Impacts of an invasive virus (CyHV-3) on established invasive populations of common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) in North America

The effects of invasive pathogens on wild fish and fish communities generally are not well documented. We compiled information on the impacts of mass mortality events due to Cyprinid Herpesvirus-3 (CyHV-3), otherwise known as Koi Herpesvirus, on wild North American populations of the invasive cyprinid, Cyprinus carpio (common carp), based on our personal experiences, discussions with North American fish ecologists and virologists, a detailed survey of technical and popular publications and a web search. We found evidence of 17 mass die-offs of carp due to CyHV-3 in North America since 2004, for 7 of which we were able to obtain information about carp before and after the events. For 6 of the events, effects of the die-offs on carp population indices appeared to be slight. Carp size-frequency distributions before and after the well-documented 2007/08 event in Ontario were also not conspicuously different. The exceptional event was at Blue Springs Lake, Missouri, in 2012, at which we estimate 65% of the carp present died as a result of CyHV-3 infections and carp abundance continues to decline. Why Blue Springs Lake differs from other events in North America is not clear. Overall, carp die-offs due to CyHV-3 in North America (1) confirm laboratory studies that only common carp are affected, (2) are of brief duration (3–6 weeks), (3) are not repeated in subsequent years and (4) cause much lower mortality (with the exception of Blue Springs Lake) than previously reported for carp in aquaculture facilities or in the laboratory. In terms of both wild carp and their effects on aquatic communities, the short and long-term effects of most die-offs appear to be slight. These features could have implications for the effectiveness of the proposed use of CyHV-3 to reduce feral carp populations in Australia.     

A common pathway for dendritic cell and early B cell development

B cells and dendritic cells (DCs) each develop from poorly described progenitor cells in the bone marrow (BM). Although a subset of DCs has been proposed to arise from lymphoid progenitors, a common developmental pathway for B cells and BM-derived DCs has not been clearly identified. To address this possibility, we performed a comprehensive analysis of DC differentiative potential among lymphoid and B lymphoid progenitor populations in adult mouse BM. We found that both the common lymphoid progenitors (CLPs), shown here and elsewhere to give rise exclusively to lymphocytes, and a down-stream early B-lineage precursor population devoid of T and NK cell precursor potential each give rise to DCs when exposed to the appropriate cytokines. This result contrasts with more mature B-lineage precursors, all of which failed to give rise to detectable numbers of DCs. Significantly, both CLP and early B-lineage-derived DCs acquired several surface markers associated with functional DCs, and CLP-derived DCs readily induced proliferation of allogeneic CD4(+) T cells. Surprisingly, however, DC differentiation from both lymphoid-restricted progenitors was accompanied by up-regulation of CD11b expression, a cell surface molecule normally restricted to myeloid lineage cells including putative myeloid DCs. Together, these data demonstrate that loss of DC developmental potential is the final step in B-lineage commitment and thus reveals a previously unrecognized link between early B cell and DC ontogeny.     

The decline in B lymphopoiesis in aged mice reflects loss of very early B-lineage precursors

The primary age-related loss in B cell progenitors is thought to be at the pro- to pre-B cell transition. However, we show that the frequencies and absolute numbers of all progenitor populations for the B cell lineage, including B-lineage-committed pro-B cells and multipotent B-lymphoid progenitors, decline in aged C57BL/6 mice. Moreover, when derived from aged mice, lymphoid progenitors within every population examined exhibited suboptimal IL-7 responsiveness, demonstrating that age-associated suboptimal IL-7R signaling is a general property of all early B-lineage precursors. Collectively, these data indicate that aging results in a previously unappreciated decline in the earliest stages of B cell development.     

Single cell volume measurement by quantitative phase microscopy (QPM): a case study of erythrocyte morphology.

