VIP and tolerance induction in autoimmunity.

Vasoactive intestinal peptide (VIP) is a potent anti-inflammatory agent with immunoregulatory properties, skewing the immune response to a Th2 pattern of cytokine production. Here, we studied the effect of treatment with VIP in the development of diabetes in nonobese diabetic (NOD) mice, an animal model of type 1 diabetes. Mice treated with VIP from 4 weeks of age did not develop diabetes and showed milder insulitis than nontreated mice. The protective mechanism of VIP was associated with a reduction in the circulating levels of Th1 cytokines. In the pancreas of VIP-treated animals, regulatory T cell markers predominate, as indicated by the upregulation of FoxP3 and transforming growth factor-beta (TGF-beta), and the downregulation of the transcription factor, T-bet. These findings indicate that VIP restores tolerance to pancreatic islets by promoting the local differentiation and function of regulatory T cells.

Vasoactive intestinal peptide modulates proinflammatory mediator synthesis in osteoarthritic and rheumatoid synovial cells.

OBJECTIVE: Vasoactive intestinal peptide (VIP) has demonstrated beneficial effects in several murine models of immune-mediated inflammation by inhibiting both the inflammatory and the autoimmune components of the disease. We investigate its potential to modulate the release of proinflammatory cytokines and chemokines by human synovial cells from patients with rheumatoid arthritis (RA). METHODS: Fresh suspensions of synovial tissue cells (STC) or cultured fibroblast-like synoviocytes (FLS) were obtained from patients with RA or osteoarthritis (OA). The effects of VIP on basal or tumour necrosis factor alpha (TNF-alpha)-stimulated production of CCL2 (MCP-1, monocyte chemotactic protein 1), CXCL8 [interleukin (IL)-8], IL-6 and TNF-alpha were studied by specific ELISAs (enzyme-linked immunosorbent assays). The mRNAs for CCL2, CXCL8 and IL-6 in FLS were analysed by real-time reverse transcription-polymerase chain reaction. RESULTS: VIP at 10 nm down-regulated chemokine production by STC and FLS from RA and OA patients. VIP also down-regulated the expression of mRNAs for CCL2, CXCL8 and IL-6. The effects of VIP were more clearly detected in RA samples and after stimulation with TNF-alpha. CONCLUSION: Our observations confirm that the proposed anti-inflammatory actions of VIP in murine models also apply to human synovial cells ex vivo. Further studies are encouraged to evaluate the use of VIP as a potential therapy for chronic inflammatory joint diseases.

Optimization by experimental design of polyacrylamide gel composition as support for enzyme immobilization by entrapment.

We have developed a methodology based on experimental design, to optimize a polyacrylamide gel as the support for enzyme immobilization, taking advantage of all the properties which this type of gel has. Monomer and crosslinking agent proportions are responsible for both the porous structure and pore size of the gel. A correct selection of those variables and suitable synthesis conditions leads to an increase in the activity retained by the gel. The path of steepest ascent method was used to obtain the relative maximum activity. The maximum retained activity was chosen with a central composite design in terms of the gel composition. The retained activity in the network, loss activity in the wash water, and loss activity due to steric impediment or blockage was modeled in terms of the variables responsible for the gel structure. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 497-506, 1997.

Immunity in fish larvae.

The route of immunization, dose and nature of antigens are relevant for induction of specific immune responses but they are particularly influenced by the state of immunomaturation of fish. It is difficult, however, to determine when the fish immune system has matured and, accordingly, when the fry can be successfully vaccinated. To establish some general parameters which can correlate the development of protective immunity with maturation of the immune system we review: (i) the appearance of first lympho-haemopoietic cell precursors; (ii) the histological and functional maturation of lymphoid organs; (iii) the relevance of phagocytosis and transport of maternal Ig to eggs for early vaccination; (iv) the appearance of both T-like and B-like cells and their correlation with the development of the cell-mediated and humoral immune responses and (v) the mechanisms which may be involved in the induction of tolerance following early immunization of fry. These data rather than a general rule for vaccination of larval fish indicate that the earliest age (size) that a fish can be vaccinated differs between species and vaccines.

