Reviewing the biological activity of chitosan in the mucosa: Focus on intestinal immunity.

Chitosan is a polymer derived from the partial deacetylation of chitin with particular characteristics, such as mucoadhesiveness, tolerability, biocompatibility and biodegradability. Biomedical uses of chitosan cover a wide spectrum of applications as dietary fiber, immunoadjuvant and regulator of the intestinal microbiota or delivery agent. Chemical modification of chitosan is feasible because its reactive amino and hydroxyl groups can be modified by a diverse array of ligands, functional groups and molecules. This gives rise to numerous derivatives that allow different formulation types influencing their activity. Considering the multiple events resulting from the interaction with mucosal tissues, chitosan is a singular candidate for strategies targeting immune stimulation (i.e., tolerance induction, vaccination). Its role as a prebiotic and probiotic carrier represents an effective option to manage intestinal dysbiosis. In the intestinal scenario where the exposure of the immune system to a wide variety of antigens is permanent, chitosan increases IgA levels and favors a tolerogenic environment, thus becoming a key ally for host homeostasis.

Lipase of Candida albicans induces activation of NADPH oxidase and L-arginine pathways on resting and activated macrophages.

Candida albicans secretes various hydrolytic enzymes which are considered to be an integral part in the pathogenesis. However, the role of lipases is far from being completely understood and the direct effects of these fungal enzymes during the host-pathogen interaction remain to be established. We recently isolated and characterized an extracellular C. albicans lipase (CaLIP), and demonstrated the ability of this fungal enzyme to interact directly with macrophages (Mvarphi) and hepatocytes and to operate as a virulence factor. Herein, we explored the effects of CaLIP on Mvarphi functions such as oxidative burst and l-arginine metabolism. The study was performed in cells with different activation status: normal-resting Mvarphis and Mvarphis primed in vivo or in vitro with C. albicans. The ability of this fungal factor to modulate the above-mentioned parameters was dependent on cells status, dose, and microenvironment, where the interaction took place. These results constitute a new finding in the biology of candidiasis and could illustrate an additional evolutive advantage for the fungus in the framework of the bidirectional host-pathogen interaction.

The biocompatible polysaccharide chitosan enhances the oral tolerance to type II collagen.

Chitosan is a mucoadhesive polysaccharide that promotes the transmucosal absorption of peptides and proteins. At mucosal sites chitosan exhibits immunomodulatory activities and stimulates the release of regulatory cytokines. Herein we evaluated the effect of the co-administration of chitosan in the tolerance to type II collagen (CII) using an experimental model of arthritis. Rats were fed diluent (acetic acid), 1 mg CII, 1 mg chitosan or 1 mg CII + 1 mg chitosan during 5 days before immunization with CII in Freund's complete adjuvant. Systemic effects were evaluated in draining lymph nodes after antigenic challenge or during the clinical evolution of arthritis. Specific antibodies, proliferation against CII and the production of interferon (IFN)-gamma and interleukin-10 were assessed. Clinical signs were observed 13-15 days after primary immunization. The CII : chitosan group presented the lowest incidence and developed moderate arthritis, with reduced levels of immunoglobulin (Ig)G2a anti-CII, a limited proliferation in draining lymph nodes and a lower release of IFN-gamma after restimulation with CII. Our results demonstrate that chitosan enhances the tolerance to an articular antigen with a decrease in the inflammatory responses and, as a consequence, an improvement in clinical signs.

What do we need to learn to optimize the SLIT alternative?

Allergen-specific immunotherapy (IT) is the only treatment of allergy in adults and children capable of modifying the immune response at early steps. As a consequence, IT improves symptoms, prevents the onset of new sensitizations, reduces the risk of developing asthma and its clinical efficacy lasts many years. The main rationale for administering sublingually IT (SLIT) is to reduce the occurrence of side effects, still yet preserving the immunological effects. SLIT with the most common allergens have been used in many studies with significant clinical effectiveness in both asthma and rhinitis. The pharmacokinetic of allergens administered through non injection routes is complex. Peptide absorption across oral mucosa occurs mainly by passive diffusion but delivery of proteins has some limitations. Moreover, the molecular mechanisms responsible for the efficacy of SLIT are poorly defined. In this review we focus on the anatomy/histology of the oral cavity as well as on the associated immunological structures to envisage what may happen when an allergen is kept in the mouth. Moreover, the induction of immune responses in this particular immunological environment is also discussed.

