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The intestine contains some of the most diverse and complex immune compartments in the body. Here we describe a method for isolating human gut-associated lymphoid tissues (GALTs) that allows unprecedented profiling of the adaptive immune system in submucosal and mucosal isolated lymphoid follicles (SM-ILFs and M-ILFs, respectively) as well as in GALT-free intestinal lamina propria (LP). SM-ILF and M-ILF showed distinct patterns of distribution along the length of the intestine, were linked to the systemic circulation through MAdCAM-1 + high endothelial venules and efferent lymphatics, and had immune profiles consistent with immune-inductive sites. IgA sequencing analysis indicated that human ILFs are sites where intestinal adaptive immune responses are initiated in an anatomically restricted manner. Our findings position ILFs as key inductive hubs for regional immunity in the human intestine, and the methods presented will allow future assessment of these compartments in health and disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The ocular surface of the white domestic pig (Sus scrofa domestica) is used as a helpful model of the human ocular surface; however, a complete histological description has yet to be published. In this work, we studied porcine eyeballs with intact eyelids to describe and characterize the different structures that form the ocular surface, including the cornea and conjunctiva that covers the bulbar sclera, tarsi, and the nictitating membrane. We determined the distribution of goblet cells of different types over the conjunctiva and analyzed the conjunctival-associated lymphoid tissue (CALT). Porcine eyeballs were obtained from a local slaughterhouse, fixed, processed, and embedded in paraffin blocks. Tissue sections (4 μm) were stained with hematoxylin/eosin, Alcian blue/Periodic Acid Schiff, and Giemsa. Slides were also stained with lectins from Arachis hypogaea (PNA) and Helix pomatia (HPA) agglutinins and immunostained with rabbit anti-CD3. We found that the porcine cornea was composed of 6-8 epithelial cell layers, stroma, Descemet's membrane, and an endothelial monolayer. The total corneal thickness was 1131.0±87.5 μm (mean±standard error of the mean) in the center and increased to 1496.9±138.2 μm at the limbus. The goblet cell density was 71.25±12.29 cells/mm, ranging from the highest density (113.04±37.21 cells/mm) in the lower palpebral conjunctiva to the lowest density (12.69±4.29 cells/mm) in the bulbar conjunctiva. The CALT was distributed in the form of intraepithelial lymphocytes and subepithelial diffuse lymphoid tissue. Lenticular-shaped lymphoid follicles, about 8 per histological section, were also present within the conjunctival areas. In conclusion, we demonstrated that the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology., Competing Interests: The authors have declared that no competing interests exist.

This study describes the mucosa-associated lymphoid tissue (MALT) in odontocetes from the Brazilian coast and freshwater systems. Seven species were evaluated and tissue samples were analyzed by light, scanning and transmission electron microscopy, and immunohistochemistry. Laryngeal tonsil was a palpable oval mass located in the larynx, composed of a lymphoepithelial complex. Dense collections of lymphocytes were found in the skin of male fetus and calf. Clusters of lymphoid tissue were found in the uterine cervix of a reproductively active juvenile female and along the pulmonary artery of an adult female. Lymphoid tissues associated with the gastrointestinal tract were characterized by diffusely arranged or organized lymphocytes. The anal tonsil was composed of an aggregate of lymphoid tissue occurring exclusively in the anal canal, being composed of squamous epithelium branches. MALT was present in different tissues and organic systems of cetaceans, providing constant protection against mucosal pathogens present in their environment., (© 2016 Wiley Periodicals, Inc.)

CD4+ T cells are the primary HIV-1 target cell, with the vast majority of these cells residing within lymphoid tissue compartments throughout the body. Predictably, HIV-1 infection, replication, localization, reservoir establishment and persistence, as well as associated host immune and inflammatory responses and disease pathology principally take place within the tissues of the immune system. By virture of the fact that the virus-host struggle is played out within lymphoid and additional tissues compartments in HIV-1 infected individuals it is critical to understand HIV-1 infection and disease within these relevant tissue sites; however, there are obvious limitations to studying these dynamic processes in humans. Nonhuman primate (NHP) research has provided a vital bridge between basic and preclinical research and clinical studies, with experimental SIV infection of NHP models offering unique opportunities to understand key processes of HIV-1 infection and disease that are either not practically feasible or ethical in HIV-1 infected humans. In this review we will discuss current approaches to studying the tissue based immunopathogenesis of AIDS virus infection in NHPs, including both analyses of tissues obtained at biopsy or necropsy and complementary non-invasive imaging approaches that may have practical utility in monitoring HIV-1 disease in the clinical setting., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Normal structure of the accessory organs of the eye is essential for normal eye physiology. Among the most important accessory organs of the eye are the eyelids, the conjunctiva-associated lymphoid tissue (CALT) and the lacrimal gland (LG). The aim of this study was to demonstrate the histological structure of the eyelids and LG by histochemical and ultrastructural analysis. The study was performed on 13 adult female Bilgorajska geese. Eyelid samples were stained with the Alcian blue (AB pH 2.5) and periodic acid-Schiff (PAS) methods. Staining methods used for LG were AB pH 2.5, aldehyde fuchsin (AF), PAS and Hale's dialysed iron (HDI). Within the connective tissue of the eyelids, well-developed, diffuse, CALT follicles were observed, mostly under the conjunctival epithelium. Numerous lymphocytes were present within loose connective tissue. Staining of the eyelids with the PAS method demonstrated the presence of goblet cells of a mucous nature, and AB pH 2.5 staining indicated the presence of sulfated acid mucopolysaccharides. PAS staining of LG revealed the presence of secretory cells containing weakly PAS-positive granules. All epithelial cells of the corpus glandulae and the duct systems reacted positively to AB pH 2.5. HDI staining detected the presence of carboxylated acid mucopolysaccharides. Transmission electron microscopy investigations revealed two types of secretory epithelial cells in LG. Both types of LG cells contained drop-like secretory vesicles of different sizes with low or high electron density in cytoplasm, as well as small and large lipid vacuoles, and numerous small primary lysosomes.

The human palatine tonsils represent a mucosa-associated lymphoid tissue with a significant function in mucosal protection against alimentary and airborne pathogens. The ultrastructure of different morphological compartments in the human palatine tonsil was studied in eighteen tonsils obtained from the patients who had undergone elective tonsillectomy due to chronic tonsillitis. The tonsillar specimens were analyzed by scanning and transmission electron microscopy. The results showed the presence of tight junctions between superficial epithelial cells of the oropharyngeal tonsillar surface. The crypt epithelium is a sponge-like structure infiltrated by non-epithelial cells, mostly lymphocytes, and is characterized by the presence of small pores - microcrypts occupied by large microvillus cells and/or lymphocytes. Antigen-presenting Langerhans cells with typical intracytoplasmic Birbeck granules were also found in the crypt epithelium. The lymphoid follicles are composed of lymphocytes and two types of non-lymphoid follicular cells: small fibroblast-like cells and large cells, morphologically consistent with antigen-bearing follicular dendritic cells or macrophages. The interfollicular areas consisted of a dense network of reticular cells and reticular fibers; many lymphocytes were interspersed between the reticular fibers. In addition to arterioles and high endothelial venules in the interfollicular lymphoid tissue, some fenestrated capillaries were seen intraepithelially and subepithelially. The complex ultrastructure of the human palatine tonsil provides a microenvironment necessary for antigen uptake, antigen processing and immune response.

