OBJECTIVETo observe the time course of interleukin (IL)-21 and related cytokines expression in rats with experimental autoimmune myocarditis (EAM).

METHODSAntigen was prepared with an emulsion of porcine cardiac myosin in complete Freund's adjuvant, plus Mycobacterium tuberculosis H37Ra strain. EAM model was made by hypodermic injection of myosin in hind legs of Lewis rats.mRNA expression of IL-21 and related cytokines (IL-21R, IL-17, TGF-β, IL-6) in different tissues (heart, liver, spleen, kidney) were determined at 2 weeks after immunization by RT-PCR and quantitative real-time RT-PCR. Furthermore, the time course of IL-21 and related cytokines expression in the acute phase of EAM (2 w, 3 w, 4 w) was determined by quantitative real-time RT-PCR, and IL-21, IL-17 protein expression was determined by Western blot and ELISA. The location of IL-21R was examined by immunohistochemistry at 2 w after immunization.

RESULTSHistopathology examination evidenced abundant mononuclear cells in the myocardium of 2 weeks EAM rats. Fibrosis and multinucleated giant cells were observed in the myocardium of 3 weeks EAM rats. Inflammation was reduced and large amount of fibrosis could be found in 4 weeks EAM rats. The heart weight/body weight ratio in normal, EAM 2 w, 3 w, 4 w group was (3.011 ± 0.117) mg/g, (4.736 ± 1.279) mg/g, (7.200 ± 0.308) mg/g and (4.622 ± 0.978) mg/g respectively. IL-21 mainly expressed in heart and spleen, IL-21R, IL-17, TGF-β mainly expressed in spleen, and IL-17, IL-6 mainly expressed in heart of EAM rats. IL-21R mainly distributed in cardiomyocytes of 2 weeks EAM rats. In line with pathological EAM course, the expression of IL-21 and related cytokines peaked at 2 weeks and then returned to normal at 4 weeks after immunization.

CONCLUSIONIL-21 and related cytokines were involved in the pathological process of EAM, upregulated IL-21 expression might promote Th17 cell differentiation and enhance Th17 cell secretion.

Animals ; Autoimmune Diseases ; immunology ; pathology ; Disease Models, Animal ; Interleukin-17 ; immunology ; Interleukin-6 ; immunology ; Interleukins ; immunology ; Male ; Myocarditis ; immunology ; pathology ; Myocardium ; immunology ; pathology ; Rats ; Rats, Inbred Lew ; Th17 Cells ; cytology ; immunology ; Transforming Growth Factor beta ; immunology

OBJECTIVEto observe the alteration of T helper cells 17(Th17) in mice with acute viral myocarditis (VMC) induced by coxsackie virus B3 (CVB3), explore the role of Th17 in mice VMC.

METHODSCVB3 or PBS was peritoneally injected to Balb/c male mice. Pathological scores were determined in hematoxylin-eosin stained sections and flow cytometric analysis was used to evaluate the frequencies of Th17 subsets in CD4(+) T cells on 7, 14, 21, 28 and 42 days after virus injection.

RESULTSthere were significant difference of the pathological scores between the VMC mice and the control ones (P < 0.05). The pathological scores of 7 d VMC subgroup were higher (1.8 ± 0.5) than those of 0 d VMC subgroup, and the scores of 14 d subgroup were highest (2.8 ± 0.4) among the six subgroup of VMC mice, and then showed a decline tendency from 21 d group. Statistical difference of the proportion of Th17 cells were seen between the VMC and controls on different time points (P < 0.05). When compared with the 0 d VMC subgroup the proportion of spleen Th17 cells increased in 7 d VMC subgroup [(2.23 ± 0.89)%], and peaked on 28 d [(5.00 ± 0.81)%]. The results of Th17 proportion were lower than those of the 28 d subgroup.

CONCLUSIONSour data show that differentiated Th17 cells might be involved in the inflammation process of CVB3 induced VMC in mice.

