Chitosan-Silica Hybrid Biomaterials for Bone Tissue Engineering: A Comparative Study of Xerogels and Aerogels.

Chitosan (CS) is a natural biopolymer that shows promise as a biomaterial for bone-tissue regeneration. However, because of their limited ability to induce cell differentiation and high degradation rate, among other drawbacks associated with its use, the creation of CS-based biomaterials remains a problem in bone tissue engineering research. Here we aimed to reduce these disadvantages while retaining the benefits of potential CS biomaterial by combining it with silica to provide sufficient additional structural support for bone regeneration. In this work, CS-silica xerogel and aerogel hybrids with 8 wt.% CS content, designated SCS8X and SCS8A, respectively, were prepared by sol-gel method, either by direct solvent evaporation at the atmospheric pressure or by supercritical drying in CO2, respectively. As reported in previous studies, it was confirmed that both types of mesoporous materials exhibited large surface areas (821 m2g-1-858 m2g-1) and outstanding bioactivity, as well as osteoconductive properties. In addition to silica and chitosan, the inclusion of 10 wt.% of tricalcium phosphate (TCP), designated SCS8T10X, was also considered, which stimulates a fast bioactive response of the xerogel surface. The results here obtained also demonstrate that xerogels induced earlier cell differentiation than the aerogels with identical composition. In conclusion, our study shows that the sol-gel synthesis of CS-silica xerogels and aerogels enhances not only their bioactive response, but also osteoconduction and cell differentiation properties. Therefore, these new biomaterials should provide adequate secretion of the osteoid for a fast bone regeneration.

Structure-Related Mechanical Properties and Bioactivity of Silica-Gelatin Hybrid Aerogels for Bone Regeneration.

We report the synthesis of mesoporous silica-gelatin hybrid aerogels with 15, 25, and 30 wt. % gelatin contents, using 3-glycidoxypropyl trimethoxysilane (GPTMS) as a coupling agent, for tissue-engineering applications. Aerogels were obtained using a one-step sol-gel process followed by CO2 supercritical drying, resulting in crack-free monolith samples with bulk densities ranging from 0.41 g cm-3 to 0.66 g cm-3. Nitrogen adsorption measurements revealed an interconnected mesopore network and a general decrease in the textural parameters: specific surface areas (651-361 m2 g-1), pore volume (1.98-0.89 cm3 g-1), and pore sizes (10.8-8.6 nm), by increasing gelatin content. Thermogravimetric analysis (TGA), Fourier-transform infrared (FTIR) spectroscopy and uniaxial compression experiments confirmed that the structure, thermal properties and mechanical behavior of these aerogels changed significantly when the concentration of gelatin reached 25 wt.%, suggesting that this composition corresponds to the percolation threshold of the organic phase. In addition, the samples exhibited hydrophilic behavior and extremely fast swelling in phosphate-buffered saline (PBS), with swelling ratios from 2.32 to 3.32. Furthermore, in vitro bioactivity studies revealed a strong relationship between the kinetics of the nucleation and growth processes of hydroxyapatite in simulated body fluid (SBF) and the gelatin content. The live/dead assay revealed no cytotoxicity in HOB® osteoblasts in vitro and a positive influence on cell growth, focal adhesion development, and cytoskeletal arrangement for cell adhesion. Mineralization assays confirmed the positive effects of the samples on osteoblast differentiation. The biomaterials described are versatile, can be easily sterilized and are suitable for a wide range of applications in bone tissue-engineering, either alone or in combination with bioactive-reinforced phases.

Robocasting and Laser Micromachining of Sol-Gel Derived 3D Silica/Gelatin/ß-TCP Scaffolds for Bone Tissue Regeneration.

