Pathoimmune polymyositis induced in C3H/HeJ mice by Trypanosoma cruzi infection

The pathogenic mechanisms related to the development of idiopathic inflammatory skeletal muscle disease are unknown. The myotropic protozoa Trypanosoma cruzi and Toxoplasma gondii are implicated in the induction of myositis in experimental animals (1) and humans (2). In the experiments reported here, a model of pathoimmune myositis is described in C3H/HeJ T. cruzi-infecteéd mice. The results showed a significant occurrence of acquired, T. cruzi antigen-dependent spleen T cell cytotoxicity to syngeneic skeletal muscle myoblasts of C3H mice which developed an apparently sterile lymphoid polymyositis. Further experiments with polymyositic C3H mice suggest that spleen B cells do not secrete antibody capable of inducing complement-mediated skeletal muscle myoblast lysis. However, the T. cruzi sensitized splenic lymphocytes did produce a lymphokine which was capable of inducing lysis of syngeneic myoblasts by chromium-51 release assay.     

Caspase-1 deficiency decreases atherosclerosis in apolipoprotein E-null mice

Background

Caspase-1 is a cysteine protease that contributes to mammalian immunity through proteolytic activation of the proinflammatory cytokines, interleukin (IL)-1β and IL-18.

Methods

To determine if caspase-1 deficiency can protect apolipoprotein E-null (Apoe^−/−) mice from atherosclerosis, gender-matched, paired-littermate Apoe^−/− mice with (Casp1^+/+Apoe^−/−) or without (Casp1^−/−Apoe^−/−) a functional caspase-1 (Casp1) gene were fed either a low fat diet for 26 weeks, or a saturated fat and cholesterol-enriched diet for 8 weeks. Plasma lipids and lipoproteins were determined and atherosclerosis was quantified in the aortic sinus and aortic arch.

Results

On either diet, caspase-1 deficiency did not affect total serum cholesterol concentrations and lipoprotein-cholesterol distributions. However, caspase-1 deficiency significantly decreased atherosclerosis in the ascending aorta by 35%-45% in both sexes of mice fed either diet. We further examined atherosclerotic lesions for 2 indices of immune cell activation: Major Histocompatibility Complex (MHC) class II and interferon (IFN)-γ expression. There was a 40%-50% reduction in the number of lesion-associated cells expressing MHC class II from both sexes of Casp1^−/−Apoe^−/− mice compared with Casp1^+/+Apoe^−/− mice and, a significant reduction in lesion-associated IFN-γ in female Casp1^−/−Apoe^−/− compared with their Casp1^+/+Apoe^−/− counterparts.

Conclusions

We conclude that caspase-1 promotes atherosclerosis by enhancing the inflammatory status of the lesion through a mechanism likely involving activation of lesion-associated immune cells and IFN-γ expression.     

Cystatin C deficiency increases elastic lamina degradation and aortic dilatation in apolipoprotein E-null mice

The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves substantial proteolysis of the arterial extracellular matrix. The lysosomal cysteine proteases can exert potent elastolytic and collagenolytic activity. Human atherosclerotic plaques have increased cysteine protease content and decreased levels of the endogenous inhibitor cystatin C, suggesting an imbalance that would favor matrix degradation in the arterial wall. This study tested directly the hypothesis that impaired expression of cystatin C alters arterial structure. Cystatin C-deficient mice (Cyst C-/-) were crossbred with apolipoprotein E-deficient mice (ApoE-/-) to generate cystatin C and apolipoprotein E-double deficient mice (Cyst C-/-ApoE-/-). After 12 weeks on an atherogenic diet, cystatin C deficiency yielded significantly increased tunica media elastic lamina fragmentation, decreased medial size, and increased smooth muscle cell and collagen content in aortic lesions of ApoE-/- mice. Cyst C-/-ApoE-/- mice also showed dilated thoracic and abdominal aortae compared with control ApoE-/- mice, although atheroma lesion size, intimal macrophage accumulation, and lipid core size did not differ between these mice. These findings demonstrate directly the importance of cysteine protease/protease inhibitor balance in dysregulated arterial integrity and remodeling during experimental atherogenesis.     

