Reticular meshwork of the spleen in rats studied by electron microscopy

The reticular meshwork of the rat spleen, which consists of both fibrous and cellular reticula, was investigated by transmission electron microscopy. The fibrous reticulum of the splenic pulp is composed of reticular fibers and basement membranes of the sinuses. These reticular fibers and basement membranes are continuous with each other. The reticular fibers are enfolded by reticular cells and are composed of two basic elements: 1) peripheral basal laminae of the reticular cells, and 2) central connective tissue spaces in which microfibrils, collagenous fibrils, elastic fibers, and unmyelinated adrenergic nerve fibers are present. The basement membranes of the sinuses are sandwiched between reticular cells and sinus endothelial cells and are composed of lamina-densalike material, microfibrils, collagenous fibrils, and elastic fibers. The presence of these connective tissue fibrous components indicates that there are connective tissue spaces in these basement membranes. The basement membrane is divided into three parts: the basal lamina of the reticular cell, the connective tissue space, and the basal lamina of the sinus endothelial cell. When the connective tissue space is very small or absent, the two basal laminae may fuse to form a single, thick basement membrane of the splenic sinus wall. The fibrous reticulum having these structures is responsible for support (collagenous fibrils) and rebounding (elastic fibers). The cells of the cellular reticulum--reticular cells and their cytoplasmic processes, which possess abundant contractile microfilaments, dense bodies, hemidesmosomes, basal laminae, and a well-developed, rough-surfaced endoplasmic reticulum, and Golgi complexes, which are characteristic of both fibroblasts and smooth muscle cells--are considered to be myofibroblasts. They may play roles in splenic contraction and in fibrogenesis of the fibrous reticulum. The contractile ability may be influenced by the unmyelinated adrenergic nerve fibers that pass through the reticular fibers. The three-dimensional reticular meshwork of the spleen consists of sustentacular fibrous reticulum and contractile myofibroblastic cellular reticulum. This meshwork not only supports the organ but also contributes to a contractile mechanism in circulation regulation, in collaboration with major contractile elements in the capsulo-trabecular system.     

Fibrous framework of human skeletal muscles, fasciae and tendons

By means of scanning and transmissive electron microscopy, the construction of the fibrous framework of the human skeletal muscles, fasciae and tendons has been investigated and its morphofunctional analysis has been performed. The fibrous framework of the endomysium is presented as a complexly organized system of anastomosing fibers of the connective tissue, forming a net-like construction. The fibrous structures of the framework are united into a whole construction by connecting fibers and fibrils. Different types of structural interconnection of collagenous fibers with sarcolemma are revealed. The structure of the fibrous framework both in different muscles and within one muscle has certain peculiarities. The main constructive element of the fascial fibrous framework make large anastomosing collagenous fibers, their architectonics is stabilized by connective fibers and fibrils. The construction of the tendinous fibrous framework is characterized by a pronounced anisotropia of the largest collagenous fibers and a developed network of connective structures both on the surface and inside the collagenous fibers. Structural mechanisms, interconnecting muscles and tendons, are demonstrated. Presence of anastomoses between the fibrils in the composition of the collagenous fibers in the fascia and Achilles tendon are stated. Together with the peculiarities existing, the general principle of the structural organization of the fibrous framework of the muscle system is the net-like constructure dependent on presence of anastomoses and elements of the connective system between the fibrous structures. Depending on the organ's function, the construction of the network acquires certain specific morphological forms.     

Enzymatic and morphological response of the thymus to drugs in normal and zinc-deficient pregnant rats and their fetuses

