Eicosapentaenoic acid prevents arterial calcification in klotho mutant mice.

BACKGROUND: The klotho gene was identified as an "aging-suppressor" gene that accelerates arterial calcification when disrupted. Serum and vascular klotho levels are reduced in patients with chronic kidney disease, and the reduced levels are associated with arterial calcification. Intake of eicosapentaenoic acid (EPA), an n-3 fatty acid, reduces the risk of fatal coronary artery disease. However, the effects of EPA on arterial calcification have not been fully elucidated. The aim of this study was to determine the effect of EPA on arterial calcification in klotho mutant mice. METHODS AND RESULTS: Four-week-old klotho mutant mice and wild-type (WT) mice were given a diet containing 5% EPA (EPA food, klotho and WT: n = 12, each) or not containing EPA (control food, klotho and WT: n = 12, each) for 4 weeks. Calcium volume scores of thoracic and abdominal aortas assessed by computed tomography were significantly elevated in klotho mice after 4 weeks of control food, but they were not elevated in klotho mice after EPA food or in WT mice. Serum levels of EPA and resolvin E1, an active metabolite of EPA, in EPA food-fed mice were significantly increased compared to those in control food-fed mice. An oxidative stress PCR array followed by quantitative PCR revealed that NADPH oxidase-4 (NOX4), an enzyme that generates superoxide, gene expression was up-regulated in arterial smooth muscle cells (SMCs) of klotho mice. Activity of NOX was also significantly higher in SMCs of klotho mice than in those of WT mice. EPA decreased expression levels of the NOX4 gene and NOX activity. GPR120, a receptor of n-3 fatty acids, gene knockdown by siRNA canceled effects of EPA on NOX4 gene expression and NOX activity in arterial SMCs of klotho mice. CONCLUSIONS: EPA prevents arterial calcification together with reduction of NOX gene expression and activity via GPR120 in klotho mutant mice.

Polyclonal hypergammaglobulinemia and autoantibody production induced by vaccination in farmed Atlantic salmon.

The introduction of oil-adjuvanted vaccines in salmon aquaculture made large-scale production feasible by reducing the impact of infections. Vaccines given intraperitoneally (ip) contain oil adjuvant such as mineral oil. However, in rodents, a single ip injection of adjuvant hydrocarbon oil induces lupus-like systemic autoimmune syndrome. We have recently reported that autoimmune disease in farmed salmon, characterized by production of various autoantibodies, immune complex glomerulonephritis, liver thrombosis, and spinal deformity, are previously unrecognized side effects of vaccination. In the present study, we examined whether vaccination-induced autoantibody production in farmed Atlantic salmon is a mere result of polyclonal B-cell activation. Sera were collected from 205 vaccinated and unvaccinated Atlantic salmon (experimental, 7 farms) and wild salmon. Total IgM levels and autoantibodies to salmon blood cell (SBC) extract in sera were measured by ELISA and the relationship between hypergammaglobulinemia and autoantibody production was analyzed. Comparison of endpoint titers vs levels/units using a single dilution of sera in detection of autoantibodies to SBC showed near perfect correlation, justifying the use of the latter for screening. Both total IgM and anti-SBC antibodies are increased in vaccinated salmon compared with unvaccinated controls, however, they do not always correlate well when compared between groups or between individuals, suggesting the involvement of antigen-specific mechanisms in the production of anti-SBC autoantibodies. The primary considerations of successful vaccine for aquaculture are cost-effectiveness and safety. Vaccination-induced autoimmunity in farmed Atlantic salmon may have consequences on future vaccine development and salmon farming strategy. Evaluation for polyclonal hypergamamglobulinemia and autoimmunity should be included as an important trait when vaccine efficacy and safety are evaluated in future.

Involvement of the renin-angiotensin system in the development of vascular damage in a rat model of arthritis: effect of angiotensin receptor blockers.

OBJECTIVE: To explore the involvement of the renin-angiotensin system (RAS) in the development of vascular damage in adjuvant-induced arthritis (AIA) in rats. METHODS: Angiotensin II (Ang II; 0.25 or 1.0 mg/kg/day) was infused in control rats and rats with AIA for 21 days, and the impact of systemic inflammation on Ang II-induced hypertension, endothelial dysfunction, and vascular hypertrophy was evaluated. Expression of angiotensin II type 1 receptor (AT(1)R) and angiotensin-converting enzyme (ACE) in the aortas of rats with AIA were examined by real-time polymerase chain reaction (PCR) and Western blot analyses. Losartan (3 mg/kg/day) or irbesartan (5 mg/kg/day), both of which are AT(1)R blockers, was administered orally to rats with AIA for 21 days. In situ superoxide production in aortas was assessed according to the fluorogenic oxidation of dihydroethidium to ethidium. The expression and activity of NAD(P)H oxidases in aortas were examined by real-time PCR analysis and lucigenin chemiluminescence assay. Endothelial function in rats with AIA treated in vivo or ex vivo with AT(1)R blockers was also determined. RESULTS: The Ang II-induced hypertensive response, endothelial dysfunction, and vascular hypertrophy were exacerbated in rats with AIA. Expression of AT(1)R and ACE was increased in the aortas of rats with AIA. Both losartan and irbesartan decreased the levels of superoxide and the expression and activity NAD(P)H oxidases in the aortas of rats with AIA. The endothelial dysfunction in AIA was improved by the in vivo or ex vivo treatment with AT(1)R blockers. CONCLUSION: The locally activated RAS is involved in the increased vascular oxidative stress and endothelial dysfunction in AIA. Our findings have important implications for clinical approaches to the reduction of cardiovascular risk in patients with rheumatoid arthritis.

