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We previously reported that pegylated IFN-α2a (Peg–IFN-α2a) added to antiretroviral therapy (ART)–suppressed, HIV-infected subjects resulted in plasma HIV control and integrated HIV DNA decrease. We now evaluated whether innate NK cell activity or PBMC transcriptional profiles were associated with decreases in HIV measures. Human peripheral blood was analyzed prior to Peg–IFN-α2a administration (ART, baseline), after 5 wk of ART+Peg–IFN-α2a, and after 12 wk of Peg–IFN-α2a monotherapy (primary endpoint). After 5 wk of ART+Peg–IFN-α2a, immune subset frequencies were preserved, and induction of IFN-stimulated genes was noted in all subjects except for a subset in which the lack of IFN-stimulated gene induction was associated with increased expression of microRNAs. Viral control during Peg–IFN-α2a monotherapy was associated with 1) higher levels of NK cell activity and IFN-γ–induced protein 10 (IP-10) on ART (preimmunotherapy) and 2) downmodulation of NK cell KIR2DL1 and KIR2DL2/DL3 expression, transcriptional enrichment of expression of genes associated with NK cells in HIV controller subjects, and higher ex vivo IFN-α–induced NK cytotoxicity after 5 wk of ART+Peg–IFN-α2a. Integrated HIV DNA decline after immunotherapy was also associated with gene expression patterns indicative of cell-mediated activation and NK cytotoxicity. Overall, an increase in innate activity and NK cell cytotoxicity were identified as correlates of Peg–IFN-α2a–mediated HIV control.

The pleiotropic actions of interleukin-2 (IL-2) are essential for regulation of immune responses and maintenance of immune tolerance. The IL-2 receptor (IL-2R) is composed of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits, with defects in IL-2Rα and IL-2Rγ and their downstream signaling effectors resulting in known primary immunodeficiency disorders. Here, we report the first human defect in IL-2Rβ, occurring in two infant siblings with a homozygous IL2RB mutation in the WSXWS motif, manifesting as multisystem autoimmunity and susceptibility to CMV infection. The hypomorphic mutation results in diminished IL-2Rβ surface expression and dysregulated IL-2/15 signaling, with an anticipated reduction in regulatory T cells. However, in contrast to the IL-2Rβ−/− animal model, which lacks NK cells, these siblings demonstrate an expansion of NK cells, particularly the CD56bright subset, and a lack of terminally differentiated NK cells. Thus, the early-onset autoimmunity and immunodeficiency are linked to functional deficits arising from altered IL-2Rβ expression and signaling in T and NK cells.

Low-dose CT (LDCT) is widely accepted as the preferred method for detecting pulmonary nodules. However, the determination of whether a nodule is benign or malignant involves either repeated scans or invasive procedures that sample the lung tissue. Noninvasive methods to assess these nodules are needed to reduce unnecessary invasive tests. In this study, we have developed a pulmonary nodule classifier (PNC) using RNA from whole blood collected in RNA-stabilizing PAXgene tubes that addresses this need. Samples were prospectively collected from high-risk and incidental subjects with a positive lung CT scan. A total of 821 samples from 5 clinical sites were analyzed. Malignant samples were predominantly stage 1 by pathologic diagnosis and 97% of the benign samples were confirmed by 4 years of follow-up. A panel of diagnostic biomarkers was selected from a subset of the samples assayed on Illumina microarrays that achieved a ROC-AUC of 0.847 on independent validation. The microarray data were then used to design a biomarker panel of 559 gene probes to be validated on the clinically tested NanoString nCounter platform. RNA from 583 patients was used to assess and refine the NanoString PNC (nPNC), which was then validated on 158 independent samples (ROC-AUC = 0.825). The nPNC outperformed three clinical algorithms in discriminating malignant from benign pulmonary nodules ranging from 6–20 mm using just 41 diagnostic biomarkers. Overall, this platform provides an accurate, noninvasive method for the diagnosis of pulmonary nodules in patients with non–small cell lung cancer. Significance: These findings describe a minimally invasive and clinically practical pulmonary nodule classifier that has good diagnostic ability at distinguishing benign from malignant pulmonary nodules.

