The morphogenesis of the human Paneth cell. An immunocytochemical ultrastructural study

In human duodenal mucosa Paneth cells originate away from the base of crypts and migrate towards the base during maturation. The earliest cells in the Paneth cell lineage could be identified by labelling of lysozyme in the Golgi apparatus. Specific labelling for lysozyme was present in the rough endoplasmic reticulum, Golgi apparatus, condensing vacuoles, granules and many lysosomes of mature Paneth cells. The maturation of the Paneth cell is accompanied by an increase in the content of lysozyme in the secretory granules and with senescence lysozyme diffuses into the cytoplasm.     

A homing receptor-IgG chimera as a probe for adhesive ligands of lymph node high endothelial venules

The binding of lymphocytes to high endothelial venules (HEV) within peripheral lymph nodes (pln) is thought to be mediated by a lectinlike adhesion molecule termed the pln homing receptor (pln HR). The cloning and sequencing of cDNAs encoding both murine and human pln HR revealed that these adhesion molecules contain protein motifs that are homologous to C-type or calcium dependent lectin domains as well as to epidermal growth factor (egf) and complement-regulatory protein domains. We have produced a novel, antibody-like form of the murine HR by joining the extracellular region of the receptor to a human IgG heavy chain. This antibody-like molecule is capable of recognizing carbohydrates, blocking the binding of lymphocytes to pln HEV, and serving as a histochemical reagent for the staining of pln HEV. This murine HR-IgG chimera should prove useful in analyzing the distribution of the HR ligand(s) in normal as well as in inflammatory states.     

Interaction of lymphocytes and high endothelial venules in irradiated lymph nodes

After total-body exposure to various doses of ionizing radiation, the ability of lymphocytes to interact specifically with high endothelial venules of rat cervical and mesenteric lymph nodes was analyzed in frozen sections. Following a radiation dose of 1.5 Gy, high endothelial venules remained intact and the binding of unirradiated lymphocytes to the venules was enhanced relative to unirradiated controls. At radiation doses above 5.0 Gy, damage to high endothelial venules was observed histologically as well as assessed functionally. There was a significant decrease in specific lymphocyte-venule binding and a significant increase in nonspecific binding. These findings suggest that radiation-induced damage to high endothelial venules might play a role in radiation-induced immunosuppression by interfering with the normal passage of lymphocytes from the blood into lymph nodes via a specific interaction between lymphocytes and high endothelial venules.     

Abnormal deposition of basement membrane and connective tissue components in dimethylbenzanthracene-induced rat mammary tumours: an immunocytochemical and ultrastructural study

Summary Dimethylbenzanthracene-induced rat mammary tumours consist of lobules of tumours cells surrounded by connective tissue. The interstitial connective tissue proteins, collagen types I, III and V, fibronectin and elastin are largely restricted to the interlobular connective tissue. The tumour lobules are surrounded by a basement membrane that stains with antiserum to laminin. Electron microscopy reveals a greatly thickened basement membrane to which striated interstitial collagen fibres are closely juxtaposed. The lumina within the tumour lobules are of two types. In the first type, the luminal surface is characterized by the presence of microvilli and tight junctions are reacts with antiserum to rat milk fat globule membrane. In the second type, the luminal surface is flattened and lined by a thickened basement membrane that stains with antiserum to laminin and type IV collagen. These abnormal patterns of growth and differentiation may be partly a consequence of the disorganization of extracellular matrix components at the interface between the tumour epithelial cells and the surrounding stroma.     

Argyrophilic proteins in Dinoflagellates: An ultrastructural,cytochemical and immunocytochemical study