Adjuvant effect of liposomes in the autoimmune response to rat male accessory glands.

Wistar rats submitted to 3 intravenous (i.v.) or intraperitoneal (i.p.) immunizations with saline extract of rat male accessory glands (RAG) associated to liposomes developed a delayed type hypersensitivity (DTH) response to RAG after the first immunization, a remission state after the second immunization and a specific DTH response after the third injection. In a further study we transferred spleen mononuclear (SpM) cells from i.p. immunized rats taken 10 days after the second immunization (DTH negative) to normal or immunized recipients 24 h before or 10 days after the first immunization with RAG liposomes, respectively. The DTH response was reduced only in recipients previously immunized. Besides, it was possible to show that the transfer of SpM cells present when the response increased after the third injection in i.p. immunized donors reduced the suppression observed after the second injection. Rat accessory gland biopsies taken 10 days after the last immunization showed in nearly all cases mast cells, plasma cells and eosinophils with scarce lymphoid elements and increased acinar desquamation. This kind of infiltration had characteristics of cutaneous basophil hypersensitivity.

Effect of gangliosides in the autoimmune response induced by liposome-associated antigens.

A model of autoimmunity to rat male accessory glands (RAG) was recently developed by intraperitoneal administration of three doses of native RAG associated with liposomes. In this work we analysed the effects of gangliosides in the cellular response to RAG when they were intraperitoneally administrated prior to the second dose of liposome-associated RAG. Results show that the ganglioside treatment could modify an established DTH response. Also, gangliosides markedly reduced the number of Ia antigen-positive peritoneal exudated cells (PEC). However, they modified neither the processing of liposomes through PEC nor their viability. Moreover, we obtained cellular response by transferring PEC from immunized donors into naive receptors.

Modulation of the inflammatory response by corticotropin-releasing factor.

Peptides of the corticotropin-releasing factor (CRF) family have been shown to have either pro- or anti-inflammatory activities. CRF (10-30 micrograms/kg) administered subcutaneously or intravenously could inhibit edema and dye leakage in the rat paw produced by several injuries. These findings are opposed to some results suggesting a predominantly pro-inflammatory effect of CRF mainly in arthritic processes. The purpose of this work was to identify in vivo and in vitro the conditions for the pro- or anti-inflammatory actions of CRF in order to clarify its physiological and pharmacological function. Using the rat paw edema test we observed that only the highest doses of CRF employed (5 micrograms) induced a moderate and sustained swelling. Pre-treatment with low doses of CRF (0.5-5 ng) was able to inhibit the edema induced by Naja naja phospholipase A2, carrageenin or histamine. Higher doses (50 ng-5 micrograms) had no anti-inflammatory activity. When co-inhibited with Naja naja phospholipase A2 or histamine the peptide did not modify the swelling at doses up to 500 ng, showing at 5 micrograms an additive edema with Naja naja phospholipase A2. In vitro, CRF did not modify the release of histamine but slightly increased the release of arachidonic acid to the medium. Our findings show a clear dose dependence on the local effects of CRF in inflammatory responses. These results suggest that the mechanisms of the two dose-related phenomena may be distinct.

Anti-inflammatory effect of gangliosides in the rat hindpaw edema test.