The design and synthesis of sol-gel silica-based hybrid materials and composites offer significant benefits to obtain innovative biomaterials with controlled porosity at the nanostructure level for applications in bone tissue engineering. In this work, the combination of robocasting with sol-gel ink of suitable viscosity prepared by mixing tetraethoxysilane (TEOS), gelatin and ß-tricalcium phosphate (ß-TCP) allowed for the manufacture of 3D scaffolds consisting of a 3D square mesh of interpenetrating rods, with macropore size of 354.0 ± 17.0 µm, without the use of chemical additives at room temperature. The silica/gelatin/ß-TCP system underwent irreversible gelation, and the resulting gels were also used to fabricate different 3D structures by means of an alternative scaffolding method, involving high-resolution laser micromachining by laser ablation. By this way, 3D scaffolds made of 2 mm thick rectangular prisms presenting a parallel macropore system drilled through the whole thickness and consisting of laser micromachined holes of 350.8 ± 16.6-micrometer diameter, whose centers were spaced 1312.0 ± 23.0 µm, were created. Both sol-gel based 3D scaffold configurations combined compressive strength in the range of 2-3 MPa and the biocompatibility of the hybrid material. In addition, the observed Si, Ca and P biodegradation provided a suitable microenvironment with significant focal adhesion development, maturation and also enhanced in vitro cell growth. In conclusion, this work successfully confirmed the feasibility of both strategies for the fabrication of new sol-gel-based hybrid scaffolds with osteoconductive properties.

Effect of Washing Treatment on the Textural Properties and Bioactivity of Silica/Chitosan/TCP Xerogels for Bone Regeneration.

Silica/biopolymer hydrogel-based materials constitute very attractive platforms for various emerging biomedical applications, particularly for bone repair. The incorporation of calcium phosphates in the hybrid network allows for designing implants with interesting biological properties. Here, we introduce a synthesis procedure for obtaining silica-chitosan (CS)-tricalcium phosphate (TCP) xerogels, with CS nominal content varying from 4 to 40 wt.% and 10 to 20 wt.% TCP. Samples were obtained using the sol-gel process assisted with ultrasound probe, and the influence of ethanol or water as washing solvents on surface area, micro- and mesopore volume, and average pore size were examined in order to optimize their textural properties. Three washing solutions with different soaking conditions were tested: 1 or 7 days in absolute ethanol and 30 days in distilled water, resulting in E1, E7, and W30 washing series, respectively. Soaked samples were eventually dried by evaporative drying at air ambient pressure, and the formation of interpenetrated hybrid structures was suggested by Fourier transformed infrared (FTIR) spectroscopy. In addition the impact that both washing solvent and TCP content have on the biodegradation, in vitro bioactivity and osteoconduction of xerogels were explored. It was found that calcium and phosphate-containing ethanol-washed xerogels presented in vitro release of calcium (2-12 mg/L) and silicon ions (~60-75 mg/L) after one week of soaking in phosphate-buffered saline (PBS), as revealed by inductive coupled plasma (ICP) spectroscopy analysis. However, only the release of silicon was detected for water-washed samples. Besides, all the samples exhibited in vitro bioactivity in simulated body fluid (SBF), as well as enhanced in vitro cell growth and also significant focal adhesion development and maturation.

Chitosan-GPTMS-Silica Hybrid Mesoporous Aerogels for Bone Tissue Engineering.

This study introduces a new synthesis route for obtaining homogeneous chitosan (CS)-silica hybrid aerogels with CS contents up to 10 wt%, using 3-glycidoxypropyl trimethoxysilane (GPTMS) as coupling agent, for tissue engineering applications. Aerogels were obtained using the sol-gel process followed by CO2 supercritical drying, resulting in samples with bulk densities ranging from 0.17 g/cm3 to 0.38 g/cm3. The textural analysis by N2-physisorption revealed an interconnected mesopore network with decreasing specific surface areas (1230-700 m2/g) and pore sizes (11.1-8.7 nm) by increasing GPTMS content (2-4 molar ratio GPTMS:CS monomer). In addition, samples exhibited extremely fast swelling by spontaneous capillary imbibition in PBS solution, presenting swelling capacities from 1.75 to 3.75. The formation of a covalent crosslinked hybrid structure was suggested by FTIR and confirmed by an increase of four hundred fold or more in the compressive strength up to 96 MPa. Instead, samples synthesized without GPTMS fractured at only 0.10-0.26 MPa, revealing a week structure consisted in interpenetrated polymer networks. The aerogels presented bioactivity in simulated body fluid (SBF), as confirmed by the in vitro formation of hydroxyapatite (HAp) layer with crystal size of approximately 2 µm size in diameter. In vitro studies revealed also non cytotoxic effect on HOB® osteoblasts and also a mechanosensitive response. Additionally, control cells grown on glass developed scarce or no stress fibers, while cells grown on hybrid samples showed a significant (p < 0.05) increase in well-developed stress fibers and mature focal adhesion complexes.

