Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification.

BACKGROUND: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis. METHODS: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol. RESULTS: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P=0.04). A significant correlation (P=0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS=46% vs 75%, P=0.023; OS=66.8% vs 100%, P=0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P=0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P=0.018). CONCLUSIONS: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

Global transcriptional programs in peripheral nerve endoneurium and DRG are resistant to the onset of type 1 diabetic neuropathy in Ins2 mice.

While the morphological and electrophysiological changes underlying diabetic peripheral neuropathy (DPN) are relatively well described, the involved molecular mechanisms remain poorly understood. In this study, we investigated whether phenotypic changes associated with early DPN are correlated with transcriptional alterations in the neuronal (dorsal root ganglia [DRG]) or the glial (endoneurium) compartments of the peripheral nerve. We used Ins2(Akita/+) mice to study transcriptional changes underlying the onset of DPN in type 1 diabetes mellitus (DM). Weight, blood glucose and motor nerve conduction velocity (MNCV) were measured in Ins2(Akita/+) and control mice during the first three months of life in order to determine the onset of DPN. Based on this phenotypic characterization, we performed gene expression profiling using sciatic nerve endoneurium and DRG isolated from pre-symptomatic and early symptomatic Ins2(Akita/+) mice and sex-matched littermate controls. Our phenotypic analysis of Ins2(Akita/+) mice revealed that DPN, as measured by reduced MNCV, is detectable in affected animals already one week after the onset of hyperglycemia. Surprisingly, the onset of DPN was not associated with any major persistent changes in gene expression profiles in either sciatic nerve endoneurium or DRG. Our data thus demonstrated that the transcriptional programs in both endoneurial and neuronal compartments of the peripheral nerve are relatively resistant to the onset of hyperglycemia and hypoinsulinemia suggesting that either minor transcriptional alterations or changes on the proteomic level are responsible for the functional deficits associated with the onset of DPN in type 1 DM.

The International LAM Registry: a component of an innovative web-based clinician, researcher, and patient-driven rare disease research platform.

BACKGROUND: A relative inability to capture a sufficiently large patient population in any one geographic location has traditionally limited research into rare diseases. METHODS AND RESULTS: Clinicians interested in the rare disease lymphangioleiomyomatosis (LAM) have worked with the LAM Treatment Alliance, the MIT Media Lab, and Clozure Associates to cooperate in the design of a state-of-the-art data coordination platform that can be used for clinical trials and other research focused on the global LAM patient population. This platform is a component of a set of web-based resources, including a patient self-report data portal, aimed at accelerating research in rare diseases in a rigorous fashion. CONCLUSIONS: Collaboration between clinicians, researchers, advocacy groups, and patients can create essential community resource infrastructure to accelerate rare disease research. The International LAM Registry is an example of such an effort. 82.

Plaque characteristics and arterial remodeling in coronary and peripheral arterial systems.

BACKGROUND: Few studies have examined plaque characteristics among multiple arterial beds in vivo. The purpose of this study was to compare the plaque morphology and arterial remodeling between coronary and peripheral arteries using gray-scale and radiofrequency intravascular ultrasound (IVUS) at clinical presentation. METHODS AND RESULTS: IVUS imaging was performed in 68 patients with coronary and 93 with peripheral artery lesions (29 carotid, 50 renal, and 14 iliac arteries). Plaques were classified as fibroatheroma (VH-FA) (further subclassified as thin-capped [VH-TCFA] and thick-capped [VH-ThCFA]), fibrocalcific plaque (VH-FC) and pathological intimal thickening (VH-PIT). Plaque rupture (13% of coronary, 7% of carotid, 6% of renal, and 7% of iliac arteries; P = NS) and VH-TCFA (37% of coronary, 24% of carotid, 16% of renal, and 7% of iliac arteries; P = 0.02) were observed in all arteries. Compared with coronary arteries, VH-FA was less frequently observed in renal (P < 0.001) and iliac arteries (P < 0.006). Lesions with positive remodeling demonstrated more characteristics of VH-FA in coronary (84% vs. 25%, P < 0.001), carotid (72% vs. 20%, P = 0.001), and renal arteries (42% vs. 4%, P = 0.001) compared with those with intermediate/negative remodeling. There was positive relationship between remodeling index and percent necrotic area in all four arteries. CONCLUSIONS: Atherosclerotic plaque phenotypes were heterogeneous among four different arteries; renal and iliac arteries had more stable phenotypes compared with coronary artery. In contrast, the associations of remodeling pattern with plaque phenotype and composition were similar among the various arterial beds.

The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa.