Animals ; Coxsackievirus Infections ; immunology ; pathology ; Enterovirus ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; immunology ; pathology ; virology ; Myocardium ; pathology ; Th17 Cells ; immunology

OBJECTIVEViral myocarditis (VM) is one of the most common acquired myocardial diseases in children. However, its pathogenesis is not clear. Recent studies indicate that the cytotoxicity mediated by cytotoxic T lymphocyte (CTL) plays an important role in the development of myocardial injury involved in VM. Apoptosis mediated by Fas/FasL pathway is an essential mechanism of target cells damage by CTL. In this study, the authors investigated the regulatory effects of neutralizing anti-Fas ligand (anti-FasL) antibody on apoptosis and caspase-3 expression in experimental coxsackievirus B3 myocarditis and the role of the CTL mediated apoptosis in myocardium through Fas/FasL pathway in the development of VM.

METHODSA total of 80 BALB/c mice were used in the experiments. They were divided randomly into the following groups: normal control group (Gr1), CVB3 control group (Gr2), IgG control group (Gr3) and anti-FasL antibody therapy group (Gr4). The mice in Gr2, Gr3 and Gr4 were inoculated with 0.15 ml of TCID(50) 10(9)/ml coxsackie virus B3 (CVB3) and the mice in Gr1 with 0.15 ml of Eagle reagent. The mice in Gr3 and Gr4 were inoculated with IgG (0.1 mg/kg) and FasL antibody (0.1 mg/kg) on days 0 and days 3 after inoculation (p.i.), respectively. Eight mice in each group were sacrificed on day 10 p.i. Histopathological studies and terminal transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were used to quantify inflammation, necrosis and apoptosis in myocardium. The expression of active caspase-3 in myocardium was determined by immunohistochemistry. Caspase-3 mRNA and CVB3 mRNA were analyzed by reverse-transcription polymerase chain reaction (RT-PCR).

RESULTS(1) Caspase-3 activation and apoptosis were seen in the myocardium of mice with myocarditis. They had a significantly positive correlation with the changes of myocardial histopathologic scores (r = 0.81, P < 0.05; r = 0.73, P < 0.05). (2) The pathologic scores, average percentages of apoptotic cardiomyocytes, expression of active caspase-3 (protein and mRNA) and expression of CVB3 mRNA in myocardium of mice in Gr4, were significantly reduced compared to those in the myocardium of mice in Gr2 (P < 0.01, P < 0.01, P < 0.01, P < 0.01, and P < 0.05, respectively) and Gr3 (P < 0.01, P < 0.01, P < 0.05, P < 0.01, and P < 0.05, respectively).

CONCLUSIONMyocytic apoptosis is a key mechanism responsible for myocardial damage in viral myocarditis. Anti-FasL antibody can effectively reduce expression of active caspase-3 protein and mRNA, viral replication, cardiomyocytic apoptosis and myocardial injury in the experimental CVB3 myocarditis.

Animals ; Antibodies, Neutralizing ; therapeutic use ; Apoptosis ; Caspase 3 ; immunology ; Coxsackievirus Infections ; drug therapy ; immunology ; Enterovirus B, Human ; physiology ; Fas Ligand Protein ; immunology ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; immunology ; virology ; Myocardium ; immunology ; pathology ; Virus Replication

OBJECTIVETo observe the alteration of interleukin-17 and interferon-γ double positive cells (IL-17(+)/IFN-γ(+) cells) in mice with coxsackie virus B3 (CVB3) induced acute viral myocarditis (VMC).

METHODSVMC was induced in male Balb/c mice by peritoneal injection of CVB3. Control mice received PBS injection. At 0, 1, 2, 3, 4 and 6 weeks after injection, pathological scores were determined on hematoxylin-eosin stained heart sections and flow cytometric analysis was performed to evaluate the percent of IL-17(+)/IFN-γ(+) cells among CD4(+) T cells.

RESULTSCompared to control mice, the pathological scores of VMC mice were higher on CVB3 infection week 1 (1.8 ± 0.5), peaked on week 2 (2.8 ± 0.5) and declined thereafter. However, the proportion of IL-17(+)/IFN-γ(+) cells remained steadily at a low level throughout the observation period and was similar between VMC and control mice.

CONCLUSIONSOur data shows that IL-17(+)/IFN-γ(+) cells might not be involved in the inflammation process of CVB3 induced VMC mice model.

Animals ; Coxsackievirus Infections ; immunology ; pathology ; Disease Models, Animal ; Enterovirus ; Interferon-gamma ; metabolism ; Interleukin-17 ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; immunology ; pathology ; virology ; Myocardium ; pathology ; Th17 Cells ; immunology ; metabolism

In this study of the inhibitory effects of angiopeptin and aspirin on the development of accelerated graft atherosclerosis (AGAS), 22 B10.BR mice received intra-abdominal heterotopic heart transplants from B10.A mice, without immunosuppression. Group 1 (n = 5) received no pharmacological intervention, Group 2 (n = 6) was treated with angiopeptin, Group 3 (n = 5) with aspirin, and Group 4 (n = 6) with both. There was no significant difference in the incidence of AGAS among these groups. The magnitude of intimal lesion development showed less narrowing of large vessels (> 100 microns in diameter) in groups 2 and 4--i.e. the groups received angiopeptin (Group 1 = 46.9 +/- 9.3%, Group 2 = 28.5 +/- 9.2%, Group 3 = 44.1 +/- 10.9%, Group 4 = 24.2 +/- 5.9%; p < 0.01). Comparison of the fraction of tropomyosin-positive staining cells in the intima revealed a lesser degree of staining in Group 2 (p < 0.01). No intervention was effective in preventing smooth muscle cell proliferation in the media or inflammatory cell infiltration in the adventitia. In conclusion, our data suggest that angiopeptin is effective in the direct inhibition of intimal smooth muscle cell proliferation in relatively large vessels, whereas aspirin exhibits no inhibitory role in the progression of AGAS. Angiopeptin appears to be a potential therapeutic agent for inhibiting the progression of postoperative AGAS in clinical heart transplantation.
Animal ; Aspirin/pharmacology* ; Cardiovascular Agents/pharmacology* ; Coronary Arteriosclerosis/pathology ; Coronary Arteriosclerosis/immunology* ; Coronary Vessels/pathology ; Coronary Vessels/drug effects ; Heart/drug effects* ; Heart Transplantation/immunology* ; Immunohistochemistry ; Mice ; Mice, Inbred Strains ; Myocardium/pathology ; Myocardium/immunology ; Oligopeptides/pharmacology* ; Somatostatin/pharmacology ; Somatostatin/analogs & derivatives* ; Transplantation, Homologous/immunology ; Tropomyosin/metabolism

OBJECTIVETo investigate the importance of autoimmunity against beta(1)-adrenoreceptor in the pathogenesis of dilated cardiomyopathy (DCM).

METHODSFourteen rabbits were divided equally into two groups. Rabbits in the immunized group (n = 7) were immunized monthly for one year with synthetic peptide corresponding to the second extracellular loop of the beta(1)-adrenoreceptor and adjuvant. Control rabbits received the mixture with the same procedure as described except with a substitution of saline for the corresponding peptide. During the study period, all rabbits were bled to assay the titers of antipeptide antibody and left ventricular ejection fractions (LVEFs) were measured by emission computed tomography. At the end of experiment, invasive cardiac function was measured and morphologic examinations were done.

RESULTSHigh titers of antipeptide antibody were found in the sera from immunized rabbits throughout the study period in contrast to those from control rabbits. LVEFs were significantly higher in immunized rabbits than those of the control group at the 4th and 6th month. At the end of the experiment, the maximal rates of rise and decline of ventricular pressure of the immunized group were significantly lower than those of the control group. Morphological changes were found in immunized rabbits such as the enlargement of ventricles, myofibrillar lysis and necrosis, mitochondria swelling and condensation. No obvious alterations were noted in hearts of control rabbits.

CONCLUSIONAutoimmunity against the beta(1)-adrenoreceptor may be involved in the pathogenesis of dilated cardiomyopathy and beta(1)-adrenoreceptor antibody may play a role in the process.

Animals ; Cardiomyopathy, Dilated ; etiology ; immunology ; pathology ; Heart ; drug effects ; physiopathology ; Immunization ; Male ; Microscopy, Electron ; Myocardium ; pathology ; ultrastructure ; Peptide Fragments ; administration & dosage ; chemical synthesis ; immunology ; Rabbits ; Receptors, Adrenergic, beta-1 ; administration & dosage ; chemistry ; immunology ; Ventricular Dysfunction, Left ; etiology ; physiopathology

The ischemic heart disease has been endangering human health seriously. Although there are many kinds of anti-ischemic drugs, most of them are lacking in tissue specificity, which together with a remarkably reduced blood circulation in the ischemic zone often lead to a quite low drug distribution in the targets. Myocardial ischemia can cause a lot of pathophysiological changes, such as the enhanced permeability of the endothelial cell membrane, the up-regulated expression of various cell adhesion molecules on endothelium, the exposure of intracellular antigenic components, the decrease of pH within the ischemic zone, and so on. To date, some of these changes have been exploited with limited success to gain the passive, active and physicochemical targeting of diagnostic or therapeutic drugs to myocardial ischemic regions. However, more effective delivery strategies are still eagerly needed. Here, we reviewed and discussed the potential targeting-delivery mechanisms and strategies, used or may be used in the future, for myocardial ischemic regions.
Animals ; Antibodies, Monoclonal ; immunology ; Capillary Permeability ; Drug Carriers ; Drug Delivery Systems ; methods ; Genetic Therapy ; Humans ; Liposomes ; chemistry ; metabolism ; Myocardial Ischemia ; therapy ; Myocardium ; metabolism ; pathology ; Polyethylene Glycols ; metabolism ; Ultrasonics

Endomyocardial biopsy often fails to show myocardial inflammation for patients with clinically suspected myocarditis. The serum isoforms of troponin T (cTnT) level is a very sensitive marker of myocardial injury and it is elevated even in the absence of myocardial inflammation. We investigated the correlations for myocardial injury, virus titers and inflammation in acute viral infection. Using the murine coxsackievirus group B3 (CVB3) myocarditis model, the histopathologic findings and virus titers in mouse hearts were compared with the serum cTnT levels measured by ELISA at various time points. Viable virus titers in the hearts peaked at 3 days after infection (8.22+/-0.13 log10 PFU/100 mg of heart); they decreased at day 7 and no viable virus was detected from day 14. Myocardial inflammation was minimal at day 3, peaked at day 7 and markedly decreased at day 14. The individual serum TnT levels were significantly increased at day 3 (7.37+/-1.46 ng/ml), persisted to day 7 (0.73+/-0.08 ng/ml), and normalized at day 14. Serum cTnT levels were correlatable with virus titers in the heart (r=0.744, P <0.01), but the serum cTnT levels were not correlated with the degrees of inflammation. Using the less myocarditic strain of CVB3, similar relationships were observed between the changes for the serum cTnT levels and the heart virus titers. During the course of viral infection, myocardial injury precedes the pathologic evidence of inflammation, and the elevated cTnT levels provide evidence of myocardial injury even in the absence of any histologic findings of myocarditis.
Acute Disease ; Animals ; Coxsackievirus Infections/*pathology ; Enterovirus B, Human/isolation & purification/pathogenicity/*physiology ; Female ; Heart/*virology ; Hela Cells ; Humans ; Inflammation/*immunology ; Mice ; Mice, Inbred BALB C ; Myocardial Infarction/immunology/*pathology ; Myocarditis/immunology/pathology/*virology ; *Myocardium/immunology/pathology ; Research Support, Non-U.S. Gov't ; Troponin T/blood ; Virus Replication

OBJECTIVEIn order to explore the possible roles played by the autoimmune mechanism in the progression of myocarditis into dilated cardiomyopathy (DCM) using an animal model, we investigated whether autoimmune myocarditis might develop into DCM.