Royal Jelly prolongs the life span of C3H/HeJ mice: correlation with reduced DNA damage

In this study, we investigate the effect of dietary Royal Jelly (RJ) on tissue DNA oxidative damage and on the life span of C3H/HeJ mice. In C3H/HeJ mice that were fed a dietary supplement of RJ for 16 weeks, the levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative stress, were significantly reduced in kidney DNA and serum. Secondly, we determined the effect of dietary RJ on the life span in C3H/HeJ mice. The 50% mice survivals of intermediate- (about 6 mg/kg weight) and high-dose groups (about 60 mg/kg weight) were reached at significantly longer times than that of the control group according to the generalized Wilcoxon test (p<0.05). The average survival times were 88 weeks for the control group vs. 79 weeks for the low-dose group (about 0.6 mg/kg weight), 112 weeks for the intermediate-dose group and 110 weeks for the high-dose group, respectively, showing that RJ extended the average survival time by about 25% compared to the control group. However, RJ did not extend the total life span. These results indicated that dietary RJ increased the average life span of C3H/HeJ mice, possibly through the mechanism of reduced oxidative damage.     

Dermal oncogenicity study of 2-ethylhexyl acrylate by epicutaneous application in male C3H/HeJ mice

Summary A carcinogenicity bioassay of 2-ethylhexyl acrylate (2-EHA) was conducted by applying 25 l 86.5%, 21%, or 2.5% 2-EHA in acetone three times a week to the clipped dorsal skin of male C2H/HeJ mice (80 per group) over their lifetime. Another group was treated with a 43% 2-EHA solution for 24 weeks and thereafter observed for lifetime (stop-test). An untreated group and a group that received only the diluent acetone served as controls. Treatment-related changes in the skin indicative of irritation (scaling, scabbing, hyperkeratosis, hyperplasia) were found in all 2-EHA-treated groups. These lesions were reversible in the 43% group immediately after treatment was stopped, and in the 2.5% group after the 11 th week of treatment. Only in the 86.5% and 21% test groups showing chronic irritative skin damage was there a high incidence of nepolastic skin lesions (papillomas, carcinomas, and melanomas) with no dose dependency. In contrast, no skin tumors were found in the control groups, in the group treated with 2.5% 2-EHA for lifetime or in the group treated with 43% 2-EHA for about 6 months and observed for lifetime.Supported by BASF Aktiengesellschaft, BASF Corporation, Celanese Corporation, Rohm and Haas Company, and Union Carbide Corporation     

Inheritance of susceptibility to induced Escherichia coli bladder and kidney infections in female C3H/HeJ mice

In the present study, the inheritance of resistance and susceptibility to bladder and kidney infections in BALB/c, C3H/HeJ, F(1), and backcross mice was investigated, and the number of genes contributing to the phenotypes was estimated. Infections were induced in female mice by intravesical inoculation with Escherichia coli, and the number of bacteria in bladder and kidneys was quantified at 10 days. The (BALB/c x C3H/HeJ) F(1) mice had bladder and kidney infection intensities equivalent to those observed in the resistant BALB/c parents. Twelve percent of the (F(1) x C3H/HeJ) backcross mice had severe bladder infections, similar to the susceptible C3H/HeJ parents. Kidney infections ranging in intensity between those observed in BALB/c and C3H/HeJ parents were present in one-half of the backcross mice. Statistical analyses indicated that >/=1 gene is responsible for the increased susceptibility of C3H/HeJ mice and that the trait appears to be recessive.     

Lipopolysaccharide (LPS)-induced cell death of C3H mouse peritoneal macrophages in the presence of cycloheximide: different susceptibilities of C3H/HeN and C3H/HeJ mice macrophages

Lipopolysaccharide (LPS) induced cytotoxicity toward mouse peritoneal macrophages from C3H/HeN mice but not C3H/HeJ mice in vitro in the presence of cycloheximide (CHX). More than 1 ng/ml LPS induced a significant time-dependent release of a cytoplasmic enzyme, lactate dehydrogenase (LDH), while even 1000 ng/ml LPS failed to induce it in LPS-non-responsive C3H/HeJ mouse macrophages. Although similar LPS-induced cytotoxicity was observed in a murine macrophage-like cell line, J774.1, but not in an LPS-resistant mutant of J774.1, the LPS1916 cell line, these results suggest that the induction of this cytotoxicity is linked to the LPS-sensitivity of mouse macrophages. A recombinant TNF-alpha (rTNF-alpha) at 100 ng/ml augmented LDH release from both C3H/HeN and C3H/HeJ macrophages treated with LPS and CHX, while rTNF-alpha alone or in combination with LPS or CHX failed to induce LDH release. These results suggest that this cytotoxicity might be partially regulated by high concentrations of exogenous TNF-alpha in both C3H/HeN and C3H/HeJ macrophages, implying a possibility of paracrine regulation of TNF-alpha in mice toward LPS-treated macrophages under impaired protein synthesis.     

