Development and use of an Arctic charr cell line to study antiviral responses at extremely low temperatures.

Arctic charr (Salvelinus alpinus) are the northernmost distributed freshwater fish and can grow at water temperatures as low as 0.2 °C. Other teleost species have impaired immune function at temperatures that Arctic charr thrive in, and thus, charr may maintain immune function at these temperatures. In this study, a fibroblastic cell line, named ACBA, derived from the bulbus arteriosus (BA) of Arctic charr was developed for use in immune studies at various temperatures. ACBA has undergone more than forty passages at 18 °C over 3 years, while showing no signs of senescence-associated ß-galactosidase activity and producing nitric oxide. Remarkably, ACBA cells survived and maintained some mitotic activity even at 1 °C for over 3 months. At these low temperatures, ACBA also continued to produce MH class I proteins. After challenge with poly I:C, only antiviral Mx proteins were induced while MH proteins remained constant. When exposed to live viruses, ACBA was shown to permit viral infection and replication of IPNV, VHSV IVa and CSV at 14 °C. Yet at the preferred temperature of 4 °C, only VHSV IVa was shown to replicate within ACBA. This study provides evidence that Arctic charr cells can maintain immune function while also resisting infection with intracellular pathogens at low temperatures.

Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb.

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD + poly I:C, lipopolysaccharide (LPS), LPS + poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11 days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P < 0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression.

Use of cell lines and primary cultures to explore the capacity of rainbow trout to be a host for frog virus 3 (FV3).

The capacity of rainbow trout, Oncorhynchus mykiss, to be a host for frog virus 3 (FV3) was evaluated at the cellular level. Cell cultures from this species were tested for their ability to express FV3 major capsid protein (MCP) gene, to develop cytopathic effect (CPE), and to produce FV3. After FV3 addition, MCP transcripts were detected in six of six cell lines and in primary macrophage cultures. CPE developed in all cell culture systems, except primary lymphocytes. For the macrophage cell line, RTS11, and primary macrophages, cell death was by apoptosis because DNA laddering and Annexin staining were detected. By contrast, markers of apoptosis did not accompany CPE in three epithelial cell lines from the gill (RTgill-W1), intestine (RTgut-GC), and liver (RTL-W1) and in two fibroblast cell lines from gonads (RTG-2) and skin (RTHDF). Therefore, FV3 was able to enter and begin replicating in several cell types. Yet, FV3 was produced in only two cell lines, RTG-2 and RTL-W1, and only modestly. Overall, these results suggest that if tissue accessibility were possible, FV3 would have the capacity to induce injury, but the ability to replicate would be limited, likely making rainbow trout a poor host for FV3.

Corexit 9500 inactivates two enveloped viruses of aquatic animals but enhances the infectivity of a nonenveloped fish virus.

The effects of Corexit 9500, a dispersant used to clean up oil spills, on invertebrates, lower vertebrates, birds, and human health have been examined, but there is a significant lack of study of the effect of this dispersant on aquatic viruses. In this study, the effects of Corexit 9500 on four aquatic viruses of differing structural composition were examined. Corexit 9500 reduced the titer of the enveloped viral hemorrhagic septicemia virus (VHSV) at all concentrations (10% to 0.001%) examined. The titer of frog virus 3 (FV3), a virus with both enveloped and nonenveloped virions, was reduced only at the high Corexit 9500 concentrations (10% to 0.1%). Corexit 9500 was unable to reduce the titer of nonenveloped infectious pancreatic necrosis virus (IPNV) but enhanced the titer of chum salmon reovirus (CSV) by 2 to 4 logs. With the ability to inactivate enveloped viruses and possibly enhance some nonenveloped viruses, Corexit 9500 has the potential to alter the aquatic virosphere.

Differential effects of viral hemorrhagic septicaemia virus (VHSV) genotypes IVa and IVb on gill epithelial and spleen macrophage cell lines from rainbow trout (Oncorhynchus mykiss).