Very early onset inflammatory bowel disease (VEO-IBD), diagnosed

Background Mutations in ITCH, which encodes an E3 ubiquitin-protein ligase, can result in systemic autoimmunity and immunodeficiency. The clinical phenotype and mechanism of disease have not been fully characterized, resulting in a paucity of therapeutic options for this potentially fatal disease. Objective We aimed to (1) expand the understanding about the phenotype of human ITCH deficiency (2) further characterize the associated immune dysregulation, and (3) report the first successful hematopoietic cell transplant (HCT) in a patient with ITCH deficiency. Methods Disease profiling was performed in a patient with multisystem immune dysregulation. Whole exome sequencing with trio analysis and functional validation of candidate disease variants were performed, including mRNA and protein expression. Analyses to further delineate the immunophenotype included quantitative evaluation of lymphoid and myeloid subsets with flow cytometry and mass cytometry. Results A patient with multisystem immune dysregulation presenting with growth failure, very-early-onset inflammatory bowel disease, arthritis, uveitis, psoriasis, and type 1 diabetes mellitus underwent whole exome sequencing, which identified novel compound heterozygous mutations in ITCH. Reduced expression of ITCH mRNA and absent ITCH protein were found. Abnormalities in both lymphoid and myeloid lineages were identified. The patient underwent HCT. He demonstrated excellent immune reconstitution and resolution of many manifestations of his systemic disease. Conclusions Here we report ITCH deficiency with unique clinical features of colonic very-early-onset inflammatory bowel disease, arthritis, and uveitis in the setting of immune dysregulation and further characterize the underlying immune dysregulation. We demonstrate that HCT can be an effective, and potentially curative, therapy for ITCH deficiency.

OBJECTIVES Advances in surgery and perioperative care have contributed to improved outcomes after pancreas transplant. However, the development of peripancreatic infections carries a poor prognosis. It is not clear whether abdominal drainage is helpful in collection prevention. MATERIALS AND METHODS A retrospective review of adult consecutive pancreas transplants at a single institution between January 2017 and December 2018 was undertaken. Postoperative outcomes were compared between patients in whom prophylactic intraoperative drains were placed and patients with no drains. RESULTS We identified 83 patients who underwent pancreas transplant with a median age of 45 years; 54.2% were males, and median body mass index was 25.8. Thirty patients had 1 or 2 drains placed (36.1%). There was no difference in the readmission rate (70.0% vs 60.4%; P = .48), reoperation (20.0% vs 30.2%; P = .44), or percutaneous drainage of peripancreatic infections (20.0% vs 15.1%; P = .56) between patients with drains and no drains, respectively. However, prophylactic drainage was associated with a lower rate of reoperation for peripancreatic infections compared with those who were not drained (0.0% vs 13.2%; P < .05). No graft loss occurred in the drain group. CONCLUSIONS Prophylactic drainage after pancreas transplant may be helpful for reduction in the infection rate after reoperation. The risks of drain placement should be weighed against those of drain avoidance.

Background. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive plasma biomarker to evaluate for transplant allograft rejection. The relationship between infectious complications in kidney allografts and dd-cfDNA has received cursory attention in prior publications. Methods. Retrospective review of all renal transplant recipients who underwent dd-cfDNA testing between November 2017 and August 2019. Results. We report on 7 cases in whom infections affecting the transplanted kidney were associated with elevation in dd-cfDNA without concomitant rejection or elevation in serum creatinine. Five patients had BK viremia, and 2 patients had urinary tract infection associated with elevated dd-cfDNA levels. Conclusions. These observations suggest that elevations in dd-cfDNA are not specific to kidney allograft rejection and can be associated with infections affecting the transplanted kidney. This biomarker may be valuable in evaluating infectious complications of kidney allografts.

The RAG1 and RAG2 proteins are essential for the assembly of Ag receptor genes in the process known as VDJ recombination, allowing for an immense diversity of lymphocyte Ag receptors. Congruent with their importance, RAG1 and RAG2 have been a focus of intense study for decades. To date, RAG1 has been studied as a single isoform; however, our identification of a spontaneous nonsense mutation in the 5′ region of the mouse Rag1 gene lead us to discover N-truncated RAG1 isoforms made from internal translation initiation. Mice homozygous for the RAG1 nonsense mutation only express N-truncated RAG1 isoforms and have defects in Ag receptor rearrangement similar to human Omenn syndrome patients with truncating 5′ RAG1 frameshift mutations. We show that the N-truncated RAG1 isoforms are derived from internal translation initiation start sites. Given the seemingly inactivating Rag1 mutation, it is striking that homozygous mutant mice do not have the expected SCID. We propose that evolution has garnered RAG1 and other important genes with the ability to form truncated proteins via internal translation to minimize the deleterious effects of 5′ nonsense mutations. This mechanism of internal translation initiation is particularly important to consider when interpreting nonsense or frameshift mutations in whole-genome sequencing, as such mutations may not lead to loss of protein.