Adipose-derived mesenchymal stem cells (AdMSC) for the treatment of secondary-progressive multiple sclerosis: A triple blinded, placebo controlled, randomized phase I/II safety and feasibility study.

BACKGROUND: Currently available treatments for secondary progressive multiple sclerosis(SPMS) have limited efficacy and/or safety concerns. Adipose-mesenchymal derived stem cells(AdMSCs) represent a promising option and can be readily obtained using minimally invasive procedures. PATIENTS AND METHODS: In this triple-blind, placebo-controlled study, cell samples were obtained from consenting patients by lipectomy and subsequently expanded. Patients were randomized to a single infusion of placebo, low-dose(1x106cells/kg) or high-dose(4x106cells/kg) autologous AdMSC product and followed for 12 months. Safety was monitored recording adverse events, laboratory parameters, vital signs and spirometry. Expanded disability status score (EDSS), magnetic-resonance-imaging, and other measures of possible treatment effects were also recorded. RESULTS: Thirty-four patients underwent lipectomy for AdMSCs collection, were randomized and thirty were infused (11 placebo, 10 low-dose and 9 high-dose); 4 randomized patients were not infused because of karyotype abnormalities in the cell product. Only one serious adverse event was observed in the treatment arms (urinary infection, considered not related to study treatment). No other safety parameters showed changes. Measures of treatment effect showed an inconclusive trend of efficacy. CONCLUSION: Infusion of autologous AdMSCs is safe and feasible in patients with SPMS. Larger studies and probably treatment at earlier phases would be needed to investigate the potential therapeutic benefit of this technique.

Preconditioning of microglia by α-synuclein strongly affects the response induced by toll-like receptor (TLR) stimulation.

In recent years, it has become accepted that α-synuclein (αSyn) has a key role in the microglia-mediated neuroinflammation, which accompanies the development of Parkinson's disease and other related disorders, such as Dementia with Lewy Bodies and Alzheimer's disease. Nevertheless, the cellular and molecular mechanisms underlying its pathological actions, especially in the sporadic forms of the diseases, are not completely understood. Intriguingly, several epidemiological and animal model studies have revealed a link between certain microbial infections and the onset or progression of sporadic forms of these neurodegenerative disorders. In this work, we have characterized the effect of toll-like receptor (TLR) stimulation on primary murine microglial cultures and analysed the impact of priming cells with extracellular wild-type (Wt) αSyn on the subsequent TLR stimulation of cells with a set of TLR ligands. By assaying key interleukins and chemokines we report that specific stimuli, in particular Pam3Csk4 (Pam3) and single-stranded RNA40 (ssRNA), can differentially affect the TLR2/1- and TLR7-mediated responses of microglia when pre-conditioned with αSyn by augmenting IL-6, MCP-1/CCL2 or IP-10/CXCL10 secretion levels. Furthermore, we report a skewing of αSyn-primed microglia stimulated with ssRNA (TLR7) or Pam3 (TLR2/1) towards intermediate but at the same time differential, M1/M2 phenotypes. Finally, we show that the levels and intracellular location of activated caspase-3 protein change significantly in αSyn-primed microglia after stimulation with these particular TLR agonists. Overall, we report a remarkable impact of non-aggregated αSyn pre-sensitization of microglia on TLR-mediated immunity, a phenomenon that could contribute to triggering the onset of sporadic α-synuclein-related neuropathologies.

Glial innate immunity generated by non-aggregated alpha-synuclein in mouse: differences between wild-type and Parkinson's disease-linked mutants.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated alpha-synuclein (α-Syn). Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular α-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma. However, the effect of extracellular α-Syn on cellular activation and immune mediators, as well as the impact of familial PD-linked α-Syn mutants on this stimulation, are still largely unknown. METHODS AND FINDINGS: In this work, we have compared the activation profiles of non-aggregated, extracellular wild-type and PD-linked mutant α-Syn variants on primary glial and microglial cell cultures. After stimulation of cells with α-Syn, we measured the release of Th1- and Th2- type cytokines as well as IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1α/CCL3 chemokines. Contrary to what had been observed using cell lines or for the case of aggregated α-Syn, we found strong differences in the immune response generated by wild-type α-Syn and the familial PD mutants (A30P, E46K and A53T). CONCLUSIONS: These findings might contribute to explain the differences in the onset and progression of this highly debilitating disease, which could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD.

