Expression of tumor necrosis factor ligand superfamily costimulatory molecules CD27L, CD30L, OX40L and 4-1BBL in the heart of patients with acute myocarditis and dilated cardiomyopathy.

BACKGROUND: T-cell-mediated myocardial damage is known to be involved in acute myocarditis and dilated cardiomyopathy. Recently, we found that tumor necrosis factor (TNF) ligand superfamily costimulatory molecules, especially 4-1BBL, played an important role in the myocardial damage of murine acute viral myocarditis. METHODS AND RESULTS: To investigate the roles for CD27L, CD30L, OX40L and 4-1BBL, which belong to TNF ligand superfamily, in the development of acute myocarditis and dilated cardiomyopathy, we analyzed the expression of these antigens in the myocardial tissues of patients with acute myocarditis and dilated cardiomyopathy. We also examined expression of the receptors for these molecules, CD27, CD30, OX40 and 4-1BB, which belong to TNF receptor superfamily, on the infiltrating cells. Strong expression of CD27L, CD30L and 4-1BBL and weak to moderate expression of OX40L was found in the cardiac myocytes of patients with acute myocarditis. Moderate expression of CD27L, CD30L and 4-1BBL and weak expression of OX40L was found on the cardiac myocytes of patients with dilated cardiomyopathy. Most of the infiltrating cells expressed CD27, CD30 and 4-1BB and a part of the infiltrating cells expressed OX40. CONCLUSIONS: Our findings suggest that expression of TNF ligand superfamily costimulatory molecules on cardiac myocytes may play a role in the cell-mediated myocardial damage in patients with acute myocarditis and dilated cardiomyopathy as in murine viral myocarditis.     

Expression of tumour necrosis factor (TNF) receptor/ligand superfamily co-stimulatory molecules CD40, CD30L,CD27L,and OX40L in murine hearts with chronic ongoing myocarditis caused by Coxsackie virus B3

T-cell-mediated myocardial damage has been shown to be involved in acute myocarditis and dilated cardiomyopathy. It is necessary for T-cells to receive a co-stimulatory signal as well as the main signal through the T-cell receptor for antigen-specific T-cell activation to occur. To investigate the roles of the co-stimulatory molecules CD40/CD40L, CD30/CD30L, CD27/CD27L, and OX40/OX40L, which belong to the tumour necrosis factor (TNF) receptor/ligand superfamily, in the development of chronic ongoing myocarditis, the expression of CD40, CD30L, CD27L, and OX40L was analysed in the hearts of A/J mice with myocarditis induced by Coxsackie virus B3 (CVB3). The expression of CD40L, CD30, CD27, and OX40 was also examined on the infiltrating cells. Furthermore, the induction of CD40, CD30L, CD27L, and OX40L was evaluated on cultured cardiac myocytes treated with interferon (IFN)-γ. CVB3-induced myocarditis resulted in the induction of CD40 and CD30L on the surface of cardiac myocytes. Induction of CD40 and CD30L on cardiac myocytes was confirmed by treatment with IFN-γ in vitro. CD27L and OX40L were expressed on cardiac myocytes in vivo and in vitro. The expression of CD27L and OX40L on cardiac myocytes was increased, at least partly, by CVB3-induced myocarditis in vivo. Many infiltrating cells expressed CD27 and OX40, whereas much smaller numbers expressed CD40L and CD30. The induction of these molecules, especially CD40 and CD30L, on cardiac myocytes strongly suggests that cardiac myocytes may co-stimulate T-cells and induce cytokine production by T-cells and humoral immune responses. This may play an important role in the pathogenesis of the resulting myocardial damage. Copyright © 1999 John Wiley & Sons, Ltd.     