Background: Lymphoid tissue remodeling is characteristic of chronic simian immunodeficiency virus infection., Methods: A rhesus macaque infected with SIVmac239 was necropsied and its lymphoid tissues subjected to histopathology characterization., Results: Germinal centers in spleen and lymph nodes contained PAS-positive, non-amyloid extracellular deposits, decreased T follicular helper cells, and normal density of Ki67(+) B cells., Conclusions: A possible mechanism for PAS-positive deposits includes exaggerated involution of SIV-induced follicular hyperplasia secondary to virus-associated immune reaction., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Paneth cells (PCs) contribute to the host defense against indigenous bacteria in the small intestine. We found Paneth cell-like cells (PLCs) in the rat ascending colon, but the nature of PLCs is never clarified. Therefore, the present study aimed to clarify the cytological characteristics of PLCs and discuss their cellular differentiation. PLCs were localized in the bases of intestinal crypts, especially follicle-associated intestinal crypts in proximal colonic lymphoid tissue, but were very seldom found in the ordinary intestinal crypts of the ascending colon. PLCs possessed specific granules with highly electron-dense cores and haloes, as well as PCs in the small intestine. The secretory granules of PLCs were positive for PAS reaction, lysozyme and soluble phospholipase A2, but negative for Alcian blue staining, β-defensin-1 and -2, as well as the ones of PCs. Furthermore, intermediate cells possessing both the PLC-specific granules and the mucus granules similar to those of goblet cells (GCs) were occasionally found in the vicinity of PLCs. Intermediate cells ranged from goblet cell-like cells rich in mucus granules to PLC-like cells with few mucus granules. The cellular condensation and fragmentation were exclusively found in PLCs but never seen in intermediate cells or GCs. The PLCs, which were identified as PC, were suggested to be transformed from GCs through intermediate cells and finally to die by apoptosis in intestinal crypts of proximal colonic lymphoid tissue in the rat ascending colon., (Copyright © 2014 Wiley Periodicals, Inc.)

Secondary lymphoid tissues are the sites of both innate and adaptive host defense. Aside from the relatively static nonhematopoietic stromal elements and some macrophages and dendritic cells, most of the cells in these tissues are in constant movement, but the organs maintain a defined microanatomy with preferred locations for the bulk of T cells, B cells, and other lymphocytes and subsets of myeloid cells. Here we describe both the cell dynamics and spatial organization of lymph nodes and review how both physical features and molecular cues guide cell movement to optimize host defense. We emphasize the role of locality in improving the efficiency of a system requiring rare cells to find each other and interact productively through membrane-bound or short-range secreted mediators and highlight how changes in steady-state cell positioning during an infectious challenge contribute to rapid generation of productive responses.

Purpose: Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model., Methods: Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy., Results: Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min., Conclusions: CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.

We studied the effect of acute emotional stress on functional status of lymphoid epithelial structures of the jejunum in rats with different behavioral activity. Morphological and functional characteristics of lymphoid tissue were assessed using morphometric, histological and electron microscopic methods. In behaviorally active and passive rats, reduction in villus height, area of the epithelium and lymphoid tissue of the jejunum was recorded on the third day after acute emotional impact. At that, the largest number of destructively modified lymphoid cells was identified by comparison with the other time points. Moreover, destruction of the apical part of the jejunal villi was observed on days 3 and 7 after stress exposure. Recovery of the lymphoepithelial structures of the jejunum after acute stress exposure was detected on day 14.

Objective: To characterize conjunctival lymphoid nodules obtained from the nictitans of healthy cats to determine if the follicle-associated epithelium (FAE) of conjunctiva-associated lymphoid tissue (CALT) in this species contains membranous (M)-cells analogous to those described in other regions of mucosa-associated lymphoid tissue (MALT)., Methods: Lymphoid follicles from nictitan bulbar surfaces of 10 healthy cats (20 eyes total) were examined. Nictitans from five cats were harvested immediately post-mortem and a minimum of 12 lymphoid nodules from each third eyelid were isolated using a Zeiss operating microscope. At least three lymphoid follicles from each eye were examined using light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) using standard fixation and embedding protocols. Nictitan-lymphoid follicles from another five healthy cats were processed for immunohistochemistry to characterize the distribution of T- and B-lymphocytes present beneath the FAE., Results: The FAE overlying CALT from 10 healthy cats demonstrated morphology characteristic of M-cells including attenuated apical cell surface with blunted microvilli and microfolds, invaginated basolateral membrane forming a cytoplasmic pocket, and diminished distance between the apical and pocket membrane. Immunohistochemistry of lymphoid tissue subtending the FAE demonstrated B-cell dependent regions in the germinal centers surrounded by T-cell dependent interfollicular zones., Conclusions: Healthy feline CALT contains morphologic features analogous to those described in other regions of MALT. Documentation of feline conjunctival M-cells is of clinical relevance in the study of primary infectious, allergic, and autoimmune ocular diseases, as well as a potential means of vaccination or drug delivery., (© 2011 American College of Veterinary Ophthalmologists.)

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.

In the last decade, it has been accepted the formation of tertiary lymphoid organs in the renal parenchyma during inflammatory conditions. These organized cellular aggregates contain B- and T-lymphocytes, dendritic cells, surrounded by neo-lymphatic vessels. They have been described in renal allografts, acute and chronic interstitial nephritis, IgA and membranous nephropathies. The functional characteristics of these lymphoid nodules remained still under consideration. After investigating the renal biopsies of 268 patients with primary and secondary nephropathies, we have selected 20 cases showing lymphoid-like cellular aggregates located just beneath the renal capsule and having close contacts with this kidney envelope. All of these cases also showed an associated medium sized lymphatic vessel. The ultrastructure of these nodules proved to contain more or less the same cellular composition: lymphocytes, dendritic cells, seldom plasma cells and macrophages. We consider these particular subcapsular lymphoid-like nodules to be tertiary lymphatic structures in close association with the perirenal lymphatics, and the first to develop in any type of inflammatory and autoimmune renal condition.

