Amelioration of myocarditis by statin through inhibiting cross-talk between antigen presenting cells and lymphocytes in rats.

Statins, inhibitors of 3-hydroxy-3-methylglutary-coenzyme A (HMG-CoA) reductase, have been recognized as a new type of immunomodulator and reported to have anti-inflammatory effect. To investigate the effect of simvastatin, a lipophilic statin, on myocarditis, we explored whether simvastatin is able to inhibit experimental autoimmune myocarditis (EAM) and adoptive transfer of EAM in rats. We found that administration of simvastatin not only interfered with the development of EAM, but also inhibited the transfer. Antigen presenting cells (APCs) were proved to be important for the development of EAM. The ability of myocarditic splenocytes to transfer myocarditis was enhanced after co-culture with APCs. During co-culture of the myocarditic splenocytes and the APCs, simvastatin not only decreased percentages of CD28 expression in CD4-positive myocarditic splenocytes, and CD80 and CD86 expressions in APCs, but also inhibited the production of tumor necrosis factor (TNF)-partial differential in the CD4-positive myocarditic splenocytes and the APCs. These results indicate that simvastatin was able to ameliorate EAM through the inhibition of cross-talk between lymphocytes and APCs, suggesting beneficial role of simvastatin in the treatment of autoimmune myocarditis.

Multiple shRNA expressions in a single plasmid vector improve RNAi against the XPA gene.

To improve the efficiency of stable knockdown with short hairpin RNA (shRNA), we inserted multiple shRNA expression sequences into a single plasmid vector. In this study, the DNA repair factor XPA was selected as a target gene since it is not essential for cell viability and it is easy to check the functional knockdown of this gene. The efficiency of knockdown was compared among single and triple expression vectors. The single shRNA-expressing vector caused limited knockdown of the target protein in stable transfectants, however, the multiple expression vectors apparently increased the frequency of knockdown transfectants. There were correlations between the knockdown level and marker expression in multiple-expressing transfectants, whereas poorer correlations were observed in single vector transfectants. Multiple-transfectants exhibited reduced efficiency of repair of UV-induced DNA damage and an increased sensitivity to ultraviolet light-irradiation. We propose that multiple shRNA expression vectors might be a useful strategy for establishing knockdown cells.

Involvement of thymus in amiodarone-treated autoimmune myocarditis in rats.

We previously showed that amiodarone, an iodine-rich benzofuranic derivative, interferes with the progression of myocarditis using a rat model of experimental autoimmune myocarditis. Further studies have also revealed that intraperitoneal treatment with 12.5 mg/kg amiodarone, which is below the range of its therapeutic plasma concentration, also effectively interferes with the progression of myocarditis using the same model. The relationship between myocarditis and the thymus has not been studied in either patients or animal models. To investigate whether the thymus is involved in the effect of amiodarone on experimental autoimmune myocarditis, we examined its phenotypic distribution in thymocytes and peripheral blood lymphocytes using flow cytometry. We found no significant differences in the proportions of CD4(+) and CD8(+) T cells and the CD4(+)/CD8(+) T cell ratio in the control group compared with amiodarone-treated group. However, amiodarone-treated group induced a decrease in the proportion of CD4(+)TNF(+) and CD4(+)IL-4(+) T cells and an increase in CD4(+)IFN(+) T cells, resulting in a significant reduction of the CD4(+)TNF(+)/CD4(+)IFN(+) and CD4(+)IL-4(+)/CD4(+)IFN(+) T cell ratios. These results suggest for the first time that the thymus is actively involved when myocarditis is treated with amiodarone.

Pathological importance of anti-G-protein coupled receptor autoantibodies.

Anti-cardiac receptor autoantibodies have been studied intensively last 10 years. A growing evidence, both in vitro and in vivo and both clinical and experimental studies, have supported that these anti-cardiac receptor autoantibodies, mainly anti-beta1 adrenoceptor autoantibodies, play a very important pathophysiological role, at least partly, in the development of idiopathic dilated cardiomyopathy. This concise review article will only summarize studies done in rabbits with consideration of other articles in this focused issue.

Specific removal of beta1-adrenoceptor autoantibodies by immunoabsorption in rabbits with autoimmune cardiomyopathy improved cardiac structure and function.