Effects of in vivo administration of anti-B7-1/B7-2 monoclonal antibodies on the survival of mice with chronic ongoing myocarditis caused by Coxsackievirus B3

In acute myocarditis and dilated cardiomyopathy, it has previously been reported that antigen-specific T-cells infiltrate the heart and play an important role in the myocardial damage involved. For antigen-specific T-cell activation to occur, it is necessary for the T-cell to receive co-stimulatory signals provided by co-stimulatory molecules expressed on the antigen-presenting cell (APC), as well as the main signal provided by binding of the T-cell receptor (TCR) to the antigen. To investigate the roles for the co-stimulatory molecules B7-1 and B7-2 in the development of chronic ongoing viral myocarditis, firstly the expression of B7-1/B7-2 was analysed in the hearts of A/J mice with myocarditis induced by Coxsackievirus B3 (CVB3). Secondly the induction of B7-1/B7-2 on cultured cardiac myocytes treated with interferon (IFN)-γ was evaluated. Thirdly the effects of the in vivo administration of anti-B7-1/B7-2 monoclonal antibodies (MAbs) on the survival of mice with viral myocarditis were examined. CVB3-induced myocarditis resulted in enhanced expression of B7-1/B7-2 on cardiac myocytes. The expression of B7-1/B7-2 on cardiac myocytes could be induced by IFN-γ in vitro. In vivo anti-B7-1 MAb treatment significantly prolonged the survival of mice with myocarditis, whereas anti-B7-2 MAb treatment abrogated the protective effect of anti-B7-1. These findings indicate that distinct roles for B7-1 and B7-2 antigens are involved in the development of viral myocarditis and raise the possibility of immunotherapy with anti-B7-1 MAb to prevent T-cell-mediated cardiac myocyte injury and to improve the prognosis of viral myocarditis. Copyright © 1999 John Wiley & Sons, Ltd.     

The capacity of the TNF family members 4-1BBL, OX40L, CD70, GITRL, CD30L and LIGHT to costimulate human T cells

Activating signals generated by members of the tumour necrosis factor receptor superfamily upon interaction with their cognate ligands play important roles in T-cell responses. Members of the tumour necrosis factor family namely 4-1BBL, OX40L, CD70, GITRL, LIGHT and CD30L have been described to function as costimulatory molecules by binding such receptors on T cells. Using our recently described system of T-cell stimulator cells we have performed the first study where all these molecules have been assessed and compared regarding their capacity to costimulate proliferation and cytokine production of human T cells. 4-1BBL, which we found to be the most potent molecule in this group, was able to mediate sustained activation and proliferation of human T cells. OX40L and CD70 were also strong inducers of T-cell proliferation, whereas the costimulatory capacity of human GITRL was significantly lower. Importantly CD30L and LIGHT consistently failed to act costimulatory on human T cells, and we therefore suggest that these molecules might be functionally distinct from the costimulatory members of this family.     

Contribution of CD30/CD153 but not of CD27/CD70, CD134/OX40L, or CD137/4-1BBL to the optimal induction of protective immunity to Mycobacterium avium

A panel of monoclonal antibodies specific for CD27 ligand (CD70), CD30 ligand (CD153), CD134 ligand (OX40L), and CD137 ligand (4-1BBL) were screened in vivo for their ability to affect the control of Mycobacterium avium infection in C57Bl/6 mice. Only the blocking of CD153 led to increased mycobacterial burdens. We then used CD30-deficient mice and found an increase in the proliferation of two strains of M. avium in these mice as compared with control animals. The increased mycobacterial growth was associated with decreased T cell expansion and reduced interferon-gamma (IFN-gamma) responses as a result of reduced polarization of the antigen-specific, IFN-gamma-producing T cells. At late times but not early in infection, the lymphoid cuff surrounding granulomas was depleted in the CD30-deficient animals. This report expands our knowledge about tumor necrosis factor superfamily members involved in the immune responses to mycobacterial infection by identifying CD30-CD153 interactions as required for optimal immune control of M. avium infection.     