The two most prominent genotypes of viral hemorrhagic septicemia virus (VHSV) are -I in the Northeastern Atlantic region and -IV in North America, but much more is known about the cellular pathogenesis of genotype -I than -IV. VHSV genotype -IV is divided into -IVa from the Northeast Pacific Ocean and -IVb from the Great Lakes and both of which are less virulent to rainbow trout than genotype -I. In this work, infections of VHSV-IVa and -IVb have been studied in two rainbow trout cell lines, RTgill-W1 from the gill epithelium, and RTS11 from spleen macrophages. RTgill-W1 produced infectious progeny of both VHSV-IVa and -IVb. However, VHSV-IVa was more infectious than -IVb toward RTgill-W1: -IVa caused cytopathic effect (CPE) at a lower viral titre, elicited CPE earlier, and yielded higher titres. By contrast, no CPE and no increase in viral titre were observed in RTS11 cultures infected with either genotype. Yet in RTS11 all six VHSV genes were expressed and antiviral genes, Mx2 and Mx3, were up regulated by VHSV-IVb and -IVa. However, replication appeared to terminate at the translational stage as viral N protein, presumably the most abundant of the VSHV proteins, was not detected in either infected RTS11 cultures. In RTgill-W1, Mx2 and Mx3 were up regulated to similar levels by both viral genotypes, while VHSV-IVa induced higher levels of IFN1, IFN2 and LGP2A than VHSV-IVb.

Isolation of a nonendotoxic antitumor preparation from Serratia marcescens.

White-type polysaccharide preparation (WPS) obtained from Serratia marcescens bacteria by hot 0.2 N acetic acid extraction was shown to have antitumor effects. These were manifested by enhanced resistance to the take of TA3 transplantable murine adenocarcinoma and by the induction of regression of Meth A sarcoma in mice. Optimal conditions for the liberation and isolation of these substances were sought to achieve the highest antitumor activity and the lowest endotoxin (ET) content. Simultaneously, the activities of the WPS preparations were tested in various tests which are frequently used as in vitro correlates of in vivo antitumor effects, such as the activation of macrophage cytotoxicity, activation of natural killer (NK) cells, and tumor necrosis factor (TNF) generation. We found that the enhanced resistance to the take of TA3 tumor correlated with ET content of the WPS preparations. Preparations with reduced or no ET content showed diminishing activity in this assay or were without any measurable effect. The induction of TNF production and NK activation did not show such close relationship with the ET content. This was particularly evident if testing WPS samples obtained after 60 or 120 min hydrolysis at 90 degrees C. The greatest discrepancy was found between ET content and the Meth A regression induction. Samples with no detectable ET content and no activity in the macrophage, NK, or TNF tests were potent inducers of Meth A regression. Partial purification of such WPS samples could be achieved and a preparation was obtained with high Meth A regression capacity. Preliminary chemical analysis of this preparation showed 25.5% amino acid, 53.7% neutral carbohydrate, less than 0.4% KDO, 0.8% hexosamine, less than 0.1% phosphorous, and less 1.0% long-chain carboxylic acid content. The above chemical analytical data are not consistent with designating such preparations as ET or ET derivatives, such as Lipid A or its split products. This conclusion was confirmed by the lack of endotoxic properties as determined by biological assays on this preparation.

Biological effects of White-type polysaccharides of gram-negative bacteria.

The White-type polysaccharide (WPS), often called Freeman polysaccharide, was obtained by hydrolyzing gram-negative bacteria in 0.2 N acetic acid at 100 degrees C for 2 h. The crude product contained partially degraded O-antigens as well as other components that were active as immune adjuvants, enhancers of macrophage cytotoxicity of tumor target cells, and inducers of osteoclastic bone resorption. The same WPS preparation augmented the tumor cytotoxicity of normal mouse spleen cells and slightly retarded the take of L1210 leukemia in mice. The WPS preparations generated colony-stimulating factor in mice but were not active in lymphoproliferative (mitogenicity) tests. Chemical analyses of the WPS preparations did not detect the presence of components characteristic of the lipid moiety of endotoxins. The WPS samples were also negative in biological assays of endotoxicity such as local Shwartzman and toxicity tests. These findings indicate that gram-negative bacteria contain nonendotoxin components that are potent modifiers of some biological responses.

Biological activities of lipopolysaccharide fractionated by preparative acrylamide gel electrophoresis.

