Can Human Oral Mucosa Stem Cells Differentiate to Corneal Epithelia?

Human oral mucosa stem cells (hOMSCs) arise from the neural crest, they can self-renew, proliferate, and differentiate to several cell lines and could represent a good source for application in tissue engineering. Because of their anatomical location, hOMSCs are easy to isolate, have multilineage differentiation capacity and express embryonic stem cells markers such as-Sox2, Oct3/4 and Nanog. We have used SHEM (supplemented hormonal epithelial medium) media and cultured hOMSCs over human amniotic membrane and determined the cell's capacity to differentiate to an epithelial-like phenotype and to express corneal specific epithelial markers-CK3, CK12, CK19, Pan-cadherin and E-cadherin. Our results showed that hOMSCs possess the capacity to attach to the amniotic membrane and express CK3, CK19, Pan-Cadherin and E-Cadherin without induction with SHEM media and expressed CK12 or changed the expression pattern of E-Cadherin to a punctual-like feature when treated with SHEM media. The results observed in this study show that hOMSCs possess the potential to differentiate toward epithelial cells. In conclusion, our results revealed that hOMSCs readily express markers for corneal determination and could provide the ophthalmology field with a therapeutic alternative for tissue engineering to achieve corneal replacement when compared with other techniques. Nevertheless, further studies are needed to develop a predictable therapeutic alternative for cornea replacement.

Differential Expression of O-Glycans in CD4(+) T Lymphocytes from Patients with Systemic Lupus Erythematosus.

T cells from patients with systemic lupus erythematosus (SLE) show a decreased activation threshold and increased apoptosis. These processes seem to be regulated by glycosylated molecules on the T cell surface. Here, we determined through flow cytometry the expression of mucin-type O-glycans on T helper cells in peripheral blood mononuclear cells (PBMC) from 23 SLE patients and its relation with disease activity. We used lectins specific for the disaccharide Gal-GalNAc, such as Amaranthus leucocarpus lectin (ALL), Artocarpus integrifolia lectin (jacalin) and Arachis hypogaea lectin (peanut agglutinin, PNA), as well as lectins for sialic acid such as Sambucus nigra agglutinin (SNA) and Maakia amurensis agglutinin (MAA). The results showed that ALL, but not jacalin or PNA, identified significant differences in O-glycan expression on T helper cells from active SLE patients (n = 10). Moreover, an inverse correlation was found between the frequency of T helper cells recognized by ALL and SLE Disease Activity Index (SLEDAI) score in SLE patients. In contrast, SNA and MAA lectins did not identify any differences between CD4(+) T cells from SLE patients. There was no difference in the recognition by ALL on activated T helper cells and T regulatory (Treg) cells. Our findings point out that activation of SLE disease diminishes the expression of O-glycans in T helper cells; ALL could be considered as a marker to determine activity of the disease.

Expression Dynamics of the O-Glycosylated Proteins Recognized by Amaranthus leucocarpus Lectin in T Lymphocytes and Its Relationship With Moesin as an Alternative Mechanism of Cell Activation.

T lymphocyte activation begins with antigen/MHC recognition by the TCR/CD3 complex followed by a costimulatory signal provided by CD28. The search for novel costimulatory molecules has been extensive due to their potential use as immunotherapeutic targets. Although some molecules have been identified, they are unable to provide sustainable signaling to allow for proper T cell activation and proliferation. It has been shown that the Amaranthus leucocarpus lectin (ALL) can be used as an in vitro costimulator of CD4+ lymphocytes in the presence of anti-CD3 mAb; this lectin specifically recognizes O-glycans of the Galß1-3GalNAc-O-Ser/Thr type, including a 70-kDa moesin-like protein that has been suggested as the costimulatory molecule. However, the identity of this molecule has not been confirmed and such costimulation has not been analyzed in CD8+ lymphocytes. We show herein that the expression kinetics of the glycoproteins recognized by ALL (gpALL) is different in CD4+ and CD8+ T cells, unlike moesin expression. Results from IP experiments demonstrate that the previously described 70-kDa moesin-like protein is an O-glycosylated form of moesin (O-moesin) and that in vitro stimulation with anti-CD3 and anti-moesin mAb induces expression of the activation molecules CD69 and CD25, proliferation and IL-2 production as efficiently as cells costimulated with ALL or anti-CD28. Overall, our results demonstrate that O-moesin is expressed in CD4+ and CD8+ T lymphocytes and that moesin provides a new costimulatory activation signal in both T cell subsets.

