Prenatal Alcohol Exposure Impairs the Placenta-Cortex Transcriptomic Signature, Leading to Dysregulation of Angiogenic Pathways.

Although alcohol consumption during pregnancy is a major cause of behavioral and learning disabilities, most FASD infants are late- or even misdiagnosed due to clinician's difficulties achieving early detection of alcohol-induced neurodevelopmental impairments. Neuroplacentology has emerged as a new field of research focusing on the role of the placenta in fetal brain development. Several studies have reported that prenatal alcohol exposure (PAE) dysregulates a functional placenta-cortex axis, which is involved in the control of angiogenesis and leads to neurovascular-related defects. However, these studies were focused on PlGF, a pro-angiogenic factor. The aim of the present study is to provide the first transcriptomic "placenta-cortex" signature of the effects of PAE on fetal angiogenesis. Whole mouse genome microarrays of paired placentas and cortices were performed to establish the transcriptomic inter-organ "placenta-cortex" signature in control and PAE groups at gestational day 20. Genespring comparison of the control and PAE signatures revealed that 895 and 1501 genes were only detected in one of two placenta-cortex expression profiles, respectively. Gene ontology analysis indicated that 107 of these genes were associated with vascular development, and String protein-protein interaction analysis showed that they were associated with three functional clusters. PANTHER functional classification analysis indicated that "intercellular communication" was a significantly enriched biological process, and 27 genes were encoded for neuroactive ligand/receptors interactors. Protein validation experiments involving Western blot for one ligand-receptor couple (Agt/AGTR1/2) confirmed the transcriptomic data, and Pearson statistical analysis of paired placentas and fetal cortices revealed a negative correlation between placental Atg and cortical AGTR1, which was significantly impacted by PAE. In humans, a comparison of a 38WG control placenta with a 36WG alcohol-exposed placenta revealed low Agt immunolabeling in the syncytiotrophoblast layer of the alcohol case. In conclusion, this study establishes the first transcriptomic placenta-cortex signature of a developing mouse. The data show that PAE markedly unbalances this inter-organ signature; in particular, several ligands and/or receptors involved in the control of angiogenesis. These data support that PAE modifies the existing communication between the two organs and opens new research avenues regarding the impact of placental dysfunction on the neurovascular development of fetuses. Such a signature would present a clinical value for early diagnosis of brain defects in FASD.

Deep intronic NIPBL de novo mutations and differential diagnoses revealed by whole genome and RNA sequencing in Cornelia de Lange syndrome patients.

Cornelia de Lange syndrome (CdLS; MIM# 122470) is a rare developmental disorder. Pathogenic variants in 5 genes explain approximately 50% cases, leaving the other 50% unsolved. We performed whole genome sequencing (WGS) ± RNA sequencing (RNA-seq) in 5 unsolved trios fulfilling the following criteria: (i) clinical diagnosis of classic CdLS, (ii) negative gene panel sequencing from blood and saliva-isolated DNA, (iii) unaffected parents' DNA samples available and (iv) proband's blood-isolated RNA available. A pathogenic de novo mutation (DNM) was observed in a CdLS differential diagnosis gene in 3/5 patients, namely POU3F3, SPEN, and TAF1. In the other two, we identified two distinct deep intronic DNM in NIPBL predicted to create a novel splice site. RT-PCRs and RNA-Seq showed aberrant transcripts leading to the creation of a novel frameshift exon. Our findings suggest the relevance of WGS in unsolved suspected CdLS cases and that deep intronic variants may account for a proportion of them.

Blood functional assay for rapid clinical interpretation of germline TP53 variants.