A novel two-component system, CbrA-CbrB, was discovered in Pseudomonas aeruginosa; cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino-terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy-terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot-argR operon was not induced by arginine, indicating an essential role for this two-component system in the expression of the ArgR-dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild-type strain. When succinate or other C4-dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate-dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA-CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB-NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.

Inflammation And Autoimmune Responses Are Independent Of Peripheral Mhc Class Ii Expression Driven By Ciita Piv In Collagen Induced Arthritis

Background In rheumatoid arthritis (RA), non-professional antigen presenting cells (APCs) such as fi broblast-like synoviocytes (FLS) can express MHC class II (MHCII) molecules and function as non-professional APCs in vitro.Objective To examine the regulation of MHCII expression in FLS and to investigate the role of FLS as non-professional APCs in collagen-induced arthritis (CIA). Methods Expression of MHCII, CIITA and Ciita isoforms pI, pIII and pIV was examined by RT-qPCR, immunohistochemistry and fl ow cytometry in human synovial tissues, arthritic mouse joints and human as well as mouse FLS. CIA was induced in mice knockout for the isoform IV of Ciita (pIV-/-), in pIV-/- mice transgenic for CIITA in the thymus (pIV-/- K14 CIITA) and in control littermates in the DBA/1 background by immunising with bovine collagen type II (CII) in complete Freund's adjuvant.Results HLA-DRA, total CIITA and CIITA pIII mRNA levels were signifi cantly increased in the synovial tissues from RA compared to osteoarthritis patients. Human FLS expressed surface MHCII via CIITA pIII and pIV, while MHCII expression in murine FLS was entirely mediated by pIV. pIV-/- mice lacked both inducible MHCII expression on non-professional APCs including FLS, and in the thymic cortex. The thymic defect in pIV-/- mice impaired CD4+ positive selection, thus protecting pIV-/- mice from CIA by preventing CD4+ T cells immune responses against CII and blocking the release of IFN-γ and IL-17 in ex vivo stimulated lymph node cells. The production of T dependent, arthritogenic anti-CII antibodies was also impaired in pIV-/- mice. A normal thymic expression of MHCII and CD4+ T cell repertoire was obtained in pIV-/- K14 CIITA Tg mice. Immune responses against CII were restored in pIV-/- K14 CIITA Tg mice, as well as the arthritis incidence and clinical severity despite the lack of MHCII expression by mouse FLS. At histology, infl ammation andneutrophils infi ltration scores were not reduced in pIV-/- K14 CIITA Tg mice, while the bone erosion score was signifi cantly lower than in controls.Conclusion Over expression of MHCII is tightly correlated with CIITA pIII in the arthritic human synovium. MHCII is induced via CIITA pIII and pIV in human FLS. In the mouse, MHCII expression in the thymic cortex and in FLS is strictly dependent upon Ciita pIV. The lack of Ciita pIV in the periphery of pIV-/- K14 CIITA Tg mice lowered the bone erosion score but did not signifi cantly protect from infl ammation and autoimmune responses in CIA.

HCFC1 is a common component of active human CpG-island promoters and coincides with ZNF143, THAP11, YY1, and GABP transcription factor occupancy.

In human transcriptional regulation, DNA-sequence-specific factors can associate with intermediaries that orchestrate interactions with a diverse set of chromatin-modifying enzymes. One such intermediary is HCFC1 (also known as HCF-1). HCFC1, first identified in herpes simplex virus transcription, has a poorly defined role in cellular transcriptional regulation. We show here that, in HeLa cells, HCFC1 is observed bound to 5400 generally active CpG-island promoters. Examination of the DNA sequences underlying the HCFC1-binding sites revealed three sequence motifs associated with the binding of (1) ZNF143 and THAP11 (also known as Ronin), (2) GABP, and (3) YY1 sequence-specific transcription factors. Subsequent analysis revealed colocalization of HCFC1 with these four transcription factors at ∼90% of the 5400 HCFC1-bound promoters. These studies suggest that a relatively small number of transcription factors play a major role in HeLa-cell transcriptional regulation in association with HCFC1.

Divergent effect of cobalt and beryllium salts on the fate of peripheral blood monocytes and T lymphocytes.

Occupational exposure to metals such as cobalt and beryllium represents a risk factor for respiratory health and can cause immune-mediated diseases. However, the way they act may be different. We show here that the two metals have a divergent effect on peripheral T lymphocytes and monocytes: BeSO(4) induces cell death in monocytes but not in T lymphocytes, which instead respond by producing Interferon gamma (IFN-γ); conversely, CoCl(2) induces apoptosis in T lymphocytes but not in monocytes. Interestingly, both metals induce p53 overexpression but with a dramatic different outcome. This is because the effect of p53 in CoCl(2)-treated monocytes is counteracted by the antiapoptotic activity of cytoplasmic p21(Cip1/WAF1), the activation of nuclear factor κB, and the inflammasome danger signaling pathway leading to the production of proinflammatory cytokines. However, CoCl(2)-treated monocytes do not fully differentiate into macrophage or dendritic cells, as inferred by the lack of expression of CD16 and CD83, respectively. Furthermore, the expression of HLA-class II molecules, as well as the capability of capturing and presenting the antigens, decreased with time. In conclusion, cobalt keeps monocytes in a partially activated, proinflammatory state that can contribute to some of the pathologies associated with the exposure to this metal.

