The Peritoneum: Health, Disease, and Perspectives regarding Tissue Engineering and Cell Therapies.

There are several pathologies associated with the peritoneum, such as mesothelioma and peritonitis. Moreover, the peritoneum is widely used in ultrafiltration procedures, i.e., peritoneal dialysis, presenting advantages over hemodialysis. On the other hand, ultrafiltration failure may lead to dialysis-induced fibrosis and hypervolemia. Therefore, the pathophysiological study of this tissue is of extreme biomedical importance. Studies investigating the biology of the cells dwelling in the peritoneum wall provide evidence of their plasticity and progenitor features. For instance, both mesothelial and submesothelial cells present characteristics similar to mesenchymal stem cells, including osteogenic and adipogenic differentiation potential, support of extramedullary hematopoiesis, modulation of inflammatory responses, and regulation of tumor progression. Indeed, the participation of each cell type in peritoneal pathological and physiological phenomena is still under debate, especially regarding a possible differentiation pathway connecting these peritoneal cells. The primary aim of this review is to raise this discussion. In order to do so, we will firstly provide an overview of the peritoneum anatomy, histology, and ontology, and finally we will address how a better understanding of peritoneal cell biology may contribute to future cell therapy and tissue engineering approaches.

Lack of galectin-3 up-regulates IgA expression by peritoneal B1 lymphocytes during B cell differentiation.

Galectin-3 is a ß-galactoside-binding protein with an inhibitory role in B cell differentiation into plasma cells in distinct lymphoid tissues. We use a model of chronic schistosomiasis, a well-characterized experimental disease hallmarked by polyclonal B cell activation, in order to investigate the role of galectin-3 in controlling IgA production through peritoneal B1 cells. Chronically infected, galectin-3-deficient mice (Lgals3(-/-)) display peritoneal fluid hypercellularity, increased numbers of atypical peritoneal IgM(+)/IgA(+) B1a and B1b lymphocytes and histological disturbances in plasma cell niches when compared with Lgals3(+/+) mice. Similar to our infection model, peritoneal B1 cells from uninfected Lgals3(-/-) mice show enhanced switching to IgA after in vitro treatment with interleukin-5 plus transforming growth factor-ß (IL-5 + TGF-ß1). A higher number of IgA(+) B1a lymphocytes was found in the peritoneal cavity of Lgals3(-/-)-uninfected mice at 1 week after i.p. injection of IL-5 + TGF-ß1; this correlates with the increased levels of secreted IgA detected in the peritoneal fluid of these mice after cytokine treatment. Interestingly, a higher number of degranulated mast cells is present in the peritoneal cavity of uninfected and Schistosoma mansoni-infected Lgals3(-/-) mice, indicating that, at least in part, mast cells account for the enhanced differentiation of B1 into IgA-producing B cells found in the absence of galectin-3. Thus, a novel role is revealed for galectin-3 in controlling the expression of surface IgA by peritoneal B1 lymphocytes; this might have important implications for manipulating the mucosal immune response.

Lack of galectin-3 disturbs mesenteric lymph node homeostasis and B cell niches in the course of Schistosoma mansoni infection.

Galectin-3 is a ß-galactoside-binding protein that has been shown to regulate pathophysiological processes, including cellular activation, differentiation and apoptosis. Recently, we showed that galectin-3 acts as a potent inhibitor of B cell differentiation into plasma cells. Here, we have investigated whether galectin-3 interferes with the lymphoid organization of B cell compartments in mesenteric lymph nodes (MLNs) during chronic schistosomiasis, using WT and galectin-3(-/-) mice. Schistosoma mansoni synthesizes GalNAcß1-4(Fucα1-3)GlcNAc(Lac-DiNAc) structures (N-acetylgalactosamine ß1-4 N-acetylglucosamine), which are known to interact with galectin-3 and elicit an intense humoral response. Antigens derived from the eggs and adult worms are continuously drained to MLNs and induce a polyclonal B cell activation. In the present work, we observed that chronically-infected galectin-3(-/-) mice exhibited a significant reduced amount of macrophages and B lymphocytes followed by drastic histological changes in B lymphocyte and plasma cell niches in the MLNs. The lack of galectin-3 favored an increase in the lymphoid follicle number, but made follicular cells more susceptible to apoptotic stimuli. There were an excessive quantity of apoptotic bodies, higher number of annexin V(+)/PI(-) cells, and reduced clearance of follicular apoptotic cells in the course of schistosomiasis. Here, we observed that galectin-3 was expressed in non-lymphoid follicular cells and its absence was associated with severe damage to tissue architecture. Thus, we convey new information on the role of galectin-3 in regulation of histological events associated with B lymphocyte and plasma cell niches, apoptosis, phagocytosis and cell cycle properties in the MLNs of mice challenged with S.mansoni.