Summary In the thymus of normally fed pregnant rats the plasma membrane enzymes dipeptidyl peptidase IV (DPP IV) and alkaline phosphatase (alP) were found in cortical and medullary lymphocytes (thymocytes). Plasma membrane aminopeptidase A (APA) and adenosine monophosphate hydrolysing phosphatase (AMPP) were present in cortical reticular cells. In medullary reticular cells, aminopeptidase M (APM), -glutamyl transferase (GGT), adenosine triphosphate (ATPP) and thiamine pyrophosphate (TPPP) cleaving phosphatases were detected. Medullary reticular cells did not contain APA. Lysosomal DPP I and II, acid phosphatase, acid -d-galactosidase, -d-N-acetylglucosaminidase, -d-glucuronidase and non-specific esterases occurred especially in macrophages at the corticomedullary junction. The 21-day-old fetal thymus showed a similar reaction pattern as the maternal organ except for APA which was absent before birth.—After treatment of the pregnant rats with valproic acid (VPA), salicylic acid (SA), streptozotocin (ST) and retinoic acid (RA) APA showed an increase in activity in the thymic cortex. In addition, ST and RA induced AMPP, ATPP and TPPP activity in cortical reticular cells up to the same pattern as in medullary reticular cells. After ethanol (ET) administration severe damages occurred. The thymic cortex was free of DPP IV-positive lymphocytes; the medullary reticular cells showed reduced or no GGT and occasionally an increased APM activity. Dexamethasone (DEXA) given to normal or zinc-deficient rats produced the most severe lesions; thymocytes with DPP IV activity were completely absent in the cortex and medulla. In Zn-deficient pregnant rats similar alterations were observed as after ET. When the drugs were applied to Zn-deficient pregnant rats, the alterations resembled those observed after drug treatment alone. In all cases of severe thymus degeneration, i.e. ET and DEXA treatment and Zn-deficiency, the number of macrophages and activities of lysosomal hydrolases in macrophages and reticular cells were increased; the lysosomal hydrolases were often homogeneously distributed over the cortex. Cell contacts between reticular cells and lymphocytes were reduced. Vacuoles occurred within the reticular cells.—The fetal thymus was reduced in size and the number of macrophages and the activities of their lysosomal enzymes were increased after Zn-deficiency, DEXA treatment and Zn-deficiency combined with ET administration.Supported by the Deutsche Forschungsgemeinschaft (Sfb 174)     

Scanning electron microscopy of swine lymphoid organs

The aim of this investigation was to study by scanning electron microscopy the structure of several swine lymphoid organs (lymph nodes, Peyer's patches, and tonsil). Two groups of animals were used: six-month-old pigs and six- to nine-day-old piglets. Samples were jet-washed to eliminate most free cells in order to observe the reticular framework of these organs more clearly. Peyer's patches in piglets showed two types of villi. In one of them the cellular types were absorptive cells and goblet cells. The second type of villi were shorter and wider, with M cells characterized by presenting long, thick microvilli over their surfaces. Peyer's patches of pigs did not show this second type of villi but were usually covered by absorptive villi. The soft palate tonsil was similar in both groups of animals with its surface epithelial cells full of microfolds, partially and frequently obscured by microorganisms. The appearance of the surface epithelium in the same crypt was different depending on the area. There was a large number of holes through which cells apparently passed towards the crypt lumen. The medulla in the lymph nodes was at the periphery and showed a dense reticular framework. Cortex-like lymphoid tissue was formed by lymphoid follides and diffuse lymphoid tissue with high endothelid venules and lymphatic sinuses. The serosal surface of lymphoid organs was formed either by a typical mesothelial cell layer (small intestine) or by loosely arranged connective fibers (lymph nodes).     

Fluoride resistant alpha-naphthyl acetate esterase and combined enzyme histochemistry in the study of normal and pathologic lymphoid tissues

Alpha-Naphthyl acetate esterase (ANAE) and fluoride resistant alpha-naphthyl acetate esterase (FRANAE) have been compared as histochemical methods to identify T lymphocytes in sections of normal and pathological human lymphoid tissues. In addition, the FRANAE method was combined with alkaline phosphatase (ALP) in order to simultaneously evaluate the relationship between T lymphocytes and fibroblastic reticular cells (ALP) positive). The "dot like" esterase positivity of T lymphocyte was better evaluated by using FRANAE when compared to ANAE because of fluoride inhibitor of the strong esterase activity of dendritic cells and most macrophages. The combined ALP-FRANAE method clearly demonstrated a large number of fibroblastic reticular cells within the T-areas in various normal and pathological tissues such as hyperplastic lymph nodes and especially in the lymph nodes and spleens from patients with Hodgkin's disease.     

Effect of neonatal thymectomy on rabbit tonsils.

An intensive lymphocyte migration takes place through the venules lined with high endothelium and situated in the extrafollicular areas of the tonsils. After thymectomy the migration of lymphocytes is less marked and in the areas surrounding the vessels lymphocyte depletion, connective tissue proliferation and a disintegration of the intimate contact between reticular cells and reticular fibres can be observed. A similar lymphocyte depletion appears also in the epithelium. Similarly as the deep cortical substance of the lymph nodes the extrafollicular regions of the tonsils belong to the peripheral lymphoid organs.     

