RESUMO
Hydrolyzed soybean isolates SP-A and SP-B (Abbott Laboratories, OH), developed for use in enteral nutritional products, were tested in cultures of C2BBe cells, a colonic adenocarcinoma cell line with enterocytic differentiation, to evaluate effects on cell growth, maturation and ability to resist infection by enteric bacteria. SP-A delayed formation of confluent monolayers by 10 d compared with cells cultured without SP-A. SP-A also caused a retardation in the development of intercellular tight junctions as measured by transmonolayer electrical resistance (TER). SP-B had no effect on cell proliferation or TER of intestinal cell cultures. SP-A and SP-B enhanced the development of the brush border enzymes alkaline phosphatase and isomaltase over a 28 d period. By these criteria, SP-A and SP-B appear to affect intestinal epithelial cell development in culture. When C2BBe monolayers were exposed to the enteric bacteria, Salmonella typhimurium or Salmonella typhi, an inhibition of the passage of S. typhi was seen in cultures with SP-A and SP-B. No effect on the passage of S. typhimurium was seen with either soy isolate. Partially purified soy isolates therefore impart resistance to selected enteroinvasive bacteria. Addition of soy hydrolysates to the media of cultured intestinal cells may serve as a rapid and economical screening mechanism for preclinical trials that would test the therapeutic benefits of soybean isolates.
Assuntos
Translocação Bacteriana/efeitos dos fármacos , Hidrolisados de Proteína/farmacologia , Salmonella/fisiologia , Proteínas de Soja/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Adenocarcinoma/microbiologia , Adenocarcinoma/patologia , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/microbiologia , Neoplasias do Colo/patologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Humanos , Salmonella/efeitos dos fármacos , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/microbiologiaRESUMO
Bacterial growth chambers of Transwell units bearing intestinal epithelial monolayers (C2BBe) was consistently observed to be stagnant during the course of transmigration studies with Salmonella typhi. This limitation could not be explained by varying the bacterial load in the inoculum. Conditioned media produced by cultured C2BBe cells would not support bacterial growth. Growth support of the media was restored by heating to 95 C for 10 min. C2BBe conditioned media had bacteriostatic activity for a large variety of gram-negative, enteropathogenic bacteria but had no effect on gram-positive bacteria.
Assuntos
Antibacterianos/farmacologia , Meios de Cultivo Condicionados/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Células Cultivadas , Escherichia coli/efeitos dos fármacos , Bactérias Gram-Negativas/patogenicidade , Mucosa Intestinal/citologia , Pseudomonas aeruginosa/efeitos dos fármacos , Salmonella/efeitos dos fármacosRESUMO
This study characterizes the transmigration of enteroinvasive Salmonella typhi in vitro, using a human intestinal epithelial cell line as a model of small intestinal epithelium. C2BBe cells, a subclone of CACO-2 with a highly differentiated enterocytic phenotype, were grown to maturity on Transwell filters. S. typhi Ty2 and the vaccine strain, Ty21a, the S. typhi mutant X7344 and parent strain SB130, and S. typhimurium 5771 in logarithmic phase were introduced to the upper chamber of the filter units. Numbers of bacteria in the lower chamber, TER and permeability of the monolayer to mannitol were measured over time. Monolayers were examined by light, electron and confocal microscopy to determine the pathway of bacterial transmigration, and intracellular bacteria were estimated by gentamicin assay. Epithelial cell injury was quantified by light microscopy. S. typhi transmigrated earlier and in larger numbers than S. typhimurium, inducing marked changes in electrical resistance and permeability. Unlike S. typhimurium, S. typhi selected epithelial cells in small number and caused their death and extrusion from the monolayers leaving holes through which S. typhi transmigrated. Ty2 consistently transmigrated in larger numbers and with more injury to monolayers than Ty21a. S. typhi crosses the monolayers of C2BBe cells by a paracellular route in contrast to the transcellular pathway described for other Salmonellae. This may be related to the unique pathophysiology of S. typhi infection and the restricted host specificity of this pathogen. In these assays the vaccine strain, Ty21a, is slightly less invasive than its parent, though more invasive than S. typhimurium.