The measurement of the volume of intact, viable cells presents challenging problems in many areas of experimental and diagnostic science involved in the evaluation of cellular morphology, growth and function. This investigation details the implementation of a recently developed quantitative phase microscopy (QPM) method to measure the volume of erythrocytes under a range of osmotic conditions. QPM is a computational approach which utilizes simple bright field optics to generate cell phase maps which, together with knowledge of the cellular refractive index, may be used to measure cellular volume. Rat erythrocytes incubated in imidazole-buffered solutions (22 degrees C) of graded tonicity were analysed using QPM (n=10 cells/group, x63, 0.8 NA objective). Erythrocyte refractive index (1.367) was measured using a combination of phase and morphological data obtained from cells adopting spherical geometry under hypotonic conditions. Phase-computed volume increased with decreasing solution osmolality: 42.8 +/- 2.4, 48.7 +/- 2.3, 62.6 +/- 2.3, 90.8 +/- 7.7 microm3 in solutions of 540, 400, 240, and 170 mosmol/kg respectively. These volume changes were associated with crenated, bi-concave and spherical morphological states associated with increasing tonicity. This investigation demonstrates that QPM is a valid, simple and non-destructive approach for measuring cellular phase properties and volume. QPM cell volume analysis represents a significant advance in viable cell experimental capability and provides for acquisition of 'real-time' data - an option not previously available using other approaches.     

Androgen receptors containing expanded polyglutamine tracts exhibit progressive toxicity when stably expressed in the neuroblastoma cell line,SH-SY 5Y

The pathogenesis of X-linked spinal and bulbar muscular atrophy (SBMA) has been traced to an expansion of repeated glutamine (Gln) residues within the amino terminus of the human androgen receptor (AR). To examine the mechanisms by which these expanded repeat ARs (Exp-ARs) are toxic to neurons, we have established and characterized a cell culture model by stably transfecting SH-SY 5Y neuroblastoma cells with cDNAs containing either normal AR (81 series; 23 Glns) or Exp-AR (902 series; 56 Glns). At a low passage number, no differences in cell morphology, growth properties, or susceptibility to toxic insults were observed between clones expressing normal AR or Exp-AR. Initially, both types of cultures were found to express similar levels of specific hormone binding in monolayer binding assays. Immunohistochemical studies demonstrated the vast majority of both the normal AR and Exp-AR were localized to the nucleus in the absence and presence of androgen. As the 902 series of clones were propagated, the Exp-AR content in the cells appeared to decline progressively. However, this decrease actually reflects a gradual disappearance of the Exp-AR cell population. No such selection occurred during the propagation of cells expressing the normal AR. This selection against cells expressing physiological levels of Exp-AR occurs in the absence of intracellular aggregates and suggests that mechanisms other than those involving the formation of aggregates underlie the observed toxicity of Exp-ARs.     

Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB

The ex vivo priming and expansion of human cytotoxic T lymphocytes (CTLs) has potential for use in immunotherapy applications for cancer and infectious diseases. To overcome the difficulty in obtaining sufficient numbers of CTLs, we have developed artificial antigen-presenting cells (aAPCs) expressing ligands for the T-cell receptor (TCR) and the CD28 and 4-1BB co-stimulatory surface molecules. These aAPCs reproducibly activate and rapidly expand polyclonal or antigen-specific CD8(+) T cells. The starting repertoire of CD8+ T cells was preserved during culture. Furthermore, apoptosis of cultured CD8(+) T cells was diminished by this approach. This approach may have important therapeutic implications for adoptive immunotherapy.     

Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma gondii

The intracellular protozoan Toxoplasma gondii is among the most widespread parasites. The broad host cell range of the parasite can be explained by carbohydrate microarray screening analyses that have demonstrated the ability of the T. gondii adhesive protein, TgMIC1, to bind to a wide spectrum of sialyl oligosaccharide ligands. Here, we investigate by further microarray analyses in a dose-response format the differential binding of TgMIC1 to 2-3- and 2-6-linked sialyl carbohydrates. Interestingly, two novel synthetic fluorinated analogs of 3′SiaLacNAc_1–4 and 3′SiaLacNAc_1–3 were identified as highly potent ligands. To understand the structural basis of the carbohydrate binding specificity of TgMIC1, we have determined the crystal structures of TgMIC1 micronemal adhesive repeat (MAR)-region (TgMIC1-MARR) in complex with five sialyl-N-acetyllactosamine analogs. These crystal structures have revealed a specific, water-mediated hydrogen bond network that accounts for the preferential binding of TgMIC1-MARR to arrayed 2-3-linked sialyl oligosaccharides and the high potency of the fluorinated analogs. Furthermore, we provide strong evidence for the first observation of a C—F···H—O hydrogen bond within a lectin-carbohydrate complex. Finally, detailed comparison with other oligosaccharide-protein complexes in the Protein Data Bank (PDB) reveals a new family of sialic-acid binding sites from lectins in parasites, bacteria, and viruses.     

Characterization of bacteria by multiparameter flow cytometry.

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.