Regulatory roles of the GacS/GacA two-component system in plant-associated and other gram-negative bacteria.

The sensor kinase GacS and the response regulator GacA are members of a two-component system that is present in a wide variety of gram-negative bacteria and has been studied mainly in enteric bacteria and fluorescent pseudomonads. The GacS/GacA system controls the production of secondary metabolites and extracellular enzymes involved in pathogenicity to plants and animals, biocontrol of soilborne plant diseases, ecological fitness, or tolerance to stress. A current model proposes that GacS senses a still-unknown signal and activates, via a phosphorelay mechanism, the GacA transcription regulator, which in turn triggers the expression of target genes. The GacS protein belongs to the unorthodox sensor kinases, characterized by an autophosphorylation, a receiver, and an output domain. The periplasmic loop domain of GacS is poorly conserved in diverse bacteria. Thus, a common signal interacting with this domain would be unexpected. Based on a comparison with the transcriptional regulator NarL, a secondary structure can be predicted for the GacA sensor kinases. Certain genes whose expression is regulated by the GacS/GacA system are regulated in parallel by the small RNA binding protein RsmA (CsrA) at a posttranscriptional level. It is suggested that the GacS/GacA system operates a switch between primary and secondary metabolism, with a major involvement of posttranscriptional control mechanisms.

Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.

Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.

Consequences of modified lipid and glucose metabolism on peripheral nervous system structure and function

