Exposure to the complement C5b-9 complex sensitizes 661W photoreceptor cells to both apoptosis and necroptosis.

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]-N-5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease

Retinal changes precede visual dysfunction in the complement factor h knockout mouse

We previously reported that aged mice lacking complement factor H (CFH) exhibit visual defects and structural changes in the retina. However, it is not known whether this phenotype is age-related or is the consequence of disturbed development. To address this question we investigated the effect of Cfh gene deletion on the retinal phenotype of young and mid-age mice. Cfh (-/-) mouse eyes exhibited thickening of the retina and reduced nuclear density, but relatively normal scotopic and photopic electroretinograms. At 12 months there was evidence of subtle astroglial activation in the Cfh (-/-) eyes, and significant elevation of the complement regulator, decay-accelerating factor (DAF) in Müller cells. In the retinal pigment epithelium (RPE) of young control and Cfh (-/-) animals mitochondria and melanosomes were oriented basally and apically respectively, whereas the apical positioning of melanosomes was significantly perturbed in the mid-age Cfh (-/-) RPE. We conclude that deletion of Cfh in the mouse leads to defects in the retina that precede any marked loss of visual function, but which become progressively more marked as the animals age. These observations are consistent with a lifelong role for CFH in retinal homeostasis

Advanced Perkinsus Marinus Infections In Crassostrea Ariakensis Maintained Under Laboratory Conditions

The Suminoe oyster, Crassostrea ariakensis, has been under investigation since the early 1990s for potential use in restoring the commercial harvest or for aquaculture of oysters in the Chesapeake Bay, USA. Initial studies focusing on C. ariakensis documented a significant level of tolerance to the protozoan parasite Perkinsus marinus, a pathogen found in almost all reaches of the Bay and widely acknowledged as one of the main reasons for the decline in the eastern oyster, Crassostrea virginica, harvest since the late 1980s. Crassostrea ariakensis was demonstrated to acquire P. marinus. however infection intensities, as measured using Ray\u27s thioglycollate medium assay indices, generally were found to be light. As part of a series of experiments to study potential impacts on the Chesapeake Bay region of pathogens found in C. ariakensis in Asia, a challenge experiment was conducted to study the pathogenicity of Perkinsus olseni to C. ariakensis. During this study, we observed the acquisition of moderate and heavy infection intensities of P. marinus in triploid C. ariakensis oysters being maintained in the laboratory. Results suggest that there may be some risk of mortality from P. marinus if C. ariakensis is held under stressful conditions at least in hatchery or laboratory settings

Aging and aerobic fitness affect the contribution of noradrenergic sympathetic nerves to the rapid cutaneous vasodilator response to local heating

Sedentary aging results in a diminished rapid cutaneous vasodilator response to local heating. We investigated whether this diminished response was due to altered contributions of noradrenergic sympathetic nerves; assessing 1) the age-related decline and, 2) the effect of aerobic fitness. We measured skin blood flow (SkBF)(laser-Doppler flowmetry) in young (24±1 yr) and older (64±1 yr) endurance-trained and sedentary men (n=7 per group) at baseline and during 35 min of local skin heating to 42 °C at three forearm sites: 1) untreated; 2) bretylium tosylate (BT), preventing neurotransmitter release from noradrenergic sympathetic nerves; and 3) yohimbine and propranolol (YP), antagonising α- and β-adrenergic receptors. SkBF was converted to cutaneous vascular conductance (CVC) (SkBF/mean arterial pressure) and normalized to maximal CVC (%CVCmax) achieved by skin heating to 44 °C. Pharmacological agents were administered using microdialysis. In the young trained, the rapid vasodilator response was reduced at the BT and YP sites (P0.05) but treatment with BT did (P>0.05). Neither BT nor YP treatments affected the rapid vasodilator response in the older sedentary group (P>0.05). These data suggest that the age-related reduction in the rapid vasodilator response is due to an impairment of sympathetic-dependent mechanisms, which can be partly attenuated with habitual aerobic exercise. Rapid vasodilation involves noradrenergic neurotransmitters in young trained men, and non-adrenergic sympathetic cotransmitters (e.g., neuropeptide Y) in young sedentary and older trained men, possibly as a compensatory mechanism. Finally, in older sedentary men, the rapid vasodilation appears not to involve the sympathetic system