Change in the cells that express connective tissue growth factor in acute Coxsackievirus-induced myocardial fibrosis in mouse

Cardiac fibrosis and inflammation are major pathologic conditions that result from viral myocarditis. Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and induces production of extracellular matrix molecules. We studied the correlation between CTGF and cardiac fibrosis in an acute Coxsackievirus B3 (CVB3) myocarditis animal model. Eight-week-old BALB/c mice were infected intraperitoneally with 10(4) plaque forming units (PFU) of CVB3. Myocardial inflammation peaked on day 7 and decreased markedly by day 14 post-infection (pi); cardiac fibrosis was noted from day 7 and peaked on day 14. By contrast, CTGF was weakly expressed by the interstitial cells in uninfected control hearts and also in the hearts of day 3 pi. CTGF expression measured by real-time PCR was elevated on day 3 and peaked on day 7 pi. TGF-beta expression peaked at day 7 pi. The cell type of CTGF expression changed from interstitial cells to myocytes after virus infection. On day 7, CTGF was strongly expressed by myocytes and inflammatory cells surrounding calcified necrotic areas. In addition, cardiac myocytes expressed CTGF on day 14. Our results, based on an acute CVB3 model of myocarditis, provide evidence that CTGF may mediate the development of fibrosis after viral myocarditis, and that the cells expressed CTGF changes during the course of viral myocarditis.     

Role of T cell costimulation in anti-viral immunity

Members of both the CD28 and TNFR families can have costimulatory roles in T cell activation. Gene targeted mice as well as in vivo blocking experiments have established distinct roles for CD28/B7; ICOS/ICOSL; CD27/CD70; 4-1BB/4-1BBL and OX40/OX40L during viral infection. Many issues remain to be addressed, including the timing and location of the interactions, the possibility of partial redundancy between related family members and the molecular basis for the specific phenotypes observed in the different gene targeted mice.     

Nasal cardiac myosin peptide treatment and OX40 blockade protect mice from acute and chronic virally-induced myocarditis

Myocarditis poses a severe health problem, can lead to dilated cardiomyopathy (DCM) and death, and is thought to be triggered by infections. Enteroviruses such as Coxsackie virus B 3 (CVB3) have been implicated as a culprit, since they can cause acute and chronic heart disease in susceptible mice. CVB was detected in human cardiac myocytes in some cases, whereas acute CVB infection was thought to have caused death. Here we studied, whether nasal administration of cardiac myosin (CM) major histocompatibility class (MHC) II peptides CM_947–960 and CM_735–747 and OX40 blockade would be able to ameliorate immunopathology and heart disease in BALB/C mice infected with CVB3. We found that nasal CM-peptide prophylactic treatment significantly reduced myocarditis and mortality by enhancing Treg and IL-10 induction and that blockade of OX40 signaling could reduce heart inflammation when administered late during pathogenesis. Altogether, these results chart the way for novel prevention and intervention strategies for viral myocarditis.     

In situ immune autoradiographic identification of cells in heart tissues of mice with coxsackievirus B3-induced myocarditis.

In adolescent CD-1 male mice inoculated with a myocarditic coxsackievirus B3 (CVB3m) acute focal lesions containing necrotic myocytes, infiltrating mononuclear cells, and fibroblasts develop. With the use of an in situ immune autoradiographic method with rat monoclonal antibodies (MAb) and an 35S-labeled antibody, viral antigens were detected outside of lesions. Macrophages, T lymphocytes, and natural killer (NK) cells were identified within myocarditic lesions during the acute phase of the disease. Macrophages detected by anti-Mac-1 MAb were in focal areas within myocarditic lesions on Days 4-7 after inoculation. T lymphocytes were detected in myocarditic lesions on Days 4-10, with MAb to Thy-1 and Lyt-1 antigens showing diffuse reaction patterns, suggesting random distribution of these cells in lesions. Focal areas of reactivity were detected with MAbs to L3T4 and Lyt-2 antigens, suggesting clusters of helper and cytotoxic/suppressor T lymphocytes, respectively. NK cells were presumptively detected by asialo GM1 surface marker in lesions at all times. The presence of activated NK cells in lesions was confirmed by assay of mechanically dissociated heart tissues on Day 8. These data describe the temporal sequence and identity of leukocytes entering into CVB3-induced focal myocarditic lesions during the acute phase of disease in CD-1 mice.     

