James W. Nagle

A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

Missense mutations in desmin associated with familial cardiac and skeletal myopathy.

Desmin-related myopathy (OMIM 601419) is a familial disorder characterized by skeletal muscle weakness associated with cardiac conduction blocks, arrhythmias and restrictive heart failure, and by intracytoplasmic accumulation of desmin-reactive deposits in cardiac and skeletal muscle cells. The underlying molecular mechanisms are unknown. Involvement of the desmin gene (DES) has been excluded in three families diagnosed with desmin-related myopathy. We report two new families with desmin-related cardioskeletal myopathy associated with mutations in the highly conserved carboxy-terminal end of the desmin rod domain. A heterozygous A337P mutation was identified in a family with an adult-onset skeletal myopathy and mild cardiac involvement. Compound heterozygosity for two other mutations, A360P and N393I, was detected in a second family characterized by childhood-onset aggressive course of cardiac and skeletal myopathy.

Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients

Clinical trials have indicated that autologous hematopoietic stem cell transplantation (HSCT) can persistently suppress inflammatory disease activity in a subset of patients with severe multiple sclerosis (MS), but the mechanism has remained unclear. To understand whether the beneficial effects on the course of disease are mediated by lympho-depletive effects alone or are sustained by a regeneration of the immune repertoire, we examined the long-term immune reconstitution in patients with MS who received HSCT. After numeric recovery of leukocytes, at 2-yr follow-up there was on average a doubling of the frequency of naive CD4+ T cells at the expense of memory T cells. Phenotypic and T cell receptor excision circle (TREC) analysis confirmed a recent thymic origin of the expanded naive T cell subset. Analysis of the T cell receptor repertoire showed the reconstitution of an overall broader clonal diversity and an extensive renewal of clonal specificities compared with pretherapy. These data are the first to demonstrate that long-term suppression of inflammatory activity in MS patients who received HSCT does not depend on persisting lymphopenia and is associated with profound qualitative immunological changes that demonstrate a de novo regeneration of the T cell compartment.

Immunodominance of a low-affinity major histocompatibility complex-binding myelin basic protein epitope (residues 111-129) in HLA-DR4 (B1*0401) subjects is associated with a restricted T cell receptor repertoire.

The pathogenesis of multiple sclerosis (MS) is currently ascribed in part to a T cell-mediated process targeting myelin components. The T cell response to one candidate autoantigen, myelin basic protein (MBP), in the context of HLA-DR15Dw2, has been previously studied in detail. However, the characteristics of cellular immunity in the context of other MS-associated HLA-DR haplotypes are scarcely known. MBP-specific T cell lines (TCL) were generated from HLA-DR4 (B1*0401)-positive MS subjects. Out of 275 MBP-specific TCL, 178 (64. 7%) specifically recognized region MBP(111-129), predominantly in the context of DRB1*0401. The major T cell epitope for MBP recognition corresponded to residues MBP(116-123). These TCL expressed disparate profiles of cytokine secretion and cytotoxicity. T cell receptor analysis, on the other hand, revealed a strikingly limited heterogeneity of rearrangements. In contrast to MBP(81-99), which binds with high affinity to HLA-DR15 and is recognized by a diverse T cell repertoire, MBP(111-129) binds weakly to DRB1*0401, suggesting that only high affinity T cell receptors might be able to efficiently engage such unstable MHC/peptide complexes, thus accounting for the T cell receptor restriction we observed. This study provides new insight about MBP recognition and proposes an alternative mechanism for immunodominance of self-antigen T cell epitopes in humans.

Myelin transcription factor 1 (Myt1) of the oligodendrocyte lineage, along with a closely related CCHC zinc finger, is expressed in developing neurons in the mammalian central nervous system

The establishment and operation of the nervous system requires genetic regulation by a network of DNA‐binding proteins, among which is the zinc finger superfamily of transcription factors. We have cloned and characterized a member of the unusual Cys‐Cys‐His‐Cys (also referred to as Cys2HisCys, CCHC, or C2HC) class of zinc finger proteins in the developing nervous system. The novel gene, Myt1‐like (Myt1l), is highly homologous to the original representative of this class, Myelin transcription factor 1 (Myt1) (Kim and Hudson, 1992). The MYT1 gene maps to human chromosome 20, while MYT1L maps to a region of human chromosome 2. Both zinc finger proteins are found in neurons at early stages of differentiation, with germinal zone cells displaying intense staining for MyT1. Unlike Myt1, Myt1l has not been detected in the glial lineage. Neurons that express Myt1l also express TuJ1, which marks neurons around the period of terminal mitosis. The Myt1l protein resides in distinct domains within the neuronal nucleus, analogous to the discrete pattern previously noted for Myt1 (Armstrong et al.: 14:303–321, 1995). The developmental expression and localization of these two multifingered CCHC proteins suggests that each may play a role in the development of neurons and oligodendroglia in the mammalian central nervous system. J. Neurosci. Res. 50:272–290, 1997.

Progressive skeletal myopathy, a phenotypic variant of desmin myopathy associated with desmin mutations

Desmin myopathy is a familial or sporadic disorder characterized by the presence of desmin mutations that cause skeletal muscle weakness associated with cardiac conduction block, arrhythmia and heart failure. Distinctive histopathologic features include intracytoplasmic accumulation of desmin-reactive deposits and electron-dense granular aggregates in skeletal and cardiac muscle cells. We describe two families with features of adult-onset slowly progressive skeletal myopathy without cardiomyopathy. N342D point mutation was present in the desmin helical rod domain in patients of family 1, and I451M mutation was found in the non-helical tail domain in patients of family 2. Of interest, the same I451M mutation has previously been reported in patients with cardiomyopathy and no signs of skeletal myopathy. Some carriers of the I451M mutation did not develop any disease, suggesting incomplete penetrance. Expression studies demonstrated inability of the N342D mutant desmin to form cellular filamentous network, confirming the pathogenic role of this mutation, but the network was not affected by the tail-domain I451M mutation. Progressive skeletal myopathy is a rare phenotypic variant of desmin myopathy allelic to the more frequent cardio-skeletal form.

Rapid identification of local T cell expansion in inflammatory organ diseases by flow cytometric T cell receptor Vβ analysis

Oligoclonal expansion of antigen-specific T cells occurs frequently during inflammatory diseases. These cells may persist for a long time at high frequency in the body and be enriched in the affected tissues. As a screening test for expanded cell T cell populations at sites of inflammation, we developed an optimized methodology for flow-cytometry-based quantification of T cell receptor Vbeta (TCRBV) expression. We first validated the specificity of a TCRBV-specific monoclonal antibody set by direct comparison with PCR-based analysis of mono- and polyclonal T cell samples. This monoclonal antibody (mAb) panel recognized approximately two thirds of the T cell receptor alpha/beta repertoire in a group of 64 healthy donors and allowed defining TCR usage in the CD4+ and CD8+ subsets. The reliable detection of expanded Vbeta gene families in T cell populations was confirmed in experiments on superantigen-stimulated T cells. Through differential TCR analysis on T cell subpopulations in cerebrospinal fluid and blood in patients with acute encephalitis, we were able to identify locally expanded CD8+ T cells. The power of this approach affords not only high-throughput comparative TCR analysis for immunological studies in vitro, but also rapid ex vivo identification of cell populations enriched in organ compartments during inflammatory diseases.

Structural organization, expression and chromosomal mapping of the mouse cystatin-C-encoding gene (Cst3)

Abstract Cystatin C (CstC) is a potent cysteine-proteinase inhibitor. The structure of the mouse CstC-encoding gene (Cst3) was examined by sequencing a 6.1-kb genomic DNA containing the entire gene, as well as 0.9 kb of 5′ flanking and 1.7 kb of its 3′ flanking region. The sequence revealed that the overall organization of the gene is very similar to those of the genes encoding human CstC and other type-2 Cst, with two introns at positions identical to those in the human gene. The promoter area does not contain typical TATA or CAAT ☐es. Two copies of a Spl-binding motif, GGGCGG, are present in the 5′ flanking region within 300 bp upstream from the initiation codon. A hexa-nucleotide, TGTTCT, which is a core sequence of the androgen-responsive element (ARE), is found in the promoter region. This region also contains a 21-nucleotide sequence, 5′-AGACTAGCAGCTGACTGAAGC, which contains two potential binding sites for the transcription factor, AP-1. The mouse Cst3 mRNA was detected in all of thirteen tissues examined by Northern blot analysis. Cst3 was mapped in the mouse to a position on distal chromosome 2.

Respiratory insufficiency in desminopathy patients caused by introduction of proline residues in desmin c-terminal alpha-helical segment

Mutations in desmin gene have been identified in patients with cardiac and skeletal myopathy characterized by intracytoplasmic accumulation of desmin‐reactive deposits and electron‐dense granular aggregates. We characterized two new desminopathy families with unusual features of adult‐onset, slowly progressive, diffuse skeletal myopathy and respiratory insufficiency. Progressive reduction of respiratory muscle strength became clinically detectable between the 3rd and the 8th years of illness and led to recurrent chest infections and death in one of the patients. Novel mutations, A357P and L370P, predicted to introduce proline residue into a highly conserved α‐helical region of desmin, were identified. Proline is known to disrupt the α‐helix. In addition, the A357P mutation distorts a unique stutter sequence that is considered to be critically important for proper filament assembly. Functional assessment in two cell‐lines, one of which does and the other of which does not constitutively produce type III intermediate filaments, demonstrated the inability of mutant desmin carrying either the A357P or the L370P mutation to polymerize and form an intracellular filamentous network. The results of this study indicate that respiratory insufficiency is an intrinsic feature of disease associated with specific desmin mutations; in some patients, respiratory weakness may present as a dominant clinical manifestation and a major cause of disability and death. Muscle Nerve 27: 669–675, 2003

Genetic and phylogenetic analyses of hantaviral sequences amplified from archival tissues of deer mice (Peromyscus maniculatus nubiterrae) captured in the eastern United States

SummaryThe S and M segments of a hantavirus, enzymatically amplified from tissues of Cloudland deer mice (Peromyscus maniculatus nubiterrae) captured during 1985 in West Virginia, diverged from strains of Four Corners virus from the southwestern United States by more than 16% and 6% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis suggested that this virus strain (designated Monogahela) forms a possible evolutionary link between the Four Corners and New York hantaviruses.