OBJECTIVES: Life-long, strict gluten-free diet (GFD) is the only treatment for celiac disease (CD). Because there is still uncertainty regarding the safety of oats for CD patients, the aim was to investigate whether dietary oats influence the immune status of their intestinal mucosa.

METHODS: Paired small intestinal biopsies, before and after >11 months on a GFD, were collected from children with CD who were enrolled in a randomized, double-blind intervention trial to either of two diets: standard GFD (GFD-std; n=13) and noncontaminated oat-containing GFD (GFD-oats; n=15). Expression levels of mRNAs for 22 different immune effector molecules and tight junction proteins were determined by quantitative reverse transcriptase (RT)-PCR.

RESULTS: The number of mRNAs that remained elevated was higher in the GFD-oats group (P=0.05). In particular, mRNAs for the regulatory T cell (Treg) signature molecules interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1), the cytotoxicity-activating natural killer (NK) receptors KLRC2/NKG2C and KLRC3/NKG2E, and the tight junction protein claudin-4 remained elevated. Between the two groups, most significant differences were seen for claudin-4 (P=0.003) and KLRC3/NKG2E (P=0.04).

CONCLUSIONS: A substantial fraction of pediatric CD patients seem to not tolerate oats. In these patients, dietary oats influence the immune status of the intestinal mucosa with an mRNA profile suggesting presence of activated cytotoxic lymphocytes and Tregs and a stressed epithelium with affected tight junctions. Assessment of changes in levels of mRNA for claudin-4 and KLC3/NKG2E from onset to after a year on oats containing GFD shows promise to identify these CD patients.

We have previously shown that protein kinase C (PKC) activation has distinct effects on the structure and barrier properties of cultured epithelial cells (HT29 and MDCK I). Since the claudin family of tight junction (TJ)-associated proteins is considered to be crucial for the function of mature TJ, we assessed their expression patterns and cellular destination, detergent solubility and phosphorylation upon PKC stimulation for 2 or 18 h with phorbol myristate acetate (PMA). In HT29 cells, claudins 1, 3, 4 and 5 and possibly claudin 2 were redistributed to apical cell-cell contacts after PKC activation and the amounts of claudins 1, 3 and 5, but not of claudin 2, were increased in cell lysates. By contrast, in MDCK I cells, PMA treatment resulted in redistribution of claudins 1, 3, 4 and 5 from the TJ and in reorganization of the proteins into more insoluble complexes. Claudins 1 and 4 were phosphorylated in both MDCK I and HT29 cells, but PKC-induced changes in claudin phosphorylation state were detected only in MDCK I cells. A major difference between HT29 and MDCK I cells, which have low and high basal transepithelial electrical resistance, respectively, was the absence of claudin 2 in the latter. Our findings show that PKC activation targets in characteristic ways the expression patterns, destination, detergent solubility and phosphorylation state of claudins in epithelial cells with different capacities to form an epithelial barrier.

Histone H1 is an important constituent of chromatin which undergoes major structural rearrangements during mitosis. However, the role of H1, multiple H1 subtypes and H1 phosphorylation is still unclear. In normal human fibroblasts, phosphorylated H1 was found located in nuclei during prophase and in both cytoplasm and condensed chromosomes during metaphase, anaphase and telophase as detected by immunocytochemistry. Moreover, we detected remarkable differences in the distribution of the histone H1 subtypes H1.2, H1.3 and H1.5 during mitosis. H1.2 was found in chromatin during prophase, and almost solely in the cytoplasm of metaphase and early anaphase cells. In late anaphase it appeared in both chromatin and cytoplasm, and again in chromatin during telophase. H1.5 distribution pattern resembled that of H1.2, but some H1.5 remained situated in chromatin during metaphase and early anaphase. H1.3 was detected in chromatin in all cell cycle phases. We propose therefore, that H1 subtype translocation during mitosis is controlled by phosphorylation, in combination with H1 subtype inherent affinity. We conclude that H1 subtypes, or their phosphorylated variants, may be signalling molecules in mitosis or that they leave chromatin in a regulated way to give access for chromatin condensing factors or transcriptional regulators during mitosis.

Hepatocyte growth factor (HGF) is a multifunctional growth factor with potent wound-healing properties that functions in the healing of chronic injuries. However, there may be a loss of HGF activity in certain chronic cases; this might be indicated by the presence of high amounts of HGF in body fluids and by the elevated expression of the HGF receptor in tissue biopsies. In such cases, a reliable means of assessing the activity of endogenous HGF would be valuable in allowing clinicians to decide if treatment with HGF would be useful. In this study, we developed an in vitro wound assay that used a mouse skin epithelial cell line to evaluate the biological activity of HGF. We showed that HGF accelerated the motility of the epithelial cells in a dose-dependent fashion with high sensitivity and specificity. This in vitro assay might be used to determine the activity of both endogenous and recombinant HGF.

