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ABSTRACT: We examined the short-course expression of various parameters involved in the adenosinergic signalling of a human T cell line during in vitro decrease of the medium culture oxygen tension mimicking in vivo hypoxia. Fall of 92 mmHg in oxygen tension of culture medium induced in CEM, a CD4+ human T cell line, a continuous production of hypoxia-inducing factor-1α with a plateau value at 9 h, a rapid increase in adenosine production peaking at 3 h and a decrease in adenosine deaminase peaking at 6 h. The adenosine A(2A) receptor (A(2A)R) protein level of CEM cells was enhanced with a peak at 6 h. Intracellular 3',5'-cyclic adenosine monophosphate accumulated in CEM cells with a maximal level at 9 h. These results show that a human-cultured T cells line can upregulate its own adenosine production and A(2A)R expression during exposure to acute hypoxia. Hypoxia-increased stimulation of the adenosinergic signalling of T cells may have immunosuppressive properties and, consequently, A(2A)R agonists may have therapeutic relevance.
Purinergic Signalling 02/2012; 8(4):661-7. · 3.16 Impact Factor
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ABSTRACT: Adenosine is a modulator of nociceptive pathways, both at the spinal and supraspinal levels. Adenosine A(1) and A(2A) receptors (A(1)R, A(2A)R) are expressed in the basal ganglia where they are the target of caffeine, the most widely use psychoactive drug which acts as an antagonist to both types of receptors. Given the controversial role of A(2A)R versus A(1)R in modulating pain in brain areas, mice received intracerebroventricular injection of Adonis, an agonist-like monoclonal antibody with high specificity for the A(2A)R and were subjected to behavioral tests investigating nociceptive thresholds. We report that Adonis led to a significant dose-dependent increase in hot-plate and tail-flick latencies in mice and that such increase was prevented by caffeine and ZM 241385, a specific A(2A)R antagonist. The Adonis antinociceptive effects were also inhibited by naloxone, a non selective antagonist for opioid receptors, suggesting that Adonis acts, at least in part, through the stimulation of the endogenous opioid system. These results confirm the A(2A)R as a target for pain control and Adonis as a potential drug with therapeutic interest.
Journal of neuroimmunology 01/2011; 230(1-2):178-82. · 2.84 Impact Factor
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ABSTRACT: Immunocompetent cells express various G-protein-coupled receptors that transduce extracellular signals across the plasma membrane. Among them, CXCR4 and CCR5 chemokines receptors and adenosine A(2A) receptors (A(2A)R) are involved in inflammatory processes. Considering that A(2A)R activation may have incidence on CXCR4 and CCR5 protein expression through heterologous desensitization process, we tested Adonis, an agonist-like monoclonal antibody to A(2A)R on CD4+ CEM T-cells. We found that Adonis inhibited the CEM cell growth, upregulated A(2A)R and downregulated CXCR4 and CCR5 without modifying the CD4 expression. By reducing the expression of CXCR4 and CCR5 chemokines receptors utilized as entry co-receptors by HIV-1 during viral infection of CD4 expressing cells, Adonis stimulation of A(2A)R appears as a valuable means to treat infected cells.
Human immunology 11/2010; 71(11):1073-6. · 2.55 Impact Factor
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ABSTRACT: The second extracellular loop of the A(2A) receptor (A(2A)R) of adenosine was used to immunize mice for production of Adonis, an IgM monoclonal antibody. Adonis bound to the immunogen peptide and the native receptor in ELISA with K(D) values in 6.51-12.35 nM range. It recognized a linear epitope of 7 amino acids (LFEDVVP) at the C-terminal part of the external loop. Adonis revealed a 45-kDa band in lysate of human peripheral blood mononuclear cells in Western blotting in denaturing conditions. This served to monitor the up-regulation of the A(2A)R expression by caffeine. Adonis stimulated the cAMP production and inhibited the cell proliferation of an A(2A)R transfected stable cell line. These results confirm the immunogenicity and the functional relevance of the second extracellular loop of the A(2A)R. They suggest that Adonis may be of clinical use in various pathological situations to measure the regulation of the A(2A)R expression and to act as A(2A)R agonist drug.
