Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

ANAPHYLACTICALLY - MEDIATED EPITHELIAL CHLORINE ION SECRETORY RESPONSE INISOLATED JEJUNUM OF PARASITIZED GUINEA PIGS (TRICHINELLA SPIRALIS)

Electrophysiological studies were conducted to test the hypothesis that alterations in intestinal epithelial function are associated with immunological responses directed against the enteric parasite, Trichinella spirals. Trichinella antigens were used to challenge sensitized jejunum from infected guinea pigs while monitoring ion transport properties of the tissue in an Ussing-type chamber. The addition of antigen caused increases in transepithelial PD and I(,sc) that were rapidly induced, peaked at 1.5 to 2 min after antigen-challenge, and lasted 10 to 20 min thereafter. The increase in I(,sc) ((DELTA)I(,sc)) varied in a dose-dependent manner until a maximal increase of 40 (mu)A/cm('2) was obtained by the addition of 13 (mu)g of antigenic protein per ml of serosal fluid in the Ussing chamber. Trichinella antigen did not elicit alterations in either PD or I(,sc) of nonimmune tissue. Jejunal tissue from guinea pigs immunized with ovalbumin according to a protocol that stimulated homocytotropic antibody production responded electrically to challenge with ovalbumin but not trichinella antigen. Jejunal tissue which was passively sensitized with immune serum having a passive cutaneous anaphylaxis (PCA) titer of 32 for both IgE and IgG(,1) anti-trichinella anti-bodies responded electrically after exposure to trichinella antigen. Heat treatment of immune serum abolished the anti-trichinella IgE titer as determined by the PCA test but did not decrease either the electrical response of passively sensitized tissue to antigen or the anaphylactically mediated intestinal smooth muscle contractile response to antigen in the classical Schultz-Dale assay. These results strongly support the hypothesis that immunological responses directed against Trichinella Spiralis alter intestinal epithelial function and suggest that immediate hypersensitivity is the immunological basis of the response.^ Additional studies were performed to test the hypothesis that histamine and prostaglandins that are released from mucosal mast cells during IgE or IgG(,1) - antigen stimulated degranulation mediate electrophysiological changes in the intestinal epithelium that are reflective of Cl('-) secretion and mediated intracellularly by cAMP. Pharmacological and biochemical studies were performed to determine the physiological messengers and ionic basis of electrical alterations in small intestinal epithelium of the guinea pig during in vitro anaphylaxis. Results suggest that Cl('-) secretion mediated, in part, by cAMP contributes to antigen-induced jejunal ion transport changes and that histamine and prostaglandins are involved in eliciting epithelial responses. ^

Epithelial-intrinsic IKKα expression regulates group 3 innate lymphoid cell responses and antibacterial immunity

Innate lymphoid cells (ILCs) are critical for maintaining epithelial barrier integrity at mucosal surfaces; however, the tissue-specific factors that regulate ILC responses remain poorly characterized. Using mice with intestinal epithelial cell (IEC)-specific deletions in either inhibitor of κB kinase (IKK)α or IKKβ, two critical regulators of NFκB activation, we demonstrate that IEC-intrinsic IKKα expression selectively regulates group 3 ILC (ILC3)-dependent antibacterial immunity in the intestine. Although IKKβ(ΔIEC) mice efficiently controlled Citrobacter rodentium infection, IKKα(ΔIEC) mice exhibited severe intestinal inflammation, increased bacterial dissemination to peripheral organs, and increased host mortality. Consistent with weakened innate immunity to C. rodentium, IKKα(ΔIEC) mice displayed impaired IL-22 production by RORγt(+) ILC3s, and therapeutic delivery of rIL-22 or transfer of sort-purified IL-22-competent ILCs from control mice could protect IKKα(ΔIEC) mice from C. rodentium-induced morbidity. Defective ILC3 responses in IKKα(ΔIEC) mice were associated with overproduction of thymic stromal lymphopoietin (TSLP) by IECs, which negatively regulated IL-22 production by ILC3s and impaired innate immunity to C. rodentium. IEC-intrinsic IKKα expression was similarly critical for regulation of intestinal inflammation after chemically induced intestinal damage and colitis. Collectively, these data identify a previously unrecognized role for epithelial cell-intrinsic IKKα expression and TSLP in regulating ILC3 responses required to maintain intestinal barrier immunity.

The outer mucus layer hosts a distinct intestinal microbial niche

The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host.

Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3

Salt and water secretion from intestinal epithelia requires enhancement of anion permeability across the apical membrane of Cl− secreting cells lining the crypt, the secretory gland of the intestine. Paneth cells located at the base of the small intestinal crypt release enteric defensins (cryptdins) apically into the lumen. Because cryptdins are homologs of molecules known to form anion conductive pores in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of intestinal Cl− secreting epithelial cells in culture. Of the six peptides tested, cryptdins 2 and 3 stimulated Cl− secretion from polarized monolayers of human intestinal T84 cells. The response was reversible and dose dependent. In contrast, cryptdins 1, 4, 5, and 6 lacked this activity, demonstrating that Paneth cell defensins with very similar primary structures may exhibit a high degree of specificity in their capacity to elicit Cl− secretion. The secretory response was not inhibited by pretreatment with 8-phenyltheophyline (1 μM), or dependent on a concomitant rise in intracellular cAMP or cGMP, indicating that the apically located adenosine and guanylin receptors were not involved. On the other hand, cryptdin 3 elicited a secretory response that correlated with the establishment of an apically located anion conductive channel permeable to carboxyfluorescein. Thus cryptdins 2 and 3 can selectively permeabilize the apical cell membrane of epithelial cells in culture to elicit a physiologic Cl− secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues, and suggest a previously undescribed mechanism of paracrine signaling that in vivo may involve the reversible formation of ion conductive channels by peptides released into the crypt microenvironment.

Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver

The ability to identify, isolate, and transplant progenitor cells from solid tissues would greatly facilitate the treatment of diseases currently requiring whole organ transplantation. In this study, cell fractions enriched in candidate epithelial progenitor cells from the rat pancreas were isolated and transplanted into the liver of an inbred strain of Fischer rats. Using a dipeptidyl dipeptidase IV genetic marker system to follow the fate of transplanted cells in conjunction with albumin gene expression, we provide conclusive evidence that, after transplantation to the liver, epithelial progenitor cells from the pancreas differentiate into hepatocytes, express liver-specific proteins, and become fully integrated into the liver parenchymal structure. These studies demonstrate the presence of multipotent progenitor cells in the adult pancreas and establish a role for the liver microenvironment in the terminal differentiation of epithelial cells of foregut origin. They further suggest that such progenitor cells might be useful in studies of organ repopulation following acute or chronic liver injury.

Exploitation of host cells by enteropathogenic Escherichia coli

Microbial pathogens have evolved many ingenious ways to infect their hosts and cause disease, including the subversion and exploitation of target host cells. One such subversive microbe is enteropathogenic Escherichia coli (EPEC). A major cause of infantile diarrhea in developing countries, EPEC poses a significant health threat to children worldwide. Central to EPEC-mediated disease is its colonization of the intestinal epithelium. After initial adherence, EPEC causes the localized effacement of microvilli and intimately attaches to the host cell surface, forming characteristic attaching and effacing (A/E) lesions. Considered the prototype for a family of A/E lesion-causing bacteria, recent in vitro studies of EPEC have revolutionized our understanding of how these pathogens infect their hosts and cause disease. Intimate attachment requires the type III-mediated secretion of bacterial proteins, several of which are translocated directly into the infected cell, including the bacteria's own receptor (Tir). Binding to this membrane-bound, pathogen-derived protein permits EPEC to intimately attach to mammalian cells. The translocated EPEC proteins also activate signaling pathways within the underlying cell, causing the reorganization of the host actin cytoskeleton and the formation of pedestal-like structures beneath the adherent bacteria. This review explores what is known about EPEC's subversion of mammalian cell functions and how this knowledge has provided novel insights into bacterial pathogenesis and microbe-host interactions. Future studies of A/E pathogens in animal models should provide further insights into how EPEC exploits not only epithelial cells but other host cells, including those of the immune system, to cause diarrheal disease.

Forced expression of the tumor suppressor adenomatosis polyposis coli protein induces disordered cell migration in the intestinal epithelium.

Mutations of the human adenomatosis polyposis coli (APC) gene are associated with the development of familial as well as sporadic intestinal neoplasia. To examine the in vivo function of APC, 129/Sv embryonic stem (ES) cells were transfected with DNA encoding the wild-type human protein under the control of a promoter that is active in all four of the small intestine's principal epithelial lineages during their migration-associated differentiation. ES-APC cells were then introduced into C57BL/6-ROSA26 blastocysts. Analyses of adult B6-ROSA26<-->129/Sv-APC chimeric mice revealed that forced expression of APC results in markedly disordered cell migration. When compared with the effects of forced expression of E-cadherin, the data suggest that APC-catenin and E-cadherin-catenin complexes have opposing effects on intestinal epithelial cell movement/adhesiveness; augmentation of E-cadherin-beta-catenin complexes produces a highly ordered, "adhesive" migration, whereas augmentation of APC-beta-catenin complexes produces a disordered, nonadhesive migratory phenotype. We propose that APC mutations may promote tumorigenesis by increasing the relative activity of cadherin-catenin complexes, resulting in enhanced adhesiveness and functional anchorage of initiated cells within the intestinal crypt. Our studies also indicate that chimeric mice generated from B6-ROSA26 blastocysts and genetically manipulated ES cells should be useful for auditing gene function in the gastrointestinal tract and in other tissues.