Histological changes of the mouse thymus during involution and regeneration following administration of hydrocortisone

Summary A histological study has been made of the thymus in mice during acute involution and regeneration following administration of hydrocortisone. The cortex undergoes remarkable changes in the microscopic structure during involution and regeneration. During involution the lymphocytes in the cortex rapidly decrease and are removed. Then a rapid replacement of lymphocytes occurs during regeneration. On the basis of formation and repopulation of lymphocytes the regenerative process of the cortex is divided into seven phases. The reconstitution of the cortex proceeds more rapidly in females than in males. Newly formed lymphocytes take origin from the mesenchymal cells in the cortex. Such mesenchymal cells become distinguishable from epithelial reticular cells during involution. They appear to engulf destroyed lymphocytes and debris during involution and then transform into immature lymphoid cells during early regeneration. The findings may support the recent reutilization concept that destroyed lymphocytes are phagocytized and reutilized by reticular cells in heteroplastic differentiation into immature lymphoid cells. In the cortex PAS-positive sudanophilic cells which are derived from the perivascular and subcapsular connective tissue appear with involutionary changes. They become gradually reduced again with progress of the regeneration of the cortex. During involution the medulla are temporarily filled with lymphocytes migrated from the cortex. The epithelial reticular cells in the medulla are found grouped in cords or clumps in the severely involuted thymus. In the medulla there are two types of PAS-positive epithelial reticular cells; one contains a large, colloid-like, PAS-positive inclusion within the cytoplasm and the other has cytoplasm diffusely filled with PAS-positive substance. During involution and early regeneration, the former type increases while the other shows almost no significant changes. Hassall's corpuscles somewhat increase in frequency during involution and early regeneration.     

A Modified Silver Impregnation Technique for the Observation of Thick Sections of Lymph Nodes Perfused with Colloidal Carbon

For many years, a variant of the silver impregnation technique of Bielchowsky has been used to study the lymph node because it clearly outlines the various structures which are usually hard to contrast with standard staining methods. Like other variants of silver impregnation, this method blackens the cell nuclei as well as the reticular fibers; however, it inhibits the impregnation of the nuclear chromatin immediately adjacent to fibers. Hence, this variant selectively darkens the lymphoid cell populations of the nodal structures which contain a loose fiber network.

To study the blood vascular network of the lymph node based on perfusion of colloidal carbon, a staining procedure was needed which would contrast nodal structures on thick sections, while allowing the carbon-filled small blood vessels to be distinguished from the impregnated coarse reticular fibers. In an attempt to adapt this variant of Bielchowsky's technique, 10, 20, 40 and 60 nm thick sections from rat nodes, fixed in a solution of Bouin-Hollande for 72 hr, were silver impregnated with serial dilutions (1:2 to 1:128) of the ammoniacal silver solution. Forty-micrometer thick sections impregnated with a 1:16 dilution of the original silver solution at 37 C and for 30 min provided the best results for the conditions.     