Characterization of the CD30L binding domain on the human CD30 molecule using anti-CD30 antibodies

CD30 and its counter-receptor CD30 ligand (CD30L) are members of the TNF-receptor/TNFalpha superfamily and function to regulate lymphocyte survival and differentiation. Several monoclonal antibodies (MAbs) have been developed against CD30 and, based on mutual inhibition assays, are grouped into three nonoverlapping serologic clusters. However, the relationship between the epitopes recognized by the antibodies comprising each cluster and the binding domain for CD30L is not known. Using a soluble CD30L/CD8alpha chimeric protein, we assessed the ability of anti-CD30 MAb to inhibit the binding of CD30L to CD30 expressed by the CD30+ Karpas 299 cell line. CD30L binding by CD30 is blocked by MAb that recognize epitopes belonging to cluster Group A (like Ber-H2, Ber-H8, and HRS-4) as well as cluster Group C (like HeFi-1 and M44). Cluster Group B antibodies, including M67 and Ki-1, do not affect CD30L binding to CD30. The pattern of CD30L binding inhibition shows only limited correspondence to the functional capacity of some anti-CD30 MAb to trigger CD30 signaling. Finally, we demonstrate that the anti-CD30L MAb M81 also completely inhibits CD30/CD30L interaction. This information is useful for applying these MAbs in functional studies to further investigate the CD30/CD30L system and for designing assays for soluble CD30L.     

4-1BB (CD137) differentially regulates murine in vivo protein- and polysaccharide-specific immunoglobulin isotype responses to Streptococcus pneumoniae

4-1BB (CD137) is induced on activated CD4(+) and CD8(+) T cells and delivers a costimulatory signal upon binding the 4-1BB ligand (4-1BBL) expressed on antigen-presenting cells. Induction of 4-1BB is dependent on activation via the T-cell receptor (TCR) and possibly CD28. It was previously demonstrated that both an in vivo protein (pneumococcal surface protein A [PspA])- and polysaccharide (phosphorylcholine [PC] determinant of teichoic acid)-specific immunoglobulin (Ig) isotype response to Streptococcus pneumoniae was dependent on CD4(+) TCRalphabeta(+) T cells and B7-dependent costimulation through CD28. We thus postulated that 4-1BB costimulation would also play a role in regulating the in vivo anti-PspA and anti-PC response to S. pneumoniae. We demonstrate that mice genetically deficient in 4-1BBL elicit a markedly reduced IgM and IgG anti-PC but normal primary and secondary IgG anti-PspA responses to S. pneumoniae relative to those for wild-type mice. However, injection of an agonistic anti-4-1BB monoclonal antibody (MAb), while having no significant effect on the anti-PC response, strongly inhibits the primary anti-PspA response, the generation of PspA-specific memory, and germinal center formation but does not induce a lasting state of tolerance. In contrast, anti-4-1BB MAb has no effect on the anti-PspA response when injected only at the time of secondary immunization. Delay of the addition of anti-4-1BB leads to progressively less inhibition of the primary response up to day 8. This inhibition is independent of CD8(+) T cells and is associated with the expansion of CD4(+) T cells with an activated phenotype, which is partly dependent on B7-dependent costimulation. These data are the first to suggest a stimulatory role for endogenous 4-1BB-4-1BBL interactions during a humoral immune response to a pathogen and further underscore significant differences in costimulation requirements for an in vivo protein- versus polysaccharide-specific Ig isotype response to an extracellular bacterium.     