Combination of alpha-1 antitrypsin and doxycycline suppresses collagen-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a complex disease characterized by autoimmune inflammation and joint destruction. Despite recent advances in RA treatment, current therapies require further improvement to overcome adverse events and ineffectiveness in some cases. By targeting different pathways/molecules using drug combinations, a better treatment can be obtained, whereas adverse events are reduced. In order to develop a new treatment option, the present study employs a gene therapy-based combination therapy using doxycycline and human alpha-1 antitrypsin (hAAT). METHODS: DBA/1 mice were immunized with type II collagen to induce arthritis. Four weeks before immunization, they received a doxycycline containing diet and a single injection of adeno-associated virus vector expressing hAAT under the control of a tetracycline-dependent promoter. Control groups received doxycycline alone or saline. Macroscopic arthritis development as well as histopathological changes in the joint were evaluated. In addition, the effects of hAAT and doxycycline on lipopolysaccharide (LPS)- or tumor necrosis factor-alpha-induced interleukin (IL)-6 production from mouse fibroblast cells were also determined. RESULTS: Combination therapy significantly reduced arthritis development and progression compared to the control group in respect to macroscopic as well as histopathological changes. Doxycycline and hAAT in combination also inhibited IL-6 expression from LPS-stimulated NIH/3T3 mouse fibroblast cells, indicating a contributing mechanism of arthritis inhibition. CONCLUSIONS: The results obtained in the present study indicate that a combination therapy using AAT and doxycycline holds promising potential as a new therapy for RA.

Azelnidipine exerts renoprotective effects by improvement of renal microcirculation in angiotensin II infusion rats.

BACKGROUND: Hypoxia-induced tubulointerstitial injury caused by loss of peritubular capillary (PTC) blood flow may be associated with progressive renal disease. Therefore, the maintenance of blood flow in PTCs may protect against loss of renal function. A long-acting calcium channel blocker, azelnidipine, has been shown to be useful in the treatment of progressive renal disease. However, its mechanism of action remains unclear. The aim of the present study was to elucidate whether azelnidipine maintains PTC blood flow and to compare it to nifedipine in its ability to improve tubulointerstitial injury caused by angiotensin II (AII) infusion in rats. METHODS: PTC blood flow was initially monitored using a pencil-lens interval microscope before and after intravenous AII (30 ng/kg/min) infusion with or without azelnidipine (10 microg/kg/min). Next, Wistar rats were treated with chronic infusion of AII (500 ng/kg/min) via an osmotic minipump with or without azelnidipine (3 mg/kg/day, orally) or nifedipine (60 mg/kg/day, orally) for 14 days, and tubulointerstitial damage (PTC loss, interstitial fibrosis, tubular atrophy) was examined. RESULTS: PTC blood flow was reduced after AII infusion but improved after a bolus injection of azelnidipine. Tubulointerstitial damage observed in chronically AII-treated kidneys was associated with hypoxic conditions, as indicated by the measurement of hypoxia biomarkers (intracellular hypoxyprobe-1 adducts). These tubulointerstitial injuries in AII-infused rats were more effectively reduced by azelnidipine than by nifedipine. The area showing hypoxic conditions in the kidney was also more reduced with azelnidipine than nifedipine treatment. CONCLUSIONS: Azelnidipine may increase PTC blood flow and improve renal hypoxia and tubulointerstitial injury induced by AII infusion.

Vaccination-induced systemic autoimmunity in farmed Atlantic salmon.