Histological Assessment Of The Lobster (Homarus Americanus) In The 100 Lobsters Project

The emergence of epizootic shell disease in the American lobster (Homarus americanus) has been devastating to the industry in the coastal waters of southern New England. A comprehensive assessment of the disease syndrome, known as the 100 Lobsters Project, was initiated to examine health and physiological parameters among laboratories involved in the research on lobster shell disease. A histological study of the 100 lobsters was undertaken as part of that assessment. Tissues from 90 lobsters from Rhode Island and 19 lobsters from Maine were examined as a general health assessment of the 100 lobsters. Approximately half the lobsters from Rhode Island were selected because they had frank epizootic shell disease, whereas none of the lobsters from Maine exhibited the syndrome. In addition to epizootic shell disease, the histological findings revealed 3 other idiopathic syndromes-necrotizing hepatopancreatitis, idiopathic blindness, and nonspecific granulomas-in higher prevalences in lobsters from Rhode Island compared with those from Maine. Necrotizing hepatopancreatitis, a newly described disease syndrome in lobsters, was observed in 15% of the lobsters from Rhode Island. Idiopathic blindness was present in 54% of the lobsters from Rhode Island, and 16% of the animals from Maine. This is the first report of the syndrome in lobsters from Maine. None of the idiopathic syndromes was associated with epizootic shell disease. The detection of multiple disease syndromes such as epizootic shell disease, necrotizing hepatopancreatitis, and idiopathic blindness may be indicative of exposure to environmental stressors in Narragansett Bay, RI

Genetic Diversity In U.S. Hatchery Stocks Of Crassostrea Ariakensis (Fujita, 1913) And Comparison With Natural Populations In Asia

Although several different U.S. hatchery stocks of the Asian Suminoe oyster Crassosirea ariakensis were used in laboratory and field trials assessing performance, and in comparative studies with the native oyster Crassostrea virginica, the genetic composition of these hatchery stocks has not yet been examined comprehensively. Using eight microsatellite markers we investigated the genetic variability among five hatchery stocks and compared the genetic makeup of these stocks with 8 wild populations from Asia. Results showed significant genetic differentiation among the 5 hatchery stocks that was 6-fold larger than that observed among wild populations. A significant reduction in genetic diversity was observed in hatchery stocks compared with wild source populations, indicating a genetic bottleneck. Two long-established stocks showed significant decreases in both allelic diversity and heterozygosity compared with the wild Japanese source population, whereas three recently established stocks showed less severe reductions in allelic diversity and a nonsignificant change in levels of heterozygosity compared with their source Chinese populations. These microsatellite markers also proved useful for assignment of hatchery individuals back to their source stocks with a high degree of confidence. Although assignment of wild individuals back to their population of origin proved less reliable, approximately 70% of wild individuals could be assigned either to their source population or to geographically proximal populations. Our results suggest that results obtained from experiments that used hatchery animals of a single C. ariakensis stock for biological and ecological studies should be interpreted cautiously, because they may not always accurately reflect the behavior of wild populations or of other hatchery stocks

Mitotic instability in triploid and tetraploid one-year-old eastern oyster, Crassostrea virginica, assessed by cytogenetic and flow cytometry techniques

For commercial oyster aquaculture, triploidy has significant advantages. To produce triploids, the principal technology uses diploid x tetraploid crosses. The development of tetraploid brood stock for this purpose has been successful, but as more is understood about tetraploids, it seems clear that chromosome instability is a principal feature in oysters. This paper is a continuation of work to investigate chromosome instability in polyploid Crassostrea virginica. We established families between tetraploids-apparently stable (non-mosaic) and unstable (mosaic)-and normal reference diploids, creating triploid groups, as well as tetraploids between mosaic and non-mosaic tetraploids. Chromosome loss was about the same for triploid juveniles produced from either mosaic or non-mosaic tetraploids or from either male or female tetraploids. However, there was a statistically significant difference in chromosome loss in tetraploid juveniles produced from mosaic versus non-mosaic parents, with mosaics producing more unstable progeny. These results confirm that chromosome instability, as manifested in mosaic tetraploids, is of little concern for producing triploids, but it is clearly problematic for tetraploid breeding. Concordance between the results from cytogenetics and flow cytometry was also tested for the first time in oysters, by assessing the ploidy of individuals using both techniques. Results between the two were non-concordant