BACKGROUND: The interpretation of germline TP53 variants is critical to ensure appropriate medical management of patients with cancer and follow-up of variant carriers. This interpretation remains complex and is becoming a growing challenge considering the exponential increase in TP53 tests. We developed a functional assay directly performed on patients' blood. METHODS: Peripheral blood mononuclear cells were cultured, activated, exposed to doxorubicin and the p53-mediated transcriptional response was quantified using reverse transcription-multiplex ligation probe amplification and RT-QMPSF assays, including 10 p53 targets selected from transcriptome analysis, and two amplicons to measure p53 mRNA levels. We applied this blood functional assay to 77 patients addressed for TP53 analysis. RESULTS: In 51 wild-type TP53 individuals, the mean p53 functionality score was 12.7 (range 7.5-22.8). Among eight individuals harbouring likely pathogenic or pathogenic variants, the scores were reduced (mean 4.8, range 3.1-7.1), and p53 mRNA levels were reduced in patients harbouring truncating variants. We tested 14 rare unclassified variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro191Arg), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del), c.-117G>T) and 12 yielded functionally abnormal scores. Remarkably, the assay revealed that the c.*1175A>C polymorphic variant within TP53 poly-adenylation site can impact p53 function with the same magnitude as a null variant, when present on both alleles, and may act as a modifying factor in pathogenic variant carriers. CONCLUSION: This blood p53 assay should therefore be a useful tool for the rapid clinical classification of germline TP53 variants and detection of non-coding functional variants.

TRIM33 gene somatic mutations identified by next generation sequencing in neoplasms of patients with anti-TIF1γ positive cancer-associated dermatomyositis.

OBJECTIVE: To deep sequence the TRIM33 gene in tumours from patients with cancer-associated anti-TIF1γ autoantibody-positive dermatomyositis (DM) as TRIM33 somatic mutations in tumours may trigger this auto-immune disease. METHODS: Next generation sequencing of tumour DNA samples from patients with cancer-associated anti-TIF1γ autoantibody-positive DM. Fourteen tumours from 13 anti-TIF1γ autoantibody-positive DM individuals were sequenced along with two control tumours from non-DM individuals. RESULTS: Fourteen probable somatic variants from four tumours were identified in the TRIM33 gene. CONCLUSION: These results are in accordance with the previous report of Pinal-Fernandez et al. and support the hypothesis of a role of TRIM33 gene mutations in the pathophysiology of anti-TIF1γ autoantibody-positive DM.

Effect of Neuroprotective Magnesium Sulfate Treatment on Brain Transcription Response to Hypoxia Ischemia in Neonate Mice.

MgSO4 is widely used in the prevention of preterm neurological disabilities but its modes of action remain poorly established. We used a co-hybridization approach using the transcriptome in 5-day old mice treated with a single dose of MgSO4 (600 mg/kg), and/or exposed to hypoxia-ischemia (HI). The transcription of hundreds of genes was altered in all the groups. MgSO4 mainly produced repressions culminating 6 h after injection. Bio-statistical analysis revealed the repression of synaptogenesis and axonal development. The putative targets of MgSO4 were Mnk1 and Frm1. A behavioral study of adults did not detect lasting effects of neonatal MgSO4 and precluded NMDA-receptor-mediated side effects. The effects of MgSO4 plus HI exceeded the sum of the effects of separate treatments. MgSO4 prior to HI reduced inflammation and the innate immune response probably as a result of cytokine inhibition (Ccl2, Ifng, interleukins). Conversely, MgSO4 had little effect on HI-induced transcription by RNA-polymerase II. De novo MgSO4-HI affected mitochondrial function through the repression of genes of oxidative phosphorylation and many NAD-dehydrogenases. It also likely reduced protein translation by the repression of many ribosomal proteins, essentially located in synapses. All these effects appeared under the putative regulatory MgSO4 induction of the mTORC2 Rictor coding gene. Lasting effects through Sirt1 and Frm1 could account for this epigenetic footprint.

Assessment of branch point prediction tools to predict physiological branch points and their alteration by variants.

BACKGROUND: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3'ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3'ss. RESULTS: We used a large set of constitutive and alternative human 3'ss collected from Ensembl (n = 264,787 3'ss) and from in-house RNAseq experiments (n = 51,986 3'ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3'ss (99.48 and 65.84% accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17%. CONCLUSIONS: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3'ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area.

