Raúl Chávez

Amaranthus leucocarpus Lectin Recognizes Human Naive T Cell Subpopulations

Amaranthus leucocarpus lectin (ALL) is specific for GalNAc residue found in the inner core of Gal beta 1,3GalNAc alpha 1,O-Ser/Thr disaccharide (T-antigen) or GalNAc alpha 1,O-Ser/Thr (Tn-antigen). Flow cytometric analysis using fluorescein-labeled lectin and monoclonal antibodies against human cell surface markers indicated that 5.7% of mononuclear cells from human healthy donors are recognized by ALL. These cells have the phenotype CD2+CD4+CD19- and most of the lymphocytes recognized are also CD27+, CD45RA+ and CD43+. ALL possesses mitogenic activity on lymphocytes after neuraminidase treatment. Our results indicate that the receptors recognized by ALL could be considered surface markers for naive human T lymphocyte subsets.

Specificity of the isolectins from the plant cactus Machaerocereus eruca for oligosaccharides from porcine stomach mucin.

Sugar specificity of theMachaerocereus eruca isolectins, MeAI and MeAII, has been determined by comparing the capacity of glycans with well defined structures to inhibit their haemagglutinating activity. Both are galactose-specific isolectins with high affinity for O-glycans. However, the twoM. eruca isolectins recognize different oligosaccharidic sequences belonging to O-glycosidically linked glycans from porcine stomach mucin. The minimal structure recognized by MeAI on the porcine mucin glycans is the O-glycan core Galβ1, 3GalNAc-ol, whereas MeAII has a more extended site and interacts with a biantennary O-glycan possessing the terminal trisaccharide Fucα1,2 (GalNAcα1,3) Galβ1,4.

Analysis of microRNA expression signatures in malignant pleural mesothelioma, pleural inflammation, and atypical mesothelial hyperplasia reveals common predictive tumorigenesis-related targets.

Pleural chronic inflammation (PP) and mesothelial hyperplasia (HP) may be critical to the development of malignant pleural mesothelioma (MPM). Nonetheless, studies searching for mechanistic links involving microRNA (miRNA) regulation among these interrelated processes have not been reported. Using PCR-Array, we identified the miRNAs expressed in pleural tissues diagnosed with MPM (n=5), PP (n=4) and HP (n=5), as well as in non-cancerous/non-inflammatory tissue as the normal control (n=5). We performed bioinformatics and network analysis of differentially expressed miRNAs to identify tumorigenesis-related miRNAs and their biological networks. The targets of four down-regulated miRNAs in MPM (mir-181a-5p, miR-101-3p, miR-145-5p and miR-212-3p), one in PP (mir-101-3p) and one in HP (mir-494) were significantly enriched in pathways in cancer. Interactome networks revealed that >50% of down-regulated miRNAs in MPM targeted the signaling-activation molecule MAPK1, the transcription factor ETS1 and the mesenchymal transition-associated molecule FZDA, which have been associated with oncogenic function. Comparative analysis revealed that FZD4 was an overlapping gene target of down-regulated miRNAs that were associated with pathways in cancer in MPM, PP and HP. Moreover, MAPK1, ETS1 and Cox-2, a pro-inflammatory enzyme associated with over-expression in cancers, were among the 25 overlapping target genes in MPM and PP. This network analysis revealed a potential combinatory effect of deregulated miRNAs in MPM pathogenesis and indicated potential molecular links between pleural inflammation and hyperplasia with tumorigenesis mechanisms in pleura.

Peanut and Amaranthus leucocarpus lectins discriminate between memory and naive/quiescent porcine lymphocytes.

Lectins are relevant tools to isolate and characterize different cellular sub-populations. In this work, we used the lectins Arachis hypogaea (Peanut agglutinin, PNA) and Amaranthus leucocarpus (ALL), specific for Galss1, 3GalNAc, to characterize naive and memory lymphocytes from pigs, experimentally infected with the porcine rubulavirus (RvP). Our results showed that both lectins recognized preferentially lymphocytes with the CD4(+)CD8(+) phenotype (P<0.05). The phenotypic analysis of the cells recognized by these lectins indicated that PNA(+) lymphocytes showed higher rate of the CD29 antigen (PNA(+)CD29(high)) than ALL(+) (ALL(+)CD29(low)). The number of PNA(+)CD29(high) lymphocytes increased after 8 weeks of experimental infection with RvP, and most of the ALL(+)CD29(low) cells became CD29(middle). PNA(+) lymphocytes isolated from infected pigs proliferated after stimulation with the RvP, whereas ALL(+) cells did not. In vitro assays indicated that the ALL(+) cells from previously infected pigs diminished from 7.5 +/- 2 to 0.5 +/- 0.3% after RvP stimulation; whereas PNA(+) cells increased from 4 +/- 1 to 42 +/- 2%, whereas no modification in ALL(+) or PNA(+) cellular population was identified in lymphocytes from naive animals after RvP stimulation. Our results suggest that the cellular distribution/organization of the O-glycosydically linked glycans on lymphocytes may correlate with biological functions, and that PNA could be a tool to isolate specifically porcine memory T cell subsets, whereas ALL could be useful to isolate naive/quiescent T lymphocytes.