Background. Outcomes of liver transplantation (LT) from donation after circulatory death (DCD) have been improving; however, ischemic cholangiopathy (IC) continues to be a problem. In 2014, measures to minimize donor hepatectomy time (DHT) and cold ischemic time (CIT) have been adopted to improve DCD LT outcomes. Methods. Retrospective review of all patients who underwent DCD LT between 2005 and 2017 was performed. We compared outcomes of patients who were transplanted before 2014 (historic group) with those who were transplanted between 2014 and 2017 (modern group). Results. We identified 112 patients; 44 were in the historic group and 68 in the modern group. Donors in the historic group were younger (26.5 versus 33, P = 0.007) and had a lower body mass index (26.2 versus 28.2, P = 0.007). DHT (min) and CIT (h) were significantly longer in the historic group (21.5 versus 14, P < 0.001 and 5.3 versus 4.2, P < 0.001, respectively). Fourteen patients (12.5%) developed IC, with a significantly higher incidence in the historic group (23.3% versus 6.1%, P = 0.02). There was no difference in graft and patient survival between both groups. Conclusion. In appropriately selected recipients, minimization of DHT and CIT may decrease the incidence of IC. These changes can potentially expand the DCD donor pool.

Diverting ileostomy is used as a temporizing therapy in patients with perianal Crohn disease; however, little data exist regarding its use for colonic disease. The primary aim of the present study was to determine the role of diversion in severe refractory colonic inflammatory bowel disease (IBD) in a pediatric population.Retrospective study of patients who underwent diverting ileostomy at The Children's Hospital of Philadelphia from 2000 to 2014 for the management of severe, refractory colonic IBD. Clinical variables were compared in the 1 year before ileostomy and 1 year after diversion. Surgical and disease outcomes including changes in diagnosis were reviewed through 2015.Twenty-four patients underwent diverting ileostomy for refractory colonic disease. Initial diagnoses were Crohn disease in 10 (42%), ulcerative colitis in 1 (4%), and IBD-unclassified in 13 patients (54%). Comparing data before and after surgery, there were statistically significant improvements in height and weight velocities, height velocity z score, blood transfusion requirement, hemoglobin, and hospitalization rates. Chronic steroid use decreased from 71% to 22%. At the conclusion of the study, 10 patients had undergone subsequent colectomy, 7 had successful bowel reanastomosis, and 7 remain diverted. Seven patients (29%) had a change in diagnosis. There were 13 surgical complications in 7 subjects, including prolapse reduction, stoma revision, and resection of ischemic bowel.In pediatric patients with refractory colonic IBD, diverting ileostomy can be a successful intervention to induce clinical stability. Importantly, diversion is a steroid-sparing therapy and allows additional time to clarify the diagnosis.

BACKGROUND Vedolizumab is effective for inducing and maintaining remission in adults with inflammatory bowel disease (IBD); however, there is limited pediatric data. This study aimed to describe the adverse events and clinical response to vedolizumab in refractory pediatric IBD. METHODS Disease activity indices, clinical response, concomitant medication use, and adverse events were measured over 22 weeks in an observational prospective cohort study of children with refractory IBD who had failed anti-tumor necrosis factor therapy and subsequently initiated vedolizumab therapy. RESULTS Twenty-one subjects, 16 with Crohn disease, received vedolizumab. Clinical response was observed in 6/19 (31.6%) of the evaluable subjects at week 6 and in 11/19 (57.9%) by week 22. Before induction, 15/21 (71.4%) participants were treated with systemic corticosteroids, as compared with 7/21 (33.3%) subjects at 22 weeks. Steroid-free remission was seen in 1/20 (5.0%) subjects at 6 weeks, 3/20 (15.0%) at 14 weeks, and 4/20 (20.0%) at 22 weeks. There was statistically significant improvement in serum albumin and hematocrit; however, C-reactive protein increased by week 22 (P < 0.05). There were no infusion reactions. Vedolizumab was discontinued in 2 patients because of severe colitis, requiring surgical intervention. CONCLUSIONS There is limited experience with vedolizumab therapy in pediatric IBD. There seems to be a marked number of subjects with clinical response in the first 6 weeks that increases further by week 22 despite the severity of disease in this cohort. Adverse events may not be directly related to vedolizumab. This study is limited by small sample size, and larger prospective studies are warranted.

