Blastocystis hominis modulates immune responses and cytokine release in colonic epithelial cells

An experimental in vitro model has been developed in order to determine whether Blastocystis hominis is able to trigger inflammatory cytokine response in colonic epithelial cells. After 24 h incubation of B. hominis with the cell lines HT-29 and T-84, B. hominis cells were not able to cause cytopathic effects, but significant increases in the release of the cytokines IL-8 and GM-CSF could be observed. However, after the first 6 h of co-incubation, the production of IL-8 was not increased in HT-29 cells, and even reduced when Escherichia coli (bacteria or lipopolysaccharide) was present during co-incubation. Similar effects were observed using supernatants of B. hominis culture. These data indicate that B. hominis induces as well as modulates the immune response in intestinal epithelial cells, and we conclude that different pathophysiological events may occur during B. hominis infection.     

Types of cell differentiation in colonic and rectal adenocarcinoma]

The Normal mucous membrane and tumours of the large intestine and the rectum were studied electron microscopically. Five known cell types were found in normal intestinal mucosa: intestinal epitheliocyte with striated border, goblet enterocyte, intestinal argentaffinocyte, enterocyte with acidofilic granules, borderless enterocyte. Adenocarcinoma cells differentiate in the direction of 3 cell types: intestinal epitheliocytes, goblet enterocytes, and borderless enterocytes. The level of differentiation in different cells varies considerably.     

Myoid and epithelial cell differentiation in myasthenic thymuses.

This study demonstrates that the stromal thymus elements of postcapillary venules are the source of desmin-positive mesenchyme from which both myoid and epithelial cells arise. The double staining revealed various degrees of desmin and keratin positivity in the same kind of cells in the medulla as well as in Hassall's corpuscles. Hassall's corpuscles seem to arise from several kinds of cells of which one appears to be monocytogenic and expressed S100 protein.     

Tumor necrosis factor-alpha modulates survival, proliferation, and neuronal differentiation in neonatal subventricular zone cell cultures

Tumor necrosis factor (TNF)-alpha has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF-alpha strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1-3 C57BL/6 mice. By using single-cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF-alpha resulted in increased differentiation of cells displaying a neuronal-like profile of [Ca2+](i) responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF-alpha receptor, we found that the proneurogenic effect of 1 ng/ml TNF-alpha is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein-positive neurons was increased following exposure to mouse TNF-alpha. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF-alpha induced cell proliferation, whereas 10 and 100 ng/ml TNF-alpha induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF-alpha for 15 minutes or 6 hours caused an increase in the phospho-stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF-alpha induces neurogenesis in neonatal SVZ cell cultures of mice. TNF-alpha, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infection.     

Experimental modulation of cell-cell adhesion,invasiveness and differentiation in trophoblast cells

The establishment of pregnancy in the human decisively depends on the competence of the early trophoblast to interact during implantation with (1). the uterine epithelium and subsequently (2). with the endometrial stroma and blood vessels. In the interaction with uterine epithelium cell-to-cell adhesion appears to be a critical element, involving initially (and astonishingly) apical cell poles of both epithelia. The subsequent invasion of the stroma includes both adhesive interactions with and degradation of extracellular matrix. How these different processes are regulated in detail remains largely unknown. While the invasiveness of the trophoblast is known to be regulated in local and temporal terms it has remained unclear so far whether trophoblast adhesiveness to cells and/or matrix is subject to a coupled regulation or whether both properties involve different, maybe sequentially effective, control mechanisms. It is also not known how the regulation of these activities is related to the differentiation pathways leading to the formation of noninvasive villous trophoblast serving endocrine as well as nutritive functions. This communication reviews experiments using normal cytotrophoblast cells isolated from first trimester or term placentae as well as malignant trophoblast (choriocarcinoma) cells treated with a panel of compounds known to modulate cell differentiation [retinoic acid, methotrexate, dibutyryl-cAMP, phorbol-(12-myristoyl-13-acetyl)-diester]. Parameters indicative of trophoblast differentiation [in particular chorionic gonadotrophin (hCG) secretion] as well as adhesion to uterine epithelial cells and invasion into extracellular matrix in vitro were monitored. While expression of differentiation parameters was increased by all drug treatments, adhesion to uterine epithelial cells in vitro was reduced. Modulation of invasiveness, however, followed a different pattern: while it was reduced in normal trophoblast cells it was even increased in choriocarcinoma cells with various substances. The response of cells with respect to production of extracellular matrix proteins or matrix-degrading proteinases showed a complex pattern that again lacked a stringent correlation with hCG production and adhesion, and in addition also with invasive behavior. These results suggest that adhesiveness of trophoblast to uterine epithelial cells and invasiveness into the uterine stroma (extracellular matrix) are subject to different control mechanisms. They support the view that trophoblast-endometrium interactions involve a cascade of various adhesion and migration processes whose cellular and molecular basis is complex but accessible to experimental investigation using a variety of available in vitro systems.     

