Salvador Martínez-Cairo

Intracellular expression of interleukin-4 and interferon-γ by a Mycobacterium tuberculosis antigen-stimulated CD4+ CD57+ T-cell subpopulation with memory phenotype in tuberculosis patients

In some chronic pathological conditions, antigen persistence activates and expands the CD4+ CD57+ T‐cell subset. The host immune response against tuberculosis infection is maintained through the continuous presence of antigen‐stimulated effector/memory helper T cells. To determine whether CD4+ CD57+ T cells were also expanded in human tuberculosis, we analysed (by flow cytometry) the phenotype of peripheral blood CD4+ T cells from 30 tuberculosis patients and 30 healthy controls. We observed a significant increase in the CD4+ CD57+ T‐cell subset in tuberculosis patients in comparison to healthy controls (P < 0·001). Most CD4+ CD57+ T cells exhibited a CD28− CD45RO+ CD62L− phenotype, which is associated with memory cells. In vitro, a higher number of antigen‐stimulated CD4+ CD57+ T cells produced intracellular interferon‐γ and interleukin‐4 compared with antigen‐stimulated CD4+ CD57− T cells (P < 0·001). These findings suggest that the majority of CD4+ CD57+ T cells correspond to a phenotype of activated memory T cells.

Chemical characterization of the lectin from Amaranthus leucocarpus syn. hypocondriacus by 2-D proteome analysis.

In this work, we characterized chemically the N-acetyl-D-galactosamine specific lectin from Amaranthus leucocarpus syn hypocondriacus lectin (ALL). It is a dimeric glycoprotein composed by three isoforms with pl at 4.8, 4.9, and 5.2. Circular dichroism analysis indicated that the secondary structure of ALL contains 45% of β-sheet and 5% of α-helix. Amino acid sequence of the purified lectin and its isoforms was determined from peptides obtained after trypsin digestion by MALDI-TOF (Matrix assisted laser desorption ionization-time of flight). The tryptic peptides prepared from the purified lectin and the three isoforms showed different degrees (80 to 83%) of identity with the amino acid sequence belonging to a previously described high nutritional value protein from A. hypocondriacus not shown at the time to be a lectin. Furthermore, analysis of tryptic peptides obtained from ALL previously treated with peptide N-glycosidase, revealed a 93% identity with the aforementioned protein. Presence of N-glycosidically linked glycans of the oligomannosidic type and, in minor proportion, of the N-acetyllactosaminic type glycans was determined by affinity chromatography on immobilized Con A.

Relevance of sialoglycoconjugates in murine thymocytes during maturation and selection in the thymus

Differentiation of most T lymphocytes is characterized not only by the variable expression of CD4/CD8 coreceptor molecules and increased surface density of the T cell antigen receptor, but also by changes in the glycosylation pattern of cell surface glycolipids or glycoproteins. In this work we evaluated the changes in the sialylation pattern in thymus sections from normal and dexamethasone treated mice. We used sialic acid specific lectins, such as Sambucus nigra agglutinin (SNA, NeuAcalpha2,6-Gal specific) and Maackia amurensis agglutinin (MAA, NeuAcalpha2,3-Gal specific). Our results indicate that the sialylation pattern was modified during the maturation process of thymic cells. The immature CD4-CD8- and CD4+CD8+ cortical thymocytes were recognized by SNA, whereas the mature single positive (CD4+ or CD8+) medullary cells, preferentially bound MAA lectin. However, in the corticomedullary region we found not only SNA+ cells, but also MAA+ cells. In the thymus of dexamethasone treated mice, the clusters of thymocytes undergoing apoptosis in the cortex were characteristically stained by SNA. These results suggest that in the initial stages of the differentiation pathway, a great number of thymocytes express an alpha2,6 linked sialic acid on their surface and as they progress to more mature stages there is a change in the sialylation pattern to alpha2,3 linked sialic acids probably due to a regulated expression of different sialyltransferases, which could be modulated by the thymic microenvironment.

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.

Differential expression of a 70 kDa O-glycoprotein on T cells: a possible marker for naive and early activated murine T cells.

We purified a 70 kDa O-glycoprotein that binds to the GalNAc specific lectin from Amaranthus leucocarpus (ALLr) and determined its expression pattern on T lymphocytes from different murine lymphoid organs. High level of ALLr expression was demonstrated in 95-98% of both CD4(+)8(+) and CD4(-)8(+) thymocytes, and in 80-95% of CD8(+) T cells from peripheral blood, lymph nodes, and spleen, whereas a minor fraction of CD4(+)8(-) thymocytes (46-67%) and peripheral CD4(+) T cells (9-40%) showed low ALLr expression. Peripheral CD19(+) B cells were ALLr negative and most of the peripheral ALL(+) T cells showed a CD62L(hi)CD45RB(hi)CD44(lo/-) phenotype, indicating features of naive cells. Mitogenic activation of peripheral T cells increased 3-fold the number of ALL(+)CD4(+) T cells 24 h after stimulation, as opposed to a >80% decrease in CD8(+) T cells 72 h after stimulation. Our results suggest that ALL detects a non-described surface O-glycoprotein selectively expressed by naive CD8(+) T cells and by early activated CD4(+) T cells.

Identification of lectin isoforms in juvenile freshwater prawns Macrobrachium rosenbergii (DeMan, 1879)

From the serum of juvenile freshwater prawns, we isolated by affinity chromatography on glutaraldehyde-fixed rat erythrocytes stroma, immobilized in Sephadex G-25, a sialic acid specific lectin of 9.6[emsp4 ]kDa per subunit. Comparative analysis against adult organisms purified lectin, by chromatofocusing, showed that the lectin from juvenile specimens is composed by four main isoforms with a pl of 4.2, 4.6, 5.1, and 5.6, whereas the lectin from adults is eluted at pH 4.2. The amino acid composition of the lectin obtained from adult and juvenile stages suggest identity, but the compositions are not identical since a higher content of carbohydrates was found in the lectin from younger organisms. The freshwater prawn lectin showed specificity toward N-acetylated amino sugar residues such as GlcNAc, GalNAc, Neu5Ac and Neu5,9Ac; but in juvenile organisms the lectin showed three times less hemagglutinating activity than the lectin from adults. Both lectins agglutinated rat, rabbit and chicken erythrocytes, indicating that Neu5,9Ac in specific O-glycosydically linked glycans seems to be relevant for the interaction of M. rosenbergii lectins with their specific cellular receptor. Our results suggest that the physicochemical characteristics of the lectin from the freshwater prawn are regulated through maturation.

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.

Comparative Analysis of Mononuclear Cell Surface Markers in Atopic Processes‐A Preliminary Study

Atopic disorders are driven by the Th2 cell subset. We have determined the expression of costimulatory molecules and cell surface markers on peripheral CD4 + T cells and antigen presenting cells, in different atopic diseases, and we have also tried to correlate the expression of these markers with the severity of the disease. Cells from patients with atopic and contact dermatitis, mild or severe asthma, and symptomatic and non‐symptomatic atopic rhinitis were analyzed by flow cytometry. Our results showed that CD30, CD124, and CD152 expression on CD4 + T cells was significantly higher in atopic dermatitis than in contact dermatitis patients (p < 0.05). It was interesting to observe that the cell surface expression of CD80 in T and B cells from atopic dermatitis patients was not enhanced as opposed to the other atopic diseases we analyzed. Our results suggest that there are differences in the immune mechanisms involved in the different atopic diseases, and that expression of CD30 in CD4 + T cells might be a marker of disease activity in atopic dermatitis.