BackgroundInsight into the pathogenesis of very early onset-inflammatory bowel disease (VEO-IBD) has expanded through the identification of causative monogenic defects detected in a subset of patients. However, the clinical course of this population remains uncertain. The study objective is to determine whether VEO-IBD is associated with more severe disease, defined as increased surgical intervention and growth failure, than older pediatric IBD. Secondary outcomes included therapeutic response and hospitalizations.MethodsSubjects with IBD diagnosed younger than 6 years old (VEO-IBD) were compared with children diagnosed 6 to 10 (intermediate-onset) and older than 10 years of age (older-onset IBD). Metadata obtained from the medical record included age of onset, disease phenotype and location, surgeries, medical therapy, and comorbid conditions. Length of follow-up was at least 1 year from diagnosis.ResultsThere were 229, 221, and 521 subjects with VEO, intermediate-onset, and older-onset IBD, respectively. Very early onset-inflammatory bowel disease subjects underwent more diverting ileostomies (P < 0.001) and colectomies (P < 0.001) than the older children. There was less improvement in weight- and height-for-age Z scores during the follow-up period in subjects with VEO-IBD. Additionally, subjects with VEO-IBD had higher rates of medication failure at 1 year and were more frequently readmitted to the hospital. Targeted therapy was successfully used almost exclusively in VEO-IBD.ConclusionPatients with VEO-IBD can have a more severe disease course with increased surgical interventions and poor growth as compared with older-onset IBD patients. Further, VEO-IBD patients are more likely to be refractory to conventional therapies. Strategies using targeted therapy in these children can improve outcome and, in some cases, be curative.

A novel homozygous mutation in human IL2RB results in decreased IL-2Rβ protein expression and dysregulated IL-2/15 signaling. This hypomorphic mutation leads to decreased regulatory T cell frequency and an abnormal NK cell compartment, with clinical manifestations of autoimmunity and susceptibility to CMV., The pleiotropic actions of interleukin-2 (IL-2) are essential for regulation of immune responses and maintenance of immune tolerance. The IL-2 receptor (IL-2R) is composed of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits, with defects in IL-2Rα and IL-2Rγ and their downstream signaling effectors resulting in known primary immunodeficiency disorders. Here, we report the first human defect in IL-2Rβ, occurring in two infant siblings with a homozygous IL2RB mutation in the WSXWS motif, manifesting as multisystem autoimmunity and susceptibility to CMV infection. The hypomorphic mutation results in diminished IL-2Rβ surface expression and dysregulated IL-2/15 signaling, with an anticipated reduction in regulatory T cells. However, in contrast to the IL-2Rβ−/− animal model, which lacks NK cells, these siblings demonstrate an expansion of NK cells, particularly the CD56bright subset, and a lack of terminally differentiated NK cells. Thus, the early-onset autoimmunity and immunodeficiency are linked to functional deficits arising from altered IL-2Rβ expression and signaling in T and NK cells., Graphical Abstract

Background and aims Children with very early onset inflammatory bowel disease [VEO-IBD] represent a unique cohort, often with a severe phenotype that is refractory to conventional medications, and some cases have underlying primary immunodeficiencies. Previous work has identified distinct histopathological patterns in the gastrointestinal tract in patients with primary immunodeficiencies. The aim of this study is to characterise the diagnostic histological findings in patients with VEO-IBD as compared with older onset paediatric IBD, and determine if there are unique pathological changes that can shed light on the driving forces of the disease, particularly immunodeficiencies. Methods Clinical retrospective chart review, including disease characteristics and endoscopic findings, was performed on all included subjects. Two paediatric pathologists reviewed biopsies from diagnostic upper endoscopies and colonoscopies of subjects with very early onset inflammatory bowel disease and older onset inflammatory bowel disease, to evaluate for the presence of 11 histological features previously associated with inflammatory bowel disease and primary immunodeficiencies. Results The diagnostic gastrointestinal biopsies of subjects with very early onset inflammatory bowel disease differed from those in older onset paediatric IBD, demonstrated by increased frequency of apoptosis, severe chronic architectural changes, small intestine villous blunting, and eosinophils in the crypts, lamina propria, and surface epithelium. Conclusions The diagnostic biopsies of children with very early onset inflammatory bowel disease can identify characteristic features that may be important in guiding the diagnostic work-up in this population.