The influence of total brain gangliosides on acute inflammation was investigated using the rat hind paw edema test. Total gangliosides (10 micrograms/paw) inhibited the edema produced by the injection of bee venom phospholipase A2 (5 micrograms/paw) when the lipids were co-injected or injected 15 min before the phospholipase A2. Sulphatide (10 micrograms/paw) did not inhibit the edema but potentiated it. Gangliosides (40 micrograms/paw) inhibited the edema induced by carrageenin 1% when they were injected 1 h before the agent. However, gangliosides (up to 200 micrograms/paw) failed to inhibit the dextran-induced edema. The edema test was also used to investigate the effect of gangliosides on the production of mediators of inflammation by peritoneal adherent macrophages. Gangliosides inhibited the production of mediators of inflammation only when they were incubated with these cells before the stimulation with phospholipase A2 or carrageenin. Gangliosides did not inhibit the production of mediators of inflammation when arachidonic acid was added to the cells. These results suggest that the anti-inflammatory effect observed with gangliosides is mediated by inhibition at or before endogenous phospholipase activity.

[Basic aspects of neuroendocrinoimmunology]. / Aspectos básicos de la Neuroendocrinoinmunología (NEI).

The purpose of this article is to discuss basic aspects of the interplay between the neuroendocrine and the immune systems. Two pathways link the brain and the immune system: the autonomic nervous system and the neuroendocrine outflow via the pituitary. Most of the influence of the brain on immune events is exerted through the hypothalamic-pituitary-adrenal (HPA) axis. Moreover, certain neurotransmitters, neuropeptides, and neurohormones affect immune function both in vivo and in vitro. Receptors for these molecules are present on immune cells. This cell-to-cell communication is bi-directional, since impulses from the immune system can affect many functions of the central nervous system. Cytokines released during the activation of the immune system, in turn, can alter the function of the HPA axis. In this context, we also describe our main findings working with a model of Candida albicans infection in rats exposed to chronic varied stress.

[Induction of primary immune response by rat dendritic cells]. / Inducción de respuesta inmune primaria por células dendríticas de rata.

In this work we generated dendritic cells (DC) from rat bone marrow cultures stimulated with GM-CSF and IL-4. After 10 days of culture, we obtained numerous mature DC showing morphological characteristics of DC and high levels of MHC class II molecules. Also, we isolated DC from rat spleen on the bases of their differential adherence and low-density properties. The purity of these cells was > 90% according to morphology and MHC class II expression. To evaluate the capacity of bone marrow DC, immature spleen DC or spleen DC cultured 24h with GM-CSF (mature spleen DC), to elicit an immune response to ovalbumin (OVA), DC were loaded with this antigen and transferred to normal rats. Both bone marrow and spleen DC induced delayed type hypersensitivity responses (DTH). However, mature DC from spleen induced a stronger immune response against OVA with the highest DTH values (p < 0.05). These differences in the induction of the immune response correlated with higher expression of MHC class II molecules on mature DC.

Ability of the polysaccharide chitosan to inhibit proliferation of CD4+ lymphocytes from mucosal inductive sites, in vitro and in vivo.

OBJECTIVE: After oral administration of chitosan (a copolymer of glucosamine and N-acetylglucosamine), mesenteric lymph node (MLN) lymphocytes exhibited traits of anergy, a process coupled with inability of mature T cells to proliferate. We wondered whether biological activity of chitosan could be affecting division of lymphocytes at the mucosal inductive sites. MATERIALS AND METHODS: We studied the effect of chitosan on proliferation of carboxyfluorescein diacetate-labelled MLN lymphocytes stimulated with concanavalin A in vitro. We assessed expression of CD25 and CD71 activation markers and pro-apoptotic molecule CD95L. Moreover, we studied the effect of chitosan ex vivo, in carboxyfluorescein diacetate-labelled MLN cells isolated after feeding single or repetitive doses of the polysaccharide, and we evaluated cell cycle parameters. RESULTS: Chitosan suppressed cell proliferation and down-modulated expression of CD25 in these MLN CD4+ cells isolated from normal rats. After in vivo contact, chitosan inhibited proliferation of MLN cells and reduced secretion of interferon-gamma. Furthermore, sustained feeding produced reduction in percentage of CD4+ cells in S phase of the cell cycle. CONCLUSION: Here we demonstrate the ability of chitosan to suppress proliferation of CD4+ lymphocytes from mucosal inductive sites in vivo and in vitro This effect could be relevant in modulatory activity of chitosan in the intestinal microenvironment.