Glucocorticoids paradoxically promote steroid resistance in B cell acute lymphoblastic leukemia through CXCR4/PLC signaling.

Glucocorticoid (GC) resistance in childhood relapsed B-cell acute lymphoblastic leukemia (B-ALL) represents an important challenge. Despite decades of clinical use, the mechanisms underlying resistance remain poorly understood. Here, we report that in B-ALL, GC paradoxically induce their own resistance by activating a phospholipase C (PLC)-mediated cell survival pathway through the chemokine receptor, CXCR4. We identify PLC as aberrantly activated in GC-resistant B-ALL and its inhibition is able to induce cell death by compromising several transcriptional programs. Mechanistically, dexamethasone (Dex) provokes CXCR4 signaling, resulting in the activation of PLC-dependent Ca2+ and protein kinase C signaling pathways, which curtail anticancer activity. Treatment with a CXCR4 antagonist or a PLC inhibitor improves survival of Dex-treated NSG mice in vivo. CXCR4/PLC axis inhibition significantly reverses Dex resistance in B-ALL cell lines (in vitro and in vivo) and cells from Dex resistant ALL patients. Our study identifies how activation of the PLC signalosome in B-ALL by Dex limits the upfront efficacy of this chemotherapeutic agent.

Assessment of parental mosaicism rates in neurodevelopmental disorders caused by apparent de novo pathogenic variants using deep sequencing.

While de novo variants (DNV) are overall at low risk of recurrence in subsequent pregnancies, a subset is at high risk due to parental mosaicism. Accurately identifying cases of parental mosaicism is therefore important for genetic counseling in clinical care. Some studies have investigated the rate of parental mosaics, but most were either limited by the sensitivity of the techniques (i.e. exome or genome sequencing), or focused on specific types of disease such as epileptic syndromes. This study aimed to determine the proportion of parental mosaicism among the DNV causing neurodevelopmental disorders (NDDs) in a series not enriched in epilepsy syndromes. We collected 189 patients with NDD-associated DNV. We applied a smMIP enrichment method and sequenced parental blood DNA samples to an average depth of 7000x. Power simulation indicated that mosaicism with an allelic fraction of 0.5% would have been detected for 87% of positions with 90% power. We observed seven parental mosaic variants (3.7% of families), of which four (2.1% of families) had an allelic fraction of less than 1%. In total, our study identifies a relatively low proportion of parental mosaicism in NDD-associated DNVs and raises the question of a biological mechanism behind the higher rates of parental mosaicism detected in other studies, particularly those focusing on epileptic syndromes.

Drastic effect of germline TP53 missense mutations in Li-Fraumeni patients.

In contrast to other tumor suppressor genes, the majority of TP53 alterations are missense mutations. We have previously reported that in the Li-Fraumeni syndrome (LFS), germline TP53 missense mutations are associated with an earlier age of tumor onset. In a larger series, we observed that mean age of tumor onset in patients harboring dominant negative missense mutations and clearly null mutations was 22.6 and 37.5 years, respectively. To assess the impact of heterozygous germline TP53 mutations in the genetic context of the patients, we developed a new functional assay of the p53 pathway on the basis of induction of DNA damage in Epstein-Barr-virus-immortalized lymphocytes, followed by comparative gene-expression profiling. In wild-type lymphocytes, we identified a core of 173 genes whose expression was induced more than twofold, of which 46 were known p53 target genes. In LFS lymphocytes with canonical missense mutations, the number of induced genes and the level of known p53 target genes induction were strongly reduced as compared with controls and LFS lymphocytes with null mutations. These results show that certain germline missense TP53 mutations, such as those with dominant negative effect, dramatically alter the response to DNA damage. This probably explains why TP53 alterations are predominantly missense mutations.

Isolation, characterization, and genetic profiling of subpopulations of olfactory ensheathing cells from the olfactory bulb.

