eScholarship@UMMS

This is a personal account of the discovery of the natural anti-Gal antibody, the most abundant natural antibody in humans, the reciprocal distribution of this antibody and its ligand the alpha-gal epitope in mammals and the immunological barrier this antibody has formed in porcine to human xenotransplantation. This barrier has been overcome in the recent decade with the generation of alpha1,3-galactosyltransferase gene-knockout pigs. However, anti-Gal continues to be relevant in medicine as it can be harnessed for various therapeutic effects. Anti-Gal converts tumor lesions injected with alpha-gal glycolipids into vaccines that elicit a protective anti-tumor immune response by in situ targeting of tumor cells for uptake by antigen-presenting cells. This antibody further accelerates wound and burn healing by interaction with alpha-gal nanoparticles applied to injured areas and induction of rapid recruitment and activation of macrophages. Anti-Gal/alpha-gal nanoparticle immune complexes may further induce rapid recruitment and activation of macrophages in ischemic myocardium and injured nerves, thereby inducing tissue regeneration and prevention of fibrosis.

The histone variant H2A.Z plays key roles in gene expression, DNA repair, and centromere function. H2A.Z deposition is controlled by SWR-C chromatin remodeling enzymes that catalyze the nucleosomal exchange of canonical H2A with H2A.Z. Here we report that acetylation of histone H3 on lysine 56 (H3-K56Ac) alters the substrate specificity of SWR-C, leading to promiscuous dimer exchange in which either H2A.Z or H2A can be exchanged from nucleosomes. This result was confirmed in vivo, where genome-wide analysis demonstrated widespread decreases in H2A.Z levels in yeast mutants with hyperacetylated H3K56. Our work also suggests that a conserved SWR-C subunit may function as a "lock" that prevents removal of H2A.Z from nucleosomes. Our study identifies a histone modification that regulates a chromatin remodeling reaction and provides insights into how histone variants and nucleosome turnover can be controlled by chromatin regulators.

Anti-Gal is the most abundant natural antibody in humans constituting ~1% of immunoglobulins. Anti-Gal is naturally produced also in apes and Old World monkeys. The ligand of anti-Gal is a carbohydrate antigen called the "alpha-gal epitope" with the structure Galalpha1-3Galbeta1-4GlcNAc-R. The alpha-gal epitope is present as a major carbohydrate antigen in nonprimate mammals, prosimians and New World monkeys. Anti-Gal can contribute to several immunological pathogeneses. Anti-Gal IgE produced in some individuals causes allergies to meat and to the therapeutic monoclonal antibody cetuximab, all presenting alpha-gal epitopes. Aberrant expression of the alpha-gal epitope or of antigens mimicking it in humans may result in autoimmune processes, as in Graves' Disease. alpha-Gal epitopes produced by Trypanosoma cruzi interact with anti-Gal and induce "autoimmune like" inflammatory reactions in Chagas Disease. Anti-Gal IgM and IgG further mediate rejection of xenografts expressing alpha-gal epitopes. Because of its abundance, anti-Gal may be exploited for various clinical uses. It increases immunogenicity of microbial vaccines (e.g., flu vaccine) presenting alpha-gal epitopes by targeting them for effective uptake by APC. Tumor lesions are converted into vaccines against autologous tumor associated antigens by intratumoral injection of alpha-gal glycolipids which insert into tumor cell membranes. Anti-Gal binding to alpha-gal epitopes on tumor cells targets them for uptake by APC. Accelerated wound healing is achieved by application of alpha-gal nanoparticles which bind anti-Gal, activate complement, recruit and activate macrophages that induce tissue regeneration. This therapy may be of further significance in regeneration of internally injured tissues such as ischemic myocardium and injured nerves. This article is protected by copyright. All rights reserved.

Homologous recombination is essential for productive DNA replication particularly under stress conditions. We previously demonstrated a stress-induced recruitment of Rad51 to mitochondria and a critical need for its activity in the maintenance of mitochondrial DNA (mtDNA) copy number. Using the human osteosarcoma cell line U20S, we show in the present study that recruitment of Rad51 to mitochondria under stress conditions requires ongoing mtDNA replication. Additionally, Rad51 levels in mitochondria increase in cells recovering from mtDNA depletion. Our findings highlight an important new role for Rad51 in supporting mtDNA replication, and further promote the idea that recombination is indispensable for sustaining DNA synthesis under conditions of replication stress.