Impaired cell volume regulation in intestinal crypt epithelia of cystic fibrosis mice.

Cystic fibrosis is a disease characterized by abnormalities in the epithelia of the lungs, intestine, salivary and sweat glands, liver, and reproductive systems, often as a result of inadequate hydration of their secretions. The primary defect in cystic fibrosis is the altered activity of a cAMP-activated Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) channel. However, it is not clear how a defect in the CFTR Cl- channel function leads to the observed pathological changes. Although much is known about the structural properties and regulation of the CFTR, little is known of its relationship to cellular functions other than the cAMP-dependent Cl- secretion. Here we report that cell volume regulation after hypotonic challenge is also defective in intestinal crypt epithelial cells isolated from CFTR -/- mutant mice. Moreover, the impairment of the regulatory volume decrease in CFTR -/- crypts appears to be related to the inability of a K+ conductance to provide a pathway for the exit of this cation during the volume adjustments. This provides evidence that the lack of CFTR protein may have additional consequences for the cellular function other than the abnormal cAMP-mediated Cl- secretion.

Specific modulation of airway epithelial tight junctions by apical application of an occludin peptide

Tight junctions are directly involved in regulating the passage of ions and macromolecules (gate functions) in epithelial and endothelial cells. The modulation of these gate functions to transiently regulate the paracellular permeability of large solutes and ions could increase the delivery of pharmacological agents or gene transfer vectors. To reduce the inflammatory responses caused by tight junction-regulating agents, alternative strategies directly targeting specific tight junction proteins could prove to be less toxic to airway epithelia. The apical delivery of peptides corresponding to the first extracellular loop of occludin to transiently modulate apical paracellular flux has been demonstrated in intestinal epithelia. We hypothesized that apical application of these occludin peptides could similarly modulate tight junction permeability in airway epithelia. Thus, we investigated the effects of apically applied occludin peptide on the paracellular permeability of molecular tracers and viral vectors in well differentiated human airway epithelial cells. The effects of occludin peptide on cellular toxicity, tight junction protein expression and localization, and membrane integrity were also assessed. Our data showed that apically applied occludin peptide significantly reduced transepithelial resistance in airway epithelia and altered tight junction permeability in a concentration-dependent manner. These alterations enhanced the paracellular flux of dextrans as well as gene transfer vectors. The occludin peptide redistributed occludin but did not alter the expression or distribution of ZO-1, claudin-1, or claudin-4. These data suggest that specific targeting of occludin could be a better-suited alternative strategy for tight junction modulation in airway epithelial cells compared with current agents that modulate tight junctions.

Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells

The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated. Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1-2 μM) in plastic flasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow. Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 μM 1α,25-dihydroxyvitamin D3 to promote cellular CYP3A4 expression. Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213±54.35%) compared to that of parent cells (100±6.05%). Furthermore, they retained significantly less [3H]vincristine sulphate (p<0.05), a P-gp substrate, after efflux (272.89±11.86 fmol/mg protein) than the parent cells (381.39±61.82 fmol/mg protein). The expression of CYP3A4 in parental cells after 1α,25-dihydroxyvitamin D3 treatment was quantified to be 76.2±7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0±20.0 pmol/mg protein). Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting. Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0±20.0 to 1057.0±46.0 pmol/min/mg protein. Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells. The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association.

Vascular endothelial growth factor promotes physical wound repair and is anti-apoptotic in primary distal lung epithelial and A549 cells

Objective: There is evidence to suggest a beneficial role for growth factors, including vascular endothelial growth factor (VEGF), in tissue repair and proliferation after injury within the lung. Whether this effect is mediated predominantly by actions on endothelial cells or epithelial cells is unknown. This study tested the hypothesis that VEGF acts as an autocrine trophic factor for human adult alveolar epithelial cells and that under situations of pro-apoptotic stress, VEGF reduces cell death. Design: In vitro cell culture study looking at the effects of 0.03% H2O2 on both A549 and primary distal lung epithelial cells.Measurement and Main Results: Primary adult human distal lung epithelial cells express both the soluble and membrane-associated VEGF isoforms and VEGF receptors 1 and 2. At physiologically relevant doses, soluble VEGF isoforms stimulate wound repair and have a proliferative action. Specific receptor ligands confirmed that this effect was mediated by VEGF receptor 1. In addition to proliferation, we demonstrate that VEGF reduces A549 and distal lung epithelial cell apoptosis when administered after 0.03% H2O2 injury. This effect occurs due to reduced caspase-3 activation and is phosphatidylinositol 3′–kinase dependent. Conclusion: In addition to its known effects on endothelial cells, VEGF acts as a growth and anti-apoptotic factor on alveolar epithelial cells. VEGF treatment may have potential as a rescue therapy for diseases associated with alveolar epithelial damage such as acute respiratory distress syndrome.