Olfactory ensheathing cells (OECs) play a crucial role during neurogenesis of primary olfactory neurons. Transplantation of OECs is considered as a promising new therapy for central nervous system repair. Nevertheless, OECs are constituted of distinct subpopulations and their role during neurogenesis is not clearly understood. In particular, OECs from the olfactory bulb (OB) constitute a heterogeneous, but not yet isolated and characterized, population of cells. In our study, flow cytometry analyses of primary OB cultures, based on cell surface expression of low-affinity nerve growth factor receptor (p75), reveal the presence of two distinct populations of OECs. Indeed, some of them express a high level of p75 (P75High) and the other a low level of p75 (P75Low). Effects of OB microenvironment were assessed, and we were able to show that fibroblasts mediate the induction of these two populations through the secretion of soluble factors. To characterize P75High and P75Low OECs, cells were sorted based on their differential expression of p75. Microarray analyses revealed that P75High OECs overexpress genes implicated in modulation of extracellular matrix and cell sorting, whereas P75Low OECs overexpress genes involved in regulation of the inflammatory response and axonal guidance. These results permit, for the first time, to isolate the two distinct subpopulations of OECs from OB, and suggest their specific role during neurogenesis.

Gene profiling predicts rheumatoid arthritis responsiveness to IL-1Ra (anakinra).

OBJECTIVES: The overall non-response rate to biologics remains 30-40% for patients with RA resistant to MTX. The objective of this study was to predict responsiveness to the anakinra-MTX combination by peripheral blood mononuclear cell gene profiling in order to optimize treatment choice. METHODS: Thirty-two patients treated with anakinra (100 mg/day s.c.) and MTX were categorized as responders when their 28-joint DAS (DAS-28) had decreased by ≥1.2 at 3 months. Pre-treatment blood samples had been drawn. RESULTS: For seven responders and seven non-responders, 52 microarray-identified mRNAs were expressed as a function of the response to treatment, and unsupervised hierarchical clustering correctly separated responders from non-responders. The levels of seven of these 52 transcripts, as assessed by real-time, quantitative RT-PCR, were able to accurately classify 15 of 18 other patients (8 responders and 10 non-responders), with 87.5% specificity and 77.8% negative-predictive value for responders. Among the 52 genes, 56% were associated with IL-1ß. CONCLUSION: This predictive gene expression profile was obtained with a non-invasive procedure. After further validation in other cohorts of patients, it could be proposed and used on a large scale to select likely RA responders to combined anakinra-MTX. Trial registration. Clinical Trials; NCT00213538 (http://www.clinicaltrials.gov).

New Insights into Plant Extracellular DNA. A Study in Soybean Root Extracellular Trap.

exDNA is found in various organisms, including plants. However, plant exDNA has thus far received little attention related to its origin and role in the RET (root extracellular trap). In this study, we performed the first high-throughput genomic sequencing of plant exDNA from a Fabaceae with worldwide interest: soybean (Glycine max (L.) Merr.). The origin of this exDNA was first investigated in control condition, and the results show high-coverage on organelles (mitochondria/plastid) DNA relative to nuclear DNA, as well as a mix of coding and non-coding sequences. In the second part of this study, we investigated if exDNA release was modified during an elicitation with PEP-13 (a peptide elicitor from oomycete genus Phytophthora). Our results show that treatment of roots with PEP-13 does not affect the composition of exDNA.

Comparative gene expression profiling of olfactory ensheathing cells from olfactory bulb and olfactory mucosa.

Olfactory ensheathing cells (OEC) have the ability to promote regeneration in the nervous system. Hence, they hold promise for cell therapy. Most of the experimental studies have investigated the role of OECs taken from olfactory bulb (OB). However, for a clinical human application, olfactory mucosa (OM) seems to be the only acceptable source for OECs. Many studies have compared the distinct ability of OECs from OB and OM to improve functional nerve regeneration after lesion of the nervous system. Nevertheless, the two populations of OECs may differ in several points, which might affect all fate after transplantation in vivo. We report here the first study which compares gene expression profiling between these two populations of OECs. It appears that OB-OECs and OM-OECs display distinct gene expression pattern, which suggest that they may be implicated in different physiological processes. Notably, OM-OECs overexpress genes characteristic of wound healing and regulation of extra cellular matrix. In contrast, OB-OECs gene profile suggests a prominent role in nervous system development. Hence, OB-OECs and OM-OECs fundamentally differ in their gene expression pattern, which may represent a crucial point for future clinical application.