Growing evidence suggests that the beta1-adrenoceptor-directed autoimmune mechanism may play an important role in the pathogenesis of idiopathic dilated cardiomyopathy. The aim of this study is to further study the effect of specific immunoabsorption of anti-beta1-adrenoceptor autoantibodies on cardiac structure and function in autoimmune cardiomyopathy in rabbits. Twenty-four male rabbits were divided into 2 groups: (1) one immunized with beta1-adrenoceptor peptide (beta1 group, n=16), and (2) the other receiving saline injection as a control (control group, n=8). Immunization was performed once a month for 8 months. A high concentration of anti-beta1-adrenoceptor autoantibodies was exhibited throughout the immunization period. Rabbits in the beta1 group showed increased left ventricular end-diastolic diameter (LVDd), decreased left ventricular ejection fraction (LVEF) and increased LV mass/body weight ratio after the 8th month. Immunoabsorption with beta1-adrenoceptor peptide column was able to remove up to 35% of anti-beta1-adrenoceptor autoantibodies in 2 h, resulting in decreased LVDd and increased LVEF 3 months after. Specific removal of anti-beta1-adrenoceptor autoantibodies improved cardiac structure and function in experimental autoimmune cardiomyopathy. These results suggest that anti-beta1-adrenoceptor autoantibodies are of pathogenic importance in the induction of cardiomyopathy, and that specific immunoabsorption as an emerging therapy may be considered when anti-beta1-adrenoceptor autoantibodies are pathophysiologically involved.

Transfer of immune components from rabbit autoimmune cardiomyopathy into severe combined immunodeficiency (SCID) mice induces cardiomyopathic changes.

BACKGROUND: Growing evidence suggests that autoimmune mechanism plays an important role in the pathogenesis of cardiomyopathy. The purpose of this study was to investigate whether passive transfer of IgG and/or lymphocytes from rabbits with autoimmune cardiomyopathy is able to reproduce cardiomyopathic changes in severe combined immunodeficiency (SCID) mice. METHODS AND RESULTS: SCID mice were injected intraperitoneally with IgG and/or peripheral blood lymphocytes (PBL) from either rabbits immunized with both beta1-adrenoceptor peptide and M2-muscarinic receptor peptide (beta1+M2 group) or rabbits with adjuvant (N group). Thirty five SCID mice were divided into seven groups; N-IgG, N-PBL, N-IgG & PBL, (beta1+M2)-IgG, (beta1+M2)-PBL, (beta1+M2)-IgG & PBL and control groups. Heart weight in three (beta1+M2) groups were significantly increased. All mice in three (beta1+M2) groups showed high titer of rabbit anti-beta1 adrenoceptor autoantibodies, and 4 mice in the (beta1+M2)-PBL group and 3 mice in the (beta1+M2)-IgG & PBL group showed a significant increase in titer of rabbit anti-M2-muscarinic receptor autoantibodies. Focal infiltration of inflammatory cells in the myocardium was observed in the (beta1+M2)-IgG & PBL group. In the (beta1+M2)-PBL group and (beta1+M2)-IgG & PBL group, cardiomyocyte diameters were significantly increased. Some myocytes of the (beta1+M2)-IgG & PBL group exhibited intracellular edema, clumps of Z-band and increased numbers of mitochondria by using electron microscopy. CONCLUSION: Transfer of IgG and PBL from rabbits immunized with combined beta1 and M2 peptides was able to reproduce the early stage of cardiomyopathic changes in SCID mice.

Imbalance of sex hormone levels in men with coronary artery disease.

OBJECTIVE: Sex hormones are thought to play a key role in atherogenesis, but the available evidence is inconclusive, partly because of a lack of accuracy in measurement. The aim of the study was to investigate the potential role of sex hormones in coronary atherosclerosis. METHODS: We prospectively applied a simple highly-sensitive method using solid-phase extraction followed by radioimmunoassay. Both phases were carried out using commercially available kits to determine levels of estradiol (E2). We also measured the levels of free testosterone (FT), dehydroepiandrosterone sulfate, luteinizing hormone, follicle-stimulating hormone, and progesterone in 236 consecutive male patients with angiographically-defined stable coronary artery disease and in 143 disease-free and age-matched controls. RESULTS: The levels of highly-sensitive E2 and FT in patients and controls differed slightly in opposing directions, but neither difference reached statistical significance. However, the ratio of FT to highly-sensitive E2 in patients was significantly higher than in the controls (mean +/- SD; 2.50 +/- 1.89 versus 2.06 +/- 1.14, P = 0.018), and this difference remained significant after adjustments for age and body mass index had been made. Multiple regression analysis revealed that age, the association of diabetes, and the presence of coronary atherosclerosis were significantly and independently associated with the values of the FT/highly-sensitive E2 ratio. Other hormones examined did not differ significantly between the patients and the controls. CONCLUSIONS: Highly-sensitive E2 measurement demonstrated a significant imbalance of FT to E2 in male patients with coronary artery disease, but individual sex hormone levels did not differ between the patients and the controls.

Transient glucose deprivation causes upregulation of heme oxygenase-1 and cyclooxygenase-2 expression in cardiac fibroblasts.