Assuntos
Translocação Bacteriana , Mucosa Intestinal/microbiologia , Salmonella typhi/fisiologia , Salmonella typhi/patogenicidade , Humanos , Mucosa Intestinal/fisiologia , Mucosa Intestinal/ultraestrutura , Microscopia Eletrônica , Movimento , Permeabilidade , Salmonella typhimurium/fisiologia , Células Tumorais Cultivadas , VirulênciaRESUMO
Picryl (trinitrophenyl) chloride (PCL) contact sensitization of mice induces T cells that release an antigen-binding T cell factor (PCLF) that plays an important role in the initiation of contact sensitivity responses, in part via activation of mast cells. The current study employs an in vitro indirect rosette assay to demonstrate that PCLF can interact with the mast cell surface. Sheep red blood cells (SRBC) were hapten conjugated with trinitrophenyl (TNP), dinitrophenyl (DNP), or oxazolone (OX). When TNP-conjugated SRBC were coated with PCLF, monoclonal anti-DNP IgE, or anti-DNP IgG1, they produced 40 to 50% rosettes with purified normal mouse peritoneal mast cells. Analogous antigen-binding factors, from lymphoid cells of OX and dinitrofluorobenzene contact-sensitized mice, gave similar mast cell rosetting levels with OX-SRBC and DNP-SRBC, respectively. PCLF demonstrated a high degree of hapten specificity in that it formed rosettes with TNP-SRBC but not with DNP-SRBC, unlike IgE and IgG1, or DNPF, which formed rosettes with either SRBC type. Similarly, soluble TNP-BSA could inhibit PCLF rosette-forming capacity, but soluble DNP-BSA could not. In addition to mouse mast cells, PCLF formed rosettes with rat basophil leukemia cells, mouse peritoneal exudate macrophages, mouse alveolar macrophages, and J 774 cultured mouse macrophages; it did not form rosettes with rat mast cells, rat alveolar macrophages, or mouse spleen cells. Thus, PCLF-formed rosettes were antigen specific, relatively species specific, and mast cell/macrophage specific. PCLF-mediated rosette-forming activity could be detected in the presence of nanogram quantities of PCLF. More than 10 times greater IgE was needed to produce IgE-mediated rosettes. Reduction and alkylation eliminated the rosetting activity of IgE, but the rosetting activity of PCLF was not affected. PCLF, but not IgE rosette-forming activity, could be removed by and eluted from affinity columns linked with a monoclonal antibody specific for T cell-derived antigen-binding factors, whereas PCLF rosetting activity was not retained by an anti-immunoglobulin affinity column. Preincubation of mast cells with rat myeloma IgE or mouse monoclonal IgE of various specificities blocked IgE rosettes but not PCLF-induced rosettes. Other immunoglobulin isotypes likewise did not block PCLF rosettes. However, PCLF rosettes could be blocked by preincubation of mast cells with OX factor (OXF),and OXF-mediated rosettes could be blocked similarly by PCLF. These results suggest that the antigen-binding T cell factor PCLF interacts with a unique receptor on the surface of mouse mast cells.
Assuntos
Epitopos/imunologia , Linfocinas/metabolismo , Mastócitos/metabolismo , Proteínas Secretadas pela Próstata , Formação de Roseta/métodos , Linfócitos T/metabolismo , Animais , Afinidade de Anticorpos , Ligação Competitiva , Imunoglobulina E/metabolismo , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Masculino , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos CBA , Cloreto de Picrila/farmacologia , Ratos , Linfócitos T/imunologiaRESUMO
Pretreatment of mast cells with various psychotropic agents was shown to permit preferential release of serotonin without substantial release of histamine or massive degranulation. Differential release involved both endogenous, granule-stored serotonin, and exogenous radiolabeled serotonin that had been taken up by the cell. This phenomenon occurred in mast cells stimulated to secrete with suboptimal concentrations of the classic mast cell secretagogue compound 48/80, was associated with drugs of several different structures and known mechanisms of action, and could be inhibited by certain prostaglandins. Furthermore, differential release of serotonin occurred in mast cells of retired breeders without the use of drugs or other exogenous agents. Light microscopic studies of mast cells undergoing differential release showed minimal degranulation, indicating that most of the serotonin release did not occur via classic exocytosis. The ability of mast cell to selectively release serotonin, by a mechanism unlike that occurring in allergic anaphylactic secretion, constitutes one of the first instances of differential release from secretory cells, suggests a new mechanism of release of secretory products, and expands the potential role of mast cells in the pathophysiology of the body.
Assuntos
Liberação de Histamina , Mastócitos/metabolismo , Serotonina/metabolismo , Fatores Etários , Animais , Peso Corporal , Calmodulina/farmacologia , Grânulos Citoplasmáticos/metabolismo , Liberação de Histamina/efeitos dos fármacos , Técnicas In Vitro , Masculino , Prostaglandinas/farmacologia , Psicotrópicos/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Previous studies have shown that T cell-dependent activation of mast cells to release serotonin is required for the elicitation of delayed-type hypersensitivity in mice. We have recently described an antigen-binding T cell factor that is a suitable candidate for participation in the mechanism by which T cells activate mast cells in delayed-type hypersensitivity. The T cell factor transfers the ability to elicit an antigen-specific immediate hypersensitivity-like ear-swelling reaction following local challenge with antigen. In the current study, alterations in the morphology of local tissue mast cells and vessels were studied by light and electron microscopy at the time of optimal swelling and increase in vascular permeability. Factor-induced reactions showed mild changes in metachromatic staining of mast cell granules compared with more profound changes that were found in reactions of antigen-challenged mice that were sensitized by intravenous injection with IgE antibody. Subtle changes in the ultrastructure of mast cells in reactions induced by the T cell factor included surface activation to form filopodial extensions, resulting in a significant increase in the surface density by stereologic analysis. The cytoplasm of these mast cells also showed signs of synthetic and metabolic activity with formation of vesicles and an increased prominence of the Golgi apparatus and mitochondria. Local vessels at sites of reactions due to the T cell factor or IgE showed intravascular accumulation of polymorphonuclear leukocytes, gaps at sites of endothelial cell junctions, and occasional emigration of the leukocytes into the perivascular area. This indicates that a vasoactive factor, such as serotonin, and chemotactic factors were released in both instances. However, in recipients of the T cell factor, this was accomplished with only moderate signs of overt mast cell degranulation and loss of granule density. Instead, there was formation of vesicles at the outer margin of the granules, in the perigranular membrane, and in the cytoplasm, accompanied by the surface activation. In contrast, mast cells from reactions in IgE-sensitized animals appeared to degranulate by a process of sequential compound exocytosis with no vesicle formation or cytoplasmic findings of increased synthetic or metabolic activity. The granules of these cells showed a great loss of density and many appeared swollen, resulting in overall swelling and increase in area of the cell.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Permeabilidade Capilar , Hipersensibilidade Imediata/imunologia , Linfocinas/fisiologia , Mastócitos/imunologia , Proteínas Secretadas pela Próstata , Linfócitos T/imunologia , Animais , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Hipersensibilidade Imediata/patologia , Imunoglobulina E/administração & dosagem , Masculino , Mastócitos/ultraestrutura , Camundongos , Camundongos Endogâmicos CBA , Neutrófilos/patologia , Cloreto de Picrila/administração & dosagem , Vênulas/patologiaRESUMO
The biologically active fragment of human parathormone (PTH) and intact bovine PTH were found to induce secretion of both serotonin and histamine from rat peritoneal mast cells in vitro. Release of serotonin and histamine was demonstrated with 25 units/ml PTH or higher. This level is within the higher limits of the elevated PTH levels found in advanced uremia. Mast cell secretion by PTH was dose, time and energy dependent and was not cytotoxic. Although mast cell activation was independent of extracellular calcium, it required intracellular calcium, thus resembling the action of certain other peptide secretagogues. Intradermal injection of PTH induced immediate increases in vascular permeability suggesting that PTH could induce mast cell secretion in vivo. Light and electron microscopic observations confirmed mast cell degranulation by exocytosis. These results demonstrate that elevated levels of PTH can induce mast cell secretion in vitro and in vivo and suggest a possible role for mast cells in the pathophysiology of non-allergic disease states.