Activity-dependent neuroprotective protein (ADNP) expression in the amyloid precursor protein/presenilin 1 mouse model of Alzheimer's disease.

A major determinant in the pathogenesis of Alzheimer's disease (AD) is the deposition of beta-amyloid (Abeta) peptides in specific areas of the central nervous system. Therefore, animal models of Alzheimer amyloidosis are excellent tools to identify candidates to facilitate drug screening and to understand the molecular pathology of AD. Activity-dependent neuroprotective protein (ADNP) plays an essential role in brain development, and NAP (NAPVSIPQ, generic name: davunetide)--a peptide derived from ADNP--is currently in clinical development for the treatment of neurodegenerative disorders. However, the link between ADNP expression and AD remains unexplored. To test whether ADNP is affected by the onset of AD and progression, we employed the PS1xAPP mouse model (PS1(M146L) x APP(751SL) transgenic mice) to analyze the mRNA expression of ADNP in the hippocampus and cerebellum in early and advanced stages of disease. Results showed that ADNP expression in 6-month-old PS1xAPP mice hippocampus was higher than in wild-type (WT) mice. ADNP was originally identified as a vasoactive intestinal peptide (VIP)-responsive gene taking part in the VIP-mediated neurotrophic pathway. Interestingly, the expression of VIP was not affected in the same experimental setting, suggesting that ADNP expression is a VIP-independent marker associated with AD. Moreover, in the cerebellum, a brain area not affected by Abeta deposition, ADNP mRNA expression in 6-month-old PS1xAPP and WT were not different. A similar extent of hippocampal ADNP expression was observed in 18-month-old WT and PS1xAPP mice, in contrast to the differential expression level at 6 months of age. However, hippocampal ADNP expression in both WT and PS1xAPP was increased with aging similar to VIP mRNA expression. Our findings support the hypothesis that ADNP expression is related to early or mild AD progression by a VIP-independent mechanism.

Chemical synthesis and characterization of silver-protected vasoactive intestinal peptide nanoparticles.

UNLABELLED: We characterized a method to conjugate functional silver nanoparticles with vasoactive intestinal peptide (VIP), which could be used as a working model for further tailor-made applications based on VIP surface functionality. Despite sustained interest in the therapeutic applications of VIP, and the fact that its drugability could be largely improved by the attachament to functionalized metal nanoparticles, no methods have been described so far to obtain them. MATERIALS & METHODS: VIP was conjugated to tiopronin-capped silver nanoparticles of a narrow size distribution, by means of proper linkers, to obtain VIP functionalized silver nanoparticles with two different VIP orientations (Ag-tiopronin-PEG-succinic-[His]VIP and Ag-tiopronin-PEG-VIP[His]). VIP intermediate nanoparticles were characterized by transmission-electron microscopy and Fourier transform infrared spectroscopy. VIP functionalized silver nanoparticles cytotoxicity was determined by lactate dehydrogenase release from mixed glial cultures prepared from cerebral cortices of 1-3 days-old C57/Bl mice. Cells were used for lipopolysaccharide stimulation at day 18-22 of culture. RESULTS: Two different types of VIP-functionalized silver nanoparticles were obtained; both expose the C-terminal part of the neuropeptide, but in the first type VIP is attached to silver nanoparticle through its free amine terminus (Ag-tiopronin-PEG-succinic-[His]VIP), while in the second type, VIP N-terminus remains free (Ag-tiopronin-PEG-VIP[His]). VIP-functionalized silver nanoparticles did not compromise cellular viability and inhibited microglia-induced stimulation under inflammatory conditions. CONCLUSION: The chemical synthesis procedure developed to obtain VIP-functionalized silver nanoparticles rendered functional products, in terms of biological activity. The two alternative orientations designed, reduced the constraints for chemical synthesis that depends on the nanosurface to be functionalized. Our study provides, for the first time, a proof of principle to enhance the therapeutic potential of VIP with the valuable properties of metal nanoparticles for imaging, targeting and drug delivery.

Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/alpha-synuclein complex by Hip.