The pleiotropic actions of interleukin-2 (IL-2) are essential for regulation of immune responses and maintenance of immune tolerance. The IL-2 receptor (IL-2R) is composed of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits, with defects in IL-2Rα and IL-2Rγ and their downstream signaling effectors resulting in known primary immunodeficiency disorders. Here, we report the first human defect in IL-2Rβ, occurring in two infant siblings with a homozygous

Background Very early onset inflammatory bowel disease, diagnosed in children ≤5 years old, can be the initial presentation of some primary immunodeficiencies. Methods In this study, we describe a 17-month-old boy with recurrent infections, growth failure, facial anomalies, and inflammatory bowel disease. Immune evaluation, whole-exome sequencing, karyotyping, and methylation array were performed to evaluate the child's constellation of symptoms and examination findings. Results Whole-exome sequencing revealed that the child was homozygous for a novel variant in ZBTB24, the gene associated with immunodeficiency, centromere instability, and facial anomalies type-2 syndrome. Conclusion This describes the first case of inflammatory bowel disease associated with immunodeficiency, centromere instability, and facial anomalies type-2 syndrome in a child with a novel disease-causing mutation in ZBTB24 found on whole-exome sequencing.

Background: Inflammatory bowel disease (IBD) is a complex disorder involving pathogen infection, host immune response, and altered enterocyte physiology. Incidences of IBD are increasing at an alarming rate in developed countries, warranting a detailed molecular portrait of IBD. Methods: We used large-scale data, bioinformatics tools, and high-throughput computations to obtain gene and microRNA signatures for Crohn's disease (CD) and ulcerative colitis (UC). These signatures were then integrated with systemic literature review to draw a comprehensive portrait of IBD in relation to autoimmune diseases. Results: The top upregulated genes in IBD are associated with diabetogenesis (REG1A, REG1B), bacterial signals (TLRs, NLRs), innate immunity (DEFA6, IDO1, EXOSC1), inflammation (CXCLs), and matrix degradation (MMPs). The downregulated genes coded tight junction proteins (CLDN8), solute transporters (SLCs), and adhesion proteins. Genes highly expressed in UC compared to CD included antiinflammatory ANXA1, transporter ABCA12, T-cell activator HSH2D, and immunoglobulin IGHV4-34. Compromised metabolisms for processing of drugs, nitrogen, androgen and estrogen, and lipids in IBD correlated with an increase in specific microRNA. Highly expressed IBD genes constituted targets of drugs used in gastrointestinal cancers, viral infections, and autoimmunity disorders such as rheumatoid arthritis and asthma. Conclusions: This study presents a clinically relevant gene-level portrait of IBD subtypes and their connectivity to autoimmune diseases. The study identified candidates for repositioning of existing drugs to manage IBD. Integration of mice and human data point to an altered B-cell response as a cause for upregulation of genes in IBD involved in other aspects of immune defense such as interferon-inducible responses. (Inflamm Bowel Dis 2012;)