Increased numbers of CD5+ B cells in the thymus of estradiol benzoate-treated rats.

In the present work we combine both flow cytometry and in situ immunohistochemical techniques to study the changes affecting a minor B cell population described within the normal rat thymus, after treatment with estradiol benzoate (EB). Our results, in agreement with previous data, show that the vast majority of these intrathymic B cells are CD5+. The existence of CD5+ B cells was confirmed flow cytometrically in both cervical lymph nodes and spleen of control, adult Wistar rats. Moreover, after EB administration intrathymic B cells increased significantly especially in those rats receiving 500 micrograms of EB, constituting cell masses around the blood vessels of cortico-medullary area and in the thymic medulla. We discuss the significance of this increased number of intrathymic CD5+ B cells, which is probably due to a selective cell migration from the periphery into the thymus, from the view of the effects of estradiol on the thymic vascular permeability.

Macrophage-lymphocyte cell clusters in the hypothalamic ventricle of some elasmobranch fish: ultrastructural analysis and possible functional significance.

BACKGROUND: Previous studies have demonstrated the existence of lympho-haemopoietic tissue in the meninges and choroid plexuses of various primitive vertebrates, including the stingray Dasyatis akajei and in early human embryos. In the present study, we extend these results analyzing macrophage-lymphocyte cell clusters found in the floor of the hypothalamic ventricle of several specimens of elasmobranchs. METHODS: After aseptical isolation of the brain from several specimens of smooth dogfish Triakis scyllia, cloudy dogfish Scyliorhinus torazame, gummy shark Mustelus manazo, and stingray Dasyatis akajei their hypothalamic regions were processed routinely by light, scanning, and transmission electron microscopy. RESULTS: The study of serial histological sections demonstrated that the macrophage-lymphocyte cell clusters proceeded from the meningeal lymphohaemopoietic tissue, reaching the ventricular lumen along large blood vessels. In this tissue, macrophages, different sized lymphocytes, lymphoblasts, granulocytes, monocytes, and developing and mature plasma cells were closely packed among a meshwork of fibroblastic reticular cell processes. It never invaded the brain parenchyma. A cell layer of glial elements and a continuous basement membrane interposed between the lymphoid tissue and the neural elements although some macrophages had migrated across the ependymal cell layer. In the ventricular lumen very irregular macrophages with long cell processes and containing abundant engulfed material of unknown origin formed big cell clusters with neighboring lymphocytes, lymphoblasts, and plasma cells, similar to those described during the immune response. Moreover, electron lucent cells which resembled the antigen-presenting cells of higher vertebrates established intimate surface cell contacts with the surrounding lymphocytes. In the third ventricle of several specimens of gummy shark, Mustelus manazo, morphologically similar cell clusters appeared but these were not connected to the meningeal lympho-haemopoietic tissue. No intraventricular cell aggregates were found in the stingray brain. CONCLUSIONS: Although we cannot rule out that these macrophage-lymphocyte cell clusters represent a permanent structure in the elasmobranch brain they rather seem to be only established after specific stimulation for preventing the entrance of noxious, foreign materials into the elasmobranch brain parenchyma.

The relevance of cell microenvironments for the appearance of lympho-haemopoietic tissues in primitive vertebrates.

In higher vertebrates, mainly in mammals, a role for the non-lymphoid components of lymphoid organs in governing the maturation and functioning of immune system has been largely demonstrated. In contrast, such a role in the evolution of the vertebrate immune system has only been evidenced indirectly. In the present review we summarize histophysiological results which emphasize the relevance of lympho-haemopoietic stromal elements in the emergence and evolution of vertebrate lymphoid organs. The most primitive vertebrates, the Agnatha, have no true lymphoid organs and, accordingly, their immune responses seem more related to the non-anticipatory defence mechanisms of invertebrates than to the immune responses of vertebrates. So, the appearance and evolution of vertebrate lymphoid organs seems closely related with the emergence of immune capacities. Thymus, spleen and gut-associated lymphoid organs appear early in phylogeny whereas lymph nodes and bone marrow are late phylogenetical adquisitions. However, bone marrowless vertebrates contain numerous organs (i.e., gonads, kidney, brain, etc...), the cell microenvironments of which support lympho-haemopoiesis mimicking the condition of higher vertebrate bone marrow. On the other hand, the lack of germinal centres, another feature of the lymphoid organs of ectothermic vertebrates which impedes the selection of B cells raised after somatic hypermutation, presumably reflects the absence of some of the elements necessary for this organization.