Summary— Dinoflagellate protists constitute an original eukaryotic phylum and have an ancestor in common with ciliates. They are important tools in studies of structure and function of the nucleus because they present a mixing of prokaryotic characteristics such as chromatin devoid of histones and nucleosomes, eukaryotic characteristics such as the presence of a nuclear membrane, nucleoli and AgNOR-like proteins and original characteristics of their own. Among them are the permanent compaction of the chromosomes, the presence of a nuclear envelope during the whole cell cycle, rare bases in their DNA, as well as an original mitosis. We have studied the distribution of the nuclear argyrophilic proteins (AgP) in three genera of Dinoflagellates (Prorocentrum, Crypthecodinium and Amphidinium) by means of light microscopy (LM) and electron microscopy (EM), using cytochemical silver staining and immunocytochemical reactions following various preparation procedures. By means of the silver staining reaction, we determined by LM the distribution of nucleoli in the three non-synchronized cell populations and localized by EM the presence of AgP. These are always found in the nucleolar fibrillo-granular compartment (FG) and partly in the chromosomes and in the nucleolar UCh (unwound region of the nucleolar chromosome corresponding to the NOR); the chromosomes and the UCh are always stained in P micans, under special conditions in C cohnii but never in A carterae. To determine whether these nucleolar and chromosomal proteins are similar or different, we modified the conditions of the silver staining reaction by acidic, alkaline or enzymatic pretreatments and changes in the reaction's temperature. Our results suggested that these proteins belong to different groups. We have characterized one of these proteins using a mammalian anti-B23 Ab in P micans cells. Positive labeling was mostly detected in chromosomes and UCh and in a smaller amount in the nucleolar FG and G compartments, co-locating with end-products of the silver staining reaction. This suggests that: i) one among the dinoflagellate chromosomal AgP is analogous to the B23 mammalian protein; and ii) this B23-like protein is probably a DNA partner.     

An experimental, NADPH-diaphorase histochemical and immunocytochemical study of Mesocestoides vogae tetrathyridia.

In order to test the role of nitric oxide in flatworms, Mesocestoides vogae tetrathyridia were incubated together with L-arginine, which is the substrate for nitric oxide synthesis, or with NG-nitro-L-arginine, which is an irreversible inhibitor of nitric oxide synthase. Normally, tetrathyridia attach to each other with the aid of their suckers, forming clusters. The rate of cluster formation was followed during the incubations. L-Arginine stimulated, and NG-nitro-L-arginine clearly inhibited, the cluster formation. This is the first time that an effect of nitric oxide has been observed in a flatworm. In addition, the pattern of the NADPH-diaphorase histochemical reaction in the nervous system and the pattern of F-actin filaments in the musculature stained with TRITC-labelled phalloidin were studied. NADPH-d staining occurred in the brain and the main nerve cords but also followed the muscle fibres stained with phalloidin. The pattern of the NADPH-d reaction was compared with that of 5-HT immunoreactivity. The implications of the results are discussed in relation to the background of data on neuronal signal substances in M. vogae.     

Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley-Liss, Inc.     

An ultrastructural and histochemical study of autoimmune aspermatogenesis in the rat testis

Summary Immune aspermatogenesis was induced in young rats by the method of Freundet al. (1954) and testes were studied by electron microscopic and histochemical methods. At time of sacrifice the testes of several animals were markedly atrophic as demonstrated by reduction in weight. Sections of seminiferous tubules exhibited primarily profiles of Sertoli cells but germinal elements were sparse or absent. The ultrastructure of Sertoli cells appeared to be normal except for the presence of areas of dilated smooth endoplasmic reticulum and fragments of phagocytized germ cells in the cytoplasm.Light microscopic sections showed an apparent hyperplasia of intertubular tissue. Electron micrographs revealed a moderate to extreme vesiculation of the smooth endoplasmic reticulum in many interstitial cells. Macrophages and lymphocytes were often observed in contact with these cells.There was increased localization of non-specific esterase and acid phosphatase associated with lipid bodies of the tubules and in intertubular areas.This work was supported in part by USPHS Grant FR 5391.     

The nervous system of the chicken proventriculus: an immunocytochemical and ultrastructural study

The proventriculus constitutes the glandular region of the chicken stomach. This organ is innervated by two parasympathetic networks, the myenteric and submucous plexus, and here we present a systematic study of this system by immunohistochemistry and electron microscopy. All the neurons and fibres were positive for the neural markers, protein gene product 9.5 and the amidating enzymes. Immunoreactivities for the constitutive neuronal isoform of the enzyme nitric oxide synthase and the vasoactive intestinal peptide were present in neuronal bodies suggesting an intrinsic origin for the similarly immunoreactive fibres found in the proventriculus. On the other hand, immunoreactivity to gastric inhibitory peptide was only found in varicose fibres making contact with the blood vessels and the glandular epithelium, but never in the neuronal somas, suggesting that this substance may be provided by an extrinsic nervous system whose neuronal bodies are located elsewhere. Electron microscopy revealed frequent neuromuscular and neuroepithelial connections in the muscle layers, the wall of the blood vessels and the epithelium. In addition, synapsis-like structures were identified in the proximity of cells belonging to the diffuse endocrine system, providing a new example of neuroendocrine contacts. No positivity was found for antibodies against other neural substances including somatostatin, peptide histidine–isoleucine, peptide tyrosine–tyrosine, neuropeptide tyrosine, bombesin, met-enkephalin, serotonin, substance P, galanin, calcitonin gene-related peptide and S-100 protein.     

The development of the human lymph node

Summary Lymph nodes of human fetuses from the 11th to the 20th gestational week (g.w.) were investigated by light- and electron microscopy under particular consideration of the development of the T-cell and the B-cell regions and their specific reticulum cells. Lymph node development begins as a mesenchymal condensation, containing capillaries and mesenchymal cells; this primordium bulges into a lymph sac. Within the primordium of the lymph node granulopoiesis and erythropoiesis occur temporarily from the 12th to the 14th g.w. The first lymphoid cells and undifferentiated blast cells are seen in the 12th g.w.; monocytes and macrophages can be found from the 13th g.w. onward.The development of the T-cell regions begins during the 13th g.w., before differentiation into cortex and medulla becomes obvious in the 14th g.w. Near the marginal sinus, cells displaying features of interdigitating reticulum cells (IDC) show similarities to monocytes. The morphological differentiation of the IDC is complete in the 17th g.w. when they are found in the paracortical region. Among the IDC, lymphoid cells with features of thymocytes are arranged in small groups.The first indication of the development of B-cell regions can be recognized in the 14th g.w. when precursors of dendritic reticulum cells (DRC) are seen near the marginal sinus; this area also displays lymphoblasts, immunoblasts, and plasmoblasts. During the 20th g.w. small primary follicles are discernible in the outer cortex; in addition to blast cells they contain small lymphocytes, none of which show features of thymocytes. The morphological development of DRCs is not entirely complete until the 20th g.w.; however, some cells already show a characteristic network of interwoven processes.The probable origin of (i) the IDC from monocytes, and (ii) the DRC and fibroblastic reticulum cells from a common type of mesenchymal precursor cells, as well as their significance for a specific micromilieu in the T-cell and the B-cell regions, are discussed.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft and the Sonderforschungsbereich 111 of the Deutsche ForschungsgemeinschaftThe authors appreciate the contribution of human fetal material from Dr. J. von Hollweg and Dr. J. Körner, Hospital Heidberg, Hamburg, and the excellent technical assistance of Mrs. O.M. Bracker, Mrs. H. Hansen, Mrs. R. Köpke, Mrs. I. Knauer, Mrs. F. Müller, Mrs. H. Siebke, and Mrs. H. Waluk     

Cutting edge: the B cell chemokine CXC chemokine ligand 13/B lymphocyte chemoattractant is expressed in the high endothelial venules of lymph nodes and Peyer's patches and affects B cell trafficking across high endothelial venules

While CCR7 ligands direct T cell trafficking into lymph nodes (LNs) and Peyer's patches (PPs), chemokines that regulate B cell trafficking across high endothelial venules (HEVs) remain to be fully elucidated. Here we report that CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant) is detected immunohistologically in the majority of HEVs in LNs and PPs of nonimmunized mice. Systemically administered anti-CXCL13 Ab bound to the surface of approximately 50% of HEVs in LNs and PPs, but not to other types of blood vessels, indicating that CXCL13 is expressed in the HEV lumen. In CXCL13-null mice, B cells rarely adhered to PP HEVs, whereas T cells did efficiently. Superfusion of CXCL13-null PPs with CXCL13 restored the luminal presentation of CXCL13 and also B cell arrest in PP HEVs at least partially. Collectively, these results indicate that CXCL13 expressed in the HEV lumen plays a crucial role in B cell trafficking into secondary lymphoid tissues such as PPs.     

An ultrastructural and histochemical study of the axial musculature in the African lungfish

Summary Red, intermediate, and white axial muscle fibres of African lungfish were studied using histochemical techniques and electron microscopy. Gross dissection revealed the presence of a small wedge of red coloured muscle along the lateral line. This wedge was shown by histochemical demonstrations of lactate and succinate dehydrogenases, of adenosine triphosphatases, and of lipid to be composed of a mosaic of red and intermediate fibres measuring 23.63 and 34.30 m in average diameter, respectively. The bulk of the myotome was composed of white fibres having an average diameter of 67.35 m. Mitochondrial density, capillarity and lipid content were very low for all fibres. These data suggest that the axial musculature is geared primarily for anaerobic function. The mosaic arrangement of fibres, and the lack of a subsarcolemmal band of mitochondria suggests that the lungfish have a muscle organisation that is transitional between lower vertebrates and amphibians.     

Human breast epithelial xenografts: An immunocytochemical and ultrastructural study of differentiation and lactogenic response

Fragments of ductal and lobular epithelium ('organoids') produced by collagenase digestion of reduction-mammoplasty specimens were injected into athymic 'nude' mice. These heterospecific tissues were accepted without rejection, and the presence of xenografts was confirmed by cytology, immunocytochemistry and chromatin staining. Lactation, as confirmed by immunocytochemical and ultrastructural criteria, was observed in the grafted human epithelium during murine pregnancy at both intra- and extra-mammary sites.     

The aboral haemal system of the sea star,Asterias rubens (Echinodermata,Asteroidea): An ultrastructural and histochemical study

Summary The aboral parts of the haemal system of the sea star Asterias rubens are described, based on light and electron microscopy. These parts are (1) the mesenteric strands along the pyloric caeca and the pyloric stomach, (2) the gastric haemal tufts, and (3) the aboral haemal ring. The mesenteric haemal strands are very limited in size and distribution and, therefore, do not seem to have a major function in nutrient translocation. The myoepithelial cells of the gastric haemal tufts have the typical features of choanocytes; their ultrastructural characteristics corroborate the possible absorptive role of the gastric haemal tufts. The myoepithelial cells of the aboral haemal ring often show distinct apical bulbs of cytoplasm suggesting apocrine secretion of PAS-positive materials which are found in the surrounding aboral coelomic ring. These cells contain large stores of particulate glycogen and typical 1–2 m electrondense globules.The ground substance of the haemal tissues, which contains collagen fibers, reticular fibrils, and numerous amoeboid phagocytes, has been analyzed histochemically. Sulfated glycosaminoglycans are almost completely absent; the predominant components are polysaccharides, proteins, and/or glycoproteins. Lipids have not been demonstrated.The possible functions of the haemal tissues and associated coelomic channels are discussed.     

An ultrastructural and histochemical study of oogenesis in the trichostrongylid nematode Heligmosomoides polygyrus

Oogenesis in trichostrongylids has been examined for the first time in a light and electron microscopic investigation of Heligmosomoides polygyrus. The female reproductive tract is a single straight tube containing small oogonia (6 micron in diameter), which are arranged in a rosette pattern around a central rachis at the anterior end of the tract. Developing oocytes separate from the rachis and pass posteriorly in single file down the growth zone. Oocytes increase rapidly in volume due to the accumulation of cytoplasmic inclusion granules. These granules are of 3 types. Type 1 granules are amorphous and probably consist primarily of lipoprotein. Type 2 granules are large lipid inclusions and type 3 granules are electron-dense lipoprotein yolk bodies, which are probably used for energy reserves in the developing embryo. Histochemical studies show a more intense reaction for DNA in the nuclei of oogonia than in the nuclei of oocytes. There is a strong reaction for RNA in the nucleoli and in the cytoplasm of oogonia and oocytes. Ultrastructural studies indicate that this RNA is probably in the form of rRNA in the abundant ribosomes. Mature oocytes are cylindrical (60 X 70 micron), have a distinct nucleus with nuclear pores, and the cytoplasm is filled with inclusion granules and ribosomes but contains only small amounts of glycogen. Prior to fertilization the plasma membrane of oocytes acquires a flocculent coat. These oocytes contain 6 distinct bivalent chromosomes in diakinesis. Thus the major changes that occur in developing germ cells are 2-fold: nuclear changes that prepare the chromosomes for fertilization by initiating reduction division, and cytoplasmic changes that involve the synthesis and storage of inclusion granules.