Lipopolysaccharide from a smooth strain of Salmonella minnesota was fractionated into two major fractions and one intermediate fraction by using sodium dodecylsulfate-polyacrylamide gel electrophoresis. On the basis of the study by Hitchcock and Brown, it was deduced that the top fraction was mainly long O-side chain LPS and the bottom fraction was O-side chain-less LPS. The middle fraction was a mixture of both short O-side chain LPS and O-side chain-less LPS. The antigenic properties and biological activities were not altered in this fractionation procedure. Comparison of the biological activities of the top fraction with those of the bottom fraction revealed that the bottom fraction had higher activity in polyclonal B-cell activation and spleen-swelling effect and that there was no significant difference in adjuvant activity, ability to render macrophages cytotoxic, induction of colony-stimulating factor and the ability to induce the Schwartzmann reaction. It was suggested that O-side chain makes no contribution to the latter biological activities including adjuvant activity of S. minnesota LPS.

Studies on the optimal conditions of CSF generation by endotoxic LPS and its PS derivative in mice.

Not only the endotoxic LPS preparations, but a non-toxic, lipid-free, non-mitogenic hydrolytic breakdown product of it (called PS) is also capable of inducing colony stimulating factor (CSF) release (1). Due to difficulties to reproduce above findings it became necessary to study the optimal conditions to obtain CSF active PS preparations. It was found that the CSF generating component of the highly heterogeneous PS mixture is sensitive to acidic hydrolyses, but it is less sensitive than the toxic site in the lipid moiety of the LPS. Carefully controlled optimal hydrolytic conditions give PS preparations which have less than one percent residual endotoxicity but maintained 40 to 80% of the original CSF generating capacity. Prolonged hydrolysis will destroy this activity too. Optimal dose of LPS and PS for CSF induction in mice differed widely. For LPS the optimal dose is 25 micrograms, injecting more gave a much reduced or non-detectable CSF level. Optimal dose for PS was 160 micrograms, and this generated a significantly higher CSF level than 25 micrograms LPS. At concentrations below 25 micrograms, LPS was clearly more active than PS. The CSF level reached its peak at 3-4 hours after other LPS or PS injection. Intravenous route was sometimes but not always more effective than intraperitoneal.

Release of toxic microvesicles by Actinobacillus actinomycetemcomitans.

Oral isolates of Actinobacillus actinomycetemcomitans (strain Y4) release spherical microvesicles in large numbers during normal growth. The biological activities of these products were studied, and it was estimated that approximately 1/10 of their dry weight was made up of heat- and proteolysis-resistant endotoxin. The chicken embryo lethality and bone-resorbing activity of the microvesicles were heat stable but proteolysis sensitive. Other laboratories have reported the presence of a heat- and proteolysis-sensitive leukotoxin in similar preparations. Accordingly, the microvesicles released by strain Y4 may contain, in addition to endotoxin, several potent substances which are highly toxic and active in bone resorption, and these may be significant factors in the pathogenesis of periodontal diseases.

Humoral immunity and reduced periodontal bone loss in Eikenella corrodens-monoassociated rats.

Germfree Sprague-Dawley rats monoassociated with Eikenella corrodens exhibited alveolar bone loss. This progressive bone loss occurred over a period of weeks, during which time the hosts developed an immune response toward the infective microorganism. By means of repeated bacterial vaccination resulting in elevated serum antibody titers, reduced bone loss was observed.

Biological activity of the slime and endotoxin of the periodontopathic organism Eikenella corrodens.

A partially purified water-soluble slime extract was obtained from two strains of the oral pathogen Eikenella corrodens, designated CS10 A and CS10 B. Endotoxin was also isolated from this organism by the phenol-water extraction procedure. Assays including the local Shwartzman skin reactivity, chicken embryo lethality, Limulus lysate clotting, spleen cell mitogenicity, and immune adjuvancy were used to test the biological properties of these two bacterial extracts. In each of these assay systems the endotoxins of the Eikenella corrodens strains demonstrated the classical endotoxic responses. In contrast, the effect of slime extract in the corresponding assays was either extremely low or absent with the exception of a very strong immunosuppressive effect. Mice treated with slime extract showed a severely reduced immune response to sheep erythrocytes as measured by the hemolytic plaque-forming cell assay.