioning, situs solitus, sometimes does not occur normaly, leading to laterality defects. Studies in animal models have shown that laterality decisions are mediated by a cascade of genes that lead to the asymmetric expression of Nodal, LEFTA, LEFTB and PITX2 in the lateral plate mesoderm. A search for mutations in genes implicated in left-right patterning in animal models allowed genes associated with heterotaxia defects in humans to be identified. However, these genes explain only a small percentage of human situs defects, suggesting that other genes must play a role. In this study, we report a consanguineous family of Turkish origin, composed of two unaffected parents and three children, two of whom presented Kartagener syndrome. On the basis of their family history, we hypothesize autosomal recessive mode of inheritance. A genotype analysis with polymorphic markers did not show linkage with any known genes or loci causing laterality disorders. Array CGH did not detect a duplication or microdeletion greater than 1 Mb as a possible cause. Genome wide screening using 10 K Affymetrix SNP chips was performed, allowing the identification of two regions of autozygosity, one in chromosome 1 and the other on chromosome 7. In the chromosome 1 locus, a strong candidate gene, encoding the kinesin-associated protein 3 (KIF3AP) was not mutated, based on SSCP/heteroduplex analysis and direct sequencing. These data provide a basis for the identification of a novel gene implicated in Kartagener syndrome.

Mutations in the glomulin gene result in dominantly inherited vascular lesions of the skin known as glomuvenous malformations (GVMs). These lesions are histologically distinguished by their distended vein-like channels containing characteristic 'glomus cells', which appear to be incompletely or improperly differentiated vascular smooth muscle cells (VSMCs). The function of glomulin is currently unknown. We studied glomulin expression during murine development (E9.5 days post-coitum until adulthood) by non-radioactive in situ hybridization. Glomulin was first detected at E10.5 dpc in cardiac outflow tracts. Later, it showed strong expression in VSMCs as well as a limited expression in the perichondrium. At E11.5-14.5 dpc glomulin RNA was most abundant in the walls of the large vessels. At E16.5 dpc expression was also detectable in smaller arteries and veins. The high expression of glomulin in murine vasculature suggests an important role for glomulin in blood vessel development and/or maintenance, which is supported by the vascular phenotype seen in GVM patients with mutations in this gene.

Susceptibility to autoimmune disorders results from the interaction of multiple genetic factors that regulate the threshold of autoreactivity. Genome-wide microsatellite screens and large-scale single nucleotide polymorphism (SNP) association studies have identified chromosomal loci that are associated with specific disorders including systemic lupus erythematosus, rheumatoid arthritis, juvenile arthritis, multiple sclerosis, and diabetes. Numerous candidate gene association studies have in turn investigated the association of specific genes within these chromosomal regions, with susceptibility to autoimmune diseases (e.g. FcgammaReceptors, TYK2 and systemic lupus). More recently, large-scale differential gene expression studies performed on selected tissues from patients with autoimmune disorders, have led to the identification of gene signatures associated with the activation of specific pathways in these diseases (e.g. interferon signature in lupus). In the future, integrated analyses of gene (and protein) expression together with SNP data will allow us to sketch an intelligible picture of the genesis of autoimmunity in humans. This review sets out to illustrate how the most recent advances in the field of systemic lupus erythematosus, rheumatoid arthritis and juvenile arthritis have led to a better understanding of these disorders.

Contains fulltext : 49619.pdf (Publisher’s version ) (Closed access) The prevalence of congenital heart defects is approximately 1% of all live births. Identifying the genes responsible for cardiac malformation is the first step to understand pathogenesis. Heterozygous mutations in the CSX/NKX2-5 (NKX2E) gene have been identified to cause atrial septal defect (ASD) and/or atrioventricular (AV) conduction disturbance in some families. However, there is great variability in expressivity of the phenotype between the patients with a CSX/NKX2-5 mutation.We screened four sporadic patients and three index cases of families with ASD and/or conduction defects. In one of them, a CSX/NKX2-5 mutation was identified. This novel mutation (p.Tyr256X) was inherited in a three-generation family causing five individuals to have cardiac anomalies ranging from ASD to arrhythmias. Interestingly, all the observed AV conduction disturbances were at the nodal level, manifesting first as an AV block of the first degree and evolving toward a second-degree block. Atrial fibrillation, previously reported in three individuals with CSX/NKX2-5 mutations, was observed in three patients.

Non-syndromic cardiac septation defects are common, yet the causative factors remain largely uncharacterised. Septation defects are an integral part of many syndromes, some of which are associated with chromosomal alterations. For the majority, the physiopathogenesis is believed to be multi-factorial, hindering the identification of causative factors. Ten mutations in the gene encoding the transcription factor CSX/NKX2-5 have been described in individuals with ASD and/or atrioventricular conduction defects. In addition, several other cardiac abnormalities were observed, yet the mildest forms are reminiscent of non-syndromic septation defects. The CSX/NKX2-5 gene is thus a good candidate for various cardiopathies. We have collected two families with inherited predisposition to cardiac abnormalities. Some members of the families presented ASD and AV block. In both families a novel CSX/NKX2-5 mutation was identified in the homeodomain. Variable expressivity in the phenotype was observed in both families. Importantly, mutation carriers did not present any symptoms at young age. In addition, anomalous venous return, a phenotype not previously associated to CSX/NKX2-5 mutations, was observed in one of the families. We also screened the CSX/NKX2-5 gene in sporadic and familial cases of other cardiopathies. As additional mutations were not found, substitutions in CSX/NKX2-5 gene seem to be a rare cause of cardiopathies without conduction defect.