Gene expression regulated by abatacept associated with methotrexate and correlation with disease activity in rheumatoid arthritis.

OBJECTIVES: Abatacept acts as a competitive inhibitor of the CD28/(CD80/86) costimulation signal required for T cell activation. Mechanisms of action of abatacept have not been fully investigated. The objective of this study was to provide detailed insight into the mode of action of Abatacept based on gene expression data. METHODS: In this ancillary study from the APPRAISE trial, we investigated the global molecular effects of Abatacept in whole blood samples collected prospectively in biologic naive rheumatoid arthritis patients (n = 19) at baseline and 6 months after the initiation of Abatacept therapy concomitant with methotrexate. Whole human genome microarrays (4x44K) were performed on both baseline and 6-month samples from responders and non-responders patients categorized according to EULAR criteria. T-test with Benjamini-Hochberg correction was performed to identify significant gene expression changes. Gene Ontology and Single Experiment Analysis tools allowed us to highlight specific biological mechanisms involved in methotrexate/Abatacept. RESULTS: In methotrexate/Abatacept responders, 672 genes were significantly (q<0.05) dysregulated at 6 months compared to baseline. Correlation analysis highlighted 19 genes whose dysregulations were significantly associated with disease activity variation (p<0.05) and whose functions were associated with proliferation, apoptosis of cells and mitochondrial metabolism, suggesting a restoration of oxidative signaling. The other 653 gene expression changes were relative to direct or indirect effects of methotrexate/Abatacept treatment and were significantly (p<0.005) involved in pathways relative to mRNA processing, proteasome, angiogenesis, apoptosis and TCR signaling. This study highlights new mechanisms of action of methotrexate/Abatacept and may provide new therapeutic targets to prevent autoimmunity in rheumatoid arthritis.

Hypoxia-Ischemia Induced Age-Dependent Gene Transcription Effects at Two Development Stages in the Neonate Mouse Brain.

Human brain lesions in the perinatal period result in life-long neuro-disabilities impairing sensory-motor, cognitive, and behavior functions for years. Topographical aspects of brain lesions depend on gestational age at the time of insult in preterm or term infants and impaired subsequent steps of brain development and maturation. In mice, the Rice-Vannucci procedure of neonate hypoxia-ischemia (HI) was used at 5 days (P5) or P10, mimicking the development of 30 week-gestation fetus/preterm newborn, or full-term infant, respectively. Transcription response to HI was assessed at 3, 6, 12, and 24 h after insult, using micro-array technology. Statistical Pathway and Gene Ontology terms enrichments were investigated using DAVID®, Revigo® and Ingenuity Pathway Analysis (IPA®) to identify a core of transcription response to HI, age-specific regulations, and interactions with spontaneous development. Investigations were based on direction, amplitude, and duration of responses, basal expression, and annotation. Five major points deserve attention; (i) inductions exceeded repressions (60/40%) at both ages, (ii) only 20.3% (393/1938 records) were common to P5 and P10 mice, (iii) at P5, HI effects occurred early and decreased 24 h after insult whereas they were delayed at P10 and increased 24 h after insult, (iv) common responses at P5 and P10 involved inflammation, immunity, apoptosis, and angiogenesis. (v) age-specific effects occurred with higher statistical significance at P5 than at P10. Transient repression of 12 genes encoding cholesterol biosynthesis enzymes was transiently observed 12 h after HI at P5. Synaptogenesis appeared inhibited at P5 while induced at P10, showing reciprocal effects on glutamate receptors. Specific involvement of Il-1 (interleukin-1) implicated in the firing of inflammation was observed at P10. This study pointed out age-differences in HI responses kinetics, e.g., a long-lasting inflammatory response at P10 compared to P5. Whether the specific strong depression of cholesterol biosynthesis genes that could account for white matter-specific vulnerability at P5 or prevent delayed inflammation needs further investigation. Determination of putative involvement of Il-1 and the identification of upstream regulators involved in the delayed inflammation firing at P10 appears promising routes of research in the understandings of age-dependent vulnerabilities in the neonatal brain.