The BBSome is a complex of seven proteins, including BBS4, that is cycled through cilia by intraflagellar transport (IFT). Previous work has shown that the membrane-associated signaling protein phospholipase D (PLD) accumulates abnormally in cilia of Chlamydomonas reinhardtii bbs mutants. Here we show that PLD is a component of wild-type cilia but is enriched approximately 150-fold in bbs4 cilia; this accumulation occurs progressively over time and results in altered ciliary lipid composition. When wild-type BBSomes were introduced into bbs cells, PLD was rapidly removed from the mutant cilia, indicating the presence of an efficient BBSome-dependent mechanism for exporting ciliary PLD. This export requires retrograde IFT. Importantly, entry of PLD into cilia is BBSome and IFT independent. Therefore, the BBSome is required only for the export phase of a process that continuously cycles PLD through cilia. Another protein, carbonic anhydrase 6, is initially imported normally into bbs4 cilia but lost with time, suggesting that its loss is a secondary effect of BBSome deficiency.

BACKGROUND: Nicotine and alcohol are the two most co-abused drugs in the world, suggesting a common mechanism of action might underlie their rewarding properties. Although nicotine elicits reward by activating ventral tegmental area dopaminergic (DAergic) neurons via high-affinity neuronal nicotinic acetylcholine receptors (nAChRs), the mechanism by which alcohol activates these neurons is unclear.

METHODS: Because most high-affinity nAChRs expressed in ventral tegmental area DAergic neurons contain the alpha4 subunit, we measured ethanol-induced activation of DAergic neurons in midbrain slices from two complementary mouse models, an alpha4 knock-out (KO) mouse line and a knock-in line (Leu9'Ala) expressing alpha4 subunit-containing nAChRs hypersensitive to agonist compared with wild-type (WT). Activation of DAergic neurons by ethanol was analyzed with both biophysical and immunohistochemical approaches in midbrain slices. The ability of alcohol to condition a place preference in each mouse model was also measured.

RESULTS: At intoxicating concentrations, ethanol activation of DAergic neurons was significantly reduced in alpha4 KO mice compared with WT. Conversely, in Leu9'Ala mice, DAergic neurons were activated by low ethanol concentrations that did not increase activity of WT neurons. In addition, alcohol potentiated the response to ACh in DAergic neurons, an effect reduced in alpha4 KO mice. Rewarding alcohol doses failed to condition a place preference in alpha4 KO mice, paralleling alcohol effects on DAergic neuron activity, whereas a sub-rewarding alcohol dose was sufficient to condition a place preference in Leu9'Ala mice.

CONCLUSIONS: Together, these data indicate that nAChRs containing the alpha4 subunit modulate alcohol reward. All rights reserved.

Cys2-His2 zinc finger proteins (ZFPs) are the largest group of transcription factors in higher metazoans. A complete characterization of these ZFPs and their associated target sequences is pivotal to fully annotate transcriptional regulatory networks in metazoan genomes. As a first step in this process, we have characterized the DNA-binding specificities of 129 zinc finger sets from Drosophila using a bacterial one-hybrid system. This data set contains the DNA-binding specificities for at least one encoded ZFP from 70 unique genes and 23 alternate splice isoforms representing the largest set of characterized ZFPs from any organism described to date. These recognition motifs can be used to predict genomic binding sites for these factors within the fruit fly genome. Subsets of fingers from these ZFPs were characterized to define their orientation and register on their recognition sequences, thereby allowing us to define the recognition diversity within this finger set. We find that the characterized fingers can specify 47 of the 64 possible DNA triplets. To confirm the utility of our finger recognition models, we employed subsets of Drosophila fingers in combination with an existing archive of artificial zinc finger modules to create ZFPs with novel DNA-binding specificity. These hybrids of natural and artificial fingers can be used to create functional zinc finger nucleases for editing vertebrate genomes.

Mutations in the gene encoding human SOD1 (hSOD1) can cause amyotrophic lateral sclerosis (ALS) yet the mechanism by which mutant SOD1 can induce ALS is not fully understood. There is currently no cure for ALS or treatment that significantly reduces symptoms or progression. To develop tools to understand the protein conformations present in mutant SOD1-induced ALS and as possible immunotherapy, we isolated and characterized eleven unique human monoclonal antibodies specific for hSOD1. Among these, five recognized distinct linear epitopes on hSOD1 that were not available in the properly-folded protein but were available on forms of protein with some degree of misfolding. The other six antibodies recognized conformation-dependent epitopes that were present in the properly-folded protein with two different recognition profiles: three could bind hSOD1 dimer or monomer and the other three were specific for hSOD1 dimer only. Antibodies with the capacity to bind hSOD1 monomer were able to prevent increased hydrophobicity when mutant hSOD1 was exposed to increased temperature and EDTA, suggesting that the antibodies stabilized the native structure of hSOD1. Two antibodies were tested in a G93A mutant hSOD1 transgenic mouse model of ALS but did not yield a statistically significant increase in overall survival. It may be that the two antibodies selected for testing in the mouse model were not effective for therapy or that the model and/or route of administration were not optimal to produce a therapeutic effect. Therefore, additional testing will be required to determine therapeutic potential for SOD1 mutant ALS and potentially some subset of sporadic ALS.