284 million people worldwide suffered from type 2 diabetes mellitus (T2DM) in 2010, which will, in approximately half of them, lead to the development of diabetic peripheral neuropathy (DPN). Although DPN is the most common complication of diabetes mellitus and the leading cause of non-traumatic amputations its pathophysiology is still poorly understood. To get more insight into the molecular mechanism underlying DPN in T2DM, I used a rodent model of T2DM, the db/db mice.¦ln vivo electrophysiological recordings of diabetic animals indicated that in addition to reduced nerve conduction velocity db/db mice also present increased nerve excitability. Further ex vivo evaluation of the electrophysiological properties of db/db nerves clearly established a presence of the peripheral nerve hyperexcitability (PNH) phenotype in diabetic animals. Using pharmacological inhibitors we demonstrated that PNH is mostly mediated by the decreased activity of Kv1 channels. ln agreement with these data 1 observed that the diabetic condition led to a reduced presence of the Kv1.2 subunits in juxtaparanodal regions of db/db peripheral nerves whereas its mANA and protein expression levels were not affected. Lmportantly, I confirmed a loss of juxtaparanodal Kv1.2 subunits in nerve biopsies from type 2 diabetic patients. Together these observations indicate that the type 2 diabetic condition leads to potassium-channel mediated changes of nerve excitability thus identifying them as potential drug targets to treat sorne of the DPN related symptoms.¦Schwann cells ensheath and isolate peripheral axons by the production of myelin, which consists of lipids and proteins in a ratio of 2:1. Peripheral myelin protein 2 (= P2, Pmp2 or FABP8) was originally described as one of the most abundant myelin proteins in the peripheral nervous system. P2, which is a member of the fatty acid binding protein (FABP) family, is a 14.8 kDa cytosolic protein expressed on the cytoplasmic side of compact myelin membranes. As indicated by their name, the principal role of FABPs is thought to be the binding and transport of fatty acids.¦To study its role in myelinating glial cells I have recently generated a complete P2 knockout mouse model (P2-/-). I confirmed the loss of P2 in the sciatic nerve of P2-/- mice at the mRNA and protein level. Electrophysiological analysis of the adult (P56) mutant mice revealed a mild but significant reduction in the motor nerve conduction velocity. lnterestingly, this functional change was not accompanied by any detectable alterations in general myelin structure. However, I have observed significant alterations in the mRNA expression level of other FABPs, predominantly FABP9, in the PNS of P2-/- mice as compared to age-matched P2+/+ mice indicating a role of P2 in the glial myelin lipid metabolism.¦Le diabète de type 2 touche 284 million de personnes dans le monde en 2010 et son évolution conduit dans la moitié des cas à une neuropathie périphérique diabétique. Bien que la neuropathie périphérique soit la complication la plus courante du diabète pouvant conduire jusqu'à l'amputation, sa physiopathologie est aujourd'hui encore mal comprise. Dans le but d'améliorer les connaissances moléculaires expliquant les mécanismes de la neuropathie liée au diabète de type 2, j'ai utilisé un modèle murin du diabète de type 2, les souris db/db.¦ln vivo, les enregistrements éléctrophysiologiques des animaux diabétiques montrent qu'en plus d'une diminution de la vitesse de conduction nerveuse, les souris db/db présentent également une augmentation de l'excitabilité nerveuse. Des mesures menées Ex­ vivo ont montré l'existence d'un phénotype d'hyperexcitabilité sur les nerfs périphériques isolés d'animaux diabétiques. Grâce à l'utilisation d'inhibiteurs pharmacologiques, nous avons pu démontrer que l'hyperexcitabilité démontrée était due à une réduction d'activité des canaux Kv1. En accord avec ces données, j'ai observé qu'une situation de diabète conduisait à une diminution des canaux Kv1.2 aux régions juxta-paranodales des nerfs périphériques db/db, alors que l'expression du transcrit et de la protéine restait stable. J'ai également confirmé l'absence de canaux Kv1.2 aux juxta-paranoeuds de biopsies de nerfs de patients diabétiques. L'ensemble de ces observations montrent que les nerfs périphériques chez les patients atteints de diabète de type 2 est due à une diminution des canaux potassiques rapides juxtaparanodaux les identifiant ainsi comme des cibles thérapeutiques potentielles.¦Les cellules de Schwann enveloppent et isolent les axones périphériques d'une membrane spécialisée, la myéline, composée de deux fois plus de lipides que de protéines. La protéine P2 (Pmp2 "peripheral myelin protein 2" ou FABP8 "fatty acid binding protein") est l'une des protéines les plus abondantes au système nerveux périphérique. P2 appartient à la famille de protéines FABP liant et transportant les acides gras et est une protéine cytosolique de 14,8 kDa exprimée du côté cytoplasmique de la myéline compacte.¦Afin d'étudier le rôle de P2 dans les cellules de Schwann myélinisantes, j'ai généré une souris knockout (P2-/-). Après avoir validé l'absence de transcrit et de protéine P2 dans les nerfs sciatiques P2-/-, des mesures électrophysiologiques ont montré une réduction modérée mais significative de la vitesse de conduction du nerf moteur périphérique. Il est important de noter que ces changements fonctionnels n'ont pas pu être associés à quelconque changement dans la structure de la myéline. Cependant, j'ai observé dans les nerfs périphériques P2-/-, une altération significative du niveau d'expression d'ARNm d'autres FABPs et en particulier FABP9. Ce dernier résultat démontre l'importance du rôle de la protéine P2 dans le métabolisme lipidique de la myéline.

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.

Tricky and terrible T-cell tumors: these are thrilling times for testing: molecular pathology of peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) encompass a group of rare and usually clinically aggressive diseases. The classification and diagnosis of these diseases are compounded by their marked pathological heterogeneity and complex clinical features. With the exception of ALK-positive anaplastic large cell lymphoma (ALCL), which is defined on the basis of ALK rearrangements, genetic features play little role in the definition of other disease entities. In recent years, hitherto unrecognized chromosomal translocations have been reported in small subsets of PTCLs, and genome-wide array-based profiling investigations have provided novel insights into their molecular characteristics. This article summarizes the current knowledge on the best-characterized genetic and molecular alterations underlying the pathogenesis of PTCLs, with a focus on recent discoveries, their relevance to disease classification, and their management implications from a diagnostical and therapeutical perspective.

Mutations in CTC1, encoding conserved telomere maintenance component 1, cause Coats plus.

Coats plus is a highly pleiotropic disorder particularly affecting the eye, brain, bone and gastrointestinal tract. Here, we show that Coats plus results from mutations in CTC1, encoding conserved telomere maintenance component 1, a member of the mammalian homolog of the yeast heterotrimeric CST telomeric capping complex. Consistent with the observation of shortened telomeres in an Arabidopsis CTC1 mutant and the phenotypic overlap of Coats plus with the telomeric maintenance disorders comprising dyskeratosis congenita, we observed shortened telomeres in three individuals with Coats plus and an increase in spontaneous γH2AX-positive cells in cell lines derived from two affected individuals. CTC1 is also a subunit of the α-accessory factor (AAF) complex, stimulating the activity of DNA polymerase-α primase, the only enzyme known to initiate DNA replication in eukaryotic cells. Thus, CTC1 may have a function in DNA metabolism that is necessary for but not specific to telomeric integrity.