CD11c+ B Cells Are Mainly Memory Cells, Precursors of Antibody Secreting Cells in Healthy Donors.

CD11c+ B cells have been reported to be increased in autoimmune diseases, but they are detected in the blood of healthy individuals as well. We aimed to characterize CD11c+ B cells from healthy donors by flow cytometry, microarray analysis, and in vitro functional assays. Here, we report that CD11c+ B cells are a distinct subpopulation of B cells, enriched in the memory subpopulation even if their phenotype is heterogeneous, with overexpression of genes involved in B-cell activation and differentiation as well as in antigen presentation. Upon activation, CD11c+ B cells can differentiate into antibody-secreting cells, and CD11c could be upregulated in CD11c- B cells by B-cell receptor activation. Finally, we show that patients with pemphigus, an autoimmune disease mediated by B cells, have a decreased frequency of CD11c+ B cell after treatment, relative to baseline. Our findings show that CD11c+ B cells are mainly memory B cells prone to differentiate into antibody secreting cells that accumulate with age, independently of gender.

Predictors of treatment response in rheumatoid arthritis.

The expanding array of drugs available for treating rheumatoid arthritis is creating challenges in drug selection for the individual patient. The identification of biomarkers that predict the treatment response prior to drug exposure is therefore a current priority. This new approach, known as theranostics, is a component of personalized medicine, which involves selecting the management strategies that are most effective for a given patient at a given point in time. Antibodies to citrullinated peptides, rheumatoid factor, and the interferon signature are the most robust and best validated biomarkers identified to date. Matrices containing clinical or laboratory parameters of diagnostic or prognostic relevance may help to select the best treatment for the individual patient. Furthermore, the development of large-scale approaches requiring no a priori knowledge, such as functional genomics and metabolomics, hold considerable promise, despite persistent difficulties in replicating findings. The complexity of the treatment response in a given patient and substantial variability across patients suggest that biomarkers may be more helpful in combination than singly. The objectives of this review article are to discuss the approaches used to identify theranostic biomarkers and to present an overview of currently available biomarkers and of their performance in everyday clinical practice. However, the range of biomarkers suitable for use in daily practice remains extremely narrow.

Modifications of the Transcriptomic Profile of Autoreactive B Cells From Pemphigus Patients After Treatment With Rituximab or a Standard Corticosteroid Regimen.

Pemphigus Vulgaris is an autoimmune disease of the skin and mucous membranes, which is due to the production of pathogenic autoantibodies targeting desmoglein (DSG) 1 and 3, which are adhesion proteins of the keratinocytes. Rituximab is an anti-CD20 mAb which induces a prolonged depletion of blood B cells. We recently showed that rituximab was more effective than a standard oral corticosteroid (CS) treatment, allowing 90% of patients to achieve complete remission (CR). Additionally, we showed that DSG-specific-B (DSG positive) cells were still detectable during the B cell recovery which follows the initial rituximab-induced B cell depletion, even in patients in CR. In order to characterize DSG positive B cells in patients in CR after rituximab or CS treatment relative to those detectable at baseline in patients with an active pemphigus, we studied the expression profile of 31 genes of interest related to inflammatory cytokines, TNF receptors and activation markers. Using quantitative Polymerase Chain Reaction performed on one cell with a microfluidic technique, we found that patients' autoreactive B cells collected at baseline had a significantly higher expression of genes encoding for IL-1ß, IL-23p19, and IL-12p35 pro-inflammatory cytokines and the IRF5 transcription factor, than non-autoreactive B cells. Surprisingly, the gene expression profile of DSG positive B cells collected after rituximab treatment in patients in CR was close to that of DSG positive B cells at baseline in patients with active pemphigus, except for the IL-1ß and the CD27 memory marker genes, which were under-expressed after rituximab compared to baseline. Conversely, we observed a decreased expression of genes encoding for IL-1ß and IL-23p19 in patients treated with CS relative to baseline. This study showed that: (i) DSG positive autoreactive B cells have a different gene expression profile than non-autoreactive B cells; (ii) rituximab and CS have different effects on the genes' expression in autoreactive DSG positive B cells from pemphigus patients.