Accelerated atherosclerosis in C57Bl/6 mice transplanted with ApoE-deficient bone marrow

Apolipoprotein E (apoE), a high affinity ligand for lipoprotein receptors, is synthesized by the liver and extrahepatic tissues, including cells of the monocyte/macrophage cell lineage. The role of monocyte/macrophage-derived apoE in atherogenesis was assessed by transplantation of apoE-deficient (apoE-/-) bone marrow into normolipidemic C57Bl/6 mice. No significant effect could be demonstrated on serum apoE levels in C57Bl/6 mice, transplanted with apoE-deficient bone marrow compared with control transplanted mice. Furthermore, no consistent effect on serum cholesteryl esters and triglyceride concentrations could be demonstrated on either a standard chow diet or a high cholesterol diet. Quantitative analysis of atherosclerosis in mice transplanted with apoE-deficient bone marrow, after two months on a high cholesterol diet, revealed a 4-fold increase in the atherosclerotic lesion area as compared to animals transplanted with apoE+/+ bone marrow. Analysis of the ability of apoE-deficient macrophages to release cholesterol after loading with acetylated LDL revealed that the release of cholesterol from apoE-deficient macrophages was impaired as compared to wild-type macrophages in the absence and the presence of specific cholesterol acceptors. In conclusion, apoE production by macrophages retards the formation of atherosclerotic plaques, possibly by mediating cholesterol efflux. We anticipate that pharmacological approaches to increase apoE synthesis and/or secretion by macrophages might be beneficial for the treatment of atherosclerosis.     

Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice

A prior study in mice has shown that the timely application of topical antibiotics to the skin at the tick bite site could eradicate Borrelia burgdorferi infection. That study, however, did not evaluate antibiotic preparations that are considered suitable for use in humans. In this murine study, topical application of 2% erythromycin and 3% tetracycline preparations that are acceptable for use in humans was found to be ineffective in eliminating B. burgdorferi from the tick bite site or in preventing dissemination to other tissues. Reasons for the discrepant findings are discussed.     

Mitogenic response of lymphocytes from C3H/HeJ mice in the presence of lipid A and lipid A fractions

Purified lipid A and eight fractions of lipid A were examined for the ability to elicit a mitogenic response in spleen lymphocytes of endotoxin-resistant C3H/HeJ mice. The pattern of results obtained with different lipid A fractions was similar to that observed with spleen cells from the endotoxin-responsive C57Bl/6J mouse. Although the absolute mitogenic response of C3H/HeJ lymphocytes to lipid A was diminished, the cells apparently were completely normal with respect to the pattern of relative mitogenic responses induced by purified fractions of lipid A.     

Lps(d)/Ran of endotoxin-resistant C3H/HeJ mice is defective in mediating lipopolysaccharide endotoxin responses.

C3H/HeJ inbred mice are defective in that they are highly resistant to endotoxic shock as compared with normal responder mice. Their B cells and macrophages do not respond significantly when exposed to lipopolysaccharide (LPS), whereas cells from the responder mice do. Using a functional assay, we previously isolated a cDNA, which encodes for Ran/TC4 GTPase. We now show that this gene is mutated in C3H/HeJ mice, which accounts for their resistance to endotoxin stimulation. Sequence analysis of independent mutant Lps(d)/Ran cDNAs isolated from splenic B cells of C3H/HeJ mice reveals a consistent single base substitution at position 870, where a thymidine is replaced with a cytidine. In situ hybridization maps the Lps(d)/Ran cDNA to mouse chromosome 4. By retroviral gene transfer, the wild-type Lps(n)/Ran cDNA but not the mutant Lps(d)/Ran cDNA can restore LPS responsiveness of C3H/HeJ cells. Adenoviral gene transfer in vivo with the mutant Lps(d)/Ran cDNA but not the wild-type Lps(n)/Ran cDNA rescues endotoxin-sensitive mice from septic shock. Thus Lps/Ran is an important target for LPS-mediated signal transduction, and the Lps(d)/Ran gene may be useful as a therapeutic sequence in gene therapy for endotoxemia and septic shock.     

Staphylococcal enterotoxin B induces hepatic injury and lethal shock in endotoxin-resistant C3H/HeJ mice despite a deficient macrophage response

Bacterial toxins, including endotoxin/LPS as well as superantigens, are major causative agents of multi-organ failure associated with sepsis and liver disease. However, the precise mechanisms initiating cell activation by the toxins have not been clarified. We compared lethal shock and cytokine production in response to LPS with responses to the superantigen staphylococcal enterotoxin B (SEB) in both LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice treated with D-galactosamine (GalN). LPS was not lethal and did not induce production of TNF-alpha in C3H/HeJ mice. In contrast, SEB produced lethal shock associated with liver failure and induced cytokines such as TNF-alpha, IFN-gamma, and IL-2 in both C3H/HeN and C3H/HeJ mice. Peritoneal macrophages from C3H/HeJ mice did not produce TNF-alpha in vitro in response to SEB or LPS. However, no significant difference was observed in production of TNF-alpha in response to stimulation in vitro by SEB between C3H/HeN and C3H/HeJ splenic lymphocytes. We have demonstrated that SEB causes lethal toxicity associated with liver injury in LPS-hyporesponsive C3H/HeJ mice and that as the underlying mechanism, the normal T-cell function in these mice still maintained the sensitivity to SEB since the genetic defect of C3H/HeJ mice unresponsive to LPS and SEB is restricted in macrophages/monocytes and does not extend to T cells.     

Passive immunization with monoclonal IgM antibodies against phosphorylcholine reduces accelerated vein graft atherosclerosis in apolipoprotein E-null mice

Phosphorylcholine (PC) headgroup is one of the neoantigens exposed by LDL oxidation that can elicit an immune response. Active immunization with Streptococcus pneumoniae, which bears PC on its cell wall, reduced atherosclerosis in hypercholesterolemic mice and this effect was attributed to an immune response to PC. In this study we tested the hypothesis that passive immunization with a monoclonal anti-PC IgM antibody can be athero-protective in a murine model of native aortic and vein graft atherosclerosis. Inferior vena cava from 16-week-old donor male apoE-null mice was grafted into right carotid artery of age-matched male recipient apoE-null mice. Anti-PC IgM titers were evaluated before and 4 weeks after surgery. For the immunization protocol, a separate group of mice received weekly intraperitoneal injection of monoclonal anti-PC IgM (400 microg) for 4 weeks, starting the day of surgery. Controls received PBS or pooled polyclonal IgM. Anti-PC IgM titres significantly increased at 4 weeks following surgery. Passive immunization with anti-PC IgM reduced vein graft plaque size and neointimal thickness resulting in a larger luminal area; in addition immunization reduced the inflammatory cell content of the plaques. There was no significant effect on the established native aortic atherosclerotic lesions. Immunization did not affect circulating cholesterol levels. Taken together our data suggest that passive immunization with anti-PC IgM significantly reduces vein graft lesion size with less inflammatory phenotype without affecting cholesterol levels, indicating an athero-protective immune response to PC. Lack of effect on established native aortic lesions may have been due to short duration of therapy and/or reduced efficacy in established lesions as compared to evolving lesions of vein graft atherosclerosis.     

Rapid reversal of endothelial dysfunction in hypercholesterolemic apolipoprotein E-null mice by recombinant apolipoprotein A-I(Milano)-phospholipid complex

OBJECTIVES: In this study, we examined whether a reconstituted high-density lipoprotein (HDL) utilizing recombinant apolipoprotein A-I(Milano) (apo A-I(M))/phospholipid complex (PC) could restore normal endothelial function in hypercholesterolemic apolipoprotein (apo) E-null mice. BACKGROUND: We have previously shown antiatherosclerotic and vasculoprotective effects of recombinant apo A-I(M). METHODS: A perfused vessel preparation was used to examine vascular responses in control wild-type, untreated, and treated apo E-null mice. Aortic tissue cholesterol content and platelet aggregation were also measured. RESULTS: Endothelium-dependent vasodilator responses to acetycholine were significantly inhibited in untreated apo E-null mice compared with control wild-type mice (p < 0.001). Treatment of the mice for five weeks with once every-other-day intravenous bolus injections of apo A-I(M)/PC restored endothelium-dependent dilation in a dose-dependent manner (p < 0.01 at 80 mg/kg dose). The improvement in endothelial function was associated with a reduction in aortic cholesterol content and reduced platelet aggregability and occurred despite severe and persistent hypercholesterolemia. Neither treatment with free protein nor phospholipid carrier alone produced any significant effects. We performed additional experiments in vitro in isolated rabbit carotid arteries to compare the effects on lysophosphatidylcholine (LPC)-induced endothelial dysfunction. Treatment with apo A-I(M)/PC prevented impairment of endothelium-dependent vasodilator responses to acetylcholine to a greater degree than either wild-type apo A-I or plasma-derived HDL. CONCLUSIONS: Our results indicate a rapid improvement in endothelial dysfunction with recombinant apo A-I(M)/PC that is associated with mobilization of tissue cholesterol. Taken together with previously established antiatherosclerotic and antithrombotic effects, these findings suggest significant vasculoprotective effects with apo A-I(M)/PC therapy.