The Reticular Cell Network: A Robust Backbone for Immune Responses

Lymph nodes are meeting points for circulating immune cells. A network of reticular cells that ensheathe a mesh of collagen fibers crisscrosses the tissue in each lymph node. This reticular cell network distributes key molecules and provides a structure for immune cells to move around on. During infections, the network can suffer damage. A new study has now investigated the network’s structure in detail, using methods from graph theory. The study showed that the network is remarkably robust to damage: it can still support immune responses even when half of the reticular cells are destroyed. This is a further important example of how network connectivity achieves tolerance to failure, a property shared with other important biological and nonbiological networks.Lymph nodes are critical sites for immune cells to connect, exchange information, and initiate responses to foreign invaders. More than 90% of the cells in each lymph node—the T and B lymphocytes of the adaptive immune system—only reside there temporarily and are constantly moving around as they search for foreign substances (antigen). When there is no infection, T and B cells migrate within distinct regions. But lymph node architecture changes dramatically when antigen is found, and an immune response is mounted. New blood vessels grow and recruit vast numbers of lymphocytes from the blood circulation. Antigen-specific cells divide and mature into “effector” immune cells. The combination of these two processes—increased influx of cells from outside and proliferation within—can make a lymph node grow 10-fold within only a few days [1]. Accordingly, the structural backbone supporting lymph node function cannot be too rigid; otherwise, it would impede this rapid organ expansion. This structural backbone is provided by a network of fibroblastic reticular cells (FRCs) [2], which secrete a form of collagen (type III alpha 1) that produces reticular fibers—thin, threadlike structures with a diameter of less than 1 μm. Reticular fibers cross-link and form a spider web–like structure. The FRCs surrounding this structure form the reticular cell network (Fig 1), which was first observed in the 1930s [3]. Interestingly, experiments in which the FRCs were destroyed showed that the collagen fiber network remained intact [4].Open in a separate windowFig 1Structure of the reticular cell network.The reticular cell network is formed by fibroblastic reticular cells (FRCs) whose cell membranes ensheathe a core of collagen fibers that acts as a conduit system for the distribution of small molecules [5]. In most other tissues, collagen fibers instead reside outside cell membranes, where they form the extracellular matrix. Inset: graph structure representing the FRCs in the depicted network as “nodes” (circles) and the direct connections between them as “edges” (lines). Shape and length of the fibers are not represented in the graph.Reticular cell networks do not only support lymph node structure; they are also important players in the immune response. Small molecules from the tissue environment or from pathogens, such as viral protein fragments, can be distributed within the lymph node through the conduit system formed by the reticular fibers [5]. Some cytokines and chemokines that are vital for effective T cell migration—and the nitric oxide that inhibits T cell proliferation [6]—are even produced by the FRCs themselves. Moreover, the network is thought of as a “road system” for lymphocyte migration [7]: in 2006, a seminal study found that lymphocytes roaming through lymph nodes were in contact with network fibers most of the time [8]. A few years before, it had become possible to observe lymphocyte migration in vivo by means of two-photon microscopy [9]. Movies from these experiments strikingly demonstrated that individual cells were taking very different paths, engaging in what appeared to be a “random walk.” But these movies did not show the structures surrounding the migrating cells, which created an impression of motion in empty space. Appreciating the role of the reticular cell network in this pattern of motion [8] suggested that the complex cell trajectories reflect the architecture of the network along which the cells walk.Given its important functions, it is surprising how little we know about the structure of the reticular cell network—compared to, for instance, our wealth of knowledge on neuron connectivity in the brain. In part this is because the reticular cells are hard to visualize. In vivo techniques like two-photon imaging do not provide sufficient resolution to reliably capture the fine-threaded mesh. Instead, thin tissue sections are stained with fluorescent antibodies that bind to the reticular fibers and are imaged with high-resolution confocal microscopy to reveal the network structure. One study [10] applied this method to determine basic parameters such as branch length and the size of gaps between fibers. Here, we discuss a recent study by Novkovic et al. [11] that took a different approach to investigating properties of the reticular cell network structure: they applied methods from graph theory.Graph theory is a classic subject in mathematics that is often traced back to Leonhard Euler’s stroll through 18th-century Königsberg, Prussia. Euler could not find a circular route that crossed each of the city’s seven bridges exactly once, and wondered how he could prove that such a route does not exist. He realized that this problem could be phrased in terms of a simple diagram containing points (parts of the city) and lines between them (bridges). Further detail, such as the layout of city’s streets, was irrelevant. This was the birth of graph theory—the study of objects consisting of points (nodes) connected by lines (edges). Graph theory has diverse applications ranging from logistics to molecular biology. Since the beginning of this century, there has been a strong interest in applying graph theory to understand the structure of networks that occur in nature—including biological networks, such as neurons in the brain, and more recently, social networks like friendships on Facebook. Various mathematical models of network structures have been developed in an attempt to understand network properties that are relevant in different contexts, such as the speed at which information spreads or the amount of damage that a network can tolerate before breaking into disconnected parts. Three well-known network topologies are random, small-world, and scale-free networks (Box 1). Novkovic et al. modeled reticular cell networks as graphs by considering each FRC to be a node and the fiber connections between FRCs to be edges (Fig 1).

Box 1. Graph Theory and the Robustness of Real Networks

After the publication of several landmark papers on network topology at the end of the previous century, the science of complex networks has grown explosively. One of these papers described “small-world” networks [16] and demonstrated that several natural networks have the amazing property that the average length of shortest paths between arbitrary nodes is unexpectedly small (making it a “small world”), even if most of the network nodes are clustered (that is, when neighbors of neighbors tend to be neighbors). The Barabasi group published a series of papers describing “scale-free” networks [17,18] and demonstrated that scale-free networks are extremely robust to random deletions of nodes—the vast majority of the nodes can be deleted before the network falls apart [15]. In scale-free networks, the number of edges per node is distributed according to a power law, implying that most nodes have very few connections, and a few nodes are hubs having very many connections. Thus, the topology of complex networks can be scale-free, small-world, or neither, such as with random networks [19]. Novkovic et al. [11] describe the clustering of the edges of neighbors and the average shortest path–length between arbitrary nodes, finding that reticular cell networks have small-world properties. Whether or not these networks have scale-free properties is not explicitly examined in the paper, but given that they are embedded in a three-dimensional space, that they “already” lose functionality when about 50% of the FRCs are ablated, and that the number of connected protrusions per FRC is not distributed according to a power law (see the data underlying their Figure 2g), reticular cell networks are not likely to be scale-free. Thus, the enhanced robustness of reticular cell networks is most likely due to their high local connectivity: Networks lose functionality when they fall apart in disconnected components, and high clustering means that the graph is unlikely to split apart when a single node is removed, because the neighbors of that node tend to stay connected [14]. Additionally, since the reticular cell network has a spatial structure (unlike the internet or the Facebook social network), its high degree of clustering is probably due to the preferential attachment to nearby FRCs when the network develops, which agrees well with Novkovic et al.’s recent classification as a small-world network with lattice-like properties [11].Some virus infections are known to damage reticular cell networks [12], either through infection of the FRCs or as a bystander effect of inflammation. It is therefore important to understand to what extent the network structure is able to survive partial destruction. Novkovic et al. first approached this question by performing computer simulations, in which they randomly removed FRC nodes from the networks they had reconstructed from microscopy images. They found that they had to remove at least half of the nodes to break the network apart into disconnected parts. To study the effect of damage on the reticular cell network in vivo rather than in silico, Novkovic et al. used an experimental technique called conditional cell ablation. In this technique, a gene encoding the diphtheria toxin receptor (DTR) is inserted after a specific promoter that leads it to be expressed in a particular cell type of interest. Administration of diphtheria toxin kills DTR-expressing cells, leaving other cells unaffected. By expressing DTR under the control of the FRC-specific Ccl19 promoter, Novkovic et al. were able to selectively destroy the reticular cell network and then watch it grow back over time. Regrowth took about four weeks, and the resulting network properties were no different from a network formed naturally during development. Thus, it seems that the reticular cell network structure is imprinted and reemerges even after severe damage. This finding ties in nicely with previous data from the same group [13], showing that reticular cell networks form even in the absence of lymphotoxin-beta receptor, an otherwise key player in many aspects of lymphoid tissue development. Together, these data make a compelling case that network formation is a robust fundamental trait of FRCs.Next, Novkovic et al. varied the dose of diphtheria toxin such that only a fraction of FRCs were destroyed, effectively removing a random subset of the network nodes. They measured in two ways how FRC loss affects the immune system: they tracked T cell migration using two-photon microscopy and they determined the amount of antiviral T cells produced by the mice after an infection. Remarkably, as predicted by their computer simulations, lymph nodes appeared capable of tolerating the loss of up to half of FRCs with little effect on either T cell migration or the numbers of activated antiviral T cells. Only when more than half of the FRCs were destroyed did T cell motion slow down significantly and the mice were no longer able to mount effective antiviral immune responses. Such a tolerance of damage is impressive—for comparison, consider what would happen if one were to close half of London’s subway stations!Robustness to damage is of interest for many different networks, from power grids to the internet [14]. In particular, the “scale-free” architecture that features rare, strongly connected “hub” nodes is highly robust to random damage [15]. Novkovic et al. did not address whether the reticular cell network is scale-free, but it is likely that it isn’t (Box 1). Instead, the network’s robustness probably arises from its high degree of clustering, which means that the neighbors of each node are likely to be themselves also neighbors. If a node is removed from a clustered network, then there is still likely a short detour available by going through two of the neighbors. Therefore, one would have to randomly remove a large fraction of the nodes before the network structure breaks down. High clustering in the network could be a consequence of the fact that multiple fibers extend from each FRC and establish connections to many FRCs in its vicinity. A question not yet addressed by Novkovic et al. is how robust reticular cell networks would be to nonrandom damage, such as a locally spreading viral infection. In fact, scale-free networks are drastically more vulnerable to targeted rather than random damage: the United States flight network can come to a grinding halt by closing a few hub airports [15]. Less is known about the robustness to nonrandom damage for other network architectures, and the findings by Novkovic et al. motivate future research in this direction.Novkovic et al. did not yet explicitly identify all mechanisms that hamper T cell responses when more than half of the FRCs are depleted. But given the reticular cell network’s many different functions, this could occur in several ways. For instance, severe depletion might prevent the secretion of important molecules, halt the migration of T cells, prevent the anchoring of antigen-presenting dendritic cells (DCs) on the network, or cause structural disarray in the tissue. In addition to the effects on T cell migration, Novkovic et al. also showed that the amount of DCs in fact decreased when FRCs were depleted, emphasizing that several mechanisms are likely at play. Disentangling these mechanisms will require substantial additional research efforts.The current reticular cell network reconstruction by Novkovic et al. is based on thin tissue slices. It will be exciting to study the network architecture when it can be visualized in the whole organ. Some aspects of the network may then look different. For instance, those FRCs that are near the border of a slice will have their degree of connectivity underestimated, as not all of their neighbors in the network can be seen. Further refinements of the network analysis may also consider that reticular fibers are real physical objects situated in a three-dimensional space (unlike abstract connections such as friendships). Migrating T cells may travel quicker via a short, straight fiber than on a long, curved one, but the network graph does not make this distinction. More generally, it would be interesting to understand conceptually how reticular cell networks help foster immune cell migration. While at first it appears obvious that having a “road system” should make it easier for cells to roam lymph node tissue, three different theoretical studies have in fact all concluded that effective T cell migration should also be possible in the absence of a network [20–22]. A related question is whether T cells are constrained to move only on the network or are merely using it for loose guidance. For instance, could migrating T cells be in contact with two or more network fibers at once or with none at all? This would make the relationship between cell migration and network structure more complex than the graph structure alone suggests.There is also some evidence that T cells can migrate according to what is called a Lévy walk [23]—a kind of random walk where frequent short steps are interspersed with few very long steps, a search strategy that appears to occur frequently in nature (though this is debated [24]). While there is so far no evidence that T cells perform a Lévy walk when roaming the lymph node [25], this may be in part due to limitations of two-photon imaging, and one could speculate that reticular cell networks might in fact be constructed in a way that facilitates this or another efficient kind of “search strategy.” Resolving this question will require substantial improvements in imaging technology, allowing individual T cells to be tracked across an entire lymph node.No doubt further studies will address these and other questions, and provide further insights on how reticular cell networks benefit immune responses. Such advances may help us design better treatments against infections that damage the network. It may also help us understand how we can best administer vaccines or tumor immune therapy treatments in a way that ensures optimal delivery to immune cells in the lymph node. As is nicely illustrated by the study of Novkovic et al., mathematical methods may well play key roles in this quest.     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

Summary In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, -D-mannose and -D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; -D-mannose and -D-glucose residues and terminal sialic acidgalactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs

Mesenchymal stromal cells are crucial components of secondary lymphoid organs (SLOs). Organogenesis of SLOs involves specialized stromal cells, designated lymphoid tissue organizer (LTo) in the embryonic anlagen; in the adult, several distinct stromal lineages construct elaborate tissue architecture and regulate lymphocyte compartmentalization. The relationship between the LTo and adult stromal cells, however, remains unclear, as does the precise number of stromal cell types that constitute mature SLOs are unclear. From mouse lymph nodes, we established a VCAM-1(+)ICAM-1(+)MAdCAM-1(+) reticular cell line that can produce CXCL13 upon LTbetaR stimulation and support primary B cell adhesion and migration in vitro. A similar stromal population sharing many characteristics with the LTo, designated marginal reticular cells (MRCs), was found in the outer follicular region immediately underneath the subcapsular sinus of lymph nodes. Moreover, MRCs were commonly observed at particular sites in various SLOs even in Rag2(-/-) mice, but were not found in ectopic lymphoid tissues, suggesting that MRCs are a developmentally determined element. These findings lead to a comprehensive view of the stromal composition and architecture of SLOs.     

Basement-membrane components associated with the extracellular matrix of the lymph node

Summary Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as reticular fibers because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.     

Microfibrils: a constitutive component of reticular fibers in the mouse lymph node

Summary Fibrous components other than collagen fibrils in the reticular fiber of mouse lymph node were studied by electron microscopy. Bundles of microfibrils not associated by elastin and single microfibrils dispersed among collagen fibrils were present. The diameter of the microfibrils was 13.29±2.43 nm (n=100). Elastin-associated microfibrils occurred at the periphery of the reticular fiber. Elastin was enclosed by microfibrils, thus forming the elastic fiber, which was clearly demonstrated by tannic acid-uranyl acetate staining. In the reticular fiber of lymph nodes, the elastic fiber consisted of many more microfibrils and a small amount of elastin. These microfibrils, together with the collagen fibrils, may contribut to the various functions of the reticular fibers.     

