白细胞介素17A对柯萨奇B3病毒性心肌炎小鼠病毒复制的影响及其机制

目的:研究白细胞介素17A(IL-17A)对柯萨奇B3(CVB3)病毒性心肌炎(VMC)小鼠病毒复制的影响及其可能的机制。方法:雄性Balb/c小鼠野生型(WT)和IL-17A基因敲除型(IL-17A-/-)分别感染CVB3建立VMC模型(WT-VMC组和IL-17A-/--VMC组),同时雄性Balb/c WT小鼠腹腔内注射PBS建立对照组(WT-PBS组)。14d后分别留取小鼠心脏及脾脏,计算心体比(HW/BW)并行HE染色观察心肌病理,心肌空斑法测定CVB3病毒滴度,RT-PCR法测定心肌中CVB3RNA、γ干扰素(IFN-γ)mRNA的表达,流式细胞分析法检测脾Th1细胞亚群的比例(CD4+Th1),Western blot法测定心肌中转录因子T-bet蛋白的表达。结果:与WT-PBS组相比,WT-VMC组小鼠心肌炎症明显,HW/BW及心肌病理积分显著升高,CVB3病毒滴度、CVB3RNA及IFN-γmRNA的表达、脾CD4+Th1细胞比例、心肌T-bet蛋白表达均明显升高(均P0.05)。与WT-VMC组相比,IL-17A-/--VMC组小鼠心肌的炎症程度明显减轻,HW/BW及病理积分减小,CVB3病毒滴度与CVB3RNA的表达明显下降(均P0.05),而IFN-γmRNA的表达、脾CD4+Th1细胞比例、心肌T-bet蛋白的表达均较WTVMC组明显升高(均P0.05)。结论:IL-17A抑制VMC小鼠的抗病毒免疫功能,促进CVB3病毒复制,其机制可能与抑制T-bet表达有关。     

癌胚抗原相关黏附分子1对CVB3感染后CAR表达及心肌损伤的影响

目的探讨癌胚抗原相关黏附分子1(CEACAM1或CC1)对柯萨奇病毒(CVB3)感染后柯萨奇病毒-腺病毒受体(CAR)表达和继发心肌损伤的影响。方法构建过表达小鼠CC1重组病毒,包装重组慢病毒pLVX-CEACAM 1-ZsGreen-Puro(rLV-CEACAM 1)并测定慢病毒生物学滴度。分为CC1正常组、CC1过表达组、CC1正常+CVB3组和CC1过表达+CVB3组;各组使用AnnxinV-PE/7-AAD双染法检测心肌细胞凋亡率,CCK8检测细胞活性;qPCR检测CAR基因表达;Western blot检测CAR蛋白表达,ELISA检测肿瘤坏死因子α(TNF-α)及白细胞介素1β(IL-1β)水平。结果 (1)CEACAM1重组载体测序提示目的基因序列连接,证明小鼠CEACAM1重组病毒载体构建成功,测定重组慢病毒滴度为1.5×10~(11) TU/L。(2)CC1过表达+CVB3组较其他组心肌细胞凋亡率明显升高,心肌细胞增殖最低(P0.05)。(3)CAR基因相对表达量在CC1过表达+CVB3组最高,而在CC1正常组最低,CAR蛋白表达也有类似结果;CC1过表达组较CC1正常组胞内CVB3相对表达量显著升高(P0.05)。(4)CC1过表达组TNF-α、IL-1β水平较高,CVB3感染后较前明显升高。结论 CC1可能可以促进CVB3感染心肌细胞后心肌组织或细胞上CAR的表达,CAR可能是CC1调控CVB3感染心肌致心肌损伤过程的潜在作用靶点。     

穿孔素在慢性心肌损伤中的表达及其意义

目的：探讨穿孔素在阿萨奇病毒B组3型（CVB3m)至T－2毒素染毒小鼠心肌损伤中的表达及其意义,方法：实验组BALB／C小鼠1．0mg/kg体重T－2毒素隔日灌胃,3周后腹腔接种0．1ml内含1000TCID50的CVB3m病毒液,对照组生理盐水灌胃,腹腔接种0．1ml 1640营养液,观察心肌病理改变,同时用RTPCR技术对心肌组织中CVB3m RNA和穿孔素mRNA表达进行检测,结果：CVB3m感染T－2毒素染毒小鼠心肌出现慢性损伤,可见纤维结缔组织增生和坏死灶癜痕修复。心肌组织中持续表达CVB34m RNA和穿孔素mRNA。结论CVB3m RNA和穿孔素mRNA的持续表达可能是CVB3m致T－2毒素染毒小鼠心肌慢性损伤的机制之一。     