Transient glucose deprivation (TGD) has been shown to induce a resistance to a subsequent ischemia and reperfusion injury in the heart. Induction of cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1) is known to mediate the powerful defensive adaptation of the heart against oxidative stress. In this study, we found that a 30-min incubation in the absence of glucose resulted in a rapid increased expression of COX-2 and HO-1 in cardiac fibroblasts as examined by real-time quantitative polymerase chain reaction (PCR) and western blot analysis. Interestingly, TGD increased the generation of reactive oxygen species (ROS) and caused the transient phosphorylation of p38 mitogen-activated protein kinase (MAPK) as well as the translocation of protein kinase C (PKC)- from the cytosolic to the membrane fraction. However, no significant change in the distribution of PKC-delta isoform was observed compared with the control. Pretreatment of the cells with an antioxidant, N-acetylcysteine (NAC), resulted in the inhibition of p38 MAPK phosphorylation and PKC- translocation during TGD. In addition, the induction of COX-2 and HO-1 expression by TGD was prevented by pretreatment with NAC or SB203580, a p38 MAPK inhibitor. Surprisingly, pretreatment with chelerythrine, an inhibitor of PKC, strongly augmented the HO-1 mRNA expression but blocked the COX-2 mRNA induction by TGD. These results demonstrate that briefly removing glucose from cultured cardiac fibroblasts induces COX-2 and HO-1 expression via generation of ROS and p38 MAPK phosphorylation, while the translocation of PKC- to the membrane fraction may participate in the induction of COX-2 but not in the HO-1 expression.

Phorbol myristate induces apoptosis of taxol-resistant sarcoma cells in vitro.

Taxol was found to induce polyploidization and apoptosis in cultured methylcholanthrene-induced sarcoma cells (Meth-A cells), but some of the cells in G1 phase were not affected. We refer to these cells as taxol-resistant cells. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) regulator, was used to test the taxol-resistant cells. Many of the taxol-resistant cells disappeared after treatment with taxol in the presence of PMA. To explore the mechanism of this effect, we employed flow cytometry to determine levels of p53, p21, bcl-2 and caspase proteins in the taxol-resistant cells, and found that the expression of the bcl-2 protein was markedly decreased and the expression of the caspase protein markedly increased after treatment with taxol in the presence of PMA. These findings suggest that PMA enhances the sensitivity of taxol-resistant cells to taxol, and taxol treatment in the presence of PMA induces the apoptosis of taxol-resistant cells by inhibiting the expression of the bcl-2 protein and increasing the expression of the caspase protein.

Effect of pretreatment vitamin D levels on in vivo effects of atorvastatin on bone metabolism in patients with heterozygous familial hypercholesterolemia.

To find the clinical variables associated with atorvastatin's effects on bone metabolism markers, 35 patients with heterozygous familial hypercholesterolemia were treated with atorvastatin for 24 weeks, and the levels of bone formation markers (bone-specific alkaline phosphatase and osteocalcin) and resorption marker (urine collagen type-1 cross-linked N-telopeptide) were determined. Pretreatment vitamin D levels showed significant and positive associations with changes in 2 bone formation markers. The serial changes in 3 markers were favorable-increased bone formation markers and unchanged bone resorption marker-but the changes occurred only in patients with pretreatment vitamin D levels >50 pg/ml.

Amiodarone minimizes experimental autoimmune myocarditis in rats.

Amiodarone, a promising drug for the treatment of tachyarrythmias, was recently found to have immunomodulatory effects in vitro. We hypothesized that amiodarone would affect the immune system in vivo and examined the effect of amiodarone on myocarditis in rats. We induced experimental autoimmune myocarditis in rats by cardiac myosin immunization and treated the animals with an intraperitoneal injection of amiodarone at 25 mg/kg/every other day, 10 times after the induction of experimental autoimmune myocarditis. In the treated group, both microscopic and macroscopic examinations showed reduced heart weights, a mild and localized infiltration of inflammatory cells and fibrosis in the myocardium, and a mild congestion in the liver and lungs as compared with the control group. The phenotypic distribution of lymphocytes in peripheral blood showed a significant decrease in the CD4/CD8a ratio in the treated group, but not in the control group. The proportion of mast cells involved in inflammatory cell infiltration was lower in the treated group than the control group. In vitro, amiodarone inhibited the proliferation of mast cells by arresting them in the G2 phase of the cell cycle. These results indicated that amiodarone minimized the progression of experimental autoimmune myocarditis, suggesting a potential therapeutic role for amiodarone treatment in patients suffering from myocarditis, especially myocarditis complicated by cardiac arrhythmias. One possible mechanism by which amiodarone minimizes the progression of experimental autoimmune myocarditis may be to affect the immune system via the immunomodulatory effects on T cell and mast cell functions.