We analyzed gene expression profiles of young and aged mouse CD8+ T cells specific for the nucleoprotein (NP) of influenza A/PR8/34 virus. CD8+ T cells were stimulated either by the NP antigen expressed in its native form or fused into the herpes virus (HSV)-1 glycoprotein D (gD) protein, which blocks signaling through the immunoinhibitory B and T lymphocyte attenuator (BTLA) and CD160 pathways. We show that NP-specific CD8+ T cells from aged mice exhibit numerous differences in gene expression compared to NP-specific CD8+ T cells from young mice, including a significant reduction of expression in genes involved in T cell receptor (TcR) and CD28 signaling. We also show that these changes can be reversed in a sub-population (~50%) of the aged mice by a BTLA/CD160 checkpoint blockade. These results suggest that BTLA/CD160 checkpoint blockade has potential value as a vaccine additive to induce better CD8+ T cell responses in the aged.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the EBV-encoded nuclear antigen and sequence-specific DNA binding protein required for viral origin binding and episome maintenance during latency. EBNA1 can also bind to numerous sites in the cellular genome and can provide a host cell survival function, but it is not yet known how EBNA1 sequence-specific binding is responsible for host cell survival. Here, we integrate EBNA1 chromatin immunoprecipitation sequencing (ChIP-Seq) with transcriptome sequencing (RNA-Seq) after EBNA1 depletion to identify cellular genes directly regulated by EBNA1 that are also essential for B-cell survival. We first compared EBNA1 ChIP-Seq patterns in four different EBV-positive cell types, including Burkitt lymphoma (BL) cells, nasopharyngeal carcinoma (NPC) cells, and lymphoblastoid cell lines (LCLs). EBNA1 binds to ∼1,000 sites that are mostly invariant among cell types and share a consensus recognition motif. We found that a large subset of EBNA1 binding sites are located proximal to transcription start sites and correlate genome-wide with transcription activity. EBNA1 bound to genes of high significance for B-cell growth and function, including MEF2B , IL6R , and EBF1 . EBNA1 depletion from latently infected LCLs results in the loss of cell proliferation and the loss of gene expression for some EBNA1-bound genes, including MEF2B , EBF1 , and IL6R . Depletion of MEF2B, EBF1, or IL6R partially phenocopies EBNA1 depletion by decreasing the cell growth and viability of cells latently infected with EBV. These findings suggest that EBNA1 binds to a large cohort of cellular genes important for cell viability and implicates EBNA1 as a critical regulator of transcription of host cell genes important for enhanced survival of latently infected cells. IMPORTANCE Epstein-Barr virus (EBV) latent infection is responsible for a variety of lymphoid and epithelial cell malignancies. EBNA1 is the EBV-encoded nuclear antigen that is consistently expressed in all EBV-associated cancers. EBNA1 is known to provide a host cell survival function, but the mechanism is not known. EBNA1 is a sequence-specific binding protein important for viral genome maintenance during latency. Here, by integrating ChIP-Seq and RNA-Seq, we demonstrate that EBNA1 binds directly to the promoter regulatory regions and upregulates the transcription of host genes that are important for the survival of EBV-infected cells. Identification of EBNA1 target genes provides potential new targets for therapeutic intervention in EBV-associated disease.

We analyzed age-related defects in B cell populations from young and aged mice. Microarray analysis of bone marrow resident antibody secreting cells (ASCs) showed significant changes upon aging, affecting multiple genes, pathways and functions including those that play a role in immune regulation, humoral immune responses, chromatin structure and assembly, cell metabolism and the endoplasmic reticulum (ER) stress response. Further analysis showed upon aging defects in energy production through glucose catabolism with reduced oxidative phosphorylation. In addition aged B cells had increased levels of reactive oxygen-species (ROS), which was linked to enhanced expression of the co-inhibitor programmed cell death (PD)-1.

Cutaneous T-cell lymphoma (CTCL) is responsive at all stages to immunotherapy. We determined whether a novel agonist for Toll-like receptor (TLR) 7/8 (3M-007) combined with either interferon-γ (IFN-γ) or interleukin-15 (IL-15) would enhance patients' immune responses in vitro. Our data demonstrate that IFN-γ or IL-15 in combination with 007 significantly increases patients' natural killer (NK) cytolytic activity against CTCL tumor cell lines and synergistically induces dendritic cell cytokines, compared to 007 alone. Microarray studies of gene expression of patients' peripheral blood mononuclear cells (PBMCs) primed with IFN-γ followed by stimulation with 007 identified significant up-regulation of the expression of IL-12 p35 (α-chain), IL-12 p40 (β-chain), and nine IFN-α genes. Importantly, the underlying mechanism of increased levels of IFN-α and IL-12 from combined treatment appears to involve IFN regulatory factor 8 (IRF-8). These results further support our hypothesis that combinations of biological modifiers activating different arms of the immune system may provide significant therapeutic benefits for patients with advanced CTCL.

