口服免疫Ag85A脂质体DNA诱导T细胞亚群应答效应研究

目的:拟采用构建的可在真核细胞内表达Ag85A基因的质粒,以阳离子脂质体为运载体,制成DNA疫苗,经口途径投予小鼠,以观察Ag85A脂质体DNA疫苗诱导免疫应答效应,为口服DNA疫苗的临床应用提供理论和实验依据.方法:ELISA方法检测Ag85A特异性抗体产生水平及血清Th1型细胞因子IFN-γ及Th2型细胞因子IL-4的分泌水平,ELISPOT技术检测口服DNA疫苗后小鼠可分泌IFN-γ和IL-4脾淋巴细胞数量.流式细胞术观察口服DNA疫苗后小鼠脾淋巴细胞CD4+T细胞及CD8+T细胞亚群的变化,从而判断口服DNA疫苗的免疫效果及脂质体是否有免疫增强作用.结果:口服自制Ag85ADNA疫苗可见血清中抗Ag85A特异性抗体的产生;下调了脾CD4+T细胞和CD8+T细胞亚群的数量;分泌IFN-γ的Th1型细胞比率和血清中的IFN-γ水平下降,而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高.口服DNA疫苗组诱导Ag85A特异性抗体产生,脂质体具有免疫佐剂作用.结论:应用阳离子脂质体为运载体,重组Ag85A DNA疫苗口服免疫C57BL/6小鼠,诱导了CD4+及CD8+T细胞亚群的下调及IFN-γ的表达减少,而玎IL-4的分泌呈增高趋势;疫苗可诱导Ag85A特异性抗体的分泌,产生了明显的体液应答.     

HBsAg重组腺病毒转染的树突状细胞诱导BALB/c小鼠抗HBV免疫的研究

目的 研究HBsAg重组腺病毒转染的小鼠树突状细胞（DC）体内外免疫刺激活性和诱导小鼠抗HBV免疫的特点。方法 BALB／c小鼠的骨髓细胞体外扩增为DC，转染HBsAg重组腺病毒Ad-S或被HBsAg蛋白冲击后，流式细胞术分析DC的表型，混合淋巴细胞反应（MLR）检测DC的刺激活性；或与pcDNA3．1（＋）-S质粒分别免疫小鼠后，LDH法测定脾细胞CTL活性，放免法检测血清抗．HBs；流式细胞术分析体外自体MLR中及免疫后小鼠脾脏T细胞内细胞因子。结果 DC／Ad-S、DC／HBsAg和各对照组DC之间的表型和MLR差异无统计学意义，并均刺激TH和Tc分泌IFN-γ。DC／Ad-S免疫后2周能诱导比DNA疫苗更强产生IFN-γ的TH（P〈0．05），以及比DC／HBsAg和DNA疫苗更强分泌IFN-γ的Tc（均P〈0．01）和特异性CTL（P〈0．05，P〈0．01）。但DC／Ad-S和DC／HBsAg诱导的CTL反应及Tc分泌IFN-γ在免疫后4周均明显减弱，且诱导抗．HBs作用弱于DNA疫苗。结论 HBsAg重组腺病毒转染的DC比HBsAg冲击的DC及DNA疫苗能诱导更强的TH1/Tcl（Ⅰ型）细胞免疫和特异性CTL反应，是诱导抗HBV细胞免疫的有效刺激细胞。     

HIV-1 B''亚型gag-env融合基因的DNA疫苗构建和免疫原性分析

目的 构建HIV-1 B′亚型中国流行株gag和env融合基因的DNA疫苗，对其免疫原性进行研究。方法 根据已报道的HIV-1 B′亚型RIA2分离株gag和env基因的氨基酸序列按哺乳动物密码子使用频率进行优化并人工合成基因，插入真核表达载体pDRVISV1．0中，构建表达RL42 gag-env，融合蛋白的DNA疫苗，pSVRL／GE。用Western blot和抗gag p55抗体细胞内染色的方法体外检测pSVRUGE的表达效率。DNA疫苗pSVRL/GE免疫BALB／c小鼠后，用ELISPOT检测小鼠的细胞免疫反应。结果 限制性内切酶鉴定表明融合基因已成功插入pDRVISV1．0载体中，Western blot证实融合基因可有效表达融合蛋白；细胞内染色结果表明，pSVRL/GE转染的293T细胞中49．8％表达gag p55，荧光强度均值为924；而空载体pDRVISV1．0转染的293T细胞中非特异背景染色只有0．5％。经免疫的小鼠脾细胞体外用H-2^d限制性表位肽AMQMIKET刺激后，ELISPOT检测显示，pSVRUGE免疫小鼠每10^6脾细胞形成226个斑点（SD=140），而空载对照组每1驴脾细胞形成29个斑点（SD=16）（P〈0．05）。结论 所构建的DNA疫苗pSVRL/GE可高效表达相应抗原蛋白，并可有效激活机体的细胞免疫反应。     

门脉高压性脾脏形态结构的实验研究

目的：研究门脉高压性脾脏形态结构。方法：用硫代乙酰胺诱发大鼠门脉高压性脾肿大，不同的时期摘脾研究其形态结构。结果：发现早期脾组织充血水肿，８周后红髓区脾窦扩大、脾索狭窄，白髓区动脉周围淋巴鞘萎缩纤维化。形态计量测定表明随着脾肿大的发展，红髓面积百分比增加，白髓面积百分比减少。白髓区内ＰＡＬＳ、生发中心面积百分比亦进行性减少。结论：门脉高压性脾脏发挥免疫功能的区域萎缩，其免疫功能低下，此时外科保脾意义不大。     

Ag85A DNA疫苗加强免疫显著提高卡介苗初免小鼠的抗结核T细胞免疫应答

目的 构建表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)免疫优势抗原Ag85A的DNA疫苗,分析其加强免疫后提高卡介苗(BCG)初免小鼠的抗结核T细胞免疫应答.方法 以Mtb毒株H37Rv基因组DNA为模板,PCR扩增Ag85A抗原编码的结构基因并克隆至真核表达载体pVAX1中构建其DNA疫苗;接着,将纯化后的该DNA疫苗加强免疫BCG初免小鼠2针,以BCG和DNA单独免疫小鼠为对照,免疫8周后无菌分离脾淋巴细胞,分别应用IFN-γ ELISPOT和多因子胞内流式细胞术(intracellular staining)分析免疫小鼠的Mtb抗原特异性效应细胞免疫水平与分泌IFN-γ/TNF-α/IL-2的多功能CD4+T细胞频率及其强度以及CD8+T细胞免疫应答.结果 与BCG免疫及DNA单独免疫组相比,Ag85A DNA加强免疫不仅能显著提高小鼠IFN-γ+TNF-α+IL-2+多功能T细胞,IFN-γ+IL-2+、IL-2+TNF-α+双功能T细胞与IL-2+单功能T细胞的频率以及IL-2的分泌能力,还能显著诱导小鼠产生更多分泌IFN-γ和IL-2的CD8+T细胞.结论 本研究成功构建了表达Mtb免疫优势抗原Ag85A的DNA疫苗并分析了其免疫原性,证实了BCG初免-DNA加强的免疫策略可同时显著增强实验小鼠的Mtb抗原特异性CD4+T和CD8+T细胞应答水平,有利于提高BCG的免疫原性,为增强BCG逐渐下降的抗结核保护效果提供新思路.     

HO-1对DXR免疫反应影响的实验研究

目的 探讨HO-1对延迟性异种心脏移植免疫反应的影响。方法 应用NIH小鼠-Wistar大鼠颈部异位心脏移植模型，用CoPP（cobalt protoporphyrin，CoPP）诱导HO-1并结合免疫抑制剂Cyclosporine A（CSA）及Cyclophosphamid（CYP）处理受体。比较移植心脏存活时间并分别用免疫组化、Westem- blot蛋白印迹杂交等检测移植心脏HO-1、IL-1β、INF-γ、TNF-α和STAT-3表达，免疫组化检测并计数分析CD68细胞，比较不同处理因素之间的差异。结果 单用CoPP诱导HO-1后移植心脏存活时间较CSA组及空白对照组长（t=2．6936，P〈0．05），CSA＋CoPP组移植心脏存活时间和CYP＋CoPP组均长于单用CSA组和CYP组（t=2．8511，P〈0．05），CoPP＋CSA＋CYP组移植心脏存活时间优于其余任何组（t=3．8644，P〈0．001）。CoPP组TNF-α、LI-1β低于在空白对照组的表达（t=2．6364，P〈0．05），但INF-γ除外。三者在CoPP＋CSA＋CYP组表达均低于其余各组。CoPP及CSA、CYP组间STAT-3表达无差异（t=1．0854，P〉0．05），但均高于空白对照组（t=10．1254，P〈0．001）。CoPP与CSA及CYP联合应用时STAT-3表达高于单用CoPP组（t=2．8164，P〈0．05），三者联合使用时高于任何组（t=2．5605，P〈0．05）。CoPP组移植心肌组织中CD68阳性细胞数低于空白对照组（t=3．1837，P〈0．01），CoPP＋CSA＋CYP组最低（t=2．5099，P〈0．05）。结论 HO-1并联合免疫抑制剂CSA、CYP可能通过上调STAT-3蛋白表达，减少CD68细胞对移植心脏的浸润，降低心脏移植后炎症细胞因子IL-1β、TNF-α和IFN-γ的产生，并可能由此推迟了DXR的发生。     

小柴胡汤对免疫抑制小鼠脾细胞凋亡的影响及其机制

目的探讨小柴胡汤对免疫抑制小鼠脾细胞凋亡的影响及其机制。方法每日给予正常小鼠及肌肉注射环磷酰胺诱导的免疫抑制小鼠小柴胡汤25g/kg,连续10日,并设免疫抑制模型组及正常对照组。于实验第11日采用末端脱氧核苷酸转移酶介导的缺口末端标记法检测脾脏细胞凋亡;免疫细胞化学法检测脾脏细胞Bcl-2和NF—KB的表达。结果与正常对照小鼠比较,免疫抑制小鼠脾细胞凋亡百分率明显升高（F=40．65,P〈0．01）,Bcl-2和NF—KB的表达均降低（F值分别为37．25、52．16,P〈0．01）;与正常对照组比较,给予小柴胡汤的正常小鼠的脾细胞凋亡率及Bcl-2和NF—KB的表达均没有统计学意义（F值分别为3．76、1．69、5．48,P〉0．05）。与免疫抑制小鼠比较,给予小柴胡汤的免疫抑制小鼠脾细胞凋亡率明显降低（F=36．84,P〈0．01）,Bcl-2和NF—KB的表达升高（F值分别为29．56、37．58,P〈0．01）。结论小柴胡汤可能通过影响免疫抑制小鼠脾细胞的凋亡进而调节机体免疫功能。     

结核分枝杆菌Ag85A基因DNA疫苗的构建及其联合人IL-12表达质粒诱导的小鼠细胞免疫应答观察

目的:构建编码结核分枝杆菌Ag85A分泌蛋白重组真核表达质粒,研究其与hIL-12联合免疫小鼠后的细胞免疫应答。方法:(1)构建质粒:采用PCR法从H37Rv菌株中扩增Ag85A编码基因,用限制性内切酶消化后,插入克隆载体PMD20-T中,经酶切鉴定与序列测定证实后,以亚克隆法构建于真核表达载体PCDNA3.1的相应酶切位点。(2)动物实验:50只C57BL/6N小鼠随机分为:①Ag85A基因疫苗+hIL-12质粒组(联合免疫组);②重组Ag85A基因疫苗组;③卡介苗BCG组(阳性对照);④空载体组(阴性对照);⑤PBS组(空白对照)。基因疫苗、空载体和PBS经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106CFU BCG免疫1次,约0.3 ml/只。第三次免疫小鼠后28天,处死各组小鼠,分离脾细胞,ELISA法检测脾细胞培养上清液中IFNγ-、IL-2、IL-4水平;乳酸脱氢酶释放法检测脾细胞杀伤活性;分离的脾细胞经TB-PPD刺激后,XTT比色法检测脾淋巴细胞增殖活性。结果:(1)成功构建结核分枝杆菌Ag85A基因DNA疫苗。(2)联合免疫组能诱导较强烈的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-r和IL-2水平显著高于Ag85A基因疫苗组,与BCG组相当,IL-4分泌减少;特异性CTL杀伤活性明显增强;淋巴细胞增殖活性也明显高于其他组别。结论:hlL-12表达质粒能够增强结核分枝杆菌Ag85A基因DNA疫苗所诱导的小鼠免疫应答。     

日常生活时间性预期记忆的提取线索探析

目的探讨日常生活时间性预期记忆的提取线索及其特点。方法采用自编的时间性预期记忆的提取问题调查表，对254名17—40岁的学生、教师等进行测试。结果（1）日常生活时间性预期记忆的提取线索主要有三个方面；记忆策略、加工产物和意图类型。（2）日常生活时间性预期记忆的提取线索存在显著的年龄差异，26-40岁年龄组在记忆策略（t=2.98，P〈0．01）、加工产物（t=2．53，P〈0．05）和意图类型（t=2．64，P〈0．01）因子上的得分均真显著高于17-25岁年龄组。（3）日常生活时间性预期记忆的提取线索存在显著的师生差异，教师在记忆策略（t=3．75，P〈0．001）、加工产物（t=2.43，P〈0．05）和意图类型（t=2．10，P〈0．05）因子上的得分真显著高于学生。（4）记忆自评偏好组在加工产物（t=5．53，P〈0．001）和意图类型（t=2．27，P〈0．05）因子上的得分显著高于记忆自评偏差组。结论个体倾向于使用记忆策略、加工产物和意图类型作为时间性预期记忆的提取线索，表现出明真真的加工性和经验性特点。     

结核杆菌免疫保护性抗原Ag85A的基因克隆与表达研究

目的结核病疫情再度增高给结核病的防治提出了新的挑战,由于卡介苗对结核病的预防效果极不稳定,发展新型结核病疫苗势在必行,本研究通过对结核杆菌免疫保护性抗原Ag85A进行克隆与表达研究,旨在为结核病新型疫苗研制提供新的靶点.方法根据结核杆菌H37Rv株免疫保护性抗原Ag85A的基因序列自行设计一对寡核苷酸引物,以结核杆菌H37Rv株基因组DNA为模板通过PCR扩增获得具有完整开架读码框(ORF)的Ag85A抗原基因,并将其正向插入真核和原核穿梭表达型载体pBKCMV构建重组质粒,重组质粒转入大肠杆菌后IPTG诱导表达,并对其表达产物进行Western-blotting分析.结果①PCR扩增获得了具有完整开架读码框的Ag85A抗原基因;②构建了Ag85A抗原基因真核和原核穿梭表达型载体;③Ag85A抗原基因在大肠杆菌中实现了稳定表达.结论本研究成功地对结核杆菌免疫保护性抗原Ag85A进行了基因克隆与表达,为进一步研究其在结核病新型疫苗研制中的应用奠定了基础.     

Species variation in the structure and function of the marginal zone—an electron microscope study of cat spleen

The marginal zone in the cat spleen consisted of a characteristic mixture of lymphocytes and other blood cells located mainly between the several layers of circumferential reticulum around white pulp. A region of fine-meshed reticulum between white pulp and red pulp, as present in some species, was absent from the cat spleen. Arterial capillaries to the marginal zone were few. Some were continuations of white pulp capillaries, whereas others were red pulp capillaries that likely were continuations of axial capillaries of periarterial macrophage sheaths (PAMS) (ellipsoids). Blood cells deposited in the marginal zone could reach red pulp by passing through the numerous openings in each layer of circumferential reticulum. Lymphocytes appeared to migrate across the marginal zone both toward and away from white pulp. Macrophages lying on the circumferential reticulum of the marginal zone phagocytized cells but did not ingest Thorotrast, although it coated their surfaces. Because of the scarcity of arterial endings and the lack of a macrophage-charged reticular meshwork, the marginal zone in cat spleen is not a major site of blood clearance and phagocytosis. These functions are better served in PAMS and red pulp.     

The human spleen; a histological study in splenectomy specimens embedded in methylmethacrylate

In a series of 316 surgically removed spleens, a histological and supportive immunohistological study was performed on methylmethacrylate sections. The structure of the human white and red pulp differs from the rat spleen in many respects, e.g. the human lacks the marginal sinus and the architecture of the periarteriolar lymph sheath seen in the rat. In man, the lymphoid compartment is in both white and red pulps. In the white pulp separate periarteriolar T-cell areas contain a large lymph-vessel plexus, which was reconstructed in serial sections. The circulation in the red pulp is discussed. The area between the red and white pulp, the perifollicular zone, is not the equivalent of the marginal sinus in the rat. Its anatomy in man suggests that it is an area formed from red pulp during the expansion of new follicles. The micro-anatomy was analysed in 119 controls. In cases of traumatic rupture the white pulp showed evidence of stimulation. A pathognomonic histological picture was not found in idiopathic thrombocytopenic purpura. In haemolytic anaemia the pulp cords were engorged by erythrocytes accompanied by a decreased B/T cell ratio in autoimmune haemolytic anaemia and by an increased B/T cell ratio in congenital spherocytosis.     

Lymphocyte migration in the spleen: the effect of macrophage elimination.

To study the influence of macrophages on the migration and distribution of lymphocytes in the spleen, macrophages were eliminated from the spleen of mice by injection of liposomes in which DMDP was encapsulated. This leads to an elimination of macrophages in both the red pulp and marginal zone of the spleen within 1-2 days. In these animals the distribution of lymphocytes was determined by transfer of either syngeneic fluoresceinated or Ly 5 congeneic cells. It was found that after elimination of the macrophages the number of lymphocytes immigrating into the spleen had decreased, although a comparable mode of compartimentalization was found with an initial localization in the marginal zone and a subsequent distribution into the white pulp. After this elimination spleen macrophage subsets return with different kinetics, and in this way the influence of the red pulp macrophages, the marginal zone macrophages and the marginal metallophilic macrophages on lymphocyte immigration and redistribution could be investigated. A quantitative decrease of immigration was still found when red pulp and marginal metallophilic macrophages had repopulated their compartments, but was only fully restored when the last population to repopulate the spleen after treatment with DMDP-liposomes, the marginal zone macrophages, had returned. Experiments with isolated T and B cells showed that the elimination of macrophages had a profound effect on the localization of B cells in the white pulp, whereas it hardly affected T cells.     

Lymphocyte circulation in the spleen: Marginal zone bridging channels and their possible role in cell traffic

Histological evidence is presented for distinct, anatomically determined pathways in the spleen for cells in transit between the white pulp and the red pulp prior to entering the draining veins. In rats and mice these appear as narrow channels of lymphocytes which run between both the periarteriolar lymphatic sheath and the red pulp sinuses, and the peripheral white pulp and the red pulp sinuses, crossing the marginal zone in association with fine argentophilic fibres. These marginal zone bridging channels were found to contain labelled T or B cells 4 and 8 hours after injection which suggested that transit was occurring in the direction from white pulp to red pulp rather than the reverse.

Additional histological evidence is given to suggest that, after antigenic stimulation, germinal centre dissociation occurs by release of the germinal centre cells towards the periarteriolar lymphatic sheath before they are shed into the red pulp through marginal zone bridges occurring in the periarteriolar region.

The data are incorporated into a scheme of unidirectional lymphoid cell flow through the spleen. This proposes that the spleen is composed of many functionally discrete units in which the anatomical matrix, reflected by the reticulin fibre pattern, plays a major role. It further implies that the periarteriolar region of the spleen is not totally thymus dependent.

     

Localization in the rat spleen of carbon-laden macrophages introduced into the splenic artery: a subpopulation of macrophages entering the white pulp

Heavily carbon-laden (HC) macrophages, largely derived from the red pulp of the donor spleen, were injected into the splenic artery of recipient rats. Immediately after injection, HC macrophages were found only in the marginal sinus and in the splenic cords. With time after injection, they appeared successively at the periphery of the white pulp, in the deeper white pulp, and finally in and near the germinal centers, suggesting migration of HC macrophages from the marginal sinus towards the germinal centers. The number of HC macrophages in and near the germinal centers reached a peak at 12 h. Most of the HC macrophages in the white pulp were spherical or ovoid in shape with a diameter of 7-11 microns in sections, having an eccentric round or oval nucleus often with a distinct nucleolus and a cap-like or horseshoe-like cytoplasm filled with carbon. When immunostained with monoclonal antibodies against rat macrophage subpopulations, more than 90% of HC macrophages in the white pulp were found to be ED1+2-3-. A population of the same type of macrophages, both in morphology and phenotype, were found in the red pulp of the donor spleen. They were different from the major residents, red pulp scavenger macrophages, which were ED1+2+3- and larger in size and irregular in shape. These results suggest the presence of a distinct subpopulation of macrophages which actively migrate into the splenic white pulp including the germinal centers. A discharge of transferred macrophages from the red pulp to the general circulation is also suggested.     

The rat FcR for monomeric IgG is preferentially expressed on red pulp macrophages in the spleen.

Rat monoclonal IgG2b immunoglobulins labelled with 125I or biotin were localized in the red pulp areas but were not found in the marginal zones of the white pulp of the spleen 8 hr after their i.v. injection. In cell suspensions made from the spleen, 90% of red pulp macrophages (M phi) bound 125I-IgG2b monomeric proteins in vitro, whereas similar estimates for marginal zone M phi were 12-30%. Red pulp and marginal zone M phi were distinguished primarily by monoclonal antibodies (mAb) ED2 and ED3, but also by their ability to take up FITC-labelled Ficoll in vivo.     

The cellular reaction of the rat spleen after extensive loss of liver parenchyma (author's transl)]

Autoradiographic studies were performed to investigate the changes in cellular proliferation of splenic cells after sham operation and 2/3 partial hepatectomy. Material and Methods: 54 female Wistar rats were hepatectomized and 27 rats were sham operated. The life span of the animals ranged between 12 hours and 31 days. One hour before sacrificing, the animals were injected intraperitoneally with 3H-thymidine (2.5 muCi/Ig). Liver and spleen were embedded in paraffin and epoxy. In strippingfilm autoradiograms (Kodak, AR 10) and in semithinsection autoradiograms (Eastman Kodak, NTB 2) labeling indices of the hepatocytes and Kupgger cells of the regenerating liver and of the cells in the white and red pulp as well as in the marginal zone of the spleen were measured. Results and Discussion: After 2/3 partial hepactectomy there is a marked increase of proliferating. DNA synthezising basophilic blasts and basophilic and polychromatic erythroblasts in the red pulp of the spleen of rats during the first 5 to 10 days. After the fifth day the labeling index of the myelopoietic cells increases too. The curve of the percentages of radioactive labeled cells in the red pulp shows two peaks. The first can be observed after 24 hours and the second after 5 to 10 days. The percentages of labeled cells in the lymphatic mantle zone of the white pulp and in the marginal zone increase also during the first two postoperative days. The first maximum of labeled cells in all the zones of the spleen shows a close relationship to the maximum of proliferating DNA synthesizing cells in the regenerating liver. The second peak in the red pulp is possible caused by a nonspecific multifactorial stimulus such as operation stress, blood loss or inflammation during woundhealing. This proliferation enhancing effect in the spleen, especially on the erythropoietic cells, after partial hepatectomy, which produces a mild splenomegaly during the first 5 days, is more distinct than after sham operation, mechanical or thermical lesions on parenchymal organs.     

“Intermediate zone” of mammalian spleens: Light and electron microscopic study of three primitive mammalian species (platypus,shrew, and mole) with special reference to intrasplenic arteriovenous communication

The intermediate zone (IZ) of nonperfused and perfused spleens in three species of primitive mammals (shrew, mole, platypus) was studied morphologically. The IZ is a tissue zone consisting of plexiform vessels, probably venous capillaries, and is located transitionally between the white and red pulp. The IZ is separated from the white pulp by the arterial net (AN), in which the white pulp arteries terminate. Development of the IZ differs between the three species examined being distinctive in the platypus and shrew. The IZ is thin in the mole spleen. A closed type of arteriovenous (A-V) anastomosis was demonstrated in or around the IZ in the two Insectivora species examined. In the shrew spleen, peripheral arterial branches running within the IZ anastomose with the AN around the follicle. The AN anastomoses eventually with venous plexi-form vessels of the IZ around the nonfollicular area of the white pulp to form a closed system. In the mole spleen, A-V anastomoses were noted between white pulp arteries (follicular and AN) and veins of the red pulp, either by direct communication or through fenestrated IZ vessels compatible with the plexiform vessels of the shrew spleen. A-V anastomosis in the IZ is probable, but not confirmed, in the platypus spleen, as analysis was limited to a nonperfused specimen. Well-developed ellipsoids were noted around arterial terminals of the IZ in the shrew spleen. Ellipsoids were also noted around all arterial terminals of the mole spleen directed to the red pulp. Most ellipsoids of the mole spleen appeared located within the IZ. No ellipsoids were present around arterial terminals of the IZ in the platypus spleen. Closed circulation was noted in terminals of the pulp artery in spleens of all three species. All pulp arteries of the mole spleen are postellipsoid segments of white pulp (AN and follicle) arteries. No ellipsoids were found around terminals of the pulp artery (penicillar artery) in shrew and platypus spleens. The IZ is probably homologous to the perilymphatic sinusoid (vein) of the lungfish spleen and may be regarded as part of the red pulp. The IZ may be representative of primitive mammalian spleens that have closed circulation. The marginal zone (MZ) of common mammalian spllens is probably a modified IZ by differentiation (remodelling) of the intrasplenic vein. In this process, with drawal of venous vessels from the IZ occurrred, leaving a lymphoreticular zone with open circulation (MZ). The marginal sinus reported in some mammalian spleens is probably a modified AN formed durig this process. Possible morphological alterations of the spleen in vertebrate phylogeny are discussed.     

Human spleen microanatomy: why mice do not suffice

The microanatomical structure of the spleen has been primarily described in mice and rats. This leads to terminological problems with respect to humans and their species‐specific splenic microstructure. In mice, rats and humans the spleen consists of the white pulp embedded in the red pulp. In the white pulp, T and B lymphocytes form accumulations, the periarteriolar lymphatic sheaths and the follicles, located around intermediate‐sized arterial vessels, the central arteries. The red pulp is a reticular connective tissue containing all types of blood cells. The spleen of mice and rats exhibits an additional well‐delineated B‐cell compartment, the marginal zone, between white and red pulp. This area is, however, absent in human spleen. Human splenic secondary follicles comprise three zones: a germinal centre, a mantle zone and a superficial zone. In humans, arterioles and sheathed capillaries in the red pulp are surrounded by lymphocytes, especially by B cells. Human sheathed capillaries are related to the splenic ellipsoids of most other vertebrates. Such vessels are lacking in rats or mice, which form an evolutionary exception. Capillary sheaths are composed of endothelial cells, pericytes, special stromal sheath cells, macrophages and B lymphocytes. Human spleens most probably host a totally open circulation system, as connections from capillaries to sinuses were not found in the red pulp. Three stromal cell types of different phenotype and location occur in the human white pulp. Splenic white and red pulp structure is reviewed in rats, mice and humans to encourage further investigations on lymphocyte recirculation through the spleen.     

Butyrylcholinesterase-positive innervation of the spleen in rats

Innervation of the spleen in rats was studied. Butyrylcholinesterase-(BuChE)-positive nerve components of the organ were visualized by the direct thiocholine method. BuChE-positive nerve components enter the spleen in a common bundle with arteries. In the organ they form characteristic periarterial and periarteriolar plexiform arrangements, which are especially conspicuous around the aa. centrales running through the white pulp. Then, nerve fibres extend away from these plexuses into adjacent layers of trabeculae further into marginal layers of periarterial lymphatic sheath (PALS) as well as into the mantle zone of follicles. Several scattered periarteriolar and solitary nerve fibres can be seen in the marginal sinuses and cords of the red pulp. In the fibrous capsula BuChE-positive nerve fibres can also be seen which have an evident connection with trabecular and parenchymal nerves of the organ. Microscopic findings support the notion that BuChE-positive nerve profiles supply not only the vasculature, but also the parenchymal components of the spleen, and they may participate, to a great extent, in the regulation of the immune processes in this organ.