Fine structure and molecular content of human chondrocytes encapsulated in alginate beads.

Preservation of the chondrocytic phenotype in vitro requires a 3D (three-dimensional) culture model. Diverse biomaterials have been tested as scaffolds for culture of animal chondrocytes; however, to date, none is considered a gold standard in regenerative medicine. Here, we studied the fine structure and the GAGs (glycosaminoglycans) content of human chondrocytes encapsulated in alginate beads by using electron microscopy and radioactive sulfate [35S] incorporation, respectively. Cells were obtained from human cartilage, encapsulated in alginate beads and cultured for 28 days. [35S]Na2SO4 was added to the culture media and later isolated for quantification of the sulfated GAGs found in three compartments: IC (intracellular), IB (intra-bead) and EB (extra-bead). Round cells were seen isolated or forming small groups throughout the alginate. Human chondrocytes presented the features of active cells such as euchromatic nuclei, abundant RER (rough endoplasmic reticulum) and many transport vesicles. We observed an extracellular matrix rich in collagen fibres and electrondense material adjacent to the cells. Most of the GAGs produced (74%) were found in the culture medium (EB), indicating that alginate has a limited capacity to retain the GAGs. CS (chondroitin sulfate), the major component of aggrecan, was the most prominent GAG produced by the encapsulated cells. Human chondrocytes cultured in alginate can sustain their phenotype, confirming the potential application of this biomaterial for cartilage engineering.

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.

Biochemical characterization of heparan sulfate derived from murine hemopoietic stromal cell lines: a bone marrow-derived cell line S17 and a fetal liver-derived cell line AFT024.

Heparan sulfate (HS) present on the surface of hemopoietic stromal cells has important roles in the control of adhesion and growth of hemopoietic stem and progenitor cells. Recent studies have characterized several different heparan sulfate proteoglycans (HSPGs) from both human and murine bone marrow stromal cells. In the present study, we have compared the molecular structure of HS, metabolically labeled with [(35)S]-sulfate produced by two distinct preparations of murine hemopoietic stromal cell lines. These comprised a bone marrow-derived cell line S17 and a fetal liver-derived cell line AFT024. [(35)S]-HS was examined in the cell layers and in the culture medium. We identified and measured the relative proportions of the various glycosaminoglycans (GAGs) in the two stromal cell lines. Chondroitin sulfate (CS) was preponderantly secreted by the stromal cell lines, while HS was relatively more abundant in the cell-associated fractions. The two types of stromal cells differ in their HS composition, mainly due to different patterns of N- and O-sulfation. The two stromal cell lines expressed mRNA for different HSPGs. Data from reverse transcription PCR revealed that the two stromal cell lines expressed mRNA for glypican and syndecan4. Only AFT024 cell line expressed mRNA for betaglycan. There was no evidence for expression of mRNA for both syndecan1 and syndecan2. [(35)S]-sulfated macromolecules could be released from the cell surface of both stromal cell lines by phosphatidylinositol phospholipase C (PI-PLC), which is consistent with the expression of glypican detected by PCR experiments.

Effect of magnesium chloride and guanidinum chloride on the extraction of components of extracellular matrix from chicken cartilage

In order to evaluate the effect of chaotropic agents on proteoglycan and non-collagenous proteins, chicken xiphoid cartilage was treated with guanidine-HCI and MgCl2 in different concentrations (1M to 5M), and different periods of time (12, 24, 48 and 72hr). The maximum yield of uronic acid was obtained with 3M MgCl2 (73.3 per cent). Concentrations of 4M and 5M of MgCl2 showed that much less uronic acid was removed, 55.3 per cent and 38.1 respectively. Extraction with 3M MgCl2 and 3M guanidine-HCl resulted better efficiency when performed for 48 hr. Analysis by SDS-PAGE of the extracts obtained with guanidine-HCl and MgCl, in different concentrations pointed out that most components are equally removed with the two solvents, showing that the extraction with MgCl2 is an alternative assay to remove non-collagenous proteins from extracellular matrix