Experimental Chagas disease. Innate immune response in Balb/c mice previously vaccinated with Trypanosoma rangeli. I. The macrophage shows immunological memory: Reality or fiction?

Chagas’ disease, caused by Trypanosoma cruzi, is a major vector borne health problem in Latin America and an emerging or re-emerging infectious disease in several countries. Immune response to T. cruzi infection is highly complex and involves many components, both regulators and effectors. Although different parasites have been shown to activate different mechanisms of innate immunity, T. cruzi is often able to survive and replicate in its host because they are well adapted to resisting host defences. An experimental model for vaccinating mice with Trypanosoma rangeli, a parasite closely related to T. cruzi, but nonpathogenic to humans, has been designed in our laboratory, showing protection against challenge with T. cruzi infection. The aim of this work was to analyze some mechanisms of the early innate immune response in T. rangeli vaccinated mice challenged with T. cruzi. For this purpose, some interactions were studied between T. cruzi and peritoneal macrophages of mice vaccinated with T. rangeli, infected or not with T. cruzi and the levels of some molecules or soluble mediators which could modify these interactions. The results in vaccinated animals showed a strong innate immune response, where the adherent cells of the vaccinated mice revealed important phagocytic activity, and some soluble mediator (Respiratory Burst: significantly increase, p ≤ 0.03; NO: the levels of vaccinated animals were lower than those of the control group; Arginasa: significantly increase, p ≤ 0.04). The results showed an important role in the early elimination of the parasites and their close relation with the absence of histological lesions that these animals present with regard to the only infected mice. This behaviour reveals that the macrophages act with some type of memory, recognizing the antigens to which they have previously been exposed, in mice were vaccinated with T. rangeli, which shares epitopes with T. cruzi.     

Phagocytic cells internalize ZnO particles by FcγII/III-receptor pathway

The present study investigates the process of internalization for bulk ZnO particles in macrophages, and further elucidates the underlying mechanism. Since macrophages are active phagocytes and phagocytosis is a size dependent phenomenon, therefore we hypothesized that bulk ZnO may internalize into macrophages by phagocytic pathways. Interestingly, the phagocytic activity got enhanced in bulk ZnO treated macrophages. Moreover, the bulk ZnO treated macrophages internalized via FcγR-II/III, complement and scavenger–receptor pathways. To confirm the specificity of phagocytic pathway, the uptake was also analyzed in splenocytes where phagocytic (monocytes) and non-phagocytic cells (lymphocytes) are present. It was observed that no significant uptake of bulk ZnO in case of lymphocytes whereas significant uptake in monocytes. Henceforth, our quest for uptake mechanisms also revealed that severe plasma membrane extensions (pseudopodia), FcγR clustering over the surface of macrophages and activation of FcγR signaling were the key players for bulk ZnO uptake; whereas clathrin or caveolae mediated endocytic pathways contributed less. Uptake of these particles was further strengthened by the ZnO-induced activation of the Src-kinase p-Lyn, phospho-tyrosine kinases Syk (spleen tyrosine kinase), p-PLC-γ and PI3K (phosphatidylinositol 3-kinase). Our findings illustrate that the phagocytic nature of macrophages could have led to higher uptake of bulk ZnO.     

Effect of candesartan on the expression of sclera-choroidal intercellular adhesion molecule-1 in hypercholesterolemic models

OBJECTIVE:

To evaluate the effect of blocking the angiotensin II AT-1 receptor by the systemic administration of candesartan on the expression of intercellular adhesion molecule-1 in the sclera and choroid of hypercholesterolemic rabbits.

METHODS:

New Zealand rabbits were divided into 3 groups, as follows: GI, which was fed a rabbit standard diet; GII, which was fed a hypercholesterolemic diet; and GIII, which received hypercholesterolemic diet plus candesartan. Samples of the rabbits'' sclera and choroid were then studied by hematoxylin-eosin staining and histomorphometric and immunohistochemical analyses for intercellular adhesion molecule-1 expression.

RESULTS:

Histological analysis of hematoxylin- and eosin-stained sclera and choroid revealed that macrophages were rarely present in GI, and GII had significantly increased macrophage numbers compared to GIII. Moreover, in GII, the sclera and choroid morphometry showed a significant increase in thickness in comparison to GI and GIII. GIII presented a significant increase in thickness in relation to GI. Sclera and choroid immunohistochemical analysis for intercellular adhesion molecule-1 expression revealed a significant increase in immunoreactivity in GII in relation to GI and GIII. GIII showed a significant increase in immunoreactivity in relation to GI.

CONCLUSION:

Candesartan reduced the expression of intercellular adhesion molecule-1 and consequently macrophage accumulation in the sclera and choroid of hypercholesterolemic rabbits.     

NF-κB活化在氯化钆诱导ANP肺泡巨噬细胞凋亡中的作用

目的：观察氯化钆（GdCl3）诱导急性坏死性胰腺炎(ANP)肺泡巨噬细胞(AM)凋亡时核因子-κB（NF-κB）表达情况,探讨NF-κB在其中的作用及机制。
方法：36只SD大鼠随机分为正常对照组,ANP组,GdCl3治疗组。逆行性胰胆管注射5%牛磺胆酸钠建立ANP大鼠模型,正常对照组大鼠以同法注入生理盐水,GdCl3治疗组制模后即刻自阴茎背静脉注射GdCl3。各组大鼠于成模后6 h经支气管肺泡灌洗获取AM,检测支气管肺泡灌洗液(BALF)中TNF-α和IL-1β含量及肺组织髓过氧化物酶(MPO)水平。用琼脂糖凝胶电泳、流式细胞仪检测AM的凋亡情况;Western-blot检测AM中NF-κB活性。同时对肺组织行病理学检查。
结果：ANP组TNF-α和IL-1β含量高于对照组（P<0.05）,而治疗组显著低于ANP组（P<0.05）。仅治疗组DNA电泳见典型的细胞凋亡的梯状条带。对照组,ANP组,治疗组AM凋亡率分别为（10.81±0.86）%,（6.47±1.52）%,（17.41±3.36）%,3组间差异均有统计学意义（P<0.05）;Western-blot检测3组AM中NF-κB表达的相对灰度值分别为（0.80±0.05）,（1.96±0.15）,（1.42±0.10）,3组间差异亦有统计学意义（P<0.05）。AM的凋亡率与NF-κB活性呈负相关（r＝-0.554,P<0.01）。
结论：GdCl3可能通过抑制NF-κB的活化诱导大鼠ANP肺泡巨噬细胞凋亡,从而减轻肺损伤。     

Secondary microvascular degeneration in amyloid angiopathy of patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D)

Various secondary microvascular degenerative and inflammatory alterations may complicate cerebral amyloid angiopathy (CAA) and contribute to the morbidity of CAA-associated stroke. We have investigated the severity of CAA-associated microangiopathy in a genetically determined Dutch form of CAA (HCHWA-D) that has major similarities to the type of CAA that more commonly occurs with aging or Alzheimer’s disease (AD). The presence and extent of the following vascular abnormalities was assessed: (1) hyalinization/fibrosis, (2) microaneurysm formation, (3) chronic (especially lymphocytic) inflammation, (4) perivascular multinucleated giant cells/granulomatous angiitis, (5) macrophages/histiocytes within the vessel wall, (6) vessel wall calcification, (7) fibrinoid necrosis, and (8) mural or occlusive thrombi. (Of these, calcification of CAA-affected vessel walls has, to our knowledge, been described in only a single patient with CAA-associated cerebral hemorrhage.) Some of the changes, such as histiocytes in blood vessel walls and the relationship of vascular hyalinosis to amyloid β/A4 protein deposition, were highlighted by immunohistochemistry. By assessing the numbers of sections in which the changes were present for each case, a ‘score’ reflective of CAA-associated angiopathy could be obtained. This ‘score’ was reproducible among several observers. We suggest that it might also be applicable to quantifying severe CAA and related microvascular degenerative changes in patients with AD. β/A4 immunoreactivity was often sparse and adventitial (or almost absent) in severely hyalinized arterioles and microaneurysms. However, macrophages were prominent in the walls of such vessels and may play a role in the pathogenesis and progression of CAA-related microvasculopathy. Received: 18 June 1997 / Revised, accepted: 22 September 1997     

九种中药多糖含药血清对小鼠腹腔巨噬细胞释放细胞因子的影响

目的 研究黄芪等中药多糖干预小鼠免疫功能的部分机制.方法 常规提取黄芪、防风、当归、柴胡、枸杞、地黄、板蓝根、蒲公英、香菇等9味中药多糖组分,分别按大、中、小剂量灌胃给药,制备小鼠含药血清;选择浓度为20％的含药血清干预体外培养的小鼠腹腔巨噬细胞(Mφ),并用酶联免疫吸附法(ELISA)检测Mφ释放白细胞介素(IL)-1β、IL-6、IL-8、肿瘤坏死因子(TNF)-α等细胞因子(CK)的水平.结果 除柴胡多糖外,其余8种中药多糖含药血清对IL-1β、IL-6、IL-8、TNF-α中一个或多个CK水平具有明显升高或降低作用(P＜0.05或P＜0.01),且具有浓度差异性(P ＜0.05或P＜0.01).结论 黄芪等8味中药调节机体免疫功能的机制,可能与这些中药的多糖组分在体内干预巨噬细胞释放IL-1β、IL-6、IL-8、TNF-α等CK有关.     

神经导向因子Netrin-1调节单核巨噬细胞迁移与动脉粥样斑块关系的研究进展

动脉粥样硬化是动脉壁的一种慢性炎症性疾病,单核巨噬细胞在其发生发展中起着关键作用。动脉粥样斑块中单核巨噬细胞迁移能力受损,滞留于斑块内,增加了斑块不稳定性,加速动脉粥样硬化病变的进展。目前研究表明动脉粥样斑块中巨噬细胞分泌的神经导向因子Netrin-1通过与巨噬细胞表面相应受体结合,可以抑制巨噬细胞迁出斑块,促进动脉粥样硬化的进展。但在动脉粥样硬化形成初期,血管内皮细胞表达的Netrin-1却被发现对动脉粥样硬化起到保护作用。     

miR-20a-5p调控结核分枝杆菌诱导巨噬细胞凋亡相关基因表达的研究

目的 明确小非编码RNA(microRNA,miR-20a-5p)对结核分枝杆菌(MTB)诱导人巨噬细胞凋亡相关基因表达的调控作用。方法 构建可表达或抑制miR-20a-5p、转染miR-20a-5p抑制剂(miR-20a-5p-inhibitor,简称“miR-20a-5p-inh”)、作为慢病毒载体的阴性对照(LV1-NC)的慢病毒载体,由oligo-dT引物通过固相亚磷酰胺法合成茎环寡核苷酸,并克隆到含绿色荧光蛋白(GFP)的慢病毒载体pGLV3-GFP内,将构建的miR-20a-5p、miR-20a-5p-inh、LV1-NC转染THP-1人巨噬细胞3h,培养72h后用流式细胞仪分选出GFP+THP-1细胞,并分别采用减毒MTB菌株(H37Ra)感染8h和过氧化氢(H₂O₂)处理30min 两种方法诱导。通过实时定量PCR技术测定细胞中线粒体相关抗凋亡基因Bcl-2,以及促凋亡基因Bax、Bim和Bad的转录水平。同时,用蛋白免疫印迹法检测细胞裂解物中相关蛋白的表达。结果 慢病毒转染细胞后,在无刺激的THP-1细胞中miR-20a-5p的相对荧光强度值为12.21±1.29、miR-20a-5p-inh为9.68±1.38、LV1-NC为10.64±0.96,三者的表达差异均无统计学意义(q=1.815,P=0.385;q=2.072,P=0.602);但在H37Ra和H₂O₂的刺激后,miR-20a-5p过表达时其相对荧光强度值分别为7.20±0.53、8.55±0.82,明显高于LV1-NC (4.46±0.07、5.49±0.44)(q=50.250,P=0.007;q=1.041,P<0.01),miR-20a-5p受抑制时(1.88±0.08、1.44±0.21)明显低于LV1-NC(q=3.457,P=0.031;q=4.384,P=0.001)。在感染miR-20a-5p慢病毒的THP-1细胞中,H37Ra诱导Bcl-2的表达增加(相对荧光强度值由10.67±0.89增至14.98±0.88)(q=1.064,P=0.008),而感染miR-20a-5p-inh慢病毒时Bcl-2表达减少(由10.67±0.89降至6.49±0.47)(q=3.518,P=0.003);在miR-20a-5p过表达的THP-1细胞中Bim的转录水平减少(由1.22±0.05降至0.98±0.04)(q=1.240,P=0.011),当miR-20a-5p受抑制时Bim的转录水平增加(由1.22±0.05增至1.51±0.08)(q=2.460,P=0.021)。蛋白印迹法检测凋亡相关基因Bcl-2和Bim的蛋白表达水平,结果与基因转录水平相符。 结论 miR-20a-5p的表达水平影响MTB诱导的巨噬细胞凋亡相关基因Bcl-2和Bim的表达,并且与促凋亡基因Bim的水平呈负相关,提示miR-20a-5p可能通过调控Bim的表达而影响细胞凋亡。     

人参提取液对小鼠巨噬细胞的辐射防护作用

本文利用图象分析系统(TAS)定量细胞化学和环核苷酸免疫荧光细胞化学技术观察了人参提取液对小鼠巨噬细胞的辐射防护作用。定量测定表明人参可明显提高受照小鼠巨噬细胞内ANAE、AcPase、ATPase和PAS阳性物质的含量。同时,环核苷酸免疫细胞化学亦显示受照小鼠施加人参后巨噬细胞cAMP和cGMP免疫荧光的定位和强度发生变化,恢复至正常定位和强度。本文对结果的意义进行了讨论