体外培养人羊膜上皮细胞的表型及分化能力

背景：人羊膜上皮细胞具有多系分化能力,是再生医学中重要的细胞来源。目前的研究多集中于对其分化能力的考察,而体外培养过程中羊膜上皮细胞的生物学特征如何变化尚不清楚。 目的：分析体外培养对人羊膜上皮细胞生长、表型及向心肌样细胞分化的能力等生物学特性的影响,探讨原代人羊膜上皮细胞干性标志物SSEA-4的表达水平与人羊膜上皮细胞生物学特性变化之间的关联性。 方法：使用统一分离方法获得原代羊膜上皮细胞并进行体外培养。利用CCK-8、流式细胞仪及real-time PCR等手段检测不同培养阶段人羊膜上皮细胞的增殖、表型以及向心肌样细胞分化的能力。 结果与结论：不同胎儿样本来源的原代人羊膜上皮细胞的SSEA-4表达在26.7%-97%,存在很大的个体差异。并且,随着传代次数的增加,人羊膜上皮细胞的SSEA-4表达水平显著降低,其下降程度与原代SSEA-4的表达水平无关。另外,培养后人羊膜上皮细胞的心肌分化潜能也存在很大个体差异,且其差异与原代人羊膜上皮细胞的SSEA-4表达水平的高低无关。结果提示,不同胎儿样本来源的原代人羊膜上皮细胞的SSEA-4表达水平受到个体差异的影响,需要建立更准确的临床样本筛选指标来稳定获得原代高表达SSEA-4的胎儿样本,以实现对人羊膜上皮细胞的质量监控。另外,体外培养过程中SSEA-4的表达水平受到培养条件的影响,需要继续优化培养条件以维持其高表达。此外,人羊膜上皮细胞向心肌样细胞分化的能力受到样本个体差异以及培养条件的影响,在今后还需要进一步研究。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Natriuretic peptides up-regulate aquaporin 3 in a human colonic epithelial cell line

Several specialized channels termed aquaporins (AQPs) facilitate water transport in the gastrointestinal tract. AQP3 localizes to epithelial cells in the human small intestine and colon. However, the regulatory mechanisms responsible for AQP3 function in the gastrointestinal tract are not well understood. To characterize the regulation of AQP3 expression by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), we studied mRNA expression by Northern blotting, protein expression by Western blotting and DNA binding activity by electrophoretic mobility shift assay (EMSA) in the human colonic epithelial cell line HT-29. We also used several inhibitors to investigate signal transduction. AQP3 mRNA was up-regulated in addition to ANP (>100 nM) and BNP (>10 nM). The expression of AQP3 protein was enhanced at 1 h after the addition of ANP and BNP. The combination of protein kinase-A (PK-A) and protein kinase-G (PK-G) inhibitors completely inhibited the expression of AQP3 mRNA enhanced by ANP or BNP to its basal level. The EMSA of the cyclic-AMP response element (CRE) in HT-29 cells revealed a single band. These results indicate that ANP and BNP up-regulated the expression of AQP3 mRNA and protein, and both PK-A and PK-G dependent pathways mediated this effect.     