Fibroblasts form a body-wide cellular network

Loose connective tissue forms a network extending throughout the body including subcutaneous and interstitial connective tissues. The existence of a cellular network of fibroblasts within loose connective tissue may have considerable significance as it may support yet unknown body-wide cellular signaling systems. We used a combination of histochemistry, immunohistochemistry, confocal scanning laser microscopy (confocal microscopy), and electron microscopy to investigate the extent and nature of cell-to-cell connections within mouse subcutaneous connective tissue. We found that fibroblasts formed a reticular web throughout the tissue. With confocal microscopy, 30% of fibroblasts processes could be followed continuously from one cell to another. Connexin 43 immunoreactivity was present at apparent points of cell-to-cell contact. Electron microscopy revealed that processes from adjacent cells were in close apposition to one another, but gap junctions were not observed. Our findings indicate that soft tissue fibroblasts form an extensively interconnected cellular network, suggesting they may have important and so far unsuspected integrative functions at the level of the whole body.     

Perivascular lymphoid tissue associated with the axillary lymph sinus and the lateral vein of Gehyra variegata (Reptilia:Gekkonidae)

The axillary sinus of G. variegata is formed from a perivascular lymphatic which locally invests the lateral vein. Within the sinus the wall of the vein is distended by lymphoid tissue which is itself supported by reticular fibres. Lymphocytes, reticular cells, macrophages and mast cells occur in the tissue. The overall appearance of the structure is lymph node-like. Although Cardianema sp. (Nematoda:Filarioidea) parasitised the lymphatic system of some geckos examined, the non-pathologic origin of the lymphoid tissue is indicated by its presence in both axillae of infected and uninfected geckos alike. Comparison is made with lymph nodes and node-like structures in other vertebrates.     

Ultrastructural changes during propagation of a Group D streptococcal L-form

Summary L-form colonies derived from a Group D streptococcus showed alterations in fine structure during propagation. For the first 6 months after preparation the L-forms were capable of reversion and fibrillar and lamellate inclusions were found in some cells. Fibrillar bundles, composed of fibrils with an external diameter of 12.5 nm were found attached to the cytoplasmic membrane of the large 20 m surface cells. Small, 1 m cells within the agar contained lamellate inclusions with a periodicity of 14 nm, also attached to the cytoplasmic membrane. In empty cells both inclusions appeared as hollow fibrils. The composition of these structures is not known. Long filamentous evaginations of the cytoplasmic membrane were also found in a small proportion of cells. After 15 months' propagation, when the L-form had become stable, the inclusions and filaments were no longer visible.     

Molecular complexity of the cutaneous basement membrane zone

Ultrastructural examination of the cutaneous basement membrane zone (BMZ) reveals the presence of several attachment structures, which are critical for integrity of the stable association of epidermis and dermis. These include hemidesmosomes which extend from the intracellular compartment of the basal keratinocyte to the underlying basement membrane where they complex with anchoring filaments, thread-like structures traversing the lamina lucida. At the lower portion of dermal-epidermal attachment zone, anchoring fibrils extend from the lamina densa to the papillary dermis, where they associate with basement membrane-like structures, known as anchoring plaques. Molecular cloning of the cutaneous BMZ components has allowed elucidation of the structural features of the proteins which constitute these attachment structures. Specifically, hemidesmosomes have been shown to consist of at least four distinct proteins. The intracellular hemidesmosomal inner plaque is comprised of the 230-kD bullous pemphigoid antigen (BPAG1), and plectin, a high-molecular weight cytomatrix protein, encoded by the corresponding gene, PLEC1. The transmembrane component of the hemidesmosomes consists of the 180-kD bullous pemphigoid antigen (BPAG2), a collagenous protein also known as type XVII collagen (COL17A1), as well as of the basal keratinocyte-specific integrin 64. The anchoring filaments consist predominantly of laminin 5 with three constitutive subunit polypeptides, the 3, 3 and 2 chains, which is associated with laminin 6 with the chain composition 3, 1 and 1. Also associated with anchoring filaments is a novel protein, ladinin, which serves as autoantigen in the linear IgA disease, and the corresponding gene, LAD1, has been mapped to human chromosome 1. Finally, the major, if not the exclusive, component of anchoring fibrils is type VII collagen, encoded by the gene (COL7A1) which consists of 118 distinct exons, the largest number of exons in any gene published thus far. Collectively, the cutaneous basement membrane zone is a complex continuum of macromolecules which form a network providing the stable association of the epidermis to the underlying dermis. Thus, genetic lesions resulting in abnormalities in any part of this network could result in a blistering skin disease, such as epidermolysis bullosa.Abbreviations BMZ basement membrane zone - EB epidermolysis bullosa - JEB junctional EB - GABEB generalized atrophic benign EB - EB-MD epidermolysis bullosa with muscular dystrophy - EB-PA epidermolysis bullosa with pyloric atresia     