Molecular Immunology 12/2008; 46(3):400-5. · 2.90 Impact Factor
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ABSTRACT: In the processes underlying thyroid autoimmunity, thyrocytes probably act as antigen-presenting cells exposing T-cell epitopes to intrathyroid lymphocytes. To study the interactions between lymphocytes and thyrocytes, which are arranged in a tight, polarized monolayer, we developed a new in vitro model based on human thyrocytes grown on the underside of a filter placed in a bicameral chamber. Thyrocytes from Graves' disease glands were plated onto the upper face of a 8-mum-pore polyethylene terephthalate culture insert filter placed in the inverted position and grown for 24 h before the insert was returned to the normal position for a week in the cell culture plate wells. Thyrocytes grown in the presence of thyroid stimulating hormone, forming a homogeneous monolayer on the underside of the filter, reached confluence after 8 days in vitro. The cells developed a transepithelial electrical resistance >1,000 Omega.cm(2), and the ZO-1 tight junction protein showed a junctional pattern of distribution. Thyrocytes showed a polarized pattern of thyroperoxidase and thyroid stimulating hormone receptor expression in the apical and basolateral positions, respectively. They were also found to aberrantly express DR class II human leukocyte antigen and an Fc immunoglobulin receptor (FcgammaRIIB2) in the basolateral and apical positions, respectively. Autologous intrathyroidal T lymphocytes cocultured for 24 h across the filter with the thyrocyte monolayer proliferated and remained in the upper chamber without any leakage occurring through the epithelial barrier, which makes this model particularly suitable for studying the cell-cell interactions involved in antigen processing.
AJP Cell Physiology 12/2004; 287(6):C1763-8. · 3.54 Impact Factor
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ABSTRACT: Thyroglobulin (Tg) is cleaved into several peptides during thyroid hormone synthesis, an oxidative process. P40, an iodinated C-terminal peptide from human Tg, has a molecular weight of about 40 kDa and contains two hormonogenic sites. P40 is the smallest peptide that is still recognized by monoclonal antibodies from mice immunized with human Tg directed against its immunodominant region. Since P40 also contains several T-cell epitopes, it is a good candidate for studying the primary events involved in the process of hormone synthesis leading to thyroid autoimmunity. The present results show that P40 is recognized by Tg antibodies from patients with thyroid disorders and induces Tg antibodies in CBA mice. P40 may therefore be involved in the autoimmune process, thus providing a useful tool for diagnostic and therapeutic purposes.
Peptides 07/2004; 25(6):1021-9. · 2.43 Impact Factor
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ABSTRACT: Thyroid peroxidase (TPO) is involved in autoimmune thyroid diseases and high titers of TPO autoantibodies directed to various conformational B cell epitopes are frequently present in patients' sera. Deciphering these epitopes is a difficult task, but can give insight into the structural basis of autoimmune recognition. TPO is a membrane-bound enzyme with the extracellular part organized in three protein domains, but of unknown three-dimensional structure. We previously localized a TPO B cell epitope within amino acid residues 742-848, a region encompassing the two C-terminal, extracellular domains of the protein. We found that at least one of the three tyrosine residues of the peptide 742-848 might be involved in autoantibody binding. In this study, we show by site-directed mutagenesis that the autoepitope contains tyrosine 772 located near the hinge area between the two protein domains, suggesting they are both involved in the epitope structure. The B cell epitopes of TPO are clustered in two overlapping immunodominant regions. To map the newly localized epitope with respect of these regions, competition experiments were performed using a reference panel of TPO mAb and a further mAb previously found to be specific for the TPO peptide 742-848 at variance with all the other ones. Here, we show that the tyrosine 772-bearing epitope in the peptide 742-848 maps in a region that partly overlaps the reported two immunodominant regions. These results are suggestive of a complex TPO folding that involves all the three TPO protein domains to form a highly conformational immunodominant region.
International Immunology 05/2002; 14(4):359-66. · 3.41 Impact Factor
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ABSTRACT: While studying the humoral mechanisms involved in thyroid autoimmunity, we located a B-cell autoepitope in the extracellular
C-terminal region of human thyroperoxidase. Structural modeling showed that this region encompasses both a Sushi-like and
an epidermal growth factor-like domain, the flexible arrangement of which was putatively stabilized by calcium. The recombinant
peptide was found to contain the previously identified conformational thyroperoxidase autoepitope. The occurrence of a calcium-induced
conformational change was confirmed using a recombinant peptide monoclonal antibody, the decrease of which in binding to calcium-saturated
thyroperoxidase was reversed by a chelating agent. The disease specificity of recombinant peptide, which was more frequently
recognized by Hashimoto's than by Graves' patients, adds to its potential value as a diagnostic and preventive tool in the
context of B-cell autoimmunity.
Journal of Biological Chemistry 12/1999; 274(50):35313-35317. · 4.77 Impact Factor