The ATP-dependent protein chaperone heat-shock protein 70 (Hsp70) displays broad anti-aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of alpha-synuclein (alphaSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation-prone polypeptides. We show a nucleotide-dependent interaction between Hsp70 and alphaSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with alphaSyn. Such a co-aggregation phenomenon can be prevented in vitro by the co-chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of alphaSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.

Toll-like receptor stimulation differentially regulates vasoactive intestinal peptide type 2 receptor in macrophages.

Vasoactive intestinal peptide (VIP) was originally isolated as a vasodilator intestinal peptide, then as a neuropeptide. In the immune system, VIP is described as an endogenous macrophage-deactivating factor. VIP exerts its immunological actions in a paracrine and/or autocrine manner, through specific receptors. However, very little is known about the molecular regulation of VIP type 2 receptor (VPAC(2)) in the immune system. We now report that different toll-like receptor (TLR) ligands selectively regulate the VPAC(2) receptor gene and show a gene repression system controlled by key protein kinase signalling cascades in macrophages. VPAC(2) gene expression is regulated by gram-positive (TLR2 ligands) and gram-negative bacteria wall constituents (TLR4 ligands). Moreover, VPAC(2) is tightly regulated: TLR2- or TLR2/6- but not TLR2/1-mediated mechanisms are responsible for the induction of VPAC(2). TLR stimulation by viral or bacterial nucleic acids did not modify the VPAC(2) mRNA levels. Remarkably, imiquimod--a synthetic TLR7 ligand--led to a potent up-regulation of VPAC(2) gene expression. TLR5 stimulation by flagellin present in gram-positive and gram-negative bacteria did not affect VPAC(2) mRNA. The p38 mitogen-activated protein kinase (MAPK) activity accounted for the TLR4-mediated induction of VPAC(2) gene expression. Surprisingly, our data strongly suggest for the first time a tightly repressed control of VPAC(2) mRNA induction by elements downstream of MAPK kinase 1/2, PI3K/Akt, and particularly Jun-NH(2)-terminal kinase signalling pathways.

Tiopronin monolayer-protected silver nanoparticles modulate IL-6 secretion mediated by Toll-like receptor ligands.

AIMS: Capped silver nanoparticles that can be coupled to a variety of molecules and biomolecules are of great interest owing to their potential applications in biomedicine. However, there are no data about their toxicity or functional effects on a key innate immune response, such as IL-6 secretion, after the engagement of the main group of pathogen-associated molecular patterns receptors, that is, the Toll-like receptors (TLRs). MATERIALS & METHODS: N-(2-mercaptopropionyl)glycine (tiopronin)-capped silver (Ag@tiopronin) nanoparticles of a narrow sized distribution ( approximately 5 nm) were synthesized and characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, Raman, (1)H-NMR and total correlation spectroscopy. Cytotoxicity was determined by lactate deshidrogenase and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium assays in Raw 264.7 macrophages. IL-6 was measured by ELISA. RESULTS & DISCUSSION: Ag@tiopronin nanoparticles have a narrow size distribution ( approximately 5 nm), high solubility and stability in aqueous environment with no cytotoxicity in terms of mitochondrial function or plasma-membrane integrity at concentrations as high as 200 microg/10(6) cells. Ag@tiopronin nanoparticles were not proinflammatory agents, but remarkably they specifically impaired the IL-6 secretion mediated by TLR2, TLR2/6, TLR3 or TLR9 stimulation in co-treatment experiments. However, in pretreatment experiments, nanoparticles enhanced the susceptibility of macrophages to inflammatory stimulation mediated by TLR2/1 and TLR2/6 specific ligands while severely impairing the IL-6 secretion activated by the TLR3 or TLR9 ligands. CONCLUSIONS: Contrary to what is found for bare silver nanoparticles, Ag@tiopronin nanoparticles are noncytotoxic to macrophages. Ag@tiopronin nanoparticles showed differential effects on TLR signaling of a high degree of specificity, without proinflammatory effects by themselves. These effects have to be borne in mind when using bioconjugates of Ag@tiopronin nanoparticles for future medical applications.

Alzheimer's disease beyond the genomic era: nuclear magnetic resonance (NMR) spectroscopy-based metabolomics.