核转录因子抑制剂对病毒感染心肌细胞的保护作用

目的:探索核转录因子(NF κB)抑制剂吡咯基二硫氨基甲酸酯(PDTC)对病毒感染搏动心肌细胞的作用及信号转导机制。方法:利用培养的SD乳鼠心肌细胞,分为对照组,CVB3 感染组[心肌细胞感染 coxsackieB3(CVB3)病毒],PDTC干预组(加入PDTC干预后心肌细胞再感染 CVB3 病毒)。采用 MTT法检测细胞活性,采用细胞病变法计算细胞病变;采用Westen blot测定NF kBp65蛋白表达变化。结果:CVB3 感染组与对照组比较,心肌细胞活性明显降低,细胞病变加重;NF kBp65蛋白表达明显高于对照组(P<0.01)。PDTC干预组心肌细胞活性明显低于CVB3 感染组, 细胞病变较 CVB3 感染组轻,NF kBp65 蛋白表达明显低于 CVB3 感染组(P<0.01)。结论:CVB3 病毒在感染早期可导致心肌细胞损伤,NF κB抑制剂可保护心肌细胞免受病毒侵害,NF κB信号通路激活可能为病毒性心肌炎发病机制之一。     

miRNA378*对柯萨奇B3病毒感染心肌细胞凋亡、网腔钙结合蛋白、内质网应激及信号通路因子的作用

目的:探讨上调miRNA378*表达对柯萨奇B3病毒(CVB3)感染心肌细胞凋亡、网腔钙结合蛋白、内质网应激及信号通路因子的作用。方法:实验分4组:对照组(正常细胞)、CVB3感染组(正常细胞+CVB3)、miRNA378*过表达对照组(正常细胞+CVB3+转染miRNA378*空表达质粒)、miRNA378*过表达组(正常细胞+CVB3+转染miRNA378*过表达质粒)。原代培养乳鼠心肌细胞,采用免疫组织化学方法检测培养乳鼠心室肌细胞α-SMA蛋白,慢病毒质粒转染心室肌细胞,除对照组外,其他各组心肌细胞感染CVB3,采用TUNEL技术检测各组心肌细胞凋亡率;用Western blotting技术检测各组心肌细胞网腔钙结合蛋白、内质网应激伴侣蛋白GRP78及内质网应激信号通路因子PERK、P-PERK、eIF2α、ATF4、CHOP表达。结果:与CVB3感染组比较,miRNA378*过表达组心肌细胞凋亡率明显减少,网腔钙结合蛋白表达增加,而GRP78、P-PERK、eIF2α、ATF4、CHOP表达均减少(均P0.01),PERK表达差异无统计学意义。结论:上调CVB3感染心肌细胞miRNA378*表达可引起心肌细胞凋亡减少,网腔钙结合蛋白表达增多,进而缓解内质网应激,并抑制内质网应激凋亡信号通路因子表达。     

肝素酶在人β-防御素-3抗甲型流感病毒的作用研究北大核心CSCD

目的探讨肝素酶在人β-防御素-3(Humanβ-defensin 3,HBD3)抗甲型流感病毒(Influenza A virus,IAV)中的作用。方法采用CCK8法检测肝素酶对人支气管上皮细胞(BEAS-2B)的毒性情况;经肝素酶处理BEAS-2B细胞,并在4℃下与HBD3和IAV病毒液孵育2 h,分为未处理组(HBD3+IAV),肝素酶+HBD3+IAV组,提取细胞总RNA和总蛋白,采用qRT-PCR检测感染细胞中的NP mRNA表达水平,Western blot和免疫共沉淀检测HBD3和IAV HA蛋白表达水平以及HBD3和HA蛋白的相互作用。试验设未经肝素酶处理BEAS-2B细胞对照组。结果肝素酶作用48 h,其浓度在30 U/mL以下浓度时对细胞无毒性作用;HBD3干预IAV感染细胞后细胞中的NP mRNA表达水平显著降低(t=12.81、26.13、28.68,P<0.01),经肝素酶处理IAV感染的细胞其NP mRNA表达水平升高(t=-4.55,P<0.05),结果显示HBD3可抑制病毒复制,而经肝素酶处理IAV感染的细胞其病毒未减少,表明只使用肝素酶不能降低病毒感染;与未经肝素酶处理的细胞相比,经肝素酶处理的细胞中HBD3和HA蛋白表达水平降低,以及HBD3和HA蛋白的相互作用显著降低(P<0.01),表明HBD3和肝素酶共同作用可降低HA的表达来阻止IAV进入细胞。结论肝素酶和HBD3共同作用可以抑制病毒进入细胞,具有抗甲型流感病作用。     

