Nuclear-envelope proteins have been implicated in diverse and fundamental cell functions, among them transcriptional regulation. Gene expression at the territory of the nuclear periphery is known to be repressed by epigenetic modifications such as histone deacetylation and methylation. However, the mechanism by which nuclearenvelope proteins are involved in such modifications is still obscure. We have previously shown that LAP2␤, an integral nuclear-envelope protein that contains the chromatin-binding LEM domain, was able to repress the transcriptional activity of the E2F5-DP3 heterodimer. Here, we show that LAP2␤'s repressive activity is more general, encompassing various E2F members as well as other transcription factors such as p53 and NF-B. We further show that LAP2␤ interacts at the nuclear envelope with HDAC3, a class-I histone deacetylase, and that TSA (an HDAC inhibitor) abrogates LAP2␤'s repressive activity. Finally, we show that LAP2␤ is capable of inducing histone-H4 deacetylation. Our data provide evidence for the existence of a previously unknown repressive complex, composed of an integral nuclear membrane protein and a histone modifier, at the nuclear periphery.

Insights into the ontogeny of the human fetal adaptive immune system are of great value for understanding immunocompetence of the developing fetus. However, to date, this has remained largely uncharted territory, in large part because blood samples from healthy, early gestation fetuses have been hard to come by. In a comprehensive study, we analyzed levels of T cell receptor excision circles (TRECs), signal-joint κ receptor excision circles (sjKRECs), and intron recombination signal sequence-K-deleting element (iRSS-Kde) rearrangement, and T and B lymphocyte repertoire clonality in human fetuses from 12 to 26 weeks of gestational age. Using next-generation sequencing, we analyzed the diversity and complexity of T cell receptor β (TRB) and immunoglobulin heavy chain (IGH) repertoires in four fetuses at 12, 13, 22, and 26 weeks of gestation and in healthy full-term infants. We report the progressive increase of TREC, sjKREC, and iRSS-Kde levels over time and confirm that B cell development precedes T cell development in the human fetus. Temporally and spatially regulated maturation of B and T cell repertoire diversity and complexity during human fetal development was observed, including evidence that immunoglobulin somatic hypermutation and class switch recombination occur already during intrauterine life. Our results help define physiological levels of immunodeficiency in premature infants and may serve as a reference for future studies aimed at investigating the impact of intrauterine pathologies on fetal immune development and function.

The abundant expression of PIGN in various tissues is compatible with the diverse phenotypic features observed in the patients and with the involvement of multiple body systems. The presence of developmental delay, hypotonia, and epilepsy combined with multiple congenital anomalies, especially anorectal anomalies, should lead a clinician to suspect a GPI deficiency related disorder.

A previously unknown signal transduction pathway is important in human cognitive development.

he t-cell-receptor complex consists of the a and b or g and d variant chains, paired as mutually exclusive heterodimers in association with the invariant chains CD3 g , d , e , and z . T cells with a and b chains are referred to as a / b T cells, and those with g and d chains are called g / d T cells. During development, the CD3 protein complex plays an important part in the transition of thymocytes from CD4¡CD8¡ double-negative immature precursors to a CD4+CD8+ double-positive stage and finally to the mature CD4+CD8¡ or CD4¡CD8+ single-positive T cell. 1-5 Selective deficiency of CD3 component g , d , e , or z in mice, achieved by gene knockout, causes mild-to-severe, although incomplete, blockage of T-cell development. 6-10 Similarly, CD3 g or CD3 e deficiency in humans brings about a partial arrest of T-cell maturation and only moderate immunodeficiency. 11, 12 We report a novel defect in the CD3 d gene in three members of a kindred with a form of severe combined immunodeficiency (SCID) characterized by the absence of T cells but normal numbers of B cells (T¡B+ SCID). These three patients had an early arrest in T-cell development, with a nearly complete absence of circulating mature T cells and a complete lack of g / d T cells. Our results suggest that, unlike CD3 e and CD3 g , CD3 d is essential for T-cell development.We studied a kindred of Mennonite descent that shared multiple consanguineous links across several generations. Three patients with SCID were identified in this family. SCID was diagnosed in Patient 1 immediately after birth, after an examination performed because of previous cases in the family (Patients 2 and 3). She subsequently underwent bone marrow transplantation and is alive and well, with full immune reconstitution, three years later. Patient 2, a male cousin of Patient 1, was admitted at the age of two months because of fever, tachypnea, and tachycardia. Rapidly developing respiratory arrest required assisted ventilation, and he died of multiorgan failure. Adenovirus was identified in stool, urine, and bronchial secretions.Patient 3, a male cousin of Patients 1 and 2, was well and thriving until two and a half months of age, when chronic diarrhea developed. At three and a half months of age, the patient was admitted with respiratory distress, lethargy, and jaundice. On examination, he was noted to have hepatomegaly, and liver-function tests were markedly abnormal. He was transferred from another hospital with increased respiratory distress and died 12 hours later from rapidly developing refractory hypotension, liver failure, pulmonary hemorrhage, disseminated intravascular coagulopathy, and hemorrhagic shock. Cytomegalovirus was identified in multiple tissues obtained at autopsy.Flow-cytometric analyses of peripheral-blood lymphocytes from these patients showed a slight reduction in total lymphocyte counts in Patients 1 and 2 and a marked t case reportThe New England Journal of Medicine Downloaded from nejm.org at RUTGERS UNIV ALEXANDER LIBRARY on August 11, 2015. For personal use only....