Over half of the salmon consumed globally are farm-raised. The introduction of oil-adjuvanted vaccines into salmon aquaculture made large-scale production feasible by preventing infections. The vaccines that are given i.p. contain oil adjuvant such as mineral oil. However, in rodents, a single i.p. injection of adjuvant hydrocarbon oil induces lupus-like systemic autoimmune syndrome, characterized by autoantibodies, immune complex glomerulonephritis, and arthritis. In the present study, whether the farmed salmon that received oil-adjuvanted vaccine have autoimmune syndrome similar to adjuvant oil-injected rodents was examined. Sera and tissues were collected from vaccinated or unvaccinated Atlantic salmon (experimental, seven farms) and wild salmon. Autoantibodies (immunofluorescence, ELISA, and immunoprecipitation) and IgM levels (ELISA) in sera were measured. Kidneys and livers were examined for pathology. Autoantibodies were common in vaccinated fish vs unvaccinated controls and they reacted with salmon cells/Ags in addition to their reactivity with mammalian Ags. Diffuse nuclear/cytoplasmic staining was common in immunofluorescence but some had more specific patterns. Serum total IgM levels were also increased in vaccinated fish; however, the fold increase of autoantibodies was much more than that of total IgM. Sera from vaccinated fish immunoprecipitated ferritin and approximately 50% also reacted with other unique proteins. Thrombosis and granulomatous inflammation in liver, and immune-complex glomerulonephritis were common in vaccinated fish. Autoimmunity similar to the mouse model of adjuvant oil-induced lupus is common in vaccinated farmed Atlantic salmon. This may have a significant impact on production loss, disease of previously unknown etiology, and future strategies of vaccines and salmon farming.

Fluvastatin reverses endothelial dysfunction and increased vascular oxidative stress in rat adjuvant-induced arthritis.

OBJECTIVE: To investigate the effect of statins on vascular dysfunction in rat adjuvant-induced arthritis (AIA). METHODS: Fluvastatin (5 mg/kg/day) was administered orally to rats with AIA, for 21 days after the onset of arthritis. The vasodilatory response to acetylcholine of aortic rings isolated from rats with AIA that were not treated or were treated with fluvastatin and from normal rats was determined. The amounts of 4-hydroxy-2-nonenal (HNE) and nitrotyrosine in aortas were measured by Western blotting. In vitro and in situ superoxide production in aortas was evaluated based on fluorogenic oxidation of dihydroethidium to ethidium. Expression of NAD(P)H components and endothelial nitric oxide synthase (eNOS) in aortas was examined by real-time reverse transcriptase-polymerase chain reaction and Western blotting. Serum levels of tetrahydrobiopterin, a critical eNOS cofactor, were determined by high-performance liquid chromatography. RESULTS: Fluvastatin reversed endothelial dysfunction in AIA without affecting the clinical severity of arthritis or serum cholesterol concentration. Fluvastatin reduced the amounts of HNE and nitrotyrosine in the aorta, and the levels of superoxide expressed in endothelial cells and smooth muscle cells in the tissue, in rats with AIA. NADH- or L-arginine-induced superoxide production was not observed in the aortic samples from fluvastatin-treated rats with AIA. Fluvastatin decreased the levels of expression of messenger RNA for p22phox, a NAD(P)H oxidase component, in the aortas of rats with AIA, but did not affect the expression of eNOS. Serum levels of tetrahydrobiopterin were significantly reduced in rats with AIA, and were increased by administration of fluvastatin. CONCLUSION: Our findings demonstrate that fluvastatin has potent vascular protective effects in AIA and provide additional scientific rationale for the use of statins to reduce cardiovascular mortality in patients with rheumatoid arthritis.

Induction of lupus-related specific autoantibodies by non-specific inflammation caused by an intraperitoneal injection of n-hexadecane in BALB/c mice.

A single intraperitoneal (i.p.) injection of pristane, incomplete Freund's adjuvant (IFA), or the adjuvant oil squalene, but not high molecular weight medicinal mineral oils, induces lupus-related autoantibodies to nRNP/Sm and -Su in non-autoimmune strains of mice. This ability appears to be associated with the low molecular weight and adjuvanticity of hydrocarbon. n-Hexadecane (C(16)H(34)), which is present in petroleum, has adjuvant activity and induces arthritis in rodents like other lupus-inducing oils. In addition to dietary exposure to n-hexadecane in mineral oils, exposure also occurs via inhalation of oil mist, jet fuel, or diesel exhaust or by absorption through the skin. Since n-hexadecane is a low molecular weight adjuvant hydrocarbon oil similar to other lupus-inducing hydrocarbons, the present study examined whether it can also induce lupus-related autoantibodies in mice. Female BALB/cJ mice received a single i.p. injection of 0.5 ml of n-hexadecane, pristane, or saline (control). Pathology and serology (immunoglobulin levels, autoantibodies by immunofluorescence, immunoprecipitation, and ELISA) were examined 3 months later. Unexpectedly, all n-hexadecane-treated mice, but none in the other groups, developed inflammatory ascites within 2.5 months. n-Hexadecane induced hypergammaglobulinemia (IgG1, IgG2a), antinuclear (titer>1:160, 67%) and -cytoplasmic antibodies (58%) and autoantibodies to nRNP/Sm (25%), Su (33%), ssDNA (83%), and chromatin (100%). Therefore, non-specific inflammation caused by n-hexadecane resulted in the production of a limited set of specific autoantibodies. These previously unrecognized immunological effects of n-hexadecane may have implications in monitoring human exposure to hydrocarbons and in the pathogenesis of autoimmune diseases.