Caspase-3在异丙基肾上腺素致大鼠急性心肌损伤中的作用及bFGF的影响

目的：研究半胱天冬酶-3(caspase-3)在异丙基肾上腺素(isoprenaline，Iso)致大鼠急性心肌损伤中的作用及碱性成纤维细胞生长因子(bFGF)对它的影响。方法：将Wistar雄性大白鼠随机分成3组：对照组、损伤组和bFGF保护组，光镜观察心肌组织的病理变化，TUNEL法检测心肌细胞凋亡，免疫组化和原位杂交方法检测caspase-3蛋白及mRNA的表达水平，底物降解法测定该酶的活性。结果：Iso损伤组心肌坏死较重，有显著心肌细胞凋亡，且Caspase-3基因mRNA及蛋白表达水平明显升高，酶活性增加；bFGF保护组心肌坏死明显减轻，凋亡细胞数减少，Caspase-3基因mRNA和蛋白表达水平明显下降，酶活性受到抑制。结论：bFGF可能通过抑制Caspase-3的表达和活性减少心肌细胞凋亡而减轻大鼠心肌损伤。     

中性鞘磷脂酶2对缺氧预处理间充质干细胞来源的外泌体生物学特性的影响

目的探讨中性鞘磷脂酶2对缺氧预处理骨髓间充质干细胞来源的外泌体生物学特性的影响。方法分离并体外扩增6周龄C57BL/6小鼠来源的骨髓间充质干细胞,收集细胞上清,分离来自缺氧处理的外泌体(Exo~H)和常氧下培养来源的外泌体(Exo~N),并鉴定其生物学特性。比较Exo~H和Exo~N在体内实验中对心肌细胞保护作用的差异,同时收集在缺氧预处理状态下加入中性鞘磷脂酶2抑制剂GW4869来源的外泌体(Exo~H+GW),并在体外实验中比较Exo~N、Exo~H和Exo~H+GW对心肌细胞抗凋亡及血管新生能力的作用。结果 Exo~H组小鼠心肌梗死后28 d的射血分数显著高于Exo~N组(44.12%±8.12%比20.73%±7.32%,n=20,t=11.95,P<0.05)。同时发现,缺氧增加中性鞘磷脂酶2的表达,而Exo~H+GW组在体外管腔形成及心肌细胞保护方面明显弱于Exo~H组,并且其微小RNA-210含量显著减低。结论缺氧预处理骨髓间充质干细胞来源的外泌体拥有更好的心脏保护能力,中性鞘磷脂酶2可能是主要的作用机制,可能通过影响外泌体内有益微小RNA的含量来发挥生物学作用。     

心肌细胞感染病毒后钙、钾通道电流及其表达改变和牛磺酸的作用

目的 观察心肌细胞感染病毒后心肌细胞钙，钾通道电流及通道表达改变和牛磺酸的作用。方法 （1）体外心肌细胞感染病毒；用胰蛋白酶和胶原酶消化法分别获得大鼠，乳鼠培养和分离的心肌细胞并感染柯萨奇B3病毒，用于检测Ca^2 跨膜内流，电压依赖性钙通道电流（Lca)和外向钾通道电流（Iout)。（2）在体小鼠感染病毒；BALB/c小鼠腹腔内注射柯萨奇B3病毒7d后取出心脏用于检测电压堆呀性钙通道α1亚单位抗原和电压调控性钾通道（Kv)mRNA的表达。相应各组加入不同剂量的牛磺酸并观察其作用。结果 （1）感染柯萨奇B3病毒后Ca^2 跨膜内流量及Ica增加，牛磺酸对其有抑制作用。（3）柯萨奇B3病毒感染out明显增大。牛磺酸可使病毒感染后的Iout趋于恢复正常。（3）柯萨奇B3病毒感染后小鼠心肌出现强电压依赖性钙通道α1亚基抗原阳性信号。Kv1.2,Kv2.1,Kv4.2mRNA与3种探针的杂交信号亦明显增强，牛磺酸可使增强的阳性信号减弱。结论 柯萨奇B3病毒感染后心肌细胞钙，钾离子通道电流及表达均增加，牛磺酸可通过抑制其改变而发挥对感染病毒心肌细胞的保护作用。     

钙在CVB_3诱导大鼠心肌细胞凋亡中的作用

目的　探讨细胞内游离钙 [Ca2 ]i在柯萨奇病毒 B3(CVB3)诱导培养心肌细胞凋亡中的作用。方法　 DNA裂点检测法 (3′-末端标记 )及透射电镜检测细胞凋亡。 Fluo3- AM负载心肌细胞 ,共聚焦显微镜观察[Ca2 ]i荧光强度变化。结果　感染 2 4h心肌细胞内 CVB3滴度达峰值。感染 10 h未见凋亡的心肌细胞 ,17、2 4和 36 h凋亡细胞分别为 5 %、6 0 %和 90 % ,感染 17h心肌 [Ca2 ]i浓度达峰值。电镜结果提示 CVB3感染组存在典型的凋亡心肌细胞。结论　 CVB3可诱导培养心肌细胞的凋亡过程。细胞内 Ca2 参与凋亡细胞的信息传导过程 ,并在凋亡早期发挥重要作用     

In Vitro Model Systems of Coxsackievirus B3-Induced Myocarditis: Comparison of Commonly Used Cell Lines and Characterization of CVB3-Infected iCell® Cardiomyocytes

Coxsackievirus B3 (CVB3) belongs to the enteroviruses, which are a well-known cause of acute and chronic myocarditis, primarily infecting cardiac myocytes. As primary human cardiomyocytes are difficult to obtain, viral myocarditis is quite frequently studied in vitro in different non-cardiac and cardiac-like cell lines. Recently, cardiomyocytes that have been differentiated from human-induced pluripotent stem cells have been described as a new model system to study CVB3 infection. Here, we compared iCell^® Cardiomyocytes with other cell lines that are commonly used to study CVB3 infection regarding their susceptibility and patterns of infection and the mode of cell death. iCell^® Cardiomyocytes, HeLa cells, HL-1 cells and H9c2 cells were infected with CVB3 (Nancy strain). The viral load, CVB3 RNA genome localization, VP1 expression (including the intracellular localization), cellular morphology and the expression of cell death markers were compared. The various cell lines clearly differed in their permissiveness to CVB3 infection, patterns of infection, viral load, and mode of cell death. When studying the mode of cell death of CVB3-infected iCell^® Cardiomyocytes in more detail, especially regarding the necroptosis key players RIPK1 and RIPK3, we found that RIPK1 is cleaved during CVB3 infection. iCell^® Cardiomyocytes represent well the natural host of CVB3 in the heart and are thus the most appropriate model system to study molecular mechanisms of CVB3-induced myocarditis in vitro. Doubts are raised about the suitability of commonly used cell lines such as HeLa cells, HL-1 cells and H9c2 cells to evaluate molecular pathways and processes occurring in vivo in enteroviral myocarditis.     

The Role of Sex Differences in Autophagy in the Heart During Coxsackievirus B3-Induced Myocarditis

Under normal conditions, autophagy maintains cardiomyocyte health and integrity through turnover of organelles. During stress, oxygen and nutrient deprivation, or microbial infection, autophagy prolongs cardiomyocyte survival. Sex differences in induction of cell death may to some extent explain the disparity between the sexes in many human diseases. However, sex differences in gene expression, which regulate cell death and autophagy, were so far not taken in consideration to explain the sex bias of viral myocarditis. Coxsackievirus B3 (CVB3)-induced myocarditis is a sex-biased disease, with females being substantially less susceptible than males and sex hormones largely determine this bias. CVB3 was shown to induce and subvert the autophagosome for its optimal viral RNA replication. Gene expression analysis on mouse and human, healthy and CVB3-infected, cardiac samples of both sexes, suggests sex differences in autophagy-related gene expression. This review discusses the aspects of sex bias in autophagy induction in cardiomyocytes.     

SIRT6-shRNA预处理对缺氧/复氧诱导的H9C2心肌细胞损伤的影响及其机制

目的 探讨通过RNA干扰技术抑制H9C2心肌细胞内的SIRT6基因表达对缺氧/复氧(A/R)诱导的细胞损伤的影响和机制。方法 将H9C2心肌细胞随机分为正常对照组(Con组)、A/R组、阴性对照SIRT6-shRNA质粒处理组(NC组)和SIRT6-shRNA质粒处理组(shRNA组)。检测4组H9C2心肌细胞的存活率、凋亡率、Caspase-3活性及SIRT6、核因子(NF)-κBp65、I-κBα表达水平及细胞培养液中白细胞介素(IL)-6和肿瘤坏死因子(TNF)-α水平并进行比较。结果 与Con组相比,A/R组H9C2心肌细胞存活率和细胞浆中I-κBα蛋白表达水平明显降低,凋亡率、细胞SIRT6-mRNA和蛋白、细胞核中NF-κBp65蛋白表达水平、细胞培养液中IL-6和TNF-α水平及Caspase-3活性明显升高（P<0.05）。与A/R组比较,shRNA组H9C2心肌细胞存活率、细胞SIRT6-mRNA和蛋白及细胞浆中I-κBα蛋白表达水平明显降低,细胞凋亡率、细胞核中NF-κBp65蛋白、细胞培养液中IL-6和TNF-α水平及Caspase-3活性明显升高(P<0.05)。结论 抑制H9C2心肌细胞内SIRT6基因表达能促进炎症因子的分泌,激活NF-κB信号通路,诱导细胞凋亡,加重A/R诱导的心肌细胞损伤。     

MCP-1与CVB3相互作用参与病毒性心肌炎的发生

目的　研究单核细胞趋化蛋白 - 1(MCP- 1)在 B3型柯萨奇病毒 (CVB3)诱导病毒性心肌炎 (VMC)发病中的作用。方法　构建小鼠 MCP- 1真核表达质粒 p VM。 CVB3腹腔注射雄性 BAL B/c小鼠 ,随机分四组 :单独 CVB3感染 VMC组 (A组 )、10 μg过表达 MCP- 1组 (B组 )、4 0 μg过表达 MCP- 1组 (C)和空质粒对照组 (D组 )。并设未感染生理盐水对照组 (E组 )和未感染过表达组 (F组 )。比较各组小鼠心脏重量和体重的比值 (HW/BW)、心肌组织病理学积分 (PS)、心肌组织病毒载量、血清 CK- MB水平。结果　与 E组相比 ,A组的 HW/BW、PS、血清 CK- MB和心肌组织病毒载量均有明显改变。表明 CVB3感染后小鼠出现一系列的体征改变。与 A组相比 B组的 HW/BW、PS、血清 CK- MB和心肌组织病毒载量均未呈明显改变。但 C组 CVB3载量明显降低 (P<0 .0 5 ) ,血清 CK- MB水平升高 (P<0 .0 1) ,而 HW虽然升高(P<0 .0 5 ) ,但是 HW/BW未呈明显的改变 (P>0 .0 5 ) ,PS升高 (P<0 .0 5 )。 D组与 A组相比 ,CVB3载量、血清 CK-MB水平、HW/BW和 PS均未呈明显的改变 (P>0 .0 5 )。 F组的 HW/BW和血清 CK- MB水平与 E组相比未呈现显著性差异 ,而 PS升高 ,但低于 A组 (P<0 .0 1)。结论　单独在心肌组织过表达 MCP- 1仅引起心肌组织学改变 ,不能     

Activation of AMPK restricts coxsackievirus B3 replication by inhibiting lipid accumulation

Coxsackievirus B3 (CVB3) is the major pathogen of human viral myocarditis. CVB3 has been found to manipulate and modify the cellular lipid metabolism for viral replication. The cellular AMP-activated protein kinase (AMPK) is a key regulator of multiple metabolic pathways, including lipid metabolism. Here we explore the potential roles AMPK plays in CVB3 infection. We found that AMPK is activated by the viral replication during CVB3 infection in Hela cells and primary myocardial cells. RNA interference mediated inhibition of AMPK could increase the CVB3 replication in cells, indicating that AMPK contributed to restricting the viral replication. Next, we showed that CVB3 replication could be inhibited by several different pharmacological AMPK activators including metformin, A769662 and AICAR. And the constitutively active AMPK mutant (CA-AMPK) could also inhibit the CVB3 replication. Furthermore, we found that CVB3 infection increased the cellular lipid levels and showed that the AMPK agonist AICAR both restricted CVB3 replication and reduced lipid accumulation through inhibiting the lipid synthesis associated gene expression. We further found that CVB3 infection would also induce AMPK activated in vivo. The AMPK agonist metformin, which has been widely used in diabetes therapy, could decrease the viral replication and further protect the mice from myocardial histological and functional changes in CVB3 induced myocarditis, and improve the survival rate of infected mice. Lastly, it was demonstrated that the AICAR-mediated restriction of viral replication could be rescued partially by exogenous palmitate, the first product of fatty acid biosynthesis, demonstrating that AMPK activation restricted CVB3 infection through its inhibition of lipid synthesis. Taken together, these data in the present study suggest a model in which AMPK is activated by CVB3 infection and restricts viral replication by inhibiting the cellular lipid accumulation, and inform a potential novel therapeutic strategy for CVB3-associated diseases.     

CVB3损伤Hela细胞高尔基体结构的机制研究

摘要：目的 研究CVB3损伤Hela细胞高尔基体结构的机制,为进一步探讨CVB3的致病机制奠定基础。方法 采用MOI为5的CVB3感染Hela细胞,免疫荧光双标法检测病毒感染后不同时间点VP1和GM130在细胞中的定位情况,观察细胞高尔基带的变化;同时,选择CVB3感染后不同时间点收集细胞,Bradford法蛋白定量后,Western blot测定VP1、GM130和GRASP65蛋白的表达变化。结果 正常Hela细胞中GM130 和GRASP65免疫荧光呈带状分布,高尔基体结构正常;而CVB3病毒感染Hela细胞3h后,GM130的荧光呈现散点状分布,高尔基体结构损伤。与此同时,GM130和GRASP65蛋白表达量逐渐下降;而VP1蛋白表达量持续上升,且6 h后一直维持在较高水平。结论 CVB3感染引起Hela细胞蛋白GM130和GRASP65表达量下调,导致GM130和GRASP65荧光带弥散,高尔基带断裂,这可能是CVB3感染导致宿主细胞高尔基体结构损伤的机制之一。     

Expression of Inducible Nitric Oxide Synthase in Experimental Viral Myocarditis

Nitric oxide (NO) is an important bioactive molecule with regulatory, cytotoxic or cytoprotective properties. In virus-induced myocarditis, NO mediates host defense mechanisms against the infection or causes cardiac dysfunctions. NO is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). The expression of the inducible form of the nitric oxide synthase (iNOS) is regulated by cytokines, involved in the complex myocardial immune response to enterovirus infections. The present study was undertaken to characterize the role of iNOS and NO in the murine model of viral myocarditis induced by coxsackievirus B3 (CVB3). In response to CVB3 infection we investigated the time course of iNOS induction in correlation with cytokine mRNA expression (TNF-alpha, IL-1 alpha, IFN-gamma, TGF-beta) in the heart of NMRI mice by RT-PCR. Positive PCR signals for viral RNA were found in the acute and chronic stage of disease by seminested PCR, indicating the persistence of viral genome. We found distinct expression of iNOS at all time points (1, 2, 3, 4, 7, 14, 28, 56, 98 days post infection [p.i.]). Higher iNOS mRNA levels were identified between days 4 until 28 p.i. in comparison to day 56 and 98 p.i. using densitometric values. The mRNA of the inflammatory cytokines TNF-alpha, IL-1 alpha, IFN-gamma appeared at days 1, 4, and 7 p.i., peaked at day 7 p.i. and persisted until day 98 p.i. Similar like the iNOS mRNA pattern was the expression profile of TGF-beta. Using in situ hybridization and immunohistochemistry iNOS was localized in infiltrates, vascular endothelial cells, smooth muscle cells, myocytes and throughout the interstitial spaces between myocardial fibers in the heart sections of NMRI mice. Increased levels of NO were measured as total nitrate/nitrite concentration in the sera of mice from day 7 until day 28 p.i.