Wilms' tumor (WT), the embryonic kidney malignancy, is suggested to evolve from a progenitor cell population of uninduced metanephric blastema, which typically gives rise to nephrons. However, apart from blastema, WT specimens frequently contain cells that have differentiated into renal tubular or stromal phenotypes, complicating their analysis. We aimed to define tumor-progenitor genes that function in normal kidney development using WT xenografts (WISH-WT), in which the blastema accumulates with serial passages at the expense of differentiated cells. Herein, we did transcriptional profiling using oligonucleotide microarrays of WISH-WT, WT source, human fetal and adult kidneys, and primary and metastatic renal cell carcinoma. Among the most significantly up-regulated genes in WISH-WT, we identified a surprising number of paternally expressed genes (PEG1/MEST, PEG3, PEG5/NNAT, PEG10, IGF2, and DLK1), as well as Meis homeobox genes [myeloid ecotropic viral integration site 1 homologue 1 (MEIS1) and MEIS2], which suppress cell differentiation and maintain self-renewal. A comparison between independent WISH-WT and WT samples by real-time PCR showed most of these genes to be highly overexpressed in the xenografts. Concomitantly, they were significantly induced in human fetal kidneys, strictly developmentally regulated throughout mouse nephrogenesis and overexpressed in the normal rat metanephric blastema. Furthermore, in vitro differentiation of the uninduced blastema leads to rapid down-regulation of PEG3, DLK1, and MEIS1. Interestingly, ischemic/reperfusion injury to adult mouse kidneys reinduced the expression of PEG3, PEG10, DLK1, and MEIS1, hence simulating embryogenesis. Thus, multiple imprinted and stemness genes that function to expand the renal progenitor cell population may lead to evolution and maintenance of WT. (Cancer Res 2006; 66(12): 6040-9)

Background Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity. Methods We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase–chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene. Results All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of β1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis. Conclusions Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.)

There are considerable differences in tumour biology between adult and paediatric cancers. The existence of cancer initiating cells/cancer stem cells (CIC/CSC) in paediatric solid tumours is currently unclear. Here, we show the successful propagation of primary human Wilms' tumour (WT), a common paediatric renal malignancy, in immunodeficient mice, demonstrating the presence of a population of highly proliferative CIC/CSCs capable of serial xenograft initiation. Cell sorting and limiting dilution transplantation analysis of xenograft cells identified WT CSCs that harbour a primitive undifferentiated – NCAM1 expressing – “blastema” phenotype, including a capacity to expand and differentiate into the mature renal-like cell types observed in the primary tumour. WT CSCs, which can be further enriched by aldehyde dehydrogenase activity, overexpressed renal stemness and genes linked to poor patient prognosis, showed preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family. Complete eradication of WT in multiple xenograft models was achieved with a human NCAM antibody drug conjugate. The existence of CIC/CSCs in WT provides new therapeutic targets.