Horacio M. Serra

Galectin-1 Suppresses Autoimmune Retinal Disease by Promoting Concomitant Th2- and T Regulatory-Mediated Anti-Inflammatory Responses

Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In this study, we investigated the immunoregulatory role of galectin-1 (Gal-1), an endogenous lectin found at sites of T cell activation and immune privilege, in experimental autoimmune uveitis (EAU), a Th1-mediated model of retinal disease. Treatment with rGal-1 either early or late during the course of interphotoreceptor retinoid-binding protein-induced EAU was sufficient to suppress ocular pathology, inhibit leukocyte infiltration, and counteract pathogenic Th1 cells. Administration of rGal-1 at the early or late phases of EAU ameliorated disease by skewing the uveitogenic response toward nonpathogenic Th2 or T regulatory-mediated anti-inflammatory responses. Consistently, adoptive transfer of CD4+ regulatory T cells obtained from rGal-1-treated mice prevented the development of active EAU in syngeneic recipients. In addition, increased levels of apoptosis were detected in lymph nodes from mice treated with rGal-1 during the efferent phase of the disease. Our results underscore the ability of Gal-1 to counteract Th1-mediated responses through different, but potentially overlapping anti-inflammatory mechanisms and suggest a possible therapeutic use of this protein for the treatment of human uveitic diseases of autoimmune etiology.

Quantitative Analysis of N-Linked Glycoproteins in Tear Fluid of Climatic Droplet Keratopathy by Glycopeptide Capture and iTRAQ†

Glycoproteins are potentially important biomarkers of disease and therapeutic targets. In particular, the N-linked glycoproteins are a focus of interest as they can be found in the extracellular environment and body fluids. In this study, we have sampled the tears, the extracellular fluid of the epithelial cells covering the surface of the eye, of patients with climatic droplet keratopathy (CDK) using tears of unaffected normal patients for comparison. Prefractionation of the tear sample used a hydrazide-resin capture method, and the previously N-glycosylated peptides were then subjected to two-dimensional nano-LC-nano-ESI-MS/MS analysis to obtain peptide fragmentation patterns for identification through protein database searches. We have identified a total of 43 unique N-glycoproteins, 19 of which have not previously been reported in tear fluid. In addition, we have quantitatively compared N-glycoprotein profiles in tear fluid of patients with CDK to tears of nondiseased controls using glycopeptide capture, iTRAQ labeling and 2D nano-LC-nano-ESI-MS/MS analysis. In tears of CDK patients, increased levels of four N-glycosylated proteins including haptoglobin (at sites N207, N211 and N241), polymeric immunoglobulin receptor (at sites N83, N90, N135, N186, N421, and N469), immunoglobulin J chain (at site N49) and an uncharacterized protein DKFZp686M08189 (at site N470), as well as a decrease in the N-glycosylation level of one N-glycosylated protein, lacritin (at site N119) were observed. However, the overall levels of these five proteins showed no appreciable changes between control and CDK samples. The findings could be clinically significant in terms of disease etiology and biomarkers.

Up-regulation of the chemokine CCL21 in the skin of subjects exposed to irritants

BackgroundExpression of murine CCL21 by dermal lymphatic endothelial cells (LEC) has been demonstrated to be one of the most important steps in Langerhans cell emigration from skin. Previously, our group and others have found that this chemokine is up-regulated in different human inflammatory skin diseases mediated by diverse specific immune responses. This study was carried out to investigate the involvement of CCL21 in human skin after challenge with irritant agents responsible for inducing Irritant Contact Dermatitis (ICD).ResultsEleven normal individuals were challenged with different chemical or physical irritants. Two patients with Allergic Contact Dermatitis (ACD) were also challenged with the relevant antigen in order to have a positive control for CCL21 expression. Macroscopic as well as microscopic responses were evaluated. We observed typical ICD responses with mostly mononuclear cells in perivascular areas, but a predominance of polymorphonuclear cells away from the inflamed blood vessels and in the epidermis at 24 hours. Immunohistochemical studies showed up-regulation of CCL21 by lymphatic endothelial cells in all the biopsies taken from ICD and ACD lesions compared to normal skin. Kinetic study at 10, 48, 96 and 168 hours after contact with a classical irritant (sodium lauryl sulphate) showed that the expression of CCL21 was increased in lymphatic vessels at 10 hours, peaked at 48 hours, and then gradually declined. There was a strong correlation between CCL21 expression and the macroscopic response (r = 0.69; p = 0.0008), but not between CCL21 and the number of infiltrating cells in the lesions.ConclusionsThese results provide new evidence for the role of CCL21 in inflammatory processes. Since the up-regulation of this chemokine was observed in ICD and ACD, it is tempting to speculate that this mechanism operates independently of the type of dermal insult, facilitating the emigration of CCR7+ cells.