BACKGROUND:: Proposed changes to financing of teaching hospitals and new quality-based performance incentives may differentially impact the financial health of teaching and safety-net institutions. Few data have examined the potential impact of these financial changes on teaching institutions.

OBJECTIVES:: To determine the association of hospital teaching intensity with processes and outcomes of care for the most common inpatient diagnoses in the United States.

RESEARCH DESIGN:: Cross-sectional analysis of the 2008 Hospital Quality Alliance and 2007 American Hospital Association databases, adjusted for hospital characteristics.

SUBJECTS:: A total of 2418 hospitals distributed across the country with available data on teaching intensity (resident-to-bed ratio), quality-of-care process measures, and risk-adjusted readmission and mortality rates for acute myocardial infarction (AMI), congestive heart failure (CHF), and pneumonia.

MEASURES:: Hospital-level quality-of-care process indicators and 30-day risk-adjusted readmission and mortality rates for AMI, CHF, and pneumonia.

RESULTS:: Multivariable analysis demonstrates that all hospitals perform uniformly well on quality-of-care process measures for AMI, CHF, and pneumonia. However, when compared with nonteaching hospitals, increasing hospital teaching intensity is significantly associated with improved risk-adjusted mortality for AMI and CHF, but higher risk-adjusted readmission rates for all 3 conditions. Among high teaching intensity hospitals, those with larger Medicaid populations (safety-net institutions) had particularly high readmission rates for AMI and CHF.

CONCLUSIONS:: In this nationally representative evaluation, we found significant variation in performance on risk-adjusted mortality and readmission rates, and differences in readmission rates based on safety-net status. Our findings suggest that high teaching intensity and safety-net institutions may be disproportionately affected by upcoming changes in hospital payment models.

Headache is a common accompanying symptom in cerebrovascular diseases. Several specific conditions and etiologies are reviewed with emphasis on distinguishing characteristics. Recognition of these conditions can help identify underlying causes of these "secondary headache syndromes" and facilitate disease-appropriate treatment.

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%-15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.

How innate lymphoid cells (ILCs) in the thymus and gut become specialized effectors is unclear. The prototypic innate-like gammadelta T cells (Tgammadelta17) are a major source of interleukin-17 (IL-17). We demonstrate that Tgammadelta17 cells are programmed by a gene regulatory network consisting of a quartet of high-mobility group (HMG) box transcription factors, SOX4, SOX13, TCF1, and LEF1, and not by conventional TCR signaling. SOX4 and SOX13 directly regulated the two requisite Tgammadelta17 cell-specific genes, Rorc and Blk, whereas TCF1 and LEF1 countered the SOX proteins and induced genes of alternate effector subsets. The T cell lineage specification factor TCF1 was also indispensable for the generation of IL-22 producing gut NKp46(+) ILCs and restrained cytokine production by lymphoid tissue inducer-like effectors. These results indicate that similar gene network architecture programs innate sources of IL-17, independent of anatomical origins.

The mechanisms that control the natural rate of lipofuscin accumulation in the retinal pigment epithelial (RPE) cell and its stability over time are not well understood. Similarly, the contributions of retinoids, phospholipids and oxidation to the rate of accumulation of lipofuscin are uncertain. The experiments in this study were conducted to explore the individual contribution of rod outer segments (ROS) components to lipofuscin formation and its accumulation and stability over time. During the period of 14 days incubation of ROS, lipofuscin-like autofluorescence (LLAF) determined at two wavelengths (530 and 585 nm) by fluorescence-activated cell sorting (FACS) was measured from RPE cells. The autofluorescence increased in an exponential manner with a strong linear component between days 1 and 7. The magnitude of the increase was larger in cells incubated with 4-hydroxynonenal (HNE-ROS) compared with cells incubated with either bleached or unbleached ROS, but with a different spectral profile. A small (10-15%) decrease in LLAF was observed after stopping the ROS feeding for 14 days. The phagocytosis rate of HNE-ROS was higher than that of either bleached or unbleached ROS during the first 24 h of supplementation. Among the different ROS components, the increase of LLAF was highest in cells incubated with all-trans-retinal. Surprisingly, incubation with 11-cis-retinal and 9-cis-retinal also resulted in strong LLAF increase, comparable to the increase induced by all-trans-retinal. Supplementation with liposomes containing phosphatidylethanolamine (22: 6-PE) and phosphatidylcholine (18:1-PC) also increased LLAF, while incubation with opsin had little effect. Cells incubated with retinoids demonstrated strong dose-dependence in LLAF increase, and the magnitude of the increase was 2-3 times higher at 585 nm compared to 530 nm, while cells incubated with liposomes showed little dose-dependence and similar increase at both wavelengths. Very little difference in LLAF was noted between cells incubated with either unbleached or bleached ROS under any conditions. In summary, results from this study suggest that supplementation with various ROS components can lead to an increase in LLAF, although the autofluorescence generated by the different classes of components has distinct spectral profiles, where the autofluorescence induced by retinoids results in a spectral profile closest to the one observed from human lipofuscin. Future fluorescence characterization of LLAF in vitro would benefit from an analysis of multiple wavelengths to better match the spectral characteristics of lipofuscin in vivo.

Comment on: ABVD in older patients with early-stage Hodgkin lymphoma treated within the German Hodgkin Study Group HD10 and HD11 trials. [J Clin Oncol. 2013]

The regulation of skeletal muscle gene expression during myogenesis is mediated by lineage-specific transcription factors in combination with numerous cofactors, many of which modify chromatin structure. However, the involvement of scaffolding proteins that organize chromatin and chromatin-associated regulatory proteins has not extensively been explored in myogenic differentiation. Here, we report that Scaffold attachment factor b1 (Safb1), primarily associated with transcriptional repression, functions as a positive regulator of myogenic differentiation. Knockdown of Safb1 inhibited skeletal muscle marker gene expression and differentiation in cultured C2C12 myoblasts. In contrast, over-expression resulted in the premature expression of critical muscle structural proteins and formation of enlarged thickened myotubes. Safb1 co-immunoprecipitated with MyoD and was co-localized on myogenic promoters. Upon Safb1 knockdown, the repressive H3K27me3 histone mark and binding of the Polycomb histone methyltransferase Ezh2 persisted at differentiation-dependent gene promoters. In contrast, the appearance of histone marks and regulators associated with myogenic gene activation, such as myogenin and the SWI/SNF chromatin remodelling enzyme ATPase, Brg1, was blocked. These results indicate that the scaffold protein Safb1 contributes to the activation of skeletal muscle gene expression during myogenic differentiation by facilitating the transition of promoter sequences from a repressive chromatin structure to one that is transcriptionally permissive.

Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5' splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.

Targeted biologic therapies markedly slow the progression of structural damage in rheumatoid arthritis (RA); thus, the presence of bony erosion suggests that had treatment been initiated earlier, joint damage might have been prevented. Some patients with early inflammatory arthritis, the group most likely to benefit from prompt initiation of treatment, are not classified as having RA using the 1987 American Rheumatism Association classification criteria.

BACKGROUND: Sulfonylureas have been linked to increased risk of hypoglycemia. Hypoglycemia may lead to falls, and falls may lead to fracture. However, studies quantifying the association between sulfonylureas and fractures are sparse and yield inconsistent results.

OBJECTIVE: The purpose of this article was to review the literature regarding sulfonylurea use and falls or fall-related fractures among older adults with type 2 diabetes mellitus and to delineate areas for future research. DATA SOURCES: We searched MEDLINE (1966-March 2012) and CINAHL (1937-March 2012) for studies of patients with type 2 diabetes mellitus living in the community or nursing homes.

STUDY SELECTION: The search algorithms combined three domains: (1) diabetic patients, (2) sulfonylurea medications, and (3) fractures or falls. We included only publications in English that pertained to human subjects. We found 9 randomized trials and 12 non-experimental studies that met the inclusion criteria.

STUDY APPRAISAL AND SYNTHESIS METHODS: The guidelines provided by the Cochrane handbook or Agency for Healthcare Research and Quality (AHRQ) Methods Guide are too general to distinguish the quality of included non-experimental studies, so we developed several specific domains based on those general guidelines. These domains included study design, study population, follow-up time, comparison group, exposure definition, outcome definition, induction period, confounding adjustment, and attrition or missing data. The data were not amenable to a meta-analysis.

RESULTS: No clinical trials included fracture as a primary endpoint. Most clinical trials excluded older adults. Most studies were not designed to evaluate the risk of sulfonylureas on fractures or falls. Studies did not show an increased risk of falls/fractures with sulfonylurea.

LIMITATIONS: The data available from existing studies suffer from methodological limitations including insufficient events, lack of primary endpoints, exclusion of older adults, and lack of clarity or inappropriate comparison groups.

CONCLUSION: Future studies are needed to appropriately estimate the effect of sulfonylureas on falls or fall-related fractures in older adults who are at increased risk for hypoglycemia, the hypothesized mechanism for fractures related to sulfonylurea therapy.