METHODSExperimental Balb/C mice (n = 20) were immunized with cardiac myosin with Freund's complete adjuvant at days 0, 7 and 30. The control Balb/C mice (n = 10) were immunized with Freund's complete adjuvant in the same mannere. Serum and myocardium samples were collected after the first immunization at days 15, 21 and 120. The anti-myosin antibody was examined by enzyme-linked immunosorbent assay and immunoblotting.

RESULTSPathological findings demonstrated that there was myocardial necrosis or inflammatory infiltration during acute stages and fibrosis mainly in the late phase of experimental group, but the myocardial lesions were not found in the control group. Autoimmunity could induce myocarditis and DCM in the absence of viral infection. High titer anti-myosin IgG antibodies were found in the experimental group, but not in the control group. Furthermore, the anti-myosin heavy chain (200 KD) antibody was positive in 21 of 48 patients with DCM and viral myocarditis, but only 4 of 20 patients with coronary heart disease, including 1 case and 3 cases that reacted with heavy and light chains (27.5 KD), respectively. The antibodies were not detected in healthy donors.

CONCLUSIONCardiac myosin might be an autoantigen that provokes autoimmunity and leads to the transformation of myocarditis into DCM. Detection of anti-myosin heavy chain antibody might contribute to diagnosis for DCM and viral myocarditis.

Adult ; Aged ; Animals ; Autoantibodies ; blood ; Autoimmune Diseases ; complications ; Cardiac Myosins ; immunology ; Cardiomyopathy, Dilated ; etiology ; immunology ; Female ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Middle Aged ; Myocarditis ; complications ; Myocardium ; pathology

OBJECTIVETo explore the operational mechanisms and potential approach to inducing transplantation immune tolerance of FTY720.

METHODSMouse splenocytes were incubated with FTY720, then the DNA was extracted and analyzed using gel electrophoresis. Hearts of inbred BALB/c (H-2(d)) mice were transplanted heterotopically in C57BL/6 (H-2b) mice. Recipients were randomly divided into six groups. Group-1 (n = 6) was the nil-treated control. Groups-2, 3 and 4 were given FTY720 at the dose of 3 mg.kg(-1) by oral gavage once a day with different time courses. Group-2 (n = 14) were administrated from 3 days before transplantation (day-3) to the 11th day after the transplantation (day 11); Group-3 (n = 6) from day 0 to day 14; Group-4 (n = 6) from day-3 to day 0. Group-5 (n = 5) and 6 (n = 5) were treated with Cyclosporine A (10 mg.kg(-1)) and 40-O-(2-hydroxyethyl)-rapamycin (RAD) (3 mg.kg(-1)) respectively by daily gavage from day 3 to day 11. The long survivors (> 100 d) in Group-2 were detected with their IL-4 and IFN-gamma levels and their tolerant state was challenged with second graft: the donor type skin.

RESULTSApoptosis changes of the mouse splenocytes incubated with FTY720 was showed by typical DNA ladders. The median survival time (MST) of Group-1 was 8 d. MST of Group-2 was 55 d and grafts in six mice survived more than 100 d. MST of Group-3 was 16.5 d. Group-4 has a MST of 14 d with one case exceeded 100 d. MST of Group-5 and 6 were 10 d and 13 d respectively. Long survivors of Group-2 can accept donor-type skin graft and the level of IL-4 in their serum is up-regulated while IFN-gamma remained stable.

CONCLUSIONSPretreatment of FTY720 bring about effect on the early events of transplantation immune responses. This effect might be mediated by apoptosis induction in lymphocytes using this drug. We originally designed the regime of FTY720 monotherapy, which started pre-operationally and maintained for a short period of time, and induced stable tolerance the allo-graft in mouse.

Adjuvants, Immunologic ; pharmacology ; Animals ; Apoptosis ; Fingolimod Hydrochloride ; Heart Transplantation ; immunology ; Immune Tolerance ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Myocardium ; pathology ; Propylene Glycols ; pharmacology ; Sphingosine ; analogs & derivatives ; Transplantation Immunology