Yersinia-triggered arthritis in IL-12p40-deficient mice: relevant antigens and local expression of Toll-like receptor mRNA.

OBJECTIVES: To study the role of IL-12p40 at the onset of reactive arthritis (ReA) after Yersinia enterocolitica O:3 infection, and analyse relevant microbial antigens and articular expression of Toll-like receptor (TLR) mRNA. METHODS: Wild-type C57BL/6 and IL-12p40-deficient (IL-12p40-/-) mice were orogastrically infected with Y. enterocolitica O:3. Early (day 3) and late (day 21) after infection, the number of bacteria were determined in Peyer's patches (PP), mesenteric lymph nodes (MLN), the spleen, and joints. Histological studies of joints were performed. Collagen-specific and anti-Yersinia antibodies were measured by enzyme-linked immunosorbent assay (ELISA). The presence of Yersinia antigens was studied by dot blot. Induction of articular mRNA of TLR2, TLR4, and tumour necrosis factor (TNF)-alpha was analysed by reverse transcription-polymerase chain reaction (RT-PCR). TNFalpha protein levels were measured by ELISA. RESULTS: At day 3, bacterial recovery in PP, MLN, and spleen was significantly increased in IL-12p40-/- mice. Histopathological changes were observed in IL-12p40-/- mice at day 21 after infection, and correlated with higher antibody response against type II collagen. Although live bacteria could not be isolated at day 21 after infection, articular microbial components, especially from the outer membrane (OM), were detected. Moreover, intra-articular immunoglobulins to Yersinia antigens were significantly higher in IL-12p40-/- mice. Furthermore, mRNA levels for TLR2, TLR4 and TNFalpha, and TNFalpha protein were increased in joints from IL-12p40-/- mice. CONCLUSIONS: We concluded that IL-12p40 influences the resistance against Yersinia-triggered ReA. Bacterial products such as Yersinia OM could contribute to the ReA by induction of articular TLR expression, which results in an inflammatory response in the joint.

Involvement of peritoneal dendritic cells in the induction of autoimmune prostatitis.

We have been working within a model of autoimmune prostatitis induced by the intraperitoneal administration of saline extract of rat male accessory glands (RAG) associated to liposomes. The intraperitoneal administration of RAG-liposomes elicits both primary and secondary cellular autoimmune responses to RAG as well as organ-specific lesions. To evaluate the participation of dendritic cells (DC) in the induction of the autoimmune response, we purified peritoneal DC (PDC) after a single injection of RAG-liposomes and we characterized this population by morphology and phenotype. Based on adherence and morphologic criteria, we determined that PDC comprised approximately 1% of the total peritoneal cells. The ultrastructure of the dendritic cell enriched fraction was assessed by electron microscopy. By FACS analysis, PDC showed a two to three-fold increase in expression of the IA molecule compared to macrophages. They expressed low but positive levels of the CD14 marker, and intermediate levels of both CD11b (Mac-1) and CD54 (ICAM-1) adhesion molecules. In addition, PDC transferred either intravenously or intraperitoneally efficiently elicited the autoimmune response to RAG in normal receptors. These results support the involvement of peritoneal dendritic cells in the induction of autoimmune prostatitis, modifying the idea of macrophages as the single antigen presenting cell in the peritoneal cavity.

Induction of autoimmune prostatitis using liposomes is associated to peritoneal cells activation.

PROBLEM: Study and characterization of rat peritoneal cells (PC) involved in the induction of autoimmune prostatitis after the intraperitoneal administration of native extract of accessory glands (RAG) associated with liposomes (RAGL). METHOD OF STUDY: Induction of the autoimmune response in normal recipients by transferring PC or adherent-PC loaded with RAGL (RAGL-PC), but not with PC loaded with empty liposomes (L-PC). Characterization of the morphology, the ultrastructure, and the phenotype of L-PC or RAGL-PC. Study of the respiratory burst by the nitroblue tetrazolium (NBT) reduction assay after stimulation with phorbol myristate acetate (PMA) in both L-PC and RAGL-PC. RESULTS: Liposomes attached to the cell surface of the M phi were observed by electron microscopy. FACS analyses showed a similar staining pattern with high expression of Ia molecules on L-PC and RAGL-PC compared with controls. PMA-stimulated L-PC or RAGL-PC markedly reduced the NBT compared with controls. CONCLUSION: Our results suggest that the effective uptake of liposomes and the initial activation of PC together with a prolonged stimulatory effect help to disrupt the tolerance state. The present experimental model is an interesting approach to further characterize events associated with antigenic presentation when an autoimmune response is triggered.