In vitro antigen trapping by gill cells of the rainbow trout: an immunohistochemical study.

An in vitro assay was used to study the involvement of gill cells in the trapping and processing of particulate antigens. Gills were routinely processed for light microscopy after being placed in medium containing either Yersinia ruckeri O-antigen-labelled fluorescent beads, unlabelled fluorescent beads, Y, ruckeri O-antigen or formalin-killed Y. ruckeri, for 0, 30 s, 1, 5 and 30 min. Y. ruckeri formalin-killed cells, Y. ruckeri O-antigen and fluorescent beads labelled with Y. ruckeri O-antigen were taken in by gill epithelial cells as soon as 30 s after administration. In contrast, unlabelled fluorescent beads adhered to the epithelial cell membranes, but did not occur inside the gill cells. These results are discussed principally in relationship with the specificity of antigen trapping.

Seasonal variations in the immune system of lower vertebrates.

Seasonal variation, affecting the structure and function of the ectotherm immune system, is an excellent 'natural' model of the influence of neuroendocrine rhythms on immunity. In this review, Agustín Zapata, Alberto Varas and Marta Torroba examine the correlations between seasonal changes and circulating steroid levels, and investigate the possibility of a neuroendocrine-immune network in lower vertebrates.

Changes in the thymus and spleen of the turtle Mauremys caspica after testosterone injection: a morphometric study.

To confirm a possible role of sex hormones in governing the seasonal variations affecting the reptilian lymphoid organs, a morphometric analysis was carried out on the thymus and spleen of turtles, Mauremys caspica, intraperitoneally injected with a single dose of testosterone propionate (TP) at the third week of June when physiological levels of testosterone are low. At 4 and 6 weeks, control turtles show an apparent lymphocyte mobilization both from thymus and spleen with a decrease in the percentage of thymic cortex, numbers of cortical lymphocytes, and mitotic index, but increased numbers of medullary lymphocytes. In the thymic cortex of treated turtles, there is a decrease in the same parameters but they occur in the first 2 weeks, whereas the medullary lymphocytes also undergo reduction at 4 and 6 weeks. In addition, the number of reticuloepithelial cells per area unit decreases at 2 weeks in the thymic cortex of treated turtles. These results are discussed from the point of view of a biphasic effect of testosterone on turtle lymphoid organs: In the first weeks, the changes observed could be attributed to the high levels of testosterone; after 4 and 6 weeks, variations are dependent both on long-term effects of testosterone and on those induced by the high values of corticosterone occurring in summer.

Testosterone induces lymphopenia in turtles.

Owing to the possible role of sex steroids in the immune-neuroendocrine interactions found in lower vertebrates, we attempted to delineate the effect of testosterone propionate on peripheral blood (PB) and the lymphoid organs of the turtle Mauremys caspica. A single intraperitoneal injection of 200 micrograms/g body weight produced thymic involution and intense lymphopenia in the spleen and, less severely, in the PB compartment. It is suggested that lymphocyte redistribution may occur among the various compartments of the body as the main effect of hormone-induced lymphocyte redistribution, although the mechanism in reptiles and mammals is not yet understood.

Fine structure and histochemistry of the ampullary organ of the urodele amphibian Pleurodeles.