As solid morphological knowledge of ovine tonsillar epithelia might contribute to a better understanding of the pathogenesis of several diseases including prion diseases, the epithelia of all tonsils of 7 one-year-old Texel sheep were examined using scanning and transmission electron microscopy. Major parts of the pharyngeal and tubal tonsils were covered by pseudostratified columnar ciliated epithelia that were interrupted by patches of epithelium containing cells with densely packed microfolds or microvilli, and cells with both microvilli and cilia. Smaller parts were covered by either flattened polygonal cells with densely packed microvilli or microfolds, squamous epithelial cells, or patches of reticular epithelium. The palatine and paraepiglottic tonsils were mainly lined by squamous epithelial cells with apical microplicae or short knobs. Additionally, regions of reticular epithelium containing epithelial cells with apical microvilli were seen. The lingual tonsil was uniformly covered by a keratinized squamous epithelium and devoid of microvillous cells and patches of reticular epithelium. The rostral half of the tonsil of the soft palate was lined by a pseudostratified columnar ciliated epithelium with characteristics of the pharyngeal and tubal tonsils. The epithelium of the caudal part resembled the epithelia of the palatine and paraepiglottic tonsils. Putative M cells, mainly characterized by apical microvilli or microfolds and a close association with lymphoid cells, seem manifestly present on the nasopharyngeal tonsils. The reticular epithelium of the palatine and paraepiglottic tonsils also harbor cells with small apical microvilli. The exact nature of these presumptive M cells should, however, be elucidated in functional studies.

Here, for the first time, to colocalize new type gosling viral enteritis virus (NGVEV) with histological lesions and in situ apoptosis in the digestive organs (esophagus, proventriculus, gizzard, small intestine, cecum, rectum, liver, and pancreas) and the lymphoid organs (bursa of Fabricius, thymus, Harderian gland, and spleen) of experimentally infected goslings, portions of tissues were collected at sequential infection time points and examined by histopathology for histological lesions, immunohistochemical staining for viral antigens, ultrastructural observation by transmission electron microscope (TEM) for virus particles and apoptotic cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay for in situ apoptosis. The hyperemia, hemorrhage, infiltration of lymphocytes, progressive lymphoid depletion, apoptosis, and necrosis were readily observed in the lymphoid organs and intestine tract by histopathological examination. The NGVEV particles and viral antigens widely appeared in the small intestine and bursa of Fabricius as early as 2 d postinfection (PI) by TEM and immunohistochemical staining, and the presence and quantity of it reached a maximum during 6 to 12 d PI. The principal sites for NGVEV were endothelial cells, epithelia, mucosal cells, glandular cells, fibrocytes, macrophages, and lymphocytes. A series of apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, and formation of apoptotic body were observed by TEM, and the number of apoptotic cells was largely increased from 4 d PI and peaked at 9 d PI by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling analysis. The histological organ lesions and apoptosis in vivo were generally associated with sites of NGVEV localization, which can be regarded as the cause of death. This work may shed light on the pathogenesis of new type gosling viral enteritis and put new insight into the pathogenesis of goose adenovirus.

The eye is protected by the ocular immunosurveillance system. We show that tear duct-associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/differentiation 2, retinoic acid-related orphan receptor gammat, lymphotoxin (LT) alpha1beta2-LTbetaR, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity.

Translation of small interfering RNA (siRNA)-based approaches into practical therapeutics is limited because of lack of an effective and cell-specific delivery system. Herein, we present a new method of selectively delivering siRNA to dendritic cells (DCs) in vivo using CD40 siRNA-containing immunoliposomes (siILs) that were decorated with DC-specific DEC-205 mAb. Administration of CD40 siILs resulted in DC-specific cell targeting in vitro and in vivo. On treatment with CD40 siILs, the expression of CD40 in DCs, as well allostimulatory activity was inhibited. In vivo administration resulted in selective siRNA uptake into immune organs and functional immune modulation as assessed using a model antigen. In conclusion, this is the first demonstration of DC-specific siRNA delivery and gene silencing in vivo, which highlights the potential of DC-mediated immune modulation and the feasibility of siRNA-based clinical therapy.

The use of nanometer-sized iron oxide nanoparticles and micron-sized iron oxide particles as magnetic resonance (MR) contrast agents has garnered a high degree of interest in diverse areas of biology and medicine. Applications such as cell tracking, molecular imaging, gene detection, and lymphography are being explored to provide insight into disease mechanisms, monitor therapeutic efficacy, and facilitate diagnostic imaging. What makes iron oxide so appealing is a number of favorable properties including high detectability by MR, biodegradability and low toxicity. Here we describe the recent progress on the use of magnetic nanoparticles in imaging circulating cells and lymphoid tissues. The study of the lymph system and the biodistribution of various circulating immune cells is important in the diagnosis, prognosis, and treatment of a wide range of diseases and is expected to have a profound effect on patient outcome.

The changes in the histological and three dimensional organizations of lymphoid organ and characteristics of lymphoid cells after chronic infection with yellow head virus (YHV) in Penaeus monodon were investigated. The vascular cast of infected lymphoid organ showed less branching and dramatically shortened terminal capillaries that formed the lymphoid tubules, and only large stumps of these lymphoid tubules remained. This might occur because the terminal ends of the tubules were damaged from YHV infection, as stromal cells and hemocytes in the LT wall were infected and formed foci that could give rise to lymphoid spheroids that broke away from the original lymphoid tubules. Histologically, there was a decrease of PAS-stained connective tissue materials in lymphoid spheroids as well as a decrease of stromal cells as marked by anti-vimentin antibody. This indicated that stromal cells together with type 1 fibers and associated extracellular matrix degenerated in lymphoid spheroids, while type 2 or reticular fibers proliferated and encapsulated the spheroids.

In addition to being the respiratory organ in fish, the gills form a barrier against the external milieu. Innate and adaptive immune system components have been detected in the gills, but lymphoid cell accumulations similar to that seen in the mammalian mucosa have not been described. The present investigations revealed cell accumulations on the caudal edge of interbranchial septum at the base of the gill filaments in the Atlantic salmon. Cytokeratin immunohistochemical staining and identification of a basal membrane and desmosome cell junctions by electron microscopy showed that the cell accumulation was located intraepithelially. Major histocompatibility complex (MHC) class II+ cells were detected by immunohistochemistry, and laser capture micro-dissection and subsequent RT-PCR analysis revealed expression of T-cell receptor transcripts in the investigated tissue, suggesting the presence of T cells. The intraepithelial tissue reported here may be a suitable location for immune surveillance of gill infections, as well as a target site for new vaccine approaches and investigations of epithelial immunity. This is the first description of a lymphocyte cell aggregation within a teleostian gill epithelium network, illustrating a phylogenetically early form of leukocyte accumulations in a respiratory organ.