Allergic Conjunctivitis: An Immunological Point of View

Allergic conjunctivitis (AC) is an inflammation of the conjunctiva secondary to an immune response to external antigens, usually called allergens. This inflammation could be IgEmediated and non-IgE mediated and atopy could play a significant role in clinical evolution. (Johansson et al., 2004) AC is not a single disease; in fact it is a syndrome affecting the entire ocular surface, including conjunctiva, lids, cornea, and tear film. The signs and symptoms of allergic conjunctivitis have a meaningful effect on comfort and patient health, and are influenced by genetics, environment, ocular microbiota, and immune regulation mechanisms, all of which work together in a complex immunological response. Dysregulation in such immune homeostasis could turn into a variety of allergic ocular diseases (AOD). This chapter describes the current understanding of cellular and molecular pathways involved in different AOD, the clinical characteristics of ocular allergies, the new therapies related to control of immune activation, and the importance of basic research to generate new types of immunotherapy to treat allergic conjunctivitis

Purification of a N-acetyl-d-galactosamine specific lectin from the orchid Laelia autumnalis

From the pseudobulbs of the orchid L. autumnalis a lectin was purified on immobilized porcine mucin with A + H blood group substance. This lectin is a dimeric glycoprotein of M(r) 12,000 with an Sw,20 of 2.2, showing haemagglutinating activity directed mainly to human A1 desialylated erythrocytes. The lectin possesses sugar specificity for N-acetyl-D-galactosamine and also shows high specificity for glycoproteins containing the T (galactose beta 1,3GA1NAc alpha 1,0 Ser/Thr) or the Tn antigen (GalNAc alpha 1,0 Ser/Thr).

O-Glycosylation of NnTreg Lymphocytes Recognized by the Amaranthus leucocarpus Lectin

O-glycosidically-linked glycans have been involved in development, maturation, homing, and immune regulation in T cells. Previous reports indicate that Amaranthus leucocarpus lectin (ALL), specific for glycans containing galactose-N-acetylgalactosamine and N-acetylgalactosamine, recognizes human naïve CD27+CD25+CD4+ T cells. Our aim was to evaluate the phenotype of CD4+ T cells recognized by ALL in peripheral blood mononuclear cells obtained from healthy volunteers. CD4+ T cells were isolated by negative selection using magnetic beads-labeled monoclonal antibodies; the expression of T regulatory cell phenotypic markers was assessed on ALL-recognized cells. In addition, IL-4, IL-10, IFN-γ, and TGF-β intracellular production in ALL + cells was also evaluated. The analyses of phenotypic markers and intracellular cytokines were performed through flow cytometry. ALL-recognized CD4+ T cells were mainly CD45RA+, CCR7+ cells. Although 52 ± 10% CD25+Foxp3+ cells were positive to ALL, only 34 ± 4% of ALL + cells corresponded to CD25+Foxp3− cells. Intracellular cytokines in freshly obtained ALL +CD4+ T cells exhibited 8% of IL-4, 15% of IL-10, 2% of IFN-γ, and 15% of TGF-β, whereas ALL −CD4+ T cells depicted 1% of IL-4, 2% of IL-10, <1% of IFN-γ, and 6% of TGF-β. Our results show that galactose-N-acetylgalactosamine and N-galactosamine-bearing CD4+ T cells expressed phenotypic markers of NnTreg cells.

357 Tear IL-4 is Decreased in Allergic Conjunctivitis Patients with Negative Skin Test After Dialyzable Leukocyte Extracts Treatment.