Characterization of an immunoregulatory cell subset involved in the up-regulation of the autoimmune response to rat male accessory glands.

PROBLEM: We studied the regulation of the autoimmune response to rat male accessory glands (RAG) using liposomes as adjuvants. METHOD: Adult male Wistar rats were submitted to three intraperitoneal (i.p.) immunizations with 750 micrograms of saline extract of RAG associated with liposomes. The delayed type hypersensitivity (DTH) response studied approximately 10 days after each immunization developed after the first immunization, having a remission state after the second one and a clear increase after the third injection. In a further study, spleen mononuclear (SpM) cells obtained form immunized rats 10 days after the third immunization (DTH positive) or from normal rats were separated as adherent (VV+) or nonadherent (VV-) to Vicia villosa population. In VV+ SpM cells from immunized or normal animals an enhanced percentage of OX8+ cells (P < .05 and P < .01, respectively) was found, but in VV- SpM cells from the same groups of rats an enhanced percentage of W3/25+ cells (P < .01 and P < .05, respectively) was found when they were studied by immunofluorescence. Later on, we transferred total VV+ or VV- SpM cells from i.p. immunized rats to immunized recipients 10 days after the second immunization (DTH negative). The DTH response was enhanced in recipients of total or VV+ SpM cells (P < .01). It was also observed that the transfer of VV- SpM cells from immunized rats or total or VV+ SpM cells from normal rats did not reduce the suppression state observed after the second injection (P = NS). The total SpM cells obtained 10 days after the third immunization (DTH positive) were able to transfer autoimmune response to RAG to normal animals (P < .01), whereas VV+ SpM cells did not show that capacity (P = NS).

Phenotypic and functional changes on phagocytic cells recruited at the site of Candida albicans infection after chronic varied stress exposure.

The transition of Candida albicans from commensalism to pathogenicity is associated with the immune status of the host; resistance to fungus involves macrophages (Mphi) and polymorphonuclear neutrophils (PMN), which act as effector cells. T-cell function is also involved. Previously, we found that in Wistar rats exposed to chronic varied stress (CVS) immediately after C. albicans infection (Ca-S group) some functions of phagocytic cells, such as killer activity and NO production, were strongly modified compared with unstressed, infected animals (Ca group). We examined the phenotypic and functional changes of these effector cells recruited at the site of C. albicans infection. The recruitment of peritoneal cells (PC) was markedly reduced in Ca-S animals and the arrival of Mphi and PMN was selectively diminished after CVS exposure. The integrin CD11b/CD18, implicated in migration and C. albicans phagocytosis, was downregulated in Mphi of Ca-S animals. The activation markers CD54 and MHC-II were upregulated in Mphi after fungal contact. The expression of CD54 was only changed in Ca-S rats. Finally, TNF-alpha production was reduced in PC of Ca-S animals, suggesting an impairment of functional activity. Taken together, the phenotypic and functional changes detected in effector cells may account for the decreased resistance to candidiasis seen in conjunction with CVS. The changes seen also expand our knowledge of the role of Mphi in the control of C. albicans dissemination.

Impaired activity of phagocytic cells in Candida albicans infection after exposure to chronic varied stress.