Involvement of accessory cells in the Trypanosoma cruzi-induced inhibition of the polyclonal response of T lymphocytes.

Infection with Trypanosoma cruzi is characterized by hyporesponsiveness of the immune system during the acute phase of infection. To better understand the immunological mechanisms affected by T. cruzi, we studied if a reduced T cell proliferative response could originate from an inability of T cells to proliferate or a functional deficiency at the level of accessory cells (AC). The inhibitory effect exerted by T. cruzi was during the induction phase of the lymphoproliferative response, suggesting the participation of AC in the hyporesponse. Then we further investigated the potential of the parasite to interfere with accessory cell‐dependent and ‐independent pathways of human T cell proliferation. Peripheral blood mononuclear cells and peripheral blood lymphocytes from healthy individuals, enriched for T cells, were analysed with regard to their proliferative capacity using: phytohaemagglutinin, immobilized anti‐CD3 monoclonal antibody (MoAb) and MoAb to the CD28 antigen, anti‐CD3 MoAb and recombinant IL‐2 and anti‐CD3 MoAb plus phorbol myristate acetate in the presence of parasites. Significant suppression of the proliferative response was caused by the parasite only when AC were present. The parasite markedly reduced the surface expression of HLA‐DR and CD11b antigens, key molecules in PHA‐induced proliferation. Addition of indomethacin to the culture failed to reverse the inhibitory effect of the parasites, suggesting that prostaglandin E2 was not involved. These data suggest that AC in contact with T. cruzi become incompetent as antigen presenting cell because they are unable to induce a normal proliferative response in T lymphocytes.

Secondary Lymphoid Tissue Chemokine (CCL21) Is Upregulated in Allergic Contact Dermatitis

Chemokines are important players in the development of allergic contact dermatitis (ACD). The participation of secondary lymphoid tissue chemokine (CCL21) is essential in the induction of the disease due to its expression in lymphatic vessels and in secondary lymphoid organs. Since there is no information about its participation during the effector phase of ACD, we studied this chemokine in patients already diagnosed with ACD, who were challenged with the relevant positive and negative (control) antigens. All patients showed a specific antigen-induced immune response characterized by early expression of inflammatory markers in blood endothelial cells followed by dermal accumulation of mononuclear cells with an important increase in infiltration of CXCR3+ but not of CCR7+ cells. In situ hybridization and immunohistochemistry showed low levels of CCL21 in lymphatic vessels at 2 h, whereas they were significantly increased at 10 and 48 h in all positive patch tests. In contrast, very low expression of this chemokine was observed in skin biopsies from the control site at 48 h. In addition, Langerin+ cells, which were present in dermis from positive patch tests at 2 h, were diminished in number at 10 and 48 h, but a significant number of those cells was still present in dermal areas of the control site at 48 h. We demonstrate for the first time that CCL21, a constitutively expressed chemokine, is strongly upregulated in human lymphatic vessels during a Th1/Tc1 allergic inflammatory response. This can provide the signal required for CCR7+ cells to leave the skin through CCL21-positive lymphatic vessels.

Is secondary lymphoid‐organ chemokine (SLC/CCL21) much more than a constitutive chemokine?