Relevance of glycans in the interaction between T lymphocyte and the antigen presenting cell.

The immunological synapse promotes receptors and ligands interaction in the contact interface between the T lymphocyte and the antigen presenting cell; glycosylation of the proteins involved in this biological process favors regulation of molecular interactions and development of the T lymphocyte effector response. Glycans in the immunological synapse influence cellular and molecular processes such as folding, expression, and structural stability of proteins, they also mediate ligand-receptor interaction and propagation of the intracellular signaling or inhibition of uncontrolled cellular activation that could lead to the development of autoimmunity, among others. It has been suggested that altered glycosylation of proteins that participate in the immunological synapse affects the signaling processes and cell proliferation, as well as exacerbation of the effector mechanisms of T cells that trigger systemic damage and autoimmunity. Understanding the role of glycans in the immune response has allowed for advances in the development of immunotherapies in different fields through the controlled and specific activation of the immune response. This review describes the structural and biological aspects of glycans associated with some molecules present in the immunological synapse, providing information that allows understanding the function of glycosylation in the interaction between the T lymphocyte and the antigen-presenting cell, as well as its impact on signaling and development regulation of T lymphocytes effector response.

Reduction of Foxp3+ cells by depletion with the PC61 mAb induces mortality in resistant BALB/c mice infected with Toxoplasma gondii.

Regulatory T cells (Tregs) are CD4(+)Foxp3(+) cells that modulate autoimmune responses. Tregs have been shown to be also involved during the immune response against infectious agents. The aim of this work is to study the role of Tregs during the infection with the intracellular protozoan Toxoplasma gondii. Resistant BALB/c mice were injected with 200 microg of anti-CD25 mAb (clone PC61) and 2 days later they were infected with 20 cysts of the ME49 strain of T. gondii. We observed that depleted mice showed 50-60% mortality during the acute infection. When FACS analysis was carried out, we observed that although injection of PC61 mAb eliminated 50% of Tregs, infected-depleted mice showed a similar percentage of CD25(+)Foxp3(-) (activated T cells, Tact) to those observed in infected nondepleted animals, demonstrating that in our depletion/infection system, injection of PC61 mAb did not hamper T cell activation while percentage of Tregs was reduced by 75% 10 days post infection. We concluded that Tregs are essential during protection in the acute phase of T. gondii infection.

Differential effect of sodium arsenite during the activation of human CD4+ and CD8+ T lymphocytes.