OBJECTIVE: Candidiasis is a prototypic opportunistic fungal disease that may follow severe modulations of the immune system of the host. The purpose of this study was to evaluate which innate immune mechanisms involved in the protection against fungal invasion are impaired under stress conditions. METHODS: Wistar rats were infected intraperitoneally with Candida albicans and immediately exposed to chronic varied stress (CVS) over 10 days (CVS; Ca-S); the fungal burden (CFU), histopathological lesion and ACTH levels were evaluated. Additionally, functional assessment of peritoneal cells (PC) included the phagocytic and anticandidacidal activities and the production of H(2)O(2) and NO. RESULTS: In the only infected animals (Ca), C. albicans colonization stimulated an efficient inflammatory response, while in Ca-S rats poor tissue reactions were associated with increased CFU in livers and kidneys (p < 0.05, Ca vs. Ca-S). Whereas the phagocytic process was not modified, the candidacidal activity of PC was significantly decreased after the application of CVS (p < 0.001, Ca vs. Ca-S). The H(2)O(2) production by macrophages and neutrophils was downregulated by the infection, and while at early intervals these cells possessed a residual oxidative capacity, by day 10, the production of this metabolite was blocked. Spontaneous NO production by macrophages was significantly increased in both Ca and Ca-S animals (p < 0.001), but in stressed rats, this reactive nitrogen intermediate was noticeably downregulated (p < 0.05, Ca vs. Ca-S). The hyperactivity of hypothalamus-pituitary-adrenal axis after exposure to stress was confirmed by an increase in baseline plasma ACTH levels. CONCLUSION: These results show that during infection with C. albicans, the exposure to CVS contributes to the spread of the fungus and downregulates critical functions of phagocytic cells involved in the control of this opportunistic pathogen.

Evidence of a role for galectin-1 in acute inflammation.

Galectin-1 (Gal-1), a member of a family of beta-galactoside-binding proteins, has been suggested to play key roles in immunological and inflammatory processes. The present study deals with the concept of an in vivo role for Gal-1 in acute inflammation by using the rat hind paw edema test. Local administration of Gal-1 (0.5, 2, 4 and 8 microg/ml) inhibited acute inflammation induced by bee venom phospholipase A(2) (PLA(2)) when it was injected 30 min before the enzyme or co-injected together with PLA(2). The anti-inflammatory effect was prevented by a specific antibody, but independent of its carbohydrate-binding properties. In contrast, Gal-1 failed to inhibit histamine-induced edema. Histopathological studies showed a clear reduction of the inflammatory process when Gal-1 was injected before PLA(2), evidenced by a diminished number of infiltrated polymorphonuclear neutrophils and scarce degranulated mast cells. The anti-inflammatory effect was also assessed in vitro, showing that Gal-1 treatment reduced prostaglandin E(2) secretion and arachidonic acid release from stimulated peritoneal macrophages. Results presented here provide the first evidence for a role of Gal-1 in acute inflammation and suggest that the anti-inflammatory effect involves the inhibition of both soluble and cellular mediators of the inflammatory response.

Chronic varied stress modulates experimental autoimmune encephalomyelitis in Wistar rats.

Stress disturbs homeostasis by altering the equilibrium of various hormones which have a significant impact on immune responses. Few studies have examined the influence of stressors on autoimmune disease in animal models. In our work, we studied the effects of long-term exposure (14 days) to chronic varied stress (CVS) in a model of experimental autoimmune encephalomyelitis (EAE) in Wistar rats. We studied whether the exposure to CVS before or after the immune challenge would correlate with differences in the clinical course of the disease. We also examined whether the CVS would modulate the magnitude of the cellular or the humoral immune response. We observed opposite effects on the clinical signs in animals stressed before or after the immune challenge. The clinical signs of the disease were attenuated in animals stressed before but not after the immune challenge. Relationships were found in the modulation of the clinical severity related to the time of exposure to the CVS, the histological alterations and the proliferative results. Stressed animals with milder clinical signs presented an exacerbated humoral response against myelin antigens while stressed animals with more severe clinical symptoms exhibited a significantly diminished one. Besides, we detected the presence of specific IgG1 associated with the exposure to CVS before the induction of EAE. Our results show that, depending on the timing of the exposure of Wistar rats to the CVS, the neuroendocrine disbalance favors a more pronounced humoral or cellular profile of the response.