A morphological study by light and electron microscopy on the lateral line system of the urodele amphibian Pleurodeles waltii demonstrates the presence of sensory organs other than neuromasts in the head. From their morphology, they have been called ampullary organs. The ampullary organs occur in the bottom of a groove and consist of three different types of cells: sensory, supporting and mantle cells. Histochemical analysis indicates that the last two are secretory cells, probably involved in the production of the material filling the ampulla and the groove.

Ultrastructural changes in the thymus of the turtle Mauremys caspica in relation to the seasonal cycle.

Changes in the ultrastructure of the thymus of the turtle Mauremys caspica, with special reference to its non-lymphoid components, were studied in relation to the seasonal cycle. The thymic cortex contains framework-forming epithelial-reticular cells and free macrophages, while the medulla includes, in addition, mature and presumptive pro-interdigitating cells. The ultrastructural features of these cells are generally similar to those described for non-lymphoid components of the mammalian thymus. The turtle thymus undergoes cortical involution in spring, with recovery periods in May-June and during autumn. A moderate involution occurs in winter. At the beginning of spring, cortical (but not medullary) epithelial-reticular cells show degenerative changes, probably related to high levels of circulating testosterone. In spring and autumn, mature interdigitating cells are absent, but macrophages, monocytes, and pro-interdigitating cells are found. During May-June, the cortical epithelial-reticular population recovers and macrophages, monocytes, and interdigitating cells are actively phagocytic. In summer, the epithelial-reticular cells in both cortex and medulla display normal ultrastructural features; mature and immature interdigitating cells are absent and some macrophages are detected occasionally. The results suggest that non-lymphoid components of the reptilian thymus can play a role in governing T-lymphocyte differentiation, and that the thymic cortex and medulla exhibit different cycles of seasonal activity.

Macrophages and epithelial cells of the thymus gland. An ultrastructural study in the natterjack, Bufo calamita.

In the present study, the ultrastructure of the stromal components, basically epithelial elements and macrophages, of the thymus of adult natterjacks, Bufo calamita has been analyzed. A network of stellate epithelial-reticular cells joined together by desmosomes, constitutes the main component of the thymic parenchyma in both cortex and medulla. In the medulla pale, electron-lucent epithelial cells, sometimes showing surface interdigitations, are striking elements. Moreover, uni- and multicellular epithelial cysts appear in the thymic medulla as well as granulated cells of possible endocrine significance. Remarkably, isolated or grouped gland cells whose morphology and cytoplasmic content resemble that of the skin glands, were occasionally found. Finally, macrophages, multinucleated giant cells and dendritic-like cells, the latter intimately associated to lymphocytes, occur in the thymus of Bufo calamita. The most remarkable morphologic characteristics of all those non-lymphoid cell types, as well as their possible functional significance are comparatively discussed with available information on the amphibian and higher vertebrate thymic cytoarchitecture.

Occurrence of lymphohaemopoietic tissue in the meninges of the stingray Dasyatis akajei (Elasmobranchii, chondricthyes).

The cytoarchitecture of the lymphohaemopoietic masses occurring in the "meninx primitiva" of the stingray Dasyatis akajei (Elasmobranchii, Chondricthyes) has been analyzed by light and scanning and transmission electron microscopy. Lymphohaemopoietic aggregates showing similar morphologies occurred along all the central nervous system, but they were more frequent in the telencephalon, diencephalon, and mesencephalon. In each aggregate, the granulopoietic tissue appeared in a fibroblastic stroma surrounding the large blood vessels, and the lymphoid components were present in a reticular network. Developing and mature eosinophils and heterophils--as well as lymphocytes, monocytes, macrophages, and plasma cells--are the main free cells present in these meningeal aggregates. The remarkable intimate association between macrophages and lymphoid cells to form close cell clusters suggests some immunological capacity for the meningeal lymphohaemopoietic tissue. According to their capacities, presence of lymphoid tissue, and histological organization, the meningeal lymphohemopoietic aggregates of Dasyatis akajei resemble other lymphomyeloid aggregates associated with cranium and choroid plexuses in Holocephali and Ganoidei. The phylogenetical relationships of these aggregates with mammalian bone marrow are discussed.