The purpose of this study was to characterize the ultrastructure of lymphoid tissue from HIV/AIDS patients and to evaluate it as a reservoir and source of HIV. HIV has been demonstrated in lymph nodes and tonsils and adenoids, by immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM), to be associated with germinal center (GC) follicular dendritic cells (FDC). The presence of HIV in the larger gastrointestinal tract-associated lymphoid tissue (GALT) has been much less studied. Whether FDC themselves are productively infected by HIV in any of the lymphoid sites is controversial. Lymph nodes, tonsils, and gastrointestinal biopsies were fixed in neutral buffered glutaraldehyde and prepared for TEM. Mature HIV particles were abundant in GC of hyperplastic lymph nodes, tonsils, and the GALT. They were enmeshed within an electron-dense matrix associated with an all-encompassing branching FDC network of processes. HIV particles were seen budding from both FDC and lymphocytes. The greatest numbers of particles were seen in hyperplastic lymphoid tissue from untreated individuals and in lymph nodes co-infected with opportunistic organisms, such as Mycobacterium avium complex. In addition to HIV, unidentifiable "particles" of varying sizes, possibly including other viruses, were regularly seen in association with FDC. Ultrastructural study graphically demonstrated the abundance of HIV particles associated with the complex FDC network of hyperplastic lymph nodes, tonsils, and GALT. HIV was shown to productively infect FDC, as well as lymphocytes.

The normal lymphoid organ of Penaeus monodon (which tested negative for WSSV and YHV) was composed of two parts: lymphoid tubules and interstitial spaces, which were permeated with haemal sinuses filled with large numbers of haemocytes. There were three permanent types of cells present in the wall of lymphoid tubules: endothelial, stromal and capsular cells. Haemocytes penetrated the endothelium of the lymphoid tubule's wall to reside among the fixed cells. The outermost layer of the lymphoid tubule was covered by a network of fibers embedded in a PAS-positive extracellular matrix, which corresponded to a basket-like network that covered all the lymphoid tubules as visualized by a scanning electron microscope (SEM). Argyrophilic reticular fibers surrounded haemal sinuses and lymphoid tubules. Together they formed the scaffold that supported the lymphoid tubule. Using vascular cast and SEM, the three dimensional structure of the subgastric artery that supplies each lobe of the lymphoid organ was reconstructed. This artery branched into highly convoluted and blind-ending terminal capillaries, each forming the lumen of a lymphoid tubule around which haemocytes and other cells aggregated to form a cuff-like wall. Stromal cells which form part of the tubular scaffold were immunostained for vimentin. Examination of the whole-mounted lymphoid organ, immunostained for vimentin, by confocal microscopy exhibited the highly branching and convoluted lymphoid tubules matching the pattern of the vascular cast observed in SEM.

Transmissible spongiform encephalopathies (TSEs) or prion diseases often result in accumulation of disease-associated PrP (PrP(d)) in the lymphoreticular system (LRS), specifically in association with follicular dendritic cells (FDCs) and tingible body macrophages (TBMs) of secondary follicles. We studied the effects of sheep scrapie on lymphoid tissue in tonsils and lymph nodes by light and electron microscopy. FDCs of sheep were grouped according to morphology as immature, mature or regressing. Scrapie was associated with FDC dendrite hypertrophy and electron dense deposit or vesicles. PrP(d) was located using immunogold labelling at the plasmalemma of FDC dendrites and, infrequently, mature B cells. Abnormal electron dense deposits surrounding FDC dendrites were identified as immunoglobulins suggesting that excess immune complexes are retained and are indicative of an FDC dysfunction. Within scrapie-affected lymph nodes, macrophages outside the follicle and a proportion of germinal centre TBMs accumulated PrP(d) within endosomes and lysosomes. In addition, TBMs showed PrP(d) in association with the cell membrane, non-coated pits and vesicles, and also with discrete, large and random endoplasmic reticulum networks, which co-localised with ubiquitin. These observations suggest that PrP(d) is internalised via the caveolin-mediated pathway, and causes an abnormal disease-related alteration in endoplasmic reticulum structure. In contrast to current dogma, this study shows that sheep scrapie is associated with cytopathology of germinal centres, which we attribute to abnormal antigen complex trapping by FDCs and abnormal endocytic events in TBMs. The nature of the sub-cellular changes in FDCs and TBMs differs from those of scrapie infected neurones and glial cells suggesting that different PrP(d)/cell membrane interactions occur in different cell types.

The present study was carried out to analyse the vascularization of the pigeon bursa cloacalis of Fabricius and to determine whether it undergoes age-dependent changes during its functionally most important growth period after hatching of the pigeon. Morphological assessment of vascular corrosion casts, studied qualitatively and quantitatively, was applied for the first time to investigate the vascularization of the pigeon pigeon bursa of Fabricius. This also allowed us to analyse the microvasculature and morphological aspects of the vessel interrelationships as occurring in the natural state. The casts were compared with histological sections stained by haematoxylin-eosin and by binding of the lectin e-PHA (Phaseolus vulgaris, erythroagglutinin) to blood vessels. The vascular architecture of the bursa of Fabricius of the pigeon revealed that the organ is irrigated via two pathways, first through the terminal capillary system of lymphoid follicles arising from the internal pudendal artery, and secondly through arteries originating from the cloacal vasculature of the collum of the organ supplying the periluminal capillary system of the pigeon bursa of Fabricius. Both systems are drained by a venous system which is collateral to the system of the internal pudendal artery and clearly functions as a direct link between the lumen and vasculature of the cloaca or gut, respectively, and the bursa fabricii. This could allow the lymphocytes to be confronted with antigens from the contents of the gut, and their subsequent transport into the secondary lymphoid organs of the organism. Our results demonstrate that the blood vessels, as major and supplying part of the lymphoid system of the bursa Fabricii, clearly reflect three different phases of development: the evolution phase from about day 20 until day 50 post-hatching, the mature phase from days 50 to 90, and the involution phase after day 90. During the evolution phase the density of the vessel system rapidly increases, while in the mature phase the vascular architecture is maintained. The involution phase is dominated by vascular degeneration combined with shrinkage of the whole organ. Therefore, the morphology of the vasculature distinctly reflects the functional status of this primary lymphoid organ during its lifespan.

The pyloric tonsil is a novel peripheral lymphoepithelial organ of the gastrointestinal tract in the chicken. It forms a complete lymphoid ring at the beginning of the duodenum, where crypts of Lieberkühn are transformed to tonsillar crypts with lymphoepithelial lining. The oesophageal (described previously) and pyloric tonsils are characteristic of the chicken, while they are absent in mammals. The lymphoid system develops from the middle germ layer, the mesoderm, and forms connections with the ecto- and endoderm, namely the skin and gut, respectively. These connections are based on the lymphoepithelial lining of the crypts, and provide gates for environmental antigens. Recent findings, taken together with the literature, suggest that in birds the lymphoid system forms connections with the endoderm-derived organs that are anatomically and histologically more extensive than the ectoderm-derived ones, which may be explained by the absence of regional lymph nodes, and the less developed lymphoid circulation of the skin.

Fluorescent in situ hybridization (FISH) analysis is a molecular technique allowing the detection of recurrent translocations in cancer. Several hybridization protocols were assayed in order to evaluate their performances for interphase FISH analysis of histological sections and imprints using split probes. Adult and foetal lymphoid tissues were selected. Touch imprints of fresh (EF) or frozen (EC) tissues, sections (CF) and isolated nuclei (NI) of formol-fixed paraffin-embedded tissues were performed. The cut-off values of the IGH, IGlambda, BCL-2, BCL-6, CCND1 and MYC DNA FISH split signal probes were calculated for adult reactive lymph nodes on the different histological preparations (EC, CF, CC, NI) and on several tissues for the IGH and BCL-6 probes. In reactive lymph nodes, the cut-off values of the probes were between 3 and 13% and found independent of the preparation type. Conversely, slight but significant variations of the cut-off level were observed when different foetal control tissues were assayed with the same probe set. Finally, this study provided optimized-protocols for FISH analysis of either fresh/frozen imprints or formalin-fixed paraffin-embedded sections using split signal DNA probes.

Two bacterial strains Lactobacillus fructivorans (AS17B), isolated from adult seabream (Sparus aurata L.) gut, and Lactobacillus plantarum (906), isolated from human faeces, were administered contemporaneously during seabream development using Brachionus plicatilis and/or Artemia salina and dry feed as vectors. Experimental group A received the probiotic strains already via rotifers from day 5 post-hatch (ph), whereas treatment of group B began with Artemia feeding from day 27 ph. Fish were sampled at day 28 ph (group A and control) and day 99 ph (groups A, B and control) for electron microscopy, histology and immunohistochemistry with the polyclonal antiserum ORa against homologous serum Ig and the mAb G7 specific for seabream acidophilic granulocytes. In all groups, timing and pattern of differentiation of the digestive tract did not differ. Furthermore, neither tissue damage nor manifest inflammation was provoked by probiotic administration. At day 28 ph, the developing GALT already housed mucosal leucocytes, including Ig(+) cells but no acidophilic granulocytes. No differences were seen between experimental groups. At day 99 ph, the density of Ig(+) cells (+51%) and acidophilic granulocytes (+284%) was significantly higher (p<0.05) in group A than in controls. Also group B had a higher density of Ig(+) cells (+17%) and acidophilic granulocytes (+130%) compared with controls, although less pronounced. Light and electron microscopy observations detailed the occurrence of heterogeneous populations of lymphocytes and granulocytes in the developing intestinal mucosa, and highlighted the net expansion of G7(+) acidophilic granulocytes (A +536%, B +292% vs. control) due to probiotic administration. Evidence is provided that early feeding with probiotic-supplemented diet increased the number of Ig(+) cells and acidophilic granulocytes in seabream gut and that the effects were more pronounced when administration started during gut metamorphosis. These results point to a stimulatory effect of probiotics on the gut immune system that correlates with improvement of fry survival.

Changes in the structure and cellular composition of aggregated lymphoid nodules (ALN, Peyer's patches) of the small intestine and in mesenterial lymph nodes (MLN) were studied in 82 female Wistar rats 7, 14 and 21 days following a single intraperitoneal injection of a complex of antineoplastic drugs (cyclophosphamide, adriamycine, vincristine and prednisolone). In ALN and MLN lymphoid nodules the administration of antineoplastic drugs resulted in the decrease of the number of blast cells and mitotically dividing cells, suggesting the suppression of B-cell proliferation and differentiation. At the same time, the dimensions of germinal centers in ALN lymphoid nodules were reduced, while their mantle zone size remained unchanged. On the contrary, in MLN lymphoid nodules, mantle zone was reduced, while germinal center dimensions remained constant. Possible reasons of these differences in responses of the immune organs studied to chemotherapy, are discussed.

The M cells of nasopharyngeal lymphoid tissue (NALT) have been considered to play an important role for vaccine delivery systems in humans. A number of investigations have reported particle uptake data in NALT of rodents. However, there have been no reports indicating any involvement of the nasopharyngeal lymphoid tissue in human vaccination. In the present study, we investigated whether the epithelium of human adenoid tissues might incorporate fluorescent microparticles using electron and fluorescent microscopy. The dissected adenoid tissues were incubated with various sizes and concentrations of fluorescent microparticles for 120 min at 37 degrees C. Furthermore, the effect of surface coatings of microparticles with cations on the uptake into the epithelium of adenoid tissues was investigated. Transmission electron microscopy revealed that microparticles were taken up by the M cells of human nasopharyngeal lymphoid tissues. The NALT-M cells showed greater uptake of the smallest particles, 0.2 microm in diameter, than those of 0.5, 1.0, or 2.0 microm diameter. It was also revealed that surface coatings with poly-L: -lysin or chitosan resulted in efficient uptake into the NALT. These results indicate that nasal administration of antigenic microparticles, which were coated with cationic materials, probably leads to a useful method of transnasal vaccination against respiratory and intestinal infections in humans.

In order to define the histological components of ocular defense, the conjunctiva in Japanese monkeys was studied using a whole mount method, light microscopy, and electron microscopy. We investigated the distribution of the conjunctiva-associated lymphoid tissue (CALT) using stereoscopic observations of the conjunctiva immunostained with human leukocyte antigen (HLA)-DR antibody and /or stained with alcian-blue. The outer surface of the conjunctival fornix was lined by sheets of mucus secreting goblet cells, with small epithelial patches without goblet cells, scattered among them. The patches, termed CALT, consisted of flattened epithelial cells, intraepithelial lymphocytes and dendritic cells, and lymphoid follicles with a germinal center. The CALT in Japanese monkeys was fundamentally similar in structure to those found in other animal species. CALT patches ranged in size ranging from 200 microm to 300 microm in diameter. The number of patches varied from 20 to 40 in the superior eyelid and 10 to 20 in the inferior eyelid. Latex microspheres administrated as eye drops were selectively taken up first by flattened associated epithelial cells covering the surfaces of CALT patches and then by intraepithelial dendritic cells of the CALT. These morphological findings show that CALT patches in the eyelids of primates are focal sites for particulate uptake and contact with lymphoid constituents, indicating that they are inductive sites for the common mucosal immune system as well as important components in ocular defense.

The gastrointestinal tract is a complex and dynamic ecosystem. Commensal microorganisms (C), which proliferate in the intestine from birth, are crucial for gut homeostasis while non commensal (NC) microorganisms are transient and enter the organism from the environment and foods. We studied comparatively the influence of oral administration of C and NC Lactobacillus fermentum and Lactobacilus acidophilus on the gut-associated lymphoid tissue (GALT) of conventional mice. To determine the importance of the selection of probiotic host-specificity bacteria with immunomodulating capacity, we examined the interaction with the gut by transmission electron microscopy and FITC-labelled bacteria. We compared the immunomodulation capacities of C and NC strains by studying the number of IgA secreting cells and cytokine profile. No differences were found in the number of IgA+ cells; however, the pattern of cytokine response to C and NC bacteria was different. With regard to proinflammatory cytokine (IFNgamma and TNFalpha), we found that TNFalpha was mainly produced by NC bacteria, while C bacteria were able to elicit mainly IFNgamma. The regulatory cytokines (IL-10 and IL-4) were induced with different patterns for both C and NC strains. No differences in the pathway of internalization to the gut between C and NC were found. In summary, we determined that C and NC bacteria interact with the intestine in the same way; both C and NC bacteria were able to reinforce the surveillance of the gut mucosal immune system. The cytokine profile showed that C bacteria would be involved in the regulation of intestinal homeostasis rather than in the immune activation as the NC bacteria.

Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications.

We generated a sequential reporter-enzyme luminescence (SRL) technology for in vivo detection of beta-galactosidase (beta-gal) activity. The substrate, a caged D-luciferin-galactoside conjugate, must first be cleaved by beta-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on beta-gal activity. Using this technology, constitutive beta-gal activity in engineered cells and inducible tissue-specific beta-gal expression in transgenic mice can now be visualized noninvasively over time. A substantial advantage of beta-gal as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike FLuc, which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant beta-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo. Thus, coupling the properties of FLuc to the advantages of beta-gal permits bioluminescent imaging applications that previously were not possible.

Purpose: This study investigates the presence of M cells in the rabbit conjunctiva. Resolving whether the conjunctiva contains M cells is important, because at other mucosal sites, these antigen sampling cells are known to initiate the mucosal immune response and to act as a site of entry for opportunistic pathogens., Methods: Fluorescent 0.2-microm polystyrene latex beads were either instilled into the conjunctival sac for 20 to 120 minutes in vivo or applied to flat mounts in vitro. Specimens were assessed by epi-fluorescence stereomicroscopy, widefield fluorescence microscopy, confocal scanning laser microscopy, and transmission and scanning electron microscopy., Results: Latex beads preferentially bound to a subpopulation of cells in the epithelium overlying mucosal lymphoid follicles in the fornix region. At 4 degrees C, the beads were associated with the apical surface of cells that had longer, more irregular microvilli than the surrounding epithelial cells. Within 20 minutes of an in vivo exposure, latex beads were internalized by the follicle-associated epithelial cells and with time moved into the underlying follicle region. After 120 minutes of in vivo exposure, latex beads could be found in cervical lymph nodes., Conclusions: This study demonstrates that the follicle-associated epithelium of the rabbit conjunctiva contains a cell with morphologic characteristics and the ability to bind and translocate latex beads, which make it indistinguishable from antigen sampling M cells in the rabbit cecum and tonsils. Consistent with its hypothesized antigen sampling role, beads that have been translocated by this cell are rapidly transferred to cervical lymph nodes.

Conjunctiva-associated lymphoid tissue (CALT) is a part of the eye-associated lymphoid tissue (EALT) at the ocular surface. Its lymphoid follicles are usually characterized by using light microscopy, but its ultrastructure remains largely unknown. In this study, flat whole-mount conjunctival tissues (n = 42) from 21 young adult rabbits were investigated native in reflected light, and further stained and cleared (n = 6), in paraffin histology sections (n = 6), scanning electron microscopy (SEM, n = 4) and transmission electron microscopy (TEM, n = 4). Secondary lymphoid follicles accumulated into a dense group nasally towards the lacrimal punctum of the lower lid. High endothelial venules (HEV) with typical ultrastructure occurred in the parafollicular zone. The bright germinal centre (GC) contained lymphoblasts, follicular dendritic cells, apoptotic cells and tingible body macrophages. The follicle-associated epithelium (FAE) was devoid of goblet cells and contained groups of lymphoid cells. TEM showed these cells to be located in cytoplasmic pockets of superficial electron-lucent cells with a thin cytoplasmic luminal lining that contained a fine filament meshwork and numerous endocytotic vesicles. These M-cells were sitting between and on top of the ordinary dense epithelial cells that were located basally and formed pillar-like structures. In stereoscopic SEM, the surface cells were very large, had a polygonal outline and covered cavernous spaces. The rabbit has a CALT with typical follicular morphology, including HEV for regulated lymphocyte migration and epithelial cells with ultrastructural characteristics of M-cells that allow antigen transport as indicated by the GC-reaction. The arrangement of these M-cells on top of and between epithelial pillar cells may reflect a special structural requirement of the multilayered CALT FAE.

The stratified squamous epithelium of the lingual tonsil of five young horses was infiltrated with CD4 and CD8 positive cells, which were very numerous in the crypt reticular epithelium along with macrophages and IgGb and IgA positive cells. Lymphoid follicles of the lamina propria mucosae consisted of a parafollicular area, corona and germinal centre. The parafollicular area was populated by large numbers of CD4 and CD8 positive lymphocytes as well as macrophages, inter-digitating cells, and a few B-lymphocytes. The germinal centre contained mainly IgGb and IgG(T) positive cells, plasma cells and small numbers of follicular dendritic cells, macrophages, CD4, CD8 and IgA positive cells. Some venules in the parafollicular and inter-follicular areas had features characteristic of high endothelial venules with irregular nuclei and cytoplasmic processes on the luminal surface. In addition to the normal cytoplasmic organelles, a novel vesiculo-vacuolar organelle was observed in small clusters toward the lateral, luminal and abluminal surfaces of these high endothelial venules. These vesiculo organelles along with their stomata and diaphragms, communicated with each other and with inter-endothelial clefts forming a structural basis for enhanced permeability and extravasation of macromolecules. The presence of lymphocytes in the high endothelial venules is consistent with transmural and inter-endothelial passage of these cells.

Antigen-sampling M cells are found in the follicle-associated epithelium above organized lymphoid tissue in many mucosae. They play a key role in initiating the mucosal immune response and act as a site of entry for opportunistic pathogens. This study investigates the presence of M cells in the Guinea pig conjunctiva. Maackia amurensis leukoagglutinin I and II (MAL-I and MAL-II) were identified as potential conjunctival M cell markers based on a screening of 12 lectins and 5 carbohydrate epitope antibodies on aldehyde-fixed follicles. Biotinylated or fluorescein-conjugated MAL-I was then instilled into conjunctival sacs in vivo for 15-60 min. Specimens were assessed by epi-fluorescence stereomicroscopy, confocal scanning laser microscopy and transmission and scanning electron microscopy (TEM and SEM). Selective labelling of a subset of epithelial cells overlying lymphoid follicles was observed following in vivo exposure to MAL-I. MAL-I labelling was restricted to cells with sparse, irregular microvilli. Cells preferentially labelled with MAL-I were found to internalize the lectin during a 60 min in vivo exposure. MAL-I was transcytosed to basolateral membranes of cells filled with intracellular vesicles during a 45 min in vivo incubation. This study demonstrates that the Guinea pig conjunctiva contains a cell with morphological and functional characteristics of antigen-sampling M cells.

Haemato- biochemical and immuno-pathophysiological changes following feeding of broiler chicks with 20 ppm fenvalerate (synthetic pyrethroid, SP), 2 ppm monocrotophos (organophosphate, OP) and 2 ppm endosulfan (chlorinated hydrocarbon, CH) were studied. Four groups of broiler birds (30 each) were fed poultry mash without (control) or mixed with pesticides for 8 weeks. Blood glucose, serum globulin and acetyl cholinesterase (AChE) activity level were decreased (P<0.01) in all treated groups compared to control, but not the serum albumin and BUN. The total ATPase activity was enhanced (P<0.01) in fenvalerate and monocrotophos than birds in control group. Body weight, total erythrocyte count, packed cell volume, haemoglobin, eosinophil and monocyte count did not show any changes. Total leucocytes and T-lymphocyte count was lower (P<0.01) in all treated groups as compared to control group. B-cell count (P<0.01), mean 2-4-dinitrofluorobenzene (DNFB) dermal sensitivity score and splenic indices from graft vs. host reaction (P<0.05) were decreased in fenvalarate and endosulfan but the values for monocrotophos were intermediate between control and other treated groups. Pesticide intoxication reduced nitroblue tetrazolium (NBT) positive cells (active splenic macrophages) (P<0.05) and spleen weight (P<0.01). Whereas bursal weight was reduced only with endosulfan, thymic weight was reduced on endosulfan and fenvalerate-treated feed. Microscopic examination of these organs further revealed atrophy/hypoplasia, decrease in the size of follicles with depletion of lymphocytes and haemorrhages in thymus. The study concludes that the chronic exposure of chicks to small amount of SP, OP and CH pesticide leads to deleterious effects on metabolism and immune system of birds.

The immunosuppressive properties of morbilliviruses including measles and canine distemper virus (CDV) are well known, but the host cells supporting infection are poorly characterized. To identify these cells, a recombinant CDV expressing green fluorescent protein was produced by reverse genetics based on a wild-type strain lethal for ferrets. This recombinant virus fully retained virulence and blazed three lymphocyte-based pathways through the immune system of its host: first, it infected rapidly and massively circulating B and T cells; second, it took over and damaged secondary lymphatic organs including spleen, lymph nodes, and gut-associated and mucosal lymphoid tissues; third, it infected most thymocytes. In contrast, replication in epithelial cells was initially not detectable, but substantial before host death. Thus, CDV initially infects lymphocytes and massively replicates therein, thereby causing immunosuppression and preparing systemic invasion and host escape.

This study documents the characteristics of a large series of spontaneously occurring thymomas in a laboratory colony of European hamsters (Cricetus cricetus). Thymomas are rare organotypic neoplasms originating from the thymic epithelial compartment. Because the hamster thymomas largely resembled their human counterparts, the recent World Health Organization (WHO) classification of human thymic epithelial tumors was used. Forty hamsters of both sexes aged 3-29 months were examined macroscopically and histologically. In 22 (55%) of the 40 animals, necropsy revealed enormous whitish masses in the anterior mediastinum, with a diameter ranging from 0.5 to 4.5 cm and a lobulated structure. The anatomy of the thymus region was normal in the remaining 18 hamsters. Histologically, the tumors presented as thymuslike organoid structures with areas of medullary and cortical differentiation and a predominance of lymphoid cells. A network of epithelial cells in the cortical areas, demonstrated immunohistochemically with a cross-reactive antibody against pancytokeratin, supported the diagnosis of thymoma. Cortical lymphocytes showed positive staining with cross-reacting antibodies against CD3 and terminal deoxynucleotidyl transferase, characteristic of immature T cells. On the basis of these findings, the tumors were classified as B1 thymomas, in some cases with AB or B2 components, according to the new WHO classification for human thymic epithelial tumors.

Rabbit appendix consists mainly of lymphoid follicles (LF) covered by M cells, the specialized antigen-sampling cells of the mucosal immune system, and surrounded by glandular epithelium. Until now, these M cells have been characterized morphologically and histologically by using cellular markers. Here, the adhesion and transport of pathogenic bacteria were investigated to assess the function of M cells of the appendix. We used the enteroinvasive motile Salmonella typhimurium and the rabbit enteropathogenic non-motile Escherichia coli RDEC-1, which are known to target specifically rabbit M cells of Peyer's patches (PPs). We found that S. typhimurium efficiently attached and was transported through appendix M cells in vivo. In contrast to S. typhimurium, RDEC-1 targeted M cells only ex vivo, when bacteria were allowed to have direct contact with the surface of the follicle. The difference in interaction of the two bacteria with appendix M cells led us to investigate whether this could be correlated with the lack of motility of RDEC-1. We used an aflagellate mutant of S. typhimurium and found that it had the same infection phenotype as RDEC-1. Gene complementation restored the efficiency of infection to that of S. typhimurium wild-type strain. In conclusion, we show that M cells of the appendix display features of the canonical M cells of PP, since they efficiently sample luminal pathogenic bacteria. However, due to the morphology of the appendix, motile bacteria appear to be more potent in their interactions with appendix M cells.

The objective of this study was twofold: first to investigate the ultrastructure of the lymphatic drainage unit on the costal pleura of rats by electron microscopy, and secondly to examine the effect of nitric oxide on the pleural lymphatic stomata and fluid absorption from the pleural cavity. The lymphatic drainage unit of the rat costal pleura is composed of three special components: the lymphatic stomata between the mesothelial cells, the initial part of the lymphatic vessels and the underlying connective tissue containing many foramina. The unit is the main passage to drainage fluid, particles and cells in the pleural space. To investigate the regulator of the lymph drainage, nitric oxide synthase inhibitor and nitric oxide donor were injected into the peritoneal cavity of the rats, respectively. Trypan blue was used as tracer. The ultrastructural changes of pleural lymphatic stomata were observed under scanning electron microscope and analyzed by a computer image processing system. It turned out that the area and density of the pleural lymphatic stomata were positively correlated with the nitric oxide quantity (p < 0.05). After the tracer was injected into the pleural cavity, the nitric oxide donor group exhibited a higher trypan blue concentration than the control group (p < 0.05). The ability of the pleura to absorb trypan blue was enhanced because of the larger opening of the lymphatic stomata (p < 0.05). It is suggested that nitric oxide can increase lymphatic absorption of the pleura by opening pleural lymphatic stomata.

Little is known about the role of the M cells of human nasopharyngeal lymphoid tissue in the sampling of viruses that cause respiratory infections. To clarify whether M cells could function as a gateway for influenza virus into human nasopharyngeal lymphoid tissue, excised adenoid tissue was incubated in media containing influenza A virus for 30, 60, and 90 min, respectively. Transmission electron microscopic observation revealed that many influenza viruses adhered to M cell surfaces and were taken up into the cytoplasmic vesicles of M cells after 30 min incubation; the viruses had been transported into enfolded lymphoid cells after 60 min incubation. By staining M cells with Sambucus nigra lectin, which specifically recognizes the NeuAcalpha2,6 Gal linkage of sialoprotein, it was also found that abundant receptors for the human influenza virus are present on the M cell surface. Our findings indicated that M cells of human nasopharyngeal tonsils function as a major port for influenza A virus entry and that the virus could be efficiently transferred to enfolded macrophages and lymphoid cells by M cells. The transport of influenza viruses to lymphoid cells by M cells may promote antigen delivery to the immune system, and these findings may be important for systemic delivery of those influenza viruses that have the capacity to productively infect cells outside of the respiratory tract.