Cytokeratins, markers of epithelial cell differentiation: expression in normal epithelia]

Intermediate filaments, the most stable of cytoskeleton components, are extremely diverse and usually correlate with the histological subtype since in nearly all cell types a single type of intermediate filament (IF) is found. The cytokeratins, which are specific of epithelia, are the largest and most diverse class of intermediate filaments. Twenty different cytokeratin polypeptides have been identified in humans and separated on the basis of isoelectrical pH and apparent molecular weight using two-dimensional electrophoresis. These data have been used to establish a cytokeratin catalogue which currently serves as a reference [43, 48]. The number of cytokeratin polypeptides expressed ranges from 2 to 5 for each epithelial cell and from 2 to 10 for each epithelium and even of each cell layer within a given epithelium. A broad spectrum of anticytokeratin antibodies with subgroup or single polypeptide specificity is currently available. The distribution of cytokeratins in normal epithelia is reviewed herein and commercially available anti-cytokeratin antibodies are listed.     

Comparison of vasoactive intestinal peptide-mediated protein phosphorylation in human lymphoblasts and colonic epithelial cells

Vasoactive intestinal peptide (VIP) induces phosphorylation of a basic 38,000 mol. wt protein in a human lymphoblastic cell line (Molt 4b) and a human colon carcinoma cell line (HT29). In both cell types, VIP interacts with specific high affinity receptors to activate adenylate cyclase and cAMP-dependent protein kinase. The two cell types appear to express homologous receptors with similar affinity and specificity for VIP, but the colonic epithelial cells express a greater number of receptors. HT29 colonic cells also exhibit a greater stimulation of adenylate cyclase and a higher phosphorylation index for the 38,000 mol. wt protein in response to VIP. This 38,000 mol. wt protein, which is phosphorylated in the presence of VIP, appears to be identical in both cell lines; it is phosphorylated in both lymphoblasts and colonic epithelial cells in the presence of forskolin, but not in the presence of phorbol 12-myristate 13-acetate. Phosphorylation of this 38,000 mol. wt protein may be an important step in VIP regulation of water and electrolyte secretion from colonic epithelial cells, and in VIP regulation of immunoglobulin and lymphokine secretion from lymphocytes.     

Arrested differentiation and epithelial cell degeneration in zebrafish lens mutants.

In a chemical mutagenesis screen, we identified two zebrafish mutants that possessed small pupils. Genetic complementation revealed these two lines are due to mutations in different genes. The phenotypes of the two mutants were characterized using histologic, immunohistochemical, and tissue transplantation techniques. The arrested lens (arl) mutant exhibits a small eye and pupil phenotype at 48 hr postfertilization (hpf) and lacks any histologically identifiable lens structures by 5 days postfertilization (dpf). In contrast, the disrupted lens (dsl) mutants are phenotypically normal until 5 dpf, and then undergo lens disorganization and cell degeneration that is apparent by 7 dpf. Histology reveals the arl mutant terminates lens cell differentiation by 48 hpf, whereas the dsl lens exhibits a defective lens epithelial cell population at 5 dpf. Lens transplantation experiments demonstrate both mutations are autonomous to the lens tissue. Immunohistochemistry reveals the retinal cells may suffer subtle effects, possibly due to the lens abnormalities.     

Commitment of embryonic stem cells to an epidermal cell fate and differentiation in vitro.

The epidermis develops from a stem cell population in the surface ectoderm that feeds a single vertical terminal differentiation pathway. To date, however, the limited capacity for the isolation or purification of epidermal stem or precursor cells has hampered studies on early commitment and differentiation events. We have developed a two-step culture scheme in which pluripotent mouse embryonic stem (ES) cells are induced first to a surface ectoderm phenotype and then are positively selected for putative epidermal stem cells. We show that the earliest stages of epidermal development follow an ordered sequence that is similar to that observed in vivo (expression of keratin 8, keratin 19, keratin 17, and keratin 14), suggesting that ES cell-derived surface ectoderm-like cells can be induced to follow the epidermal developmental pathway. At a low frequency, keratin 14-positive early epidermal cells progressed to keratin 1-positive and terminally differentiated cells producing a cornified envelope. This culturing protocol provides an invaluable system in which to study both the mechanisms that direct stem cells along the epidermal pathway as well as those that influence their subsequent epidermal differentiation.     