Intracellular transport of organelles, vesicles and proteins is crucial in all eukaryotic cells, and is accomplished by motor proteins that move along cytoskeletal filaments. A widely used model of intracellular transport is Xenopus laevis melanophores. These cells help the frog to change color by redistributing melanin-containing organelles in the cytoplasm. The high contrast of the pigment organelles permits changes in distribution to be observed by ordinary light microscopy; other intracellular transport systems often require fluorescence labeling. Here we have developed white light Image Correlation Spectroscopy (ICS) to monitor aggregation and dispersion of pigment. Hitherto in ICS, images of fluorescent particles from Confocal Laser Scanning Microscopy (CLSM) have been used to calculate autocorrelation functions from which the density can be obtained. In the present study we show that ICS can be modified to enable analysis of light-microscopy images; it can be used to monitor pigment aggregation and dispersion, and distinguish between different stimuli. This new approach makes ICS applicable not only to fluorescent but also to black-and-white images from light or electron microscopy, and is thus very versatile in different studies of movement of particles on the membrane or in the cytoplasm of cells without potentially harmful fluorescence labeling and activation.

The demand for exact engineering within the life sciences is growing and the Engineering Biology programme at Linköping University, Linköping, Sweden, prepares students for a career at this interface. Conceive – Design – Implement – Operate (CDIO) was recently pioneered in an introductory project course. Groups of six to seven students apply a LIPS scalable project model from traditional engineering educational environments on, for example, a cellular imaging task in a hospital setting, prior to taking courses in cell biology/optics. Besides facilitating the implementation of CDIO in higher courses, students gain early career insight and enhance their communication skills. A customer (senior teacher) needs to visualise structures in cells, and the student group is contracted to deliver an applied and optimised method to meet specified requirements. The customer reviews deliverables before the tollgates and communicates with the student project leader. Other students are responsible for documentation and subsystems. The project is allocated laboratory facilities and hardware, and two fictitious subcontractors supply samples and consumables. Extra teachers perform supervision and methodological consultation. In summary, CDIO is indeed applicable and rewarding in cellular imaging, yet is also challenging.

Objective. Recent studies report negligible toxicity of oats in the majority of coeliac disease (CD) patients. It has previously been shown that children with untreated CD have circulating antibodies to oats avenin. In this study we performed serial assessments of anti-avenin antibodies in children under investigation for CD on a gluten-free diet with or without oats.

Material and methods. The study involved 116 children, randomized to a standard gluten-free diet or a gluten-free diet supplemented with oats. Sera were obtained from 86 children, 48 in the standard gluten-free group and 38 in the gluten-free oats group, of which 33 consumed at least 10 g of oats daily. IgA and IgG anti-avenin antibodies were monitored at 0, 3, 6 and 12 months. Nitric oxide metabolites were measured in 7 patients, with deviating antibody results.

Results. There was a significant decrease in anti-avenin antibodies in both groups at the end as compared to the beginning of the study, (p<0.001), but no difference was found between the two groups. IgA titres already declined after 3 months. IgG titres, although significantly decreased, remained high in the majority of patients in both groups. Nitric oxide levels were high in four of the analysed samples.

Conclusions. Oats per se, do not seem to produce a humoral immune reaction in children with CD when given in an otherwise gluten-free diet, indicating that the reaction requires gluten challenge. Anti-avenin antibodies were equal in the two study groups, and these findings strengthen the clinical impression that oats can be tolerated by the majority of patients with CD.

Alpha-dystrobrevin (a-DB) has been described primarily as a cytoplasmic component of the dystrophin-glycoprotein complex in skeletal muscle cells. Isoforms of a-DB show different localization in cells and tissues, at basolateral membranes in epithelial cells, dystrobrevins mediate contact with the extracellular matrix, peripheral and transmembrane proteins and the filamentous actin cytoskeleton. Beside their structural role, a-DBs are assumed to be important in cell signalling and cell differentiation. We have primarily assessed the role of a-DB in two epithelial cell lines (MDCK I, HT 29), which represent different developmental stages and exhibit distinct permeability characteristics. Using a polyclonal anti-a-DB antibody, we have investigated its expression, localization and association with tight junction (TJ)- associated proteins (ZO-1, occludin) before and after protein kinase C (PKC) activation with phorbol myristate acetate. Distinct subsets of a-DB isoforms were detected in the two cell lines by immunoblotting. In both cell lines there was submembranous localization of a-DB both apically and basolaterally, shown with confocