Contrasting roles of NADPH oxidase isoforms in pressure-overload versus angiotensin II-induced cardiac hypertrophy

Increased production of reactive oxygen species (ROS) is implicated in the development of left ventricular hypertrophy (LVH). Phagocyte-type NADPH oxidases are major cardiovascular sources of ROS, and recent data indicate a pivotal role of a gp91phox-containing NADPH oxidase in angiotensin II (Ang II)-induced LVH. We investigated the role of this oxidase in pressure-overload LVH. gp91phox-/- mice and matched controls underwent chronic Ang II infusion or aortic constriction. Ang II-induced increases in NADPH oxidase activity, atrial natriuretic factor (ANF) expression, and cardiac mass were inhibited in gp91phox-/- mice, whereas aortic constriction-induced increases in cardiac mass and ANF expression were not inhibited. However, aortic constriction increased cardiac NADPH oxidase activity in both gp91phox-/- and wild-type mice. Myocardial expression of an alternative gp91phox isoform, Nox4, was upregulated after aortic constriction in gp91phox-/- mice. The antioxidant, N-acetyl-cysteine, inhibited pressure-overload-induced LVH in both gp91phox-/- and wild-type mice. These data suggest a differential response of the cardiac Nox isoforms, gp91phox and Nox4, to Ang II versus pressure overload.     

Low birth weights contribute to high rates of early-onset chronic renal failure in the Southeastern United States

BACKGROUND: The southeastern United States is a region in which rates of cardiovascular and renal diseases are excessive. Within the Southeast, South Carolina has unusually high rates of end-stage renal disease (ESRD) in young people, with more than 70% of cases attributed to hypertension and diabetes. OBJECTIVE: To determine whether the increased vulnerability to early-onset ESRD might originate through impaired renal development in utero as measured by low birth weight. METHODS: Patients who were diagnosed with renal failure and undergoing dialysis from 1991 through 1996 were identified from the ESRD registry maintained by the Southeastern Kidney Council, Raleigh, NC. Birth weights reported on birth certificates were selected for the ESRD cases and non-ESRD controls who were born in South Carolina in 1950 and later. Birth weights were compared for 1230 cases and 2460 controls who were matched for age, sex, and race. RESULTS: Low birth weight was associated with ESRD among men and women as well as blacks and whites. Among people whose birth weight was less than 2.5 kg, the odds ratio for ESRD was 1.4 (95% confidence interval, 1.1-1.8) compared with people who weighed 3 to 3.5 kg. This association was present for renal failure resulting from diabetes, hypertension, and other causes. CONCLUSIONS: Low birth weights, which reflect adverse effects on development in utero, contribute to the early onset of ESRD in South Carolina. Since low birth weight increases the risk of ESRD from multiple causes, the data suggest that an adverse environment in utero impairs kidney development and makes it more vulnerable to damage from a range of pathological processes.     

Age-dependent neurological phenotypes in a mouse model of PRRT2-related diseases

Paroxysmal kinesigenic dyskinesia is an episodic movement disorder caused by dominant mutations in the proline-rich transmembrane protein PRRT2, with onset in childhood and typically with improvement or resolution by middle age. Mutations in the same gene may also cause benign infantile seizures, which begin in the first year of life and typically remit by the age of 2 years. Many details of PRRT2 function at the synapse, and the effects of mutations on neuronal excitability in the pathophysiology of epilepsy and dyskinesia, have emerged through the work of several groups over the last decade. However, the age dependence of the phenotypes has not been explored in detail in transgenic models. Here, we report our findings in heterozygous and homozygous Prrt2 knockout mice that recapitulate the age dependence of dyskinesia seen in the human disease. We show that Prrt2 deletion reduces the levels of synaptic proteins in a dose-dependent manner that is most pronounced at postnatal day 5 (P5), attenuates at P60, and disappears by P180. In a test for foot slippage while crossing a balance beam, transient loss of coordination was most pronounced at P60 and less prominent at age extremes. Slower traverse time was noted in homozygous knockout mice only, consistent with the ataxia seen in rare individuals with biallelic loss of function mutations in Prrt2. We thus identify three age-dependent phenotypic windows in the mouse model, which recapitulate the pattern seen in humans with PRRT2-related diseases.

     

Pathoscope: Species identification and strain attribution with unassembled sequencing data

Emerging next-generation sequencing technologies have revolutionized the collection of genomic data for applications in bioforensics, biosurveillance, and for use in clinical settings. However, to make the most of these new data, new methodology needs to be developed that can accommodate large volumes of genetic data in a computationally efficient manner. We present a statistical framework to analyze raw next-generation sequence reads from purified or mixed environmental or targeted infected tissue samples for rapid species identification and strain attribution against a robust database of known biological agents. Our method, Pathoscope, capitalizes on a Bayesian statistical framework that accommodates information on sequence quality, mapping quality, and provides posterior probabilities of matches to a known database of target genomes. Importantly, our approach also incorporates the possibility that multiple species can be present in the sample and considers cases when the sample species/strain is not in the reference database. Furthermore, our approach can accurately discriminate between very closely related strains of the same species with very little coverage of the genome and without the need for multiple alignment steps, extensive homology searches, or genome assembly—which are time-consuming and labor-intensive steps. We demonstrate the utility of our approach on genomic data from purified and in silico “environmental” samples from known bacterial agents impacting human health for accuracy assessment and comparison with other approaches.The accurate and rapid identification of species and strains of pathogens is an essential component of biosurveillance from both human health and biodefense perspectives (Vaidyanathan 2011). For example, misidentification was among the issues that resulted in a 3-wk delay in accurate diagnosis of the recent outbreak of hemorrhagic Escherichia coli being due to strain O104:H4, resulting in over 3800 infections across 13 countries in Europe with 54 deaths (Frank et al. 2011). The most accurate diagnostic information, necessary for species identification and strain attribution, comes from the most refined level of biological data—genomic DNA sequences (Eppinger et al. 2011). Advances in DNA-sequencing technologies allows for the rapid collection of extraordinary amounts of genomic data, yet robust approaches to analyze this volume of data are just developing, from both statistical and algorithmic perspectives.Next-generation sequencing approaches have revolutionized the way we collect DNA sequence data, including for applications in pathology, bioforensics, and biosurveillance. Given a particular clinical or metagenomic sample, our goal is to identify the specific species, strains, or substrains present in the sample, as well as accurately estimate the proportions of DNA originating from each source genome in the sample. Current approaches for next-gen sequencing usually have read lengths between 25 and 1000 bp; however, these sequencing technologies include error rates that vary by approach and by samples. Such variation is typically less important for species identification given the relatively larger genetic divergences among species than among individuals within species. But for strain attribution, sequencing error has the potential to swamp out discriminatory signal in a data set, necessitating highly sensitive and refined computational models and a robust database for both species identification and strain attribution.Current methods for classifying metagenomic samples rely on one or more of three general approaches: composition or pattern matching (McHardy et al. 2007; Brady and Salzberg 2009; Segata et al. 2012), taxonomic mapping (Huson et al. 2007; Meyer et al. 2008; Monzoorul Haque et al. 2009; Gerlach and Stoye 2011; Patil et al. 2012; Segata et al. 2012), and whole-genome assembly (Kostic et al. 2011; Bhaduri et al. 2012). Composition and pattern-matching algorithms use predetermined patterns in the data, such as taxonomic clade markers (Segata et al. 2012), k-mer frequency, or GC content, often coupled with sophisticated classification algorithms such as support vector machines (McHardy et al. 2007; Patil et al. 2012) or interpolated Markov Models (Brady and Salzberg 2009) to classify reads to the species of interest. These approaches require intensive preprocessing of the genomic database before application. In addition, the classification rule and results can often change dramatically depending on the size and composition of the genome database.Taxonomy-based approaches typically rely on a “lowest common ancestor” approach (Huson et al. 2007), meaning that they identify the most specific taxonomic group for each read. If a read originates from a genomic region that shares homology with other organisms in the database, the read is assigned to the lowest taxonomic group that contains all of the genomes that share the homologous region. These methods are typically highly accurate for higher-level taxonomic levels (e.g., phylum and family), but experience reduced accuracy at lower levels (e.g., species and strain) (Gerlach and Stoye 2011). Furthermore, these approaches are not informative when the reads originate from one or more species or strains that are closely related to each other or different organisms in the database. In these cases, all of the reads can be reassigned to higher-level taxonomies, thus failing to identify the specific species or strains contained in the sample.Assembly-based algorithms can often lead to the most accurate strain identification. However, these methods also require the assembly of a whole genome from a sample, which is a computationally difficult and time-consuming process that requires large numbers of reads to achieve an adequate accuracy—often on the order of 50–100× coverage of the target genome (Schatz et al. 2010). Given current sequencing depths, obtaining this level of coverage is usually possible for purified samples, but coverage levels may not be sufficient for mixed samples or in multiplexed sequencing runs. Assembly approaches are further complicated by the fact that data collection at a crime scene or hospital might include additional environmental components in the biological sample (host genome or naturally occurring bacterial and viral species), thus requiring multiple filtering and alignment steps in order to obtain reads specific to the pathogen of interest.Here we describe an accurate and efficient approach to analyze next-generation sequence data for species identification and strain attribution that capitalizes on a Bayesian statistical framework implemented in the new software package Pathoscope v1.0. Our approach accommodates information on sequence quality, mapping quality, and provides posterior probabilities of matches to a known database of reference genomes. Importantly, our approach incorporates the possibility that multiple species can be present in the sample or that the target strain is not even contained within the reference database. It also accurately discriminates between very closely related strains of the same species with much less than 1× coverage of the genome and without the need for sequence assembly or complex preprocessing of the database or taxonomy. No other method in the literature can identify species or substrains in such a direct and automatic manner and without the need for large numbers of reads. We demonstrate our approach through application to next-generation DNA sequence data from a recent outbreak of the hemorrhagic E. coli (O104:H4) strain in Europe (Frank et al. 2011; Rohde et al. 2011; Turner 2011) and on purified and in silico mixed samples from several other known bacterial agents that impact human health. Software and data examples for our approach are freely available for download at https://sourceforge.net/projects/pathoscope/.     