We studied the relationship between insulitic development and function-structural changes of pancreatic lymphatics in non-obese diabetic (NOD) mice using combined 5'-nucleotidase (5'-Nase) enzyme histochemical and secondary lymphoid tissue chemokine (SLC/CCL21) immunohistochemical methods. Interlobular lymphatic vessels were positive for 5'-Nase throughout the pancreas, and dependent on both blood vessels and pancreatic ducts. Intralobular initial lymphatics were rare and occasionally ran in the neighbourhood of islets. During the non-insulitic stage, the 5'-Nase-reactive product was evenly distributed on the surface of lymphatic endothelial cells (LECs) with weak expression of CCL21. The activity of 5'-Nase on lymphatic vessels became slightly reduced as insulitis developed. The increasing blood glucose values appeared to be consistent with an increasing CCL21 expression by the endothelial lining, especially on the surface of LECs adjacent to the infiltrated islets and tissues. Lymphocytes and dendritic cells (DCs) were frequently located in the connective tissue, surrounding the lymphatic wall with deposition of 5'-Nase precipitates. As the infiltration became severe, lymphocytes and DCs accumulated within lymphatic vessels and expressed high levels of CCL21. The most significant finding was that many DCs adhered to lymphatic vessels, transmigrating via the thin and indented endothelial walls. The activity of 5'-Nase was increased on the adhesion surface between DCs (or lymphocytes) and LECs. The latter were characterized by open intercellular junctions and obvious cytoplasmic protrusions. These results suggest that LECs closely interact with DCs and lymphocytes, and play a key role in the migration of DCs and lymphocytes via lymphatic vessels during the pathological processes of insulitis in NOD mice.

Introduction: Components of the mucosal immune system (MALT) have been identified in the conjunctiva (as CALT) and the lacrimal drainage system (as LDALT). Their structural and functional relation with the established immune protection by the lacrimal gland is unclear., Material and Methods: Macroscopically normal and complete tissues of the conjunctiva, lacrimal drainage system and lacrimal gland from human body donors were investigated by analysis of translucent whole mounts, and using histology, immunohistology as well as scanning and transmission electron microscopy., Results: A typical diffuse lymphoid tissue, composed of effector cells of the immune system (T-lymphocytes and IgA producing plasma cells) under an epithelium that contains the IgA transporter SC, is not isolated in the conjunctiva and lacrimal drainage system. It is anatomically continuous from the lacrimal gland along its excretory ducts into the conjunctiva and from there via the lacrimal canaliculi into the lacrimal drainage system. Lymphoid follicles occur in a majority (about 60%) and with bilateral symmetry. The topography of CALT corresponds to the position of the cornea in the closed eye., Conclusion: These results show that the MALT of the lacrimal gland, conjunctiva and lacrimal drainage system constitute an anatomical and functional unit for immune protection of the ocular surface. Therefore it should be integrated as an "eye-associated lymphoid tissue" (EALT) into the MALT system of the body. EALT can detect ocular surface antigens by the lymphoid follicles and can supply other organs and the ocular surface including the lacrimal gland with specific effector cells via the regulated recirculation of lymphoid cells.

In this review, we consider the morphological aspects and topographical arrangement of gut-associated lymphoid tissue (GALT) (solitary and aggregate lymph nodules or Peyer's patches) and of vermiform appendix in the human child and in some mammals. The spatial arrangement of the vessels belonging to apparatus lymphaticus periphericus absorbens (ALPA) and of blood vessels within each lymphoid follicle as well as the ultrastructural characteristics of the lymphatic endothelium with high absorption capacity are considered. Particular attention is also paid to the morphological and biomolecular mechanisms inducing lymphocyte transendothelial migration to the bloodstream by means of lymphatic vessels as well as their passage from blood into lymphoid tissue through the high endothelial venules (HEVs). The preferential transendothelial passage of lymphocytes and polymorphonuclear neutrophils within ALPA vessels of the interfollicular area does not occur following the opening of intercellular contacts, but rather it occurs by means of 'intraendothelial channels'. In HEVs, on the contrary, the hypothesis is plausible that lymphocyte transendothelial migration into lymphoid tissue occurs through a channel-shaped endothelial invagination entirely independent of interendothelial contacts. The lymph of ALPA vessels of the single Peyer's patch is conveyed into precollector lymphatic vessels and into prelymph nodal collectors, totally independent of the ALPA vessels of the gut segments devoid of lymphoid tissue. The quantitative distribution of T lymphocytes in the lymph of mucosal ALPA vessels suggests a prevalent function of fluid uptake, whereas a reservoir and supply function is implicated for the vessels of interfollicular area. The precollector lymphatic vessels and prelymph nodal collectors are considered to be vessels with low absorption capacity, whose main function is lymph conduction and flow.

Mortalities of cultured shrimp, Penaeus vannamei (Boone), induced by white spot syndrome virus (WSSV) have occurred in Ecuador since May 1999. Three epidemiological surveys in Ecuadorian farms were carried out and showed an apparent association between lower temperature and increased mortality rates in commercial ponds. Infected animals showed a reddish discolouration and lethargy and occasionally, white spots in the exoskeleton. Histopathological studies revealed that infected cells presented nuclear hypertrophy with eosinophilic to basophilic inclusions. In some cases, two other pathologies were observed: (a) lymphoid organ spheroids and (b) cells with pyknotic and karyorrhectic nuclei in the lymphoid organ, haematopoietic tissue, connective tissue, heart and antennal gland. Occasionally pyknotic cells were encapsulated without apparent injury to the adjacent tissue and without melanization. Transmission electron microscopy showed the presence of WSSV particles in the cytoplasm of cells with pyknotic nuclei in the stomach hypodermis. Viral structure and morphogenesis agreed with previous descriptions by other authors in WSSV-infected shrimp. Occasionally, two nucleocapsids within one envelope were present amongst single enveloped nucleocapsids. A long rod-shaped structure that could reach 2.4 microm in length was present in the nuclei of some infected cells.

Armadillos are ancient mammals important as models for biomedical, evolutive and ecological studies, because they have adaptive and primitive morpho-physiological characteristics. In this work we study the splenic microarchitecture of the 'Patagonian Piche', Zaedyus pichiy, as an attempt to understand the relationship between the organizational plan of the organ and the particular features of this species. The organ samples were classically processed for light and electron microscopic study. The microanatomy of the organ as well as its different cell types are studied. The spleen is non-sinusoidal, with the typical arrangement for storage functions. White pulp is well defined. Red pulp is a meshwork of circulating, immunocompetent and haemopoietic cells. The general structure of the organ agrees with the semi-fossorial habit and the adaptability of the species. Comparative aspects with other armadillos or other less specialized groups are discussed. Persistence of haemopoietic spleen activity in the adult suggests the existence of specific inductive functions of the stroma. Better knowledge of spleen structure and function in ancient mammals may give important information about their phylogeny.