Regulation of C3 Activation by the Alternative Complement Pathway in the Mouse Retina

The purpose of this study was to examine the retinas of mice carrying hemizygous and null double deletions of Cfb-/- and Cfh-/-, and to compare these with the single knockouts of Cfb, Cfh and Cfd. Retinas were isolated from wild type (WT), Cfb-/-/Cfh-/-, Cfb-/-/Cfh+/-, Cfh-/-/Cfb+/-, Cfb-/-, Cfh-/- Cfd-/-, and Cfd+/- mice. Complement proteins were evaluated by western blotting, ELISA and immunocytochemistry, and retinal morphology was assessed using toluidine blue stained semi-thin sections. WT mice showed staining for C3 and its breakdown products in the retinal vasculature and the basal surface of the retinal pigment epithelium (RPE). Cfb-/- mice exhibited a similar C3 staining pattern to WT in the retinal vessels but a decrease in C3 and its breakdown products at the basal surface of the RPE. Deletion of both Cfb and Cfh restored C3 to levels similar to those observed in WT mice, however this reversal of phenotype was not observed in Cfh-/-/Cfb+/- or Cfb-/-/Cfh+/- mice. Loss of CFD caused an increase in C3 and a decrease in C3 breakdown products along the basal surface of the RPE. Overall the retinal morphology and retinal vasculature did not appear different across the various genotypes. We observed that C3 accumulates at the basal RPE in Cfb-/-, Cfb-/-/Cfh-/-, Cfb-/-/Cfh+/-, Cfd-/- and WT mice, but is absent in Cfh-/- and Cfh-/-/Cfb+/- mice, consistent with its consumption in the serum of mice lacking CFH when CFB is present. C3 breakdown products along the surface of the RPE were either decreased or absent when CFB, CFH or CFD was deleted or partially deleted

The 100 Lobsters Project: A Cooperative Demonstration Project For Health Assessments Of Lobsters From Rhode Island

The emergence of epizootic shell disease in the American lobster (Homarus americanus) has been devastating to the fishing industry in southern New England. In response, research was initiated to understand the roles of the environment, pathogens, and pollutants in the ecology and etiology of the disease. A comprehensive project was initiated in which tissues and hemolymph from 100 lobsters were collected from an endemic area of disease, Narragansett Bay, RI. The project has moved forward with the purpose of compiling, synthesizing, and propagating the findings from the 100 Lobsters Project. The resulting tissue bank and Web-based data repository and instructional tools serve as a nascent demonstration project to both the scientific community working on this disease as well as to members of the lobster industry

Continuous culture of Perkinsus mediterraneus, a parasite of the European flat oyster Ostrea edulis, and characterization of its morphology, propagation, and extracellular proteins in vitro

Continuous in vitro cultures of Perkinsus mediterraneus were established from tissues of infected European flat oysters, Ostrea edulis. The parasite proliferated in protein-free medium and divided by schizogony in vitro. Cell morphology was similar to that observed for P. mediterraneus in tissues of naturally infected O. edulis and for other Perkinsus spp. cultured in vitro. Parasite cells enlarged approximately 8-fold when placed in alternative Ray\u27s fluid thioglycollate medium, and stained black with Lugol\u27s iodine solution, a response characteristic of Perkinsus spp. DNA sequences matched those determined previously for P. mediterraneus, and phylogenetic analyses on three different data sets indicated that this was a Perkinsus species with a close relationship to another recently described species, Perkinsus honshuensis. Parasite viability was high (\u3e 90%) in vitro, but the proliferation rate was low, with densities generally increasing 2-to-6-fold between subcultures at 6-wk intervals. Enzyme analysis of cell-free culture supernatants revealed protease-, esterase-, glycosidase-, lipase-, and phosphatase-like activities. Incubation with class-specific protease inhibitors showed that P. mediterraneus produced serine proteases, and eight proteolytic bands with molecular weights ranging from 34 to 79 kDa were detected in the supernatants by gelatin sodium dodecylsulfate-polyacrylamide gel electrophoresis