Regulator of calcineurin 1 modulates cancer cell migration in vitro

Metastasis suppressors and other regulators of cell motility play an important role in tumor invasion and metastases. We previously identified that activation of the G protein coupled receptor 54 (GPR54) by the metastasis suppressor metastin inhibits cell migration in association with overexpression of Regulator of calcineurin 1 (RCAN1), an endogenous regulator of calcineurin. Calcineurin inhibitors also blocked cell migration in vitro and RCAN1 protein levels were reduced in nodal metastases in thyroid cancer. The purpose of the current study was to determine directly if RCAN1 functions as a motility suppressor in vitro. Several cancer cell lines derived from different cancer types with different motility rates were evaluated for RCAN1 expression levels. Using these systems we determined that reduction of endogenous RCAN1 using siRNA resulted in an increase in cancer cell motility while expression of exogenous RCAN1 reduced cell motility. In one cell line with a high migratory rate, the stability of exogenously expressed RCAN1 protein was reduced and was rescued by treatment with a proteasome inhibitor. Finally, overexpression of RCAN1 was associated with an increase in cell adhesion to collagen IV and reduced calcineurin activity. In summary, we have demonstrated that the expression of exogenous RCAN1 reduces migration and alters adhesion; and that the loss of endogenous RCAN1 leads to an increase in migration in the examined cancer cell lines. These results are consistent with a regulatory role for RCAN1 in cancer cell motility in vitro.     

Nance-Horan syndrome protein, NHS, associates with epithelial cell junctions

Nance-Horan syndrome, characterized by congenital cataracts, craniofacial, dental abnormalities and mental disturbances, is an X-linked disorder with significant phenotypic heterogeneity. Affected individuals have mutations in the NHS (Nance-Horan syndrome) gene typically resulting in premature truncation of the protein. This report underlines the complexity of the regulation of the NHS gene that transcribes several isoforms. We demonstrate the differential expression of the two NHS isoforms, NHS-A and NHS-1A, and differences in the subcellular localization of the proteins encoded by these isoforms. This may in part explain the pleiotropic features of the syndrome. We show that the endogenous and exogenous NHS-A isoform localizes to the cell membrane of mammalian cells in a cell-type-dependent manner and that it co-localizes with the tight junction (TJ) protein ZO-1 in the apical aspect of cell membrane in epithelial cells. We also show that the NHS-1A isoform is a cytoplasmic protein. In the developing mammalian lens, we found continuous expression of NHS that became restricted to the lens epithelium in pre- and postnatal lens. Consistent with the in vitro findings, the NHS-A isoform associates with the apical cell membrane in the lens epithelium. This study suggests that disturbances in intercellular contacts underlie cataractogenesis in the Nance-Horan syndrome. NHS is the first gene localized at TJs that has been implicated in congenital cataracts.     

Clonal selection, attenuation and differentiation in an in vitro model of hyperplasia

Observations of the growth kinetics and morphologies of clones and subclones of diploid human skin fibroblast cultures lead to the working hypothesis that these cells, presumably like their counterparts in healing wounds, constitute a differentiating system. There is attenuation of the growth of serial clones, with continual selection for more vigorous stem cells. The latter segregate daughter cells of varying growth potential, including a class of cells which may be regarded as terminally differentiating; we propose that such cells may be histiocytes or macrophages. These studies a) demonstrate extensive epigenetic heterogeneity in fibroblast cultures, b) suggest that hyperplastic foci may be monoclonal or oligoclonal, c) rule out a simple biologic clock mechanism as an explanation of clonal senescence, d) suggest a new approach to the analysis of various inborn errors of metabolism, such as Werner''s Syndrome.     

Thymus organogenesis and molecular mechanisms of thymic epithelial cell differentiation

In the mature thymus, thymocyte maturation depends on interactions with different thymic epithelial subtypes in a three-dimensional thymic architecture. However, the molecular mechanisms that generate these epithelial subtypes are not well understood. Evidence is accumulating that during fetal thymus development, epithelial cells differentiate by successive interactions with differentiating thymocytes. This review presents fetal thymus development as a process of organogenesis, the main function of which is to promote thymic epithelial cell differentiation and the generation of a functional thymic microenvironment. In this model, endoderm-derived epithelial cells are the driving force in generating the thymic primordium, with hematopoietic cells providing later signals that organize and pattern the developing thymus.