Caraterização in vitro dos efeitos tóxicos da patulina na integridade do epitélio intestinal e potenciais efeitos protetores da cisteína

A mucosa intestinal é a primeira barreira biológica encontrada pelas micotoxinas presentes nos alimentos, sendo a patulina, uma micotoxina produzida por fungos do género Penicillium spp., uma preocupação particular atendendo a que a exposição humana a esta micotoxina pode conduzir a efeitos imunológicos, neurológicos e gastrointestinais. Considerando estes efeitos para a saúde, o presente estudo tem como objetivos a avaliação do efeito tóxico da exposição intestinal a patulina, bem como a determinação do potencial efeito protetor da coadministração de patulina e cisteína na membrana intestinal, utilizando para o efeito células Caco-2. A integridade da membrana intestinal foi determinada pela medição da resistência elétrica transepitelial (TEER). Os resultados evidenciaram um decréscimo acentuado nos valores de TEER após 24 horas de exposição celular a 95 μM de patulina. Para as concentrações mais reduzidas verificou-se uma redução máxima inferior a 25% após 24 horas de exposição. A coadministração de patulina (95 μM) e cisteína (40 μM) revelou um decréscimo nos valores de TEER. O tratamento com cisteína em concentrações superiores ( 400 μM) revelou efeito protetor da membrana intestinal, tendo em conta os valores de TEER. Estes resultados contribuem para uma avaliação do risco mais precisa associada à exposição a contaminantes alimentares.

Desquamation Is A Novel Phenomenon For Collective Prostate Epithelial Cell Deletion After Castration.

The mechanism underlying castration-induced prostate regression, which is a classical physiological concept translated into the therapeutic treatment of advanced prostate cancer, involves epithelial cell apoptosis. In searching for events and mechanisms contributing to prostate regression in response to androgen modulation, we have frequently observed the collective deletion of epithelial cells. This work was undertaken to characterize this phenomenon hereafter named desquamation and to verify its presence after 17β-estradiol (E2) administration. Electron microscopy revealed that the desquamating cells had preserved cell-cell junctions and collapsed nuclear contents. The TUNEL reaction was negative for these cells, which were also negative for cleaved caspases-8, -9, -3 and nuclear apoptosis-inducing factor. Detailed analyses revealed that the condensed chromatin was first affected detaching from the nuclear lamina, which was observable after lamin A immunohistochemistry, suggesting the lack of lamin A degradation. A search in animals treated with supraphysiological E2 employed as an alternative anti-androgen treatment revealed no desquamation. The combined treatment (Cas + E2 group) caused changes particular to each treatment, including desquamation. In conclusion, desquamation appeared as a novel phenomenon contributing to collective prostate epithelial cell deletion, distinct from the classical castration-induced apoptosis and particular to the androgen deprivation resulting from surgical castration, and should be considered as part of the mechanisms promoting organ regression.

Gut Bacteria Products Prevent Aki Induced By Ischemia-reperfusion.

Short-chain fatty acids (SCFAs) are fermentation end products produced by the intestinal microbiota and have anti-inflammatory and histone deacetylase-inhibiting properties. Recently, a dual relationship between the intestine and kidneys has been unraveled. Therefore, we evaluated the role of SCFA in an AKI model in which the inflammatory process has a detrimental role. We observed that therapy with the three main SCFAs (acetate, propionate, and butyrate) improved renal dysfunction caused by injury. This protection was associated with low levels of local and systemic inflammation, oxidative cellular stress, cell infiltration/activation, and apoptosis. However, it was also associated with an increase in autophagy. Moreover, SCFAs inhibited histone deacetylase activity and modulated the expression levels of enzymes involved in chromatin modification. In vitro analyses showed that SCFAs modulated the inflammatory process, decreasing the maturation of dendritic cells and inhibiting the capacity of these cells to induce CD4(+) and CD8(+) T cell proliferation. Furthermore, SCFAs ameliorated the effects of hypoxia in kidney epithelial cells by improving mitochondrial biogenesis. Notably, mice treated with acetate-producing bacteria also had better outcomes after AKI. Thus, we demonstrate that SCFAs improve organ function and viability after an injury through modulation of the inflammatory process, most likely via epigenetic modification.