Autoimmunity induced by adjuvant hydrocarbon oil components of vaccine.

Adjuvant oils such as Bayol F (Incomplete Freund's adjuvant: IFA) and squalene (MF59) have been used in human and veterinary vaccines despite poor understanding of their mechanisms of action. Several reports suggest an association of vaccination and various autoimmune diseases, however, few were confirmed epidemiologically and the risk of vaccination for autoimmune diseases has been considered minimal. Microbial components, not the adjuvant components, are considered to be of primary importance for adverse effects of vaccines. We have reported that a single intraperitoneal injection of the adjuvant oils pristane, IFA or squalene induces lupus-related autoantibodies to nRNP/Sm and -Su in non-autoimmune BALB/c mice. Induction of these autoantibodies appeared to be associated with the hydrocarbon's ability to induce IL-12, IL-6, and TNF-alpha, suggesting a relationship with hydrocarbon's adjuvanticity. Whether this is relevant in human vaccination is a difficult issue due to the complex effects of vaccines and the fact that immunotoxicological effects vary depending on species, route, dose, and duration of administration. Nevertheless, the potential of adjuvant hydrocarbon oils to induce autoimmunity has implications in the use of oil adjuvants in human and veterinary vaccines as well as basic research.

Distinctive patterns of autoimmune response induced by different types of mineral oil.

Although mineral oils are generally considered nontoxic and have a long history of use in humans, the mineral oil Bayol F (incomplete Freund's adjuvant, IFA) and certain mineral oil components (squalene and n-hexadecane) induce lupus-related anti-nRNP/Sm or -Su autoantibodies in nonautoimmune mice. In the present study, we investigated whether medicinal mineral oils can induce other types of autoantibodies and whether structural features of hydrocarbons influence autoantibody specificity. Female 3-month-old BALB/c (16-45/group) mice each received an i.p. injection of pristane (C19), squalene (C30), IFA, three medicinal mineral oils (MO-F, MO-HT, MO-S), or PBS. Sera were tested for autoantibodies and immunoglobulin levels. Hydrocarbons were analyzed by gas chromatography/mass spectrometry. IFA contained mainly C15-C25 hydrocarbons, whereas MO-HT and MO-S contained C20-C40, and MO-F contained C15-C40. Pristane and n-hexadecane were found in IFA (0.17% and 0.10% w/v, respectively) and MOs (0.0026-0.027%). At 3 months, pristane and IFA induced mainly IgG2a, squalene IgG1, and MOs IgG3 and IgM in sera. Anti-cytoplasmic antibodies were common in mice treated with MO-F, as well as those treated with pristane, squalene, and IFA. Anti-ssDNA and -chromatin antibodies were higher in MO-F and MO-S than in untreated/PBS, squalene-, or IFA-treated mice, suggesting that there is variability in the induction of anti-nRNP/Sm versus -chromatin/DNA antibodies. The preferential induction of anti-chromatin/ssDNA antibodies without anti-nRNP/Sm/Su by MO-S and MO-F is consistent with the idea that different types of autoantibodies are regulated differently. Induction of autoantibodies by mineral oils considered nontoxic also may have pathogenetic implications in human autoimmune diseases.

Induction of lupus autoantibodies by adjuvants.

Exposure to the hydrocarbon oil pristane induces lupus specific autoantibodies in non-autoimmune mice. We investigated whether the capacity to induce lupus-like autoimmunity is a unique property of pristane or is shared by other adjuvant oils. Seven groups of 3-month-old female BALB/cJ mice received a single intraperitoneal injection of pristane, squalene (used in the adjuvant MF59), incomplete Freund's adjuvant (IFA), three different medicinal mineral oils, or saline, respectively. Serum autoantibodies and peritoneal cytokine production were measured. In addition to pristane, the mineral oil Bayol F (IFA) and the endogenous hydrocarbon squalene both induced anti-nRNP/Sm and -Su autoantibodies (20% and 25% of mice, respectively). All of these hydrocarbons had prolonged effects on cytokine production by peritoneal APCs. However, high levels of IL-6, IL-12, and TNFalpha production 2-3 months after intraperitoneal injection appeared to be associated with the ability to induce lupus autoantibodies. The ability to induce lupus autoantibodies is shared by several hydrocarbons and is not unique to pristane. It correlates with stimulation of the production of IL-12 and other cytokines, suggesting a relationship with a hydrocarbon's adjuvanticity. The potential to induce autoimmunity may complicate the use of oil adjuvants in human and veterinary vaccines.