The global gene expression analysis of cancer and healthy tissues typically results in large numbers of genes that are significantly altered in cancer. Such data, however, has been difficult to interpret due to the high level of variation of gene lists across laboratories and the small sample sizes used in individual studies. In this investigation, we compiled microarray data obtained from the same platform family from 84 laboratories, resulting in a database containing 1,043 healthy tissue samples and 4,900 cancer samples for 13 different tissue types. The primary cancers considered included adrenal gland, brain, breast, cervix, colon, kidney, liver, lung, ovary, pancreas, prostate and skin tissues. We normalized the data together and analyzed subsets for the discovery of genes involved in normal to cancer transformation. Our integrated significance analysis of microarrays approach produced top 400 gene lists for each of the 13 cancer types. These lists were highly statistically enriched with genes already associated with cancer in research publications excluding microarray studies (p < 1.31 E - 12). The genes MTIM and RRM2 appeared in nine and TOP2A in eight lists of significantly altered genes in cancer. In total, there were 132 genes present in at least four gene lists, 11 of which were not previously associated with cancer. The list contains 17 metal ions and 15 adenyl ribonucleotide binding proteins, six kinases and six transcription factors. Our results point to the value of integrating microarray data in the study of combination drug therapies targeting metastasis.

Background & Aims Very early onset inflammatory bowel disease (VEO-IBD), IBD diagnosed at 5 years of age or younger, frequently presents with a different and more severe phenotype than older-onset IBD. We investigated whether patients with VEO-IBD carry rare or novel variants in genes associated with immunodeficiencies that might contribute to disease development. Methods Patients with VEO-IBD and parents (when available) were recruited from the Children's Hospital of Philadelphia from March 2013 through July 2014. We analyzed DNA from 125 patients with VEO-IBD (age, 3 wk to 4 y) and 19 parents, 4 of whom also had IBD. Exome capture was performed by Agilent SureSelect V4, and sequencing was performed using the Illumina HiSeq platform. Alignment to human genome GRCh37 was achieved followed by postprocessing and variant calling. After functional annotation, candidate variants were analyzed for change in protein function, minor allele frequency less than 0.1%, and scaled combined annotation-dependent depletion scores of 10 or less. We focused on genes associated with primary immunodeficiencies and related pathways. An additional 210 exome samples from patients with pediatric IBD (n = 45) or adult-onset Crohn's disease (n = 20) and healthy individuals (controls, n = 145) were obtained from the University of Kiel, Germany, and used as control groups. Results Four hundred genes and regions associated with primary immunodeficiency, covering approximately 6500 coding exons totaling more than 1 Mbp of coding sequence, were selected from the whole-exome data. Our analysis showed novel and rare variants within these genes that could contribute to the development of VEO-IBD, including rare heterozygous missense variants in IL10RA and previously unidentified variants in MSH5 and CD19 . Conclusions In an exome sequence analysis of patients with VEO-IBD and their parents, we identified variants in genes that regulate B- and T-cell functions and could contribute to pathogenesis. Our analysis could lead to the identification of previously unidentified IBD-associated variants.

The Hippo-Yap signaling pathway regulates a number of developmental and adult cellular processes, including cell fate determination, tissue growth, and tumorigenesis. Members of the scaffold protein angiomotin (Amot) family interact with several Hippo pathway components, including Yap (Yes-associated protein), and either stimulate or inhibit Yap activity. We used a combination of genetic, biochemical, and transcriptional approaches to assess the functional consequences of the Amot-Yap interaction in mice and in human cells. Mice with a liver-specific Amot knockout exhibited reduced hepatic “oval cell” proliferation and tumorigenesis in response to toxin-induced injury or when crossed with mice lacking the tumor suppressor Nf2. Biochemical examination of the Amot-Yap interaction revealed that the p130 splicing isoform of Amot (Amot-p130) and Yap interacted in both the cytoplasm and nucleus, which involved binding of PPxY and LPxY motifs in Amot-p130 to WW domains of Yap. In the cytoplasm, Amot-p130 prevented the phosphorylation of Yap by blocking access of the WW domains to the kinase Lats1. Within the nucleus, Amot-p130 was associated with the transcriptional complex containing Yap and Teads (TEA domain family members) and contributed to the regulation of a subset of Yap target genes, many of which are associated with tumorigenesis. These findings indicated that Amot acts as a Yap cofactor, preventing Yap phosphorylation and augmenting its activity toward a specific set of genes that facilitate tumorigenesis.