Background It has been suggested that sublingual immunotherapy induces immune regulation, however in patients with clinical ophthalmological diagnosis as allergic conjunctivitis with negative skin test reactivity (ACNST) this treatment is not useful. Dialyzable leukocyte extracts (DLE) have been used in atopic dermatitis and asthma. The aim of this work was to evaluate treatment of ACNST with DLE and to analyze the microenvironment provided by tear and serum cytokines in patients before and after DLE treatment. Methods 10 ACNST-patients with negative skin test were included in this study. ACNST diagnosis was based on a clinical history and full ophthalmologic examination according to the diagnosis standards of the American Academy of Ophthalmology. Coproparasitoscopic negative for parasites was documented This study was approved by Scientific and Ethics Committees if Institute of Ophthalmology “Conde de la Valenciana “, Mexico City an all subjects gave their informed consent to obtain samples. Tear and Serum Samples were collected to determine cytokines IL2, IL-4, IL-5, IFN-g, TNF-a, IL-10 by cytometric bead arrays (CBA), following manufacturers instructions. Results Patients showed lower significant levels of L-4 after 6 months of treatment, without changes in IL2, IL5, TNFa and IL10. Significant Clinical improvement was also observed since 3 months of treatment and was maintained until the end of 6 months. Conclusions DLE could be an excellent therapeutic tool to improve the clinical outcome in ACNST patients; it is possible that clinical improvement could be Tear IL-4 dependent.

60 Tear IFN-G is Increased After Sublingual Immunotherapy in Allergic Conjunctivitis Patients and Correlates With Clinical Improvement.

Background Despite success of sublingual immunotherapy (SLIT) in the treatment of allergy diseases, more research is needed related to ocular allergy. Thus, the aim of this work was to analyze the ocular microenvironment provided by tear cytokines in allergic conjunctivitis (AC) patients treated with SLIT and to correlate tear and serum cytokines with ocular findings. Methods 19 AC-patients were included in this study. AC diagnosis was based on a clinical history and full ophthalmologic examination according to the diagnosis standards of the American Academy of Ophthalmology. Routine immunological studies were performed to corroborate allergic status Negative coproparasitoscopic results were documented. This study was approved by Scientific and Ethics Committees if Institute of Ophthalmology “Conde de la Valenciana,” Mexico City an all subjects gave their informed consent to obtain samples. Tear and serum samples were collected to determine cytokines IL2, IL-4, IL-5, IFN-g, TNF-a, IL-10 by cytometric bead arrays (CBA), following manufacturers instructions. Results After 6 months of treatment with SLIT we observed significant higher IFN-g concentration, without significant changes in IL2, IL4, IL5, TNFa or IL10. We observed significant clinical improvement since 3 months of treatment and it was maintained until the end of 6 months. Clinical improvement correlated with IFN-g concentration. Conclusions Clinical outcome in AC-patients treated with SLIT could be tear IFN-g dependent.

57 Increased Frequency of CD4+ CCR4+ CCR9+ Cells in Patients With Allergic Conjunctivitis.

Background Allergic conjunctivitis is one of the most common diseases affecting the ocular surface, it has been suggested that T CD4+ cells regulate immune response in allergic diseases such as asthma and rhinitis, in a predominant Th2 response. In animal models, it has been observed a selective migration of CD4+ T cells to conjunctiva directed by chemokines; however molecules involved in CD4+ T cell migration in humans is unknown, thus it was the aim of this study. Methods Peripheral blood mononuclear cells (PBMC) were isolated from blood samples of healthy donors (HD) and AC-patients. PBMC were labeled with mAbs against CD4, CCR4, CCR5, and CRR9, and then labeled cells were analyzed by flow cytometry. T test was used to perform statistical analysis, P < 0.05 were considered statistically significant. Results We observed increased frequency of CCR4+ and CCR9+ on PBMC cells; interestingly, expression of CCR4+ was 1.46 times increased on CD4+ T cells of AC-patients compared to CD4+ T cells of HD (P = 0.01). Similarly, we observed higher frequency of CCR9 expression on CD4+ cells of AC-patients than on CD4+ T cells of HD (P = 0.01). On the other hand, CCR5 expression was diminished on PBMC from AC-patients than in HD (P = 0.0002). Conclusions Increased frequency of CD4+ CCR4+ CCR9+ was observed in AC patients with diminished frequency of CCR5 expression on PBMC. CCR4 and CCR9 have been involved in inflammatory process such arthritis and asthma, both could be related to inflammatory reaction at conjunctiva. CCR5 expression is mainly on Th1 cells, diminished frequency on PBMC in allergic conjunctivitis patients could be related with imbalance of immune response favoring a Th2 chronic inflammation.