Contamination of water with arsenic is a problem affecting several regions of the world. Peripheral blood mononuclear cells (PBMC) from chronically exposed individuals show a lower replicating activity than non-exposed individuals when stimulated with phytohemagglutinin (PHA). We have previously reported that PBMC from healthy donors treated in vitro with 1 muM sodium arsenite (NaAsO2) and stimulated with PHA showed a reduction in proliferation by a delay in cell cycle entry and a decrease in the rounds of cell division. In this paper we tested the effect of 1-5 muM NaAsO2 on the proliferation, viability, blast transformation, expression of the CD4 and CD8 molecules, and during the activation and proliferation of both CD4+ and CD8+ T lymphocytes. We found a reduction in cell proliferation and an increase in non-dividing cells with higher concentrations of NaAsO2 (2-5 microM) when proliferation was studied by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution. The use of 7-aminoactinomycin D (7-AAD) in CFSE-labeled cells allowed us to detect an increase in percentage of non-dividing cells, and an increase in apoptotic/dead cells mainly in non-proliferating cells. Analysis of the expression of CD4 and CD8 molecules on these cells showed that concentrations > or = 2 microM NaAsO2 reduced the expression of the CD8 molecule and induced apoptosis/death in CD4+ cells. Analysis of blast transformation by flow cytometry showed an accumulation of CD8+ resting cells in the presence of NaAsO2. Analysis of CD25 and CD69 expression in kinetics experiments in both subtypes showed a delay in the expression of CD25 and a delay in the downregulation of the CD69 molecule, in both CD4+ and CD8+ cells. However, in the case of CD8+ cells, we detected an accumulation of a CD25- CD69- population in the presence of increasing concentrations of NaAsO2. Altogether, our results show that NaAsO2 alters the expression kinetics of the early activation molecules CD25 and CD69 similarly in both subtypes. In addition, activated and non-activated CD4+ cells die by apoptotic mechanisms and although a percentage of CD8+ cells also die by apoptosis, a subpopulation of these cells is unable to activate and thus accumulates as resting cells.

Sodium arsenite retards proliferation of PHA-activated T cells by delaying the production and secretion of IL-2.

Arsenic is a metalloid that commonly contaminates drinking water, and is a known human carcinogen. It has been shown that peripheral blood mononuclear cells (PBMCs) from healthy donors treated in vitro with NaAsO(2) and stimulated with phytohemagglutinin (PHA) show a lower proliferation than nontreated cells. We reported previously a reduction in the secretion of IL-2 in NaAsO(2)-treated PBMCs stimulated with PHA, an observation that might explain, in part, the reduction in proliferation. Since arsenic induces cytoskeleton alterations, which in turn may affect protein transport of the cell, we assumed that NaAsO(2) induced an accumulation of IL-2 inside the cells, and thus a reduction in the secretion of IL-2. In order to demonstrate this hypothesis, we assessed the intracellular IL-2 at the single cell level by flow cytometry, and unexpectedly found a reduction in the percentage of IL-2 producing T cells in the presence of NaAsO(2). We tracked the proliferation of T cells by using the 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dye and found that NaAsO(2) slows down the entrance to cell division and delays the proliferation of cells that have already entered the cell cycle. Nevertheless, the expression of the activation molecules, CD25 and CD69, was unaltered. Assessment of the intracellular and secreted IL-2 in kinetic experiments showed that in fact, NaAsO(2) delays the production of IL-2, given that a recovery of both intracellular and secreted IL-2 was detected at 72 h. Evaluation of the cell cycle showed a higher proportion of cells in G(0)/G(1) and a lower proportion in G(2)/M in the presence of NaAsO(2). We thus conclude that NaAsO(2) reduces proliferation of T cells by delaying the production and secretion of IL-2, thus blocking T cells in G(1); as a consequence, the entry to cell cycle and the rounds of cell division are retarded, and a lower proliferation of T cells is hence observed.

Depletion with PC61 mAb before Toxoplasma gondii infection eliminates mainly Tregs in BALB/c mice, but activated cells in C57BL/6J mice.

Analysis of regulatory T cells (Tregs) in vivo during infection is crucial for the understanding of immune response modulation. Depletion experiments using anti-CD25 monoclonal antibody (mAb) in order to eliminate Tregs have been widely used for this purpose despite the fact that this approach may also lead to the elimination of activated T cells. We show in this paper that treatment with anti-CD25 mAb before Toxoplasma gondii infection eliminates a different pattern of cell subsets in the resistant BALB/c and the susceptible C57BL/6J mouse strain. Injection with PC61 mAb leads to the elimination of most Tregs in BALB/c mice, while in C57BL/6J animals, treatment depletes other activated subsets [natural killer (NK), B and CD4(+) T cells]. This difference is a consequence of the dramatic cell activation observed in the latter, but not in the former strain. The different effect of the depletion reported here demonstrates that careful analysis in each model is mandatory in order to avoid misleading conclusions.