Chemokines are a superfamily of small cytokines with activities ranging from leukocyte traffick to hematopoiesis, angiogenesis, and tissue organogenesis. Secondary lymphoid‐organ chemokine (SLC/CCL21) was originally reported as a chemokine constitutively expressed by stromal cells and high endothelial venules in secondary lymphoid tissues and endothelium of afferent lymphatics, directing CCR7+ cells. More recently, others and we have demonstrated that SLC/CCL21 is up‐regulated in different skin inflammatory conditions. Thereafter, this molecule is much more than a constitutive chemokine, which could play a role in effector and regulatory immune functions.

Proteomic analysis of climatic keratopathy droplets

PURPOSE To identify the proteins in the corneal droplets of climatic droplet keratopathy (CDK), a disease that results in the formation of droplets on the cornea. Progressive accumulation of droplets in CDK leads to visual loss. METHODS Proteomic mass spectrometry of the CDK specimens was performed after fractionation of proteins in 4% to 20% SDS-polyacrylamide gels. Droplets were derived from two human donors. Immunohistochemistry with antibodies was performed to confirm the presence of identified proteins on donor tissues from patients with CDK and control subjects. RESULTS Proteomic analyses revealed identification of 105 proteins in CDK specimens. Immunohistochemical analyses confirmed localization of annexin A2 and glyceraldehyde 3-dehydrogenase (GAPDH), proteins identified by proteomic analyses in CDK specimens. The proteins were subjected to analyses with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Database which showed that a few biochemical pathways were more frequent for the identified proteins. CONCLUSIONS Approximately 105 proteins were identified in CDK specimens, and a subset of them was confirmed by immunohistochemistry. Several of these may play a role in fibril or deposit formation.

Cytokine polymorphisms in patients with pemphigus

The aim of this study was to assess whether tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) polymorphisms are among the factors influencing the development of pemphigus. Whole blood from 20 patients with pemphigus and 24 control subjects was taken. Genomic DNA was obtained and cytokine genotyping for IL-10 (−1082 G/A; −819 C/T), TGFB1 (codon 10 C/T, codon 25 G/C) and TNFA (−308 G/A) was performed using the ARMS-PCR method. The distribution of IL-10 (−819) alleles was significantly different between the pemphigus and control groups (P=0.009). In particular, allele T was associated with the disease (OR 3.291, 95% CI 1.350–8.020). Similar results were observed when only pemphigus vulgaris (PV) patients were analyzed (P=0.012, OR 3.410, 95% CI 1.346–8.639). An increased frequency of the low producer IL-10 haplotype (−1082/−819 A/T) in patients with pemphigus compared with controls was observed (OR 2.714, 95% CI 1.102–6.685) and this association was also significant when only PV patients were considered (OR 2.667, 95% CI 1.043–6.816). There were no differences between patients and controls in the frequency of any other gene polymorphism analyzed. The increased frequency of the low producer IL-10 haplotype (−1082 /−819 A/T) suggest that the carriage of this haplotype might predispose to pemphigus or the high and intermediate producer haplotypes may be protective factors. The prevalence of the allele IL-10 (−819 T) in pemphigus patients cannot be explained by the current hypothesis, according to which a particular allele of the gene is associated with a different level of cytokine production and therefore affects the predisposition to a particular disease. However, this cytokine polymorphism might be linked to an unknown susceptibility factor.

Expression of CS-1 fibronectin precedes monocyte chemoattractant protein-1 production during elicitation of allergic contact dermatitis

Background Leucocyte migration within inflammatory skin compartments in allergic contact dermatitis (ACD) is the result of a sophisticated multi‐step event where multiple molecules are involved.

Specific Suppression of Humoral and Delayed Hypersensitivity Responses by Cyclophosphamide in an Experimental Model of Autoimmunity

ABSTRACT: The aim of this report is to investigate the effect of cyclophosphamide (CY) in an experimental model of autoimmunity to rat male accessory glands. The results indicated that 100 mg/kg of this drug suppressed humoral immune response that persisted for at least 45 days when administered 3 days after the first immunization of rats with modified rat male accessory glands (MRAG) in complete Freunds adjuvant (CFA). Administration of the drug 3 days before ID injection of antigen caused a shorter suppression of antibody formation. Delayed type hypersensitivity (DTH) studied 13 days after the first immunization was suppressed only in the animals that were administered CY after the antigen.