EXPRESSION OF VASCULAR CELL ADHESION MOLECULE-1 IN MURINE HEARTS WITH ACUTE MYOCARDITIS CAUSED BY COXSACKIEVIRUS B3

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of the myocardial cell damage involved in viral myocarditis. Using a murine model of acute myocarditis caused by Coxsackievirus B3 (CVB3), perforin-expressing killer cells have been shown to infiltrate the heart, and intercellular adhesion molecular-1 (ICAM-1) together with major histocompatibility complex (MHC) antigen was induced on myocardial cells in acute viral myocarditis. To clarify the immunological mechanisms in more detail, the expression of vascular cell adhesion molecular-1 (VCAM-1) has been examined in the heart of acute myocarditis and on cultured cardiac myocytes treated with interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α). The effects of in vivo antibody treatment to VCAM-1 on myocardial damage involved in acute myocarditis were also analysed. CVB3-induced myocarditis resulted in enhanced expression of VCAM-1 on myocardial cells. VCAM-1 expression was also induced on cultured cardiac myocytes by treatment with IFN-γ and TNF-α. The in vivo antibody treatment to VCAM-1 decreased the myocardial damage to some extent, but the effects were not statistically significant. These data suggest that the expression of VCAM-1 on myocardial cells may play at least a partial role in the myocardial damage involved in acute viral myocarditis.     

Neutralizing anti-4-1BBL treatment improves cardiac function in viral myocarditis

Coxsackievirus B3 (CVB3) is the most common causative agent of infectious myocarditis. Chronic inflammation, loss of contractile tissue, and maladaptive remodeling all contribute to dilated cardiomyopathy and heart failure. The 4-1BB receptor is a costimulatory molecule expressed by T cells and cardiomyocytes. We infected mice with CVB3 to examine if virus infection triggers 4-1BB activation and whether inhibition of this pathway will reduce inflammation and improve heart function. Echocardiography was performed on days 3, 9, 30 and at 10 weeks post-infection (pi) and ejection fraction (EF), left ventricular (LV) wall thickness, contractility, and internal cardiac dimensions were measured. At day 9, reduced rate of wall thickening (30+/-17 vs 70+/-19%), increased LV wall thickness (0.15+/-0.04 vs 0.09+/-0.01 cm in diastole and 0.19+/-0.04 vs 0.15+/-0.02 cm in systole), and reduced cardiac volume (0.013+/-0.004 vs 0.023+/-0.003 ml in diastole and 0.004+/-0.002 ml vs 0.007+/-0.001 ml in systole) were observed in infected hearts as compared with shams. At 14 days pi, CVB3-infected mice were randomly assigned to receive either anti-4-1BBL neutralizing (M522) or control antibodies (Ab) for 8 weeks. Cardiac damage, fibrosis, and inflammation were assessed by histological stains and immunohistochemistry. Polymerase chain reaction (PCR) was utilized to detect matrix metalloproteinase (MMP)-2, MMP-9, and MMP-12 expressions. At 10 weeks pi, M522 treatment improved LV wall thickening rate (-10+/-13 vs -49+/-16%, expressed as percentage change from baseline) and reduced diastolic LV posterior wall thickness (17+/-10 vs 57+/-47%, expressed as percentage change from baseline), cardiac damage as assessed by histological scores (0 vs 1.3+/-1.5), fibrosis by collagen volume fraction (3.2+/-0.6 vs 4.9+/-2.2%), overall inflammation (5.9+/-1.3 vs 8.5+/-4.1%), and T-cell infiltration (1.3+/-0.9 vs 4.3+/-3.8%) as compared to control. MMP-12 was highly increased during acute and chronic myocarditis, but was significantly decreased by M522 treatment. Thus, long-term inhibition of the 4-1BB pathway reduces cardiac damage, remodeling, and inflammation during viral myocarditis.     

Vγ1+γδT,early cardiac infiltrated innate population dominantly producing IL-4, protect mice against CVB3 myocarditis by modulating IFNγ+ T response

Viral myocarditis (VMC) is an inflammation of the myocardium closely associated with Coxsackievirus B3 (CVB3) infection. Vγ1⁺γδT cells, one of early cardiac infiltrated innate population, were reported to protect CVB3 myocarditis while the precise mechanism not fully addressed. To explore cytokine profiles and kinetics of Vγ1⁺γδT and mechanism of protection against VMC, flow cytometry was conducted on cardiac Vγ1 cells in C57BL/6 mice following CVB3 infection. The level of cardiac inflammation, transthoracic echocardiography and viral replication were evaluated after monoclonal antibody depletion of Vγ1γδT. We found that Vγ1⁺γδT cells infiltration peaked in the heart at day3 post CVB3 infection and constituted a minor source of IFN-γ but major producers for early IL-4. Vγ1γδT cells were activated earlier holding a higher IL-4-producing efficiency than CD4⁺Th cells in the heart. Depletion of Vγ1⁺γδT resulted in a significantly exacerbated cardiac infiltration, increased T, macrophage and neutrophil population in heart homogenates and worse cardiomyopathy; which was accompanied by a significant expansion of peripheral IFNγ⁺CD4+ and CD8 + T cells. Neutralization of IL-4 in mice resulted in an exacerbated acute myocarditis confirming the IL-4-mediated protective mechanism of Vγ1. Our findings identify a unique property of Vγ1⁺γδT cells as one dominant early producers of IL-4 upon CVB3 acute infection which is a key mediator to protect mice against acute myocarditis by modulating IFNγ-secreting T response.     

Expression and function of 4-1BB during CD4 versus CD8 T cell responses in vivo

4-1BBL(-/-) mice have a defect in recall CD8+ T cell responses to viruses, whereas CD4+ T cell responses to virus are unimpaired in these mice. In contrast, both CD4+ and CD8+ T cells respond to 4-1BB ligand (4-1BBL) in vitro. To clarify the role of 4-1BB/4-1BBL in CD4+ versus CD8+ T cell responses in vivo, we compared CD4 (OT-II) and CD8 (OT-I) TCR transgenic T cells responding to the same antigen in an in vivo adoptive transfer model in 4-1BBL(+/+) versus 4-1BBL(-/-) mice. During primary and secondary responses, expression of 4-1BB on in vivo-activated TCR transgenic T cells was earlier and more transient than previously observed in vitro, correlating with expression of the early activation antigen CD69 and preceding the transition to the CD44hi state. Although 4-1BB is expressed early in the primary response, there was no effect of 4-1BBL deficiency on initial CD8 T cell expansion and only a minor effect on initial CD4 T cell expansion. The major effect of 4-1BB/4-1BBL interaction is on the T cell recall response. This is due to effects of 4-1BBL on maintenance of T cell numbers at the end of the primary response with additional effects of 4-1BBL on secondary expansion of T cells.     

CVB3诱导的病毒性心肌炎小鼠模型中CD4~+T细胞亚群格局及其作用

本研究主要关注BALB/c小鼠感染柯萨奇病毒B3型(CVB3)后CD4+T细胞亚群格局的变化及其对病毒性心肌炎发病的贡献度。CVB3腹腔感染小鼠后第7d,通过小鼠体重下降率、心肌损伤血清学指标肌酸激酶(CK)活性以及心肌组织病理学改变等多项指标证实小鼠心肌炎模型的成功建立。经Real-time PCR检测感染第7d脾脏CD4+T细胞亚群主要细胞因子IFN-γ、IL-4、IL-17A、IL-17F及转录因子Foxp3表达情况;以流式细胞术检测了CD4+T细胞各亚群比例,并通过多元线性回归分析法评估了各T细胞亚群对病毒性心肌炎发病的影响。结果显示,与对照组相比,CVB3感染小鼠脾脏IL-17A、IL-17F、IFN-γ表达均显著上升(分别约为12、8.5、5倍),而IL-4和Foxp3表达无明显变化。CVB3感染小鼠脾脏中CD4+IL-17+Th17细胞的上调幅度最为明显,约2.75倍,其次为IFN-γ+Th1细胞,上调约2.27倍,而Th2和Treg细胞无明显变化。进一步统计学分析显示,Th17对病毒性心肌炎发病的贡献度最高(1.808),Th1次之,贡献度为1.581,而Th2和Treg对病毒性心肌炎发病无显著影响,提示CD4+T细胞亚群格局变化与病毒性心肌炎发病密切相关,其中以Th17对心肌炎发病的贡献度尤为显著。     

Discordant expression of CD28 ligands, BB-1, and B7 on keratinocytes in vitro and psoriatic cells in vivo.