Alzheimer's disease (AD) is a complex disease, with no definitive biomarkers available that allow clinical diagnosis; this represents a major problem for the advance of efficient drug discovery programs. A successful approach towards the understanding and treatment of AD should take into consideration this complex nature. In this sense, metabolic networks are subject to severe stoichiometric restrictions. Metabolomics amplifies changes both in the proteome and the genome, and represents a more accurate approximation to the phenotype of an organism in health and disease. In this article, we will examine the current rationale for metabolomics in AD, its basic methodology and the available data in animal models and human studies. The discussed topics will highlight the importance of being able to use the metabolomic information in order to understand disease mechanisms from a systems biology perspective as a non-invasive approach to diagnose and grade AD. This could allow the assessment of new therapies during clinical trials, the identification of patients at risk to develop adverse effects during treatment and the final implementation of new tools towards a more personalized medicine.

Protective role for plasmid DNA-mediated VIP gene transfer in non-obese diabetic mice.

Studies focused on the development of diabetes in NOD mice-a model for human type 1 diabetes-have revealed that an autoimmune inflammatory process is produced by the effect of Th1 cells and their secreted cytokines. DNA vaccination has been shown to be an effective method for modulating immunity in viral infections and experimental autoimmune diseases, including diabetes. VIP's immunomodulatory properties are partly mediated by skewing the pattern of cytokines from a proinflammatory response to an anti-inflammatory response. Using gene delivery to express VIP, we interfered in the immune process leading to diabetes in prone, cyclophosphamide-treated NOD mice. Our results extend the role of VIP in the control of immunoregulatory networks and open new perspectives for immunointervention through VIP-based gene therapy.

NAP, a peptide derived from the activity-dependent neuroprotective protein, modulates macrophage function.

NAP is an eight-amino acid neuroprotective peptide NAPVSIPQ; it is the smallest active element derived from the recently cloned activity-dependent neuroprotective protein (ADNP). NAP readily enters the brain from the blood. It will be important to learn whether NAP, in addition to its neuroprotective activity, also might influence immune-mediated inflammation. Here, we report that: (a) macrophages express ADNP; (b) expression of ADNP in macrophages responds to VIP; and (c) NAP downregulates the key inflammatory cytokines tumor necrosis factor (TNF-alpha), interleukin-16 (IL-16), and IL-12 in macrophages. These findings indicate that ADNP/NAP can play an important role in immune regulation as well as in neuroprotection, which may be mutually related processes.

The modulatory role of melatonin on immune responsiveness.

Afterhe successful discovery of the melatonin molecule by Aaron B Lerner et al at Yale University in 1958, melatonin and the pineal gland, a tiny endocrine gland situated at the center of the human brain, have primarily been considered in terms of their effects on the endocrine and reproductive systems. During the last decade, a substantial body of research has defined melatonin as a remarkable molecule with pleiotropic effects on the immune system. Moreover, its synthesis cannot be considered as exclusively endocrine; key immunocompetent cells have the functional enzymatic machinery for melatonin synthesis, paving the wayfo r complex intracrine, autocrine and paracrine regulatory loops. The immunomodulatory role of melatonin, with regard to infection, inflammation and autoimmunity, is outlined here, and the evidence discussed in this review strengthens the notion that the nature of an immune response may be modified, and therefore therapeutically manipulated, by circadian effector signals.

The use of melatonin as a vaccine agent.

Molecules with immunomodulatory properties determine the magnitude and quality of immune responses specific for the coadministered antigen. Melatonin is considered a biological-response modifier of the immune system with broad application in veterinary medicine. In seasonally-breeding animals, the indolamine is able to improve reproductive performance. With the purpose of expanding new advantageous roles for melatonin, we investigated the effect of subcutaneous slow-release melatonin implants in the humoral response after a vaccination. We reported here a new feature of melatonin as an adjuvant-like system towards Dichelobacter nodosus (A1 and C serotypes)--the bacterium which cause ovine footrot--the most important cause of lameness in sheep. Antibody titres determined by both agglutination and ELISA techniques were substantially higher and were sustained for a longer duration than non-implanted animals. Remarkably, the effect of melatonin was completely dependent on the presence of aluminium hydroxide. The finding that melatonin enhances a defined immune response in vivo opens new perspectives for the improvement of Th2-biased immune responses by alum adjuvants.