Inhibition of Binding of von Willebrand Factor to the Platelet Glycoprotein Ib-IX Complex, Heparin and Sulfatides by Polyanionic Compounds. The Mechanism of Modulation of the Adhesive Function of von Willebrand Factor

The interaction of the multimeric glycoprotein von Willebrand Factor (vWF) with its platelet membrane receptor, the glycoprotein (GP) Ib-IX complex plays a key role in the initial adhesion of platelets to the vascular subendothelium at high shear blood flow. The GP Ib-IX-binding site is only expressed following activation of vWF, a process that regulates vWF-mediated platelet adhesion. Binding of vWF to the GP Ib-IX complex involves the vWF A1 internal repeat domain, which also contains distinct binding sites for sulfatides, heparin, and the non-physiological modulators of the vWF-GP Ib-IX interaction, ristocetin and botrocetin. With the ultimate aim of further defining the mechanism of vWF modulation, we have analyzed the ability of various polyanionic compounds, including aurintricarboxylic acid, Evans blue, fucoidan, and a range of sulfated and phosphorylated sugars, to inhibit specific binding of purified vWF to immobilized sulfatides and heparin, and the ristocetin- and botrocetindependent binding of vWF to the platelet GP Ib-IX complex. Firstly, it was confirmed using a solid-phase binding assay that, like sulfatides, heparin specifically bound to a purified 39/WkiloDalton fragment of vWF (Leu-480 to Gly-718) that encompasses the A1 domain. Secondly, the ability of a number of polyanionic compounds to inhibit binding of vWF to heparin, but not to immobilized sulfatides, supported previous data suggesting that heparin and sulfatides bind to distinct sites on vWF. In addition, aurintricarboxylic acid, Evans blue and fucoidan all inhibited binding of vWF to both heparin and sulfatides with similar ICso values. Thirdly, many of the compounds tested that inhibited binding of vWF to heparin also effectively inhibited both ristocetin- and botrocetin-dependent binding of vWF to the GP Ib-IX complex on platelets, whereas none of the compounds tested blocked vWF binding to sulfatides and GP Ib-IX but not heparin. The majority of compounds tested inhibited the vWF-platelet interaction to a comparable degree in the presence of ristocetin or botrocetin, suggesting a similar mechanism for inhibition irrespective of the modulator used. These combined experiments provide evidence for an electrostatic model of vWF modulation, and suggest that the heparin-binding domain of vWF may be an important regulatory site involved in the adhesion of vWF to the platelet GP Ib-IX complex.     

B-cell lymphoma after angioimmunoblastic lymphadenopathy: a case with oligoclonal gene rearrangements associated with Epstein-Barr virus

We describe a patient with angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), who subsequently developed large-cell immunoblastic lymphoma of B-cell immunophenotype. At the time of the initial diagnosis, histologic examination of an inguinal lymph node showed typical features of AILD, and there was no evidence of a monoclonal B-cell population by immunohistochemical analysis. In situ hybridization and Southern blot analysis for Epstein-Barr virus (EBV) were negative. At autopsy 2 years later, the patient had widespread lymph node and organ involvement by large-cell immunoblastic lymphoma of B-cell immunophenotype. Southern blot analysis performed on DNA extracted from lymph nodes, liver, and spleen showed two patterns of Ig heavy chain and kappa light chain gene rearrangements. The T-cell receptor beta chain gene was in the germline configuration. Analysis with an EBV terminal repeat region probe showed two clonal populations that paralleled the Ig gene rearrangement studies. Double-labeling immunohistochemistry and in situ hybridization confirmed the presence of EBV within the neoplastic B cells. The data support the hypothesis that EBV was not etiologically related to AILD in this case, and that EBV proliferation may occur after the onset of the disease. Further, the data suggest that some B-cell lymphomas that arise in the setting of AILD resemble EBV-associated B-cell lymphomas that arise in other immunodeficiency states.     

Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.     

Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase

A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony- forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA-containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas.