Distribution of horseradish peroxidase (HRP)-anti-HRP immune complexes in mouse spleen with special reference to follicular dendritic cells

The distribution of immune complexes has been studied in mouse spleen stimulated to contain many germinal centers (GC's). Horseradish peroxidase (HRP)-anti-HRP complexes were used as an appropriately precise and sensitive model. We were primarily interested in the relative abilities of three cell types to interact with complexes: lymphocytes, macrophages, and follicular dendritic cells (FDC's). The latter are distinctive, nonendocytic, stellate cells located primarily at the transition of mantle and GC zones of 2 degrees lymphoid follicles (Chen, L. L., J. C. Adams, and R. M. Steinman, 1978, J. Cell Biol. 77:148). Binding of immune complexes to lymphocytes could not be visualized in situ. Macrophages avidly interiorized complexes into lysosomes, but did not retain them extracellularly. In contrast, FDC's could retain HRP-anti-HRP extracellularly under appropriate conditions, but did not endocytose them. Cytochemical reactivity accumulated progressively on FDC's 1--6 h after administration of complexes i.v., remained stable in amount and location for 1 day, and then was progressively lost over a 1- to 5-day period. Several variables in the association of complexes with macrophages and FDC's were pursued. Only 1 microgram of complexed HRP had to be administered to visualize binding to both cell types. Macrophages interiorized complexes formed in a wide range of HRP/anti-HRP ratios, while FDC's associated with complexes formed in HRP excess only. Quantitative studies with [125I]HRP-anti-HRP demonstrated that 20% of the splenic load of HRP associated with FDC's. Complexes formed with an F(ab')2 anti-HRP were distributed primarily in macrophages. When the levels of the third component of serum complement were depleted by prior treatment with cobra venom factor, uptake of complexes by macrophages was reduced some 50% whereas association with FDC's was abolished. The fact that antigen excess complexes are retained extracellularly strengthens the idea that they are immunogenic. Finally, the association of complexes with FDC's seems to retard the entry of antigen into the GC proper.     

Uptake of colloidal thorium dioxide by the mouse connective tissue mast cell.

The ability of the mouse mast cell to phagocytize colloidal thorium dioxide, Thorotrast, was investigated employing the mouse connective tissue air pouch. The connective tissue mast cell of mouse was found to have limited capability to ingest the particulate Thorotrast in comparison to the rat peritoneal mast cell which ingested this material readily. Fibroblasts and macrophages in the connective tissue removed injected Thorotrast very rapidly in contrast to mast cells that demonstrated limited phagocytic capabilities. The tissue mast cell of the mouse, therefore, should not be considered a part of the reticuloendothelial system.     

Release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells

Summary The release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells was studied by electron microscopy. The treatment of the cells with SEB at the concentration of 20 μg/ml caused marked increase of the chromaffin granules that either bound to the plasma membrane by the characteristic rods, measuring 15 to 20 nm in length and showing a tubular structure, or budded off at the free cell surface, surrounded by a layer of rod-containing cytoplasm and enclosed by the plasma membrane. The binding between the granular and plasma membranes by the rods did not lead to membrane fusion and exocytosis of the granular content. Many of the bound granules showed vesiculation with loss of the electron-dense core material; at the same time, some of the binding rods contained intraluminal electron-dense material similar to the granular core material. These findings suggested that the electron-dense material (i.e., norepinephrine) of the bound granules was released extracellularly through channels within the rods. Although the granules were bound to the plasma membrane with equal frequency at the free and contiguous cell surfaces, the granular budding occurred only at the free cell surface, indicating that it occurred incidentally to some granules bound at the free cell surfaces. On the basis of the morphological observations, it is postulated that the electron-dense material of the bound granule is selectively released extracellularly through the rods, leaving the vesiculated granules behind in the cytoplasm. The same mode of release of the granular content was observed, though less frequently, in the untreated control cells. No morphological evidence that indicated that the granular content was released extracellularly by exocytosis was found in the treated and control cells. The present observations indicated that the SEB treatment of PC12 cells stimulated the binding of chromaffin granules to the plasma membrane by the rods and the budding of the bound granules at the free cell surface.     

Microanalytical study of thorium 232 deposits in bone marrow and liver

Summary Analytical microscopy was used to study the distribution and chemical composition of thorium deposits in bone marrow and liver after injection of thorium dioxide and thorium nitrate. Thorotrast (thorium dioxide) was identified as being localized in bone marrow macrophages of a patient who had undergone cerebral arteriography forty two years ago. Large thorotrast deposits were also present in liver cells. We show that non-colloidal thorium (thorium nitrate) injected in rats concentrates in a non soluble form in bone marrow macrophages, heptocytes and Kupffer cells. These deposits of thorium associated with phosphorus can be explained by the formation of thorium phosphate in lysosomes and we demonstrate that they remain in tissue for a long time. Microanalysis was performed with ion microscopy, and electron probe microanalysis by X ray spectrometry, which can identify and localize thorium and associated elements at cellular or intracellular level.     

Non-lymphoid cells of bronchus-associated lymphoid tissue of the rat in situ and in suspension

Immunohistochemical, enzyme-histochemical and electron-microscopical methods were used to study non-lymphoid cells of control and stimulated rat bronchus associated lymphoid tissue (BALT) in situ and in suspensions. Particular attention was paid to the so-called antigen-handling cells, i.e., the interdigitating cells (IDC), which are situated in the T-cell areas, the follicular dendritic cells (FDC), which appear to be restricted to germinal centers, and macrophages, present both in T-cell and B-cell areas. The interdigitating cells were distinguished by being Ia-positive and by the presence of acid phosphatase and non-specific esterase activity in an area near the nucleus. Follicular dendritic cells could be observed in situ by using a monoclonal antibody and by the in vitro trapping of HRP-anti-HRP complexes. Several types of macrophages were found. At the electron-microscopical level no well-developed IDC and FDC could be detected in control BALT. However, in BALT of lipopolysaccharide-stimulated and mycoplasma-infected rats, well-developed IDC and FDC were found. It can be concluded that IDC's and FDC's can be found in BALT.     

UPTAKE OF COLLOIDAL THORIUM DIOXIDE BY MAST CELLS

Mast cells from the peritoneal cavity of the rat were obtained at various times following in situ injection of a colloidal thorium dioxide preparation (Thorotrast). They were prepared for electron microscopy by aldehyde fixation, osmium tetroxide postfixation, and embedding in Epon. Thorotrast was rapidly taken up by mast cells through enhanced or newly elicited surface specializations. It was confined at first to large vesicles which moved to the Golgi area. Subsequently, in a matter of a few hours only, it became associated with progressively more mature granules, including "fully" mature ones. In addition to demonstrating a further phagocytic or pinocytotic activity of mast cells, the findings suggest that mast cell granules share a common membranous investment, and that substances from the tissue environment may theoretically percolate over and interact with the granules. Mast cell function could thus be served primarily by absorptive rather than secretory processes.     

S-100-immunoreactive giant macrophages in lymphoid tissues of the guinea pig

Giant cells containing S-100 protein of the lymphoid tissues in the guinea pig were studied by immunohistochemistry using S-100 antiserum. S-100-immunoreactive giant cells were dendritic in shape, contained one or two irregular-shaped, euchromatic nuclei, phagosomes of various diameter, numerous mitochondria and microfilaments in the perikaryon, and extended cell processes free of cell organelles. These cells predominantly lined the superficial cortex facing the subcapsular sinus, were less numerously scattered in the medulla of lymph nodes and located at the marginal zone of the spleen. They also stained with S-100 alpha monoclonal antiserum and showed active phagocytosis for aldehyde-fixed red cells or colloidal carbon in the popliteal lymph node and spleen. S-100-immunoreactive giant cells also appeared in the corticomedullary zone of the thymus and in the interfollicular area of the Peyer's patches of the gut. Small sinus macrophages, which exhibited active phagocytosis for colloidal carbon but were less active for red cells in the popliteal lymph node and spleen, were not stained with S-100 antiserum. These findings indicate that S-100-immunoreactive giant cells of the lymph node and spleen are a subpopulation of macrophages different from S-100-negative cells of the small type.     

AN ELECTRON MICROSCOPIC STUDY OF PINOCYTOSIS IN AMEBA : I. The Surface Attachment Phase

The attachment to the surface of the ameba (Chaos chaos L. (Pelomyxa carolinensis, Wilson)) of two proteins, ribonuclease and ferritin, and two colloidal suspensions, thorium dioxide and gold, was studied in the electron microscope. The initial step in the pinocytosis of ferritin and thorium dioxide particles by amebas is shown to be the attachment of these substances to the "hairlike" extensions of the plasmalemma. Ribonuclease caused alterations in the structure of the plasmalemma, but on account of its relative lack of density, it could not be definitely localized. Colloidal gold did not appear to be active with respect to pinocytosis in amebas. Since molecules in solution and particles in suspension are taken up by the same mechanism, the first step of which is their attachment to the cell surface, it is suggested that a single mechanism underlies phagocytosis, pinocytosis, ropheocytosis, cytopempsis, and potocytosis.     

Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes)

The morphological features of pinched-off presynaptic nerve terminals (synaptosomes) from rat brain were examined with electron microscope techniques; in many experiments, an extracellular marked (horseradish peroxidase or colloidal thorium dioxide) was included in the incubation media. When incubated in physiological saline, most terminals appeared approximately spherical, and were filled with small (approximately 400- A diameter) "synaptic vesicles"; mitochondria were also present in many of the terminals. In a number of instances the region of synaptic contact, with adhering portions of the postsynaptic cell membrane and postsynaptic density, could be readily discerned. Approximately 20--30% of the terminals in our preparations exhibited clear evidence of damage, as indicated by diffuse distribution of extracellular markers in the cytoplasm; the markers appeared to be excluded from the intraterminal vesicles under these circumstances. The markers were excluded from the cytoplasm in approximately 70--80% of the terminals, which may imply that these terminals have intact plasma membranes. When the terminals were treated with depolarizing agents (veratridine or K- rich media), in the presence of Ca, many new, large (600--900-A diameter) vesicles and some coated vesicles and new vacuoles appeared. When the media contained an extracellular marker, the newly formed structures frequently were labeled with the marker. If the veratridine- depolarized terminals were subsequently treated with tetrodotoxin (to repolarize the terminals) and allowed to "recover" for 60--90 min, most of the large marker-containing vesicles disappeared, and numerous small (approximately 400-A diameter) marker-containing vesicles appeared. These observations are consistent with the idea that pinched-off presynaptic terminals contain all of the machinery necessary for vesicular exocytosis and for the retrieval and recycling of synaptic vesicle membrane. The vesicle membrane appears to be retrieval primarily in the form of large diameter vesicles which are subsequently reprocessed to form new "typical" small-diameter synaptic vesicles.     

Long-term effects of alloxan-induced diabetes on the nucleus ventralis posterolateralis in the thalamus of the rat.

The present paper describes the long-term ultrastructural changes in the nucleus ventralis posterolateralis of the thalamus of male Wistar rats after alloxan-induced diabetes. Degenerating dendrites were characterized by an electron-dense cytoplasm with scattered endoplasmic reticulum and ribosomes. Degenerating axon terminals were characterized by an electron-dense cytoplasm and clustering of small spherical agranular vesicles. Degenerating axon terminals formed axosomatic synapses with seemingly normal cell bodies and axodendritic synapses with normal as well as degenerating dendrites. Degenerating axons (both myelinated and unmyelinated) were readily encountered in the neuropil. Activated microglial and astrocytic cells in the neuropil were in the process of phagocytosis or had residua in their cytoplasm.     

An Ultrastructural Study of the Hemocytes of the Pericardial Body in the Ascidian Ciona intestinalis (L.)

Abstract The hemocytes of the pericardial body of Ciona intestinalis were studied by electron microscopy. Our findings showed that stem cells, clear vesicular granulocytes, microgranulocytes, unilocular granulocytes and globular granulocytes are present at the periphery of the smaller-sized pericardial bodies. The stem cells are small round cells with a large nucleus, with or without nucleolus, and homogeneous cytoplasm containing numerous ribosomes. The clear vesicular granulocytes are characterized by an ameboid shape and cytoplasm containing several large electron-lucent vacuoles and small electron-dense granules. The microgranulocytes are variable in shape and contain numerous large electron-dense granules. The unilocular granulocytes show a single large vacuole with an electron-dense or electron-lucent content and a thin layer of peripheral cytoplasm that contains the flattened nucleus. The globular granulocytes are characterized by the presence of large vacuoles containing either fibrogranular material or electron-dense aggregates.     

Ultrastructural,cytochemical and functional studies on the eosinophilic granulocytes of larval lampreys

Summary Blood from larval lampreys (ammocoetes) contains a small number of eosinophilic granulocytes which are formed in the protospleen and kidney. Both immature and mature forms of this cell type are present in the blood and these are easily identified from other cell types due to the prominent eosinophilic granules that fill the cytoplasm. Ultrastructurally, these granules are electron-dense, largely unstructured, Golgi-derived and contain acid phosphatase but not peroxidase. Eosinophilic granulocytes ingest bacteria but fail to internalise colloidal carbon. The functional and phylogenetic significance of these cells is discussed.     

The cell surface of mouse dendritic cells: FACS analyses of dendritic cells from different tissues including thymus

The surface of dendritic cells from mouse spleen, thymus, and epidermis has been compared with a panel of monoclonal antibodies and the FACS. A method was first developed to isolate populations of large, adherent, thymic dendritic cells that were greater than 90% pure. These were released by collagenase digestion and separated from adherent macrophages after overnight culture. Enrichment was based on the facts that most macrophages remained plastic adherent and rosetted strongly with antibody-coated erythrocytes. As in spleen, thymic dendritic cells were stellate in shape, had abundant class I and II MHC products, lacked many standard macrophage and lymphocyte markers, and actively stimulated the mixed leukocyte reaction. Most spleen and thymic dendritic cells could be lysed by the 7D4 mAb, to the low-affinity IL-2 receptor, and complement but the levels of 7D4 by FACS were low and sometimes not above background. Differences among dendritic cells from different tissues were noted with other mAb. Adherent dendritic cells from thymus all expressed the J11d "B cell" antigen and the NL145 interdigitating cell marker, but lacked the 33D1 spleen dendritic cell antigen. Eighty to ninety percent of spleen dendritic cells were J11d-, NL145-, 33D1+ but the remainder expressed the J11d+, NL145+, 33D1- thymic phenotype. The latter phenotype also was identical to that of epidermal Langerhans' cells. We postulate that the major 33D1+ cell in spleen represents a migratory stage in which dendritic cells are moving from tissues to lymphoid organs.     

Microtubules are involved in maintaining the cellular polarity in pollen tubes ofNicotiana sylvestris

Summary The polarity of a growing pollen tube is clearly reflected by a distinct zonation of the cytoplasmic content. The vegetative nucleus and the generative cell (GC) are located in the tip region of the tube, and the basal cytoplasmic portion is highly vacuolated. Using pollen tubes ofNicotiana sylvestris Spegazz. & Comes grown in vitro, we examined the effects of varying concentrations of the microtubule inhibitors colchicine and propham. The depolymerization of the cortical microtubules by 25 M colchicine led to a disorganization of the cytoplasm, i.e., vacuolization of the tip region, and to a deranged position of both the vegetative nucleus and the generative cell. The same concentration of colchicine inhibited tube growth by 10–20% of the control. Mitosis of the GC was not affected. Only from concentrations of 200 M the configuration of the GC's microtubules was altered and an inhibition of mitosis was observed. At this concentration the disorganization of the cytoplasm was always reversible, but neither inhibition of mitosis nor derangement of the nuclear positioning was. At 1,800 M colchicine, pollen tube growth was inhibited by 50% of the control. Using propham, the same three steps of action were observed, although propham proved to be about a hundred times more effective than colchicine. We conclude that the cortical microtubules of the pollen tube are involved in maintaining cellular polarity, probably as a part of a heterogeneous cytoskeletal network including also microfilaments and membranous elements. Nuclear positioning seems to be dependent on both, the tube's cortical and the GC's microtubules. A possible involvement of the extracellular matrix in maintaining intracytoplasmic polarity is suggested.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EGTA ethyleneglycol-bis-(aminoethyl ether) tetraacetic acid - GC generative cell - MF microfilament - MT microtubule - PEM-buffer 50 mM PIPES, 1 mM EGTA, 2 mM MgSO₄, pH 6.9 - PBS phosphate buffered saline - PIPES piperazine-bis-ethanesulphonic acid - PTG-test pollen tube growth test - VN vegetative nucleus Dedicated to Professor Peter Sitte on the occasion of his 65th birthday     

神经节细胞群体同步放电模式编码的感受野特性

应用多电极同步记录技术,对牛蛙视网膜神经节细胞在伪随机棋盘格刺激下的放电活动进行胞外记录。依据记录到的神经节细胞放电情况,利用一种数据压缩算法,通过最大化压缩放电序列的信息熵,对多个神经节细胞进行群体划分,得到同步放电神经节细胞群体。利用基于动作电位的刺激平均法(spike triggered average,STA),分别计算出每个同步放电神经节细胞群体内单个神经节细胞放电活动所编码的感受野(单细胞感受野),以及群体内所有神经节细胞同步放电活动所编码的感受野(群体感受野)。计算结果显示,对于所有神经节细胞群体,约80%神经节细胞群体的群体感受野面积小于群体内所有单细胞感受野面积的平均值,约60%神经节细胞群体的群体感受野面积小于群体内任意单细胞感受野面积。在棋盘格刺激下,神经节细胞放电活动会发生对比度适应。进一步以群体感受野面积小于群体内任意单细胞感受野面积的神经节细胞群体为研究对象,考察群体感受野在对比度适应过程中的动态变化,结果显示,85%神经节细胞群体的群体感受野面积在适应后期变小。     

鸭病毒性肠炎病毒强毒株的形态发生学与超微病理学研究

应用透射电镜和超薄切片技术,研究鸭病毒性肠炎病毒(duck enteritis virus,DEV)CH强毒株人工感染成年鸭后,病毒在宿主细胞内的形态发生及各组织器官的超微结构变化.结果表明,感染后不同时间剖杀及发病后死亡鸭的肝、肠、脾、胸腺、法氏囊等组织器官中,均观察到典型的疱疹病毒粒子.病毒主要的靶细胞为淋巴细胞、网状内皮细胞、成纤维细胞、巨噬细胞、血管内皮细胞、肠道上皮细胞、肠道平滑肌细胞和肝细胞等.DEV的核衣壳有空心型、致密核心型、双环型和内壁附有颗粒型四种形态,存在胞核和胞浆两种装配方式.病毒核衣壳可在核内获得皮层,通过核内膜获得囊膜成为成熟病毒;也可通过内外核膜进入胞浆,在其中获得皮层,然后在各种质膜上获得囊膜,最后成熟病毒释放到细胞外.伴随着病毒的复制、装配和成熟,细胞中出现多种核内和胞浆包涵体、核内致密病毒核酸颗粒、微管和中空短管以及胞浆内膜包裹的电子致密小体、双层管等病毒相关结构.超微研究表明,组织细胞有坏死和凋亡两种变化.坏死细胞肿胀甚至破裂,线粒体肿胀空泡化,粗面内质网扩张,核糖体脱落,有的细胞器甚至完全崩解,染色质或固缩或溶解.凋亡细胞则染色质聚集,胞浆凝聚深染,细胞膜上有大量空泡,并有凋亡小体形成.细胞坏死与凋亡往往同时存在,疾病发生过程中,脾、胸腺、法氏囊以及小肠固有层中的淋巴细胞凋亡数量明显增多.     

Ultrastructure of "villose cells" in the early stages of their formation in the process of chemical carcinogenesis in the CNS]

Early stages of redistribution of cellular elements around the pill of carcinogenic agent DMBA introduced into the right hemisphere of the brain of female SHK albino rats were studied. Precursors of the ciliated cells were established to appear in the perivascular tissue within 12 h. Within 24 h they accumulate around the pill bed, and within 48 h a cellular edging is formed around DMBA. The cytoplasm of ciliated cells become richer in organelles. The main marker in identification of cells in all periods of experiment were lipoid inclusions in the cytoplasm which are different from lipids of usual macrophages occurring both in experiments and in control, in a polygonal shape and sinuous contour. Within 48 h the cytoplasm of ciliated cells form long lancet-shaped spiculae with upright walls. The cytoplasm of macrophages gives only short, somewhat sinuous processes.     

四种淡水养殖鱼类血细胞的细微结构

四种淡水鱼的血细胞形态基本相似。红血球形态与其他低等脊椎动物基本相似。淋巴球绝大部分是小淋巴球:单核球数量较少;四种鱼的嗜中性白血球形态结构差不多,胞核多为蚕豆形,很少见分叶核,分叶一般也只有二叶,这与哺乳类显然不同;嗜酸性白血球的形态结构与其他脊椎动物基本相似;在少数血涂片中看到了嗜碱性白血球。       

The Fine Structure of Endothelium of Large Arteries

Endothelium of large arteries from several species was studied in thin sections with the electron microscope. Before sacrifice, some animals received an intravenous injection of colloidal thorium dioxide which was visualized in the sections. Surface replicas were prepared by carbon evaporation on either frozen-dried endothelium or on endothelium dried by sublimation of naphthalene with which the tissue had been impregnated. Cell boundaries, stained with silver, were observed in sections and also from the surface by stripping off the inner part of the endothelium. In addition to the usual cytoplasmic organelles, the endothelial cells showed certain characteristic features, namely, large invaginated pockets communicating with the arterial lumen, numerous much smaller vesicular structures immediately under the plasma membrane and apparently also communicating with the lumen, and inclusions, into which injected thorium particles were incorporated. Intercellular boundaries appeared as regular double membranes in thin sections, and they were outlined by a double row of silver granules after silver staining. No evidence was obtained of permeation of intracellular spaces by colloidal thorium.