Autoimmune hair loss induced by alloantigen in C57BL/6 mice.

Exponentially growing Meth-A cells expressing H-2K(d).D (d) antigen were found to induce alopecia when injected intraperitoneally into normal C57BL/6 mice, which express the H-2K(b).D (b) antigen. However, the capacity to induce alopecia disappeared when Meth-A cells were treated with K252a, which inhibits protein kinases. Histologically, skin in affected areas showed dense mononuclear cell infiltration and a focal foreign-body giant-cell reaction in hair follicles. The subtyping of lymphocytes in peripheral blood demonstrated a significant difference between normal mice and Meth-A cell-injected mice. To further examine the mechanism by which the alloantigen induces alopecia, lymphocytes isolated from the peripheral blood of normal C57BL/6 mice were cultured in medium containing Meth-A cell homogenate, phytohemagglutinin (PHA) and recombinant mouse interleukin-2 (rm IL-2), and intravenously injected into normal C57BL/6 mice. The adoptive transfer of the lymphocytes induced alopecia in a similar way. These findings suggest that the protein kinase-modulated alloantigen induces alopecia by disturbing the immunological homeostasis, and that lymphokine-activated killer cells play an important role in induction of alopecia by cross-reacting with hair follicles.

Transfer of rabbit autoimmune cardiomyopathy into severe combined immunodeficiency mice.

Growing evidence suggests that the autoimmune mechanism plays an important role in the pathogenesis of dilated cardiomyopathy. The purpose of this study was to evaluate the effect on the cardiac structure and function by the transfer of immunoglobulin G (IgG) and/or lymphocytes from rabbits immunized with a synthetic peptide corresponding to the sequence of the second extracellular loop of beta1-adrenoceptor (beta peptide) into severe combined immunodeficiency (SCID) mice. CB-17 SCID mice were injected intraperitoneally with 2 mg of IgG and/or 1 x 10(7) peripheral blood lymphocytes (PBL) from either rabbits immunized with both beta1 peptide and adjuvant (beta group), and adjuvant or rabbits with adjuvant only (N group). Thirty-five SCID mice were divided into seven groups: (1) N-IgG group; (2) N-PBL group; (3) N-IgG+PBL group; (4) beta-IgG group; (5) beta-PBL group; (6) beta-IgG+PBL group; and (7) control group. Morphological, serological and endocrinological studies were performed 70 days after the transfer. Results showed that heart weight and heart weight/body weight ratio in the beta-IgG+PBL group tended to be increased as compared with those in other groups. All mice in the beta-IgG group, two in the beta-PBL group and four in the beta-IgG+PBL group showed high titer of rabbit anti-beta1-adrenoceptor antibodies. Brain natriuretic peptide in the beta-IgG+PBL group showed a significant increase as compared with those in the control group and N-IgG+PBL. Pathohistologically, focal infiltration of inflammatory cells in the myocardium was observed in one mouse of the beta-IgG+PBL group. Rabbit CD3-positive T-lymphocytes in the myocardium were observed in two mice of the beta group. In conclusion, transfer of IgG and PBL from rabbits immunized with beta1 peptide was able to induce the early stages of myocardial damage in SCID mice. These data provide direct evidence that the autoimmune mechanism is important in the pathogenesis of dilated cardiomyopathy.

Is cardiomyopathy an autoimmune disease?

Idiopathic dilated cardiomyopathy (DCM) is one of the leading causes of severe heart failure and the most common cause of heart transplantation due to its ventricular dilatations and contractile dysfuntions. Twenty percent of DCM is in the familiar form and the rest is sporadic. The clinical impact of DCM is far greater than its position in epidemiological terms. Despite recent improvements in therapy, both incidence and mortality are still very high. The main problem is its heterogeneous etiology. So far, three factors have been identified to be potentially important: enteroviral infection, immune mechanism and genetic factors. During the last 10 years there have been many investigations showing distinct autoantibodies or other immune factors in heterogeneous subsets of DCM which have contributed supportive and confounding evidence to the hypothesis that multiple autoimmune mechanisms are involved in DCM. Accumulated evidence hitherto demonstrated a variety of circulating autoantibodies in the sera of patients with DCM including antireceptor autoantibodies, myosin and ADP/ATP translocator protein, etc. Data available from both in-vitro and in-vivo studies of anti-receptor autoantibodies as well as from other autoantibodies and autoreactive lymphocytes demonstrated clearly that a subgroup of DCM is autoimmunity-mediated. This is understandable because DCM is heterogeneous, implying that different subgroups of DCM may have different pathogeneses. It may be practical in the future to separate "autoimmune cardiomyopathy" from other "idiopathic" DCM.