Tumor necrosis factor (TNF)-alpha persistently activates nuclear factor-kappaB signaling through the type 2 TNF receptor in chromaffin cells: implications for long-term regulation of neuropeptide gene expression in inflammation.

Chromaffin cells of the adrenal medulla elaborate and secrete catecholamines and neuropeptides for hormonal and paracrine signaling in stress and during inflammation. We have recently documented the action of the cytokine TNF-alpha on neuropeptide secretion and biosynthesis in isolated bovine chromaffin cells. Here, we demonstrate that the type 2 TNF-alpha receptor (TNF-R2) mediates TNF-alpha signaling in chromaffin cells via activation of nuclear factor (NF)-kappaB. Microarray and suppression subtractive hybridization have been used to identify TNF-alpha target genes in addition to those encoding the neuropeptides galanin, vasoactive intestinal polypeptide, and secretogranin II in chromaffin cells. TNF-alpha, acting through the TNF-R2, causes an early up-regulation of NF-kappaB, long-lasting induction of the NF-kappaB target gene inhibitor kappaB (IkappaB), and persistent stimulation of other NF-kappaB-associated genes including mitogen-inducible gene-6 (MIG-6), which acts as an IkappaB signaling antagonist, and butyrate-induced transcript 1. Consistent with long-term activation of the NF-kappaB signaling pathway, delayed induction of neuropeptide gene transcription by TNF-alpha in chromaffin cells is blocked by an antagonist of NF-kappaB signaling. TNF-alpha-dependent signaling in neuroendocrine cells thus leads to a unique, persistent mode of NF-kappaB activation that features long-lasting transcription of both IkappaB and MIG-6, which may play a role in the long-lasting effects of TNF-alpha in regulating neuropeptide output from the adrenal, a potentially important feedback station for modulating long-term cytokine effects in inflammation.

Genome-wide differences in hepatitis C- vs alcoholism-associated hepatocellular carcinoma.

AIM: To look at a comprehensive picture of etiology-dependent gene abnormalities in hepatocellular carcinoma in Western Europe. METHODS: With a liver-oriented microarray, transcript levels were compared in nodules and cirrhosis from a training set of patients with hepatocellular carcinoma (alcoholism, 12; hepatitis C, 10) and 5 controls. Loose or tight selection of informative transcripts with an abnormal abundance was statistically valid and the tightly selected transcripts were next quantified by qRTPCR in the nodules from our training set (12 + 10) and a test set (6 + 7). RESULTS: A selection of 475 transcripts pointed to significant gene over-representation on chromosome 8 (alcoholism) or -2 (hepatitis C) and ontology indicated a predominant inflammatory response (alcoholism) or changes in cell cycle regulation, transcription factors and interferon responsiveness (hepatitis C). A stringent selection of 23 transcripts whose differences between etiologies were significant in nodules but not in cirrhotic tissue indicated that the above dysregulations take place in tumor but not in the surrounding cirrhosis. These 23 transcripts separated our test set according to etiologies. The inflammation-associated transcripts pointed to limited alterations of free iron metabolism in alcoholic vs hepatitis C tumors. CONCLUSION: Etiology-specific abnormalities (chromo-some preference; differences in transcriptomes and related functions) have been identified in hepatocellular carcinoma driven by alcoholism or hepatitis C. This may open novel avenues for differential therapies in this disease.