Cellular composition of structural components of the lymph nodes of rats during rehabilitation after dimethyl sulfate poisoning

By means of microanatomical methods the inferior tracheobronchial lymph nodes have been investigated in 48 Wistar rats in 2 weeks and 3 months after discontinuance of inhalation of dimethylsulfate (DMS) vapours for 2 and 14 days by the animals in concentration 2.0 mg/m3, that is to say during rehabilitation period. Comparison of relative parameters of the structural components areas and cell composition of the lymph nodes has been carried out. During rehabilitation period after DMS inhalation for 2 days the cortical and medullary areas in histological preparations do not essentially differ from corresponding parameters of an acute experiment (2 days, 2.0 mg/m3, without rehabilitation). Amount (%) of cells with mitotic figures in the lymphoid nodules++ increases in 2 weeks and in 3 months. Contents of poorly differentiated cells during rehabilitation periods increase in the cortical plateau, but keeps nearly at the same low level as during the acute experiment in the lymphoid nodules++. In 2 weeks after DMS influence for 14 days, the cortical and medullary area in the histological preparations reach the control levels. In the lymphoid nodules++ a relative amount of reticular, poorly differentiated, mitotically dividing cells increases, and in the medullary cords contents of middle and small lymphocytes become greater in comparison with the acute experiment (14 days, 2.0 mg/m3, without rehabilitation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue nature of the lining of the lymph node sinuses]

The lymphatic nodes of intact albino rats were investigated electron microscopically. It was shown that the lymphatic sinuses were restricted by a layer of flattened cells; the basal membrane was absent. Certain distinctions in the structure of the cell lining sinuses and the reticular cells comprising the reticular base of the lymphoid tissue of the lymphatic node were found. The structure of the "sinus network" strands is shown. The structure of the cells of the sinuses lining is shown to be identical to the structure of cells of the vascular endothelium. It suggests the endothelial nature of the lining of the lymphatic node sinuses.     

Experimental paracoccidioidomycosis in the syrian hamster: Morphology,ultrastructure and correlation of lesions with presence of specific antigens and serum levels of antibodies

Male hamsters (134) received intratesticular injection of a live cerebriform culture of Paracoccidioides brasiliensis and were sacrificed from 6 hours up to 123 days onwards. Tissues from testis, lymph nodes, lungs, liver, spleen, kidneys and intestines were examined microscopically; presence of specific antigens was saught in lesions of testis, regional lymph nodes and liver by indirect immunofluorescence (IF); inoculation site lesions were studied electron microscopically and circulating specific antibodies measured by complement fixation and IF tests.Up to 24 hours inoculation site lesions showed fungi surrounded by PMNs; 48 hours latter macrophages accumulated forming loose nodules; epithelioid granulomata appeared after 5 days. Fungi, scarce in early lesions, increased in numbers up to the time when epithelioid granulomata dominated the picture; in young granulomata fungi were abundant and small; older granulomata contained rare, vacuolated fungi. Ultrastructurally the space between fungi and host-cells was larger around reproducing forms decreasing in size as the parasites grew larger and being a virtual slit around old degenerated fungi. Immunofluorescence studies revealed that fungal walls were brightly fluoerescent; in early lesions macrophages surrounding fungi or free in the intersticium contained fluorescent antigenic material in the cytoplasm; similar macrophages were observed in draining lymph nodes as early as 18 hours after inoculation, and latter, in macrophage nodules and Kupffer cells in the liver; epithelioid and giant cells appear to block diffusion of antigens, since in epithelioid granulomata fluorescence was limited to fungal walls.Disseminated paracoccidioidomycosis occurred in 100% of animals after day 5 of infection. Besides specific lesions (containing fungi), antigens were identified by immunofluorescence in non specific lesions in the liver (diffuse or nodular Kupffer cell hyperplasia) and in the lymph nodes (histiocytic hyperplasia). Serum antibodies appeared in low titers, up to day 20, increasing onwards. From day 70 on, titers decreased and lesions changed from confluent epithelioid to loose granulomata infiltrated by PMNs; fungi that before were large and quiescent now were small and in active reproduction. Secondary amyloidosis was present in 85% of the animals.In the hamster, Paracoccidioidomycosis develops as a chronic progressive disease and the lesions are related both to fungi and its antigens.