The process for optimal T-cell activation requires not only engagement of the T-cell receptor/CD3 complex, but also the delivery of additional co-stimulatory signals that synergize with the primary response mediated through the T-cell receptor. Thus, the regulated expression of ligands for such co-stimulatory molecules can be critical in determining whether a cell can effectively activate T cells following the presentation of a foreign antigen. The CD28 antigen has recently been shown to mediate such co-stimulatory signals by interacting with the B7/BB-1 molecule expressed on activated B cells and monocytes. We show in this study that activated keratinocytes, both in vitro and in vivo display a discordance in expression between B7 and BB-1 based on differential monoclonal antibody (MAb) reactivity. Activated keratinocytes in vitro, as well as psoriatic keratinocytes and epithelial cells in the thymus, are reactive with the BB-1 MAb but not anti-B7 MAbs. These BB-1 positive cells fail to express detectable B7 messenger RNA by Northern blot analysis. Furthermore, keratinocytes bind specifically to CD28-transfected COS7 cells, and this binding is inhibited by anti-CD28 and anti-BB-1 but not B7 MAbs. These studies suggest: 1) that the MAb against BB-1 binds a functional epitope on a molecule distinct from B7 as detected on activated keratinocytes in vitro and in vivo and 2) that keratinocytes in skin and epithelial cells in thymus can express cell-surface molecules that might mediate T-cell co-stimulation via CD28.     

STAT3、ERK、NF-κB在黄芪保护病毒感染心肌细胞中的作用

目的： 研究STAT3、ERK、NF-κB在黄芪保护感染病毒的心肌细胞中的作用。 方法: 体外分离培养心肌细胞并分为对照组；病毒感染组：病毒孵育；黄芪干预组：病毒孵育后分别给予黄芪培养浓度为300 mg/L、500 mg/L、700 mg/L。采用RT-PCR检测各组病毒RNA，采用Westen blotting检测P-STAT3、P-ERK、NF-κB p65表达。MTT法观察细胞活性。 结果： 在各组中STAT3、ERK总蛋白没有明显变化（P>0.05）,病毒组P-STAT3、P-ERK、NF-κB p65明显高于对照组（P<0.01），黄芪组P-STAT3、P-ERK、NF-κB p65明显低于病毒组（P<0.01）。黄芪组细胞存活率明显高于病毒组（P<0.01）。 结论： 在病毒性心肌炎中黄芪可以通过抑制STAT3、ERK、NF-κB保护心肌细胞。     

Dispensable role for 4-1BB and 4-1BBL in development of vaccinia virus-specific CD8 T cells

CD8 T cells are strongly induced in response to certain strains of vaccinia virus (VACV) and the generation of this population is tightly regulated by two Tumor Necrosis Factor (TNF)/TNFR superfamily members, OX40 (CD134) and CD27. In this study, we examined the role of another member of the TNFR superfamily, 4-1BB (CD137, TNFRSF9), and its ligand (4-1BBL, CD137L, TNFSF9), that have been described to control the generation of memory CD8 T cell populations elicited by other viruses such as influenza. Expression of 4-1BB and 4-1BBL was observed in wild-type mice during the primary infection, but we found that both 4-1BB and 4-1BBL deficient mice generated normal numbers of VACV-specific effector CD8 T cells that produced IFN-γ and TNF. Additionally, CD8 T cells deficient in 4-1BB were able to expand and persist comparably to wild-type T cells in response to VACV infection. Furthermore, the knockout mice also showed no defect in development of VACV-specific CD8 memory T cell populations. Lastly, showing alternate control mechanisms were not active in the gene-deficient environments that masked any activity, blocking 4-1BB/4-1BBL interactions using neutralizing antibody also had no effect on the number of VACV-specific memory CD8 T cells induced. Thus, our data demonstrate that 4-1BB and 4-1BBL do not play a strong or dominant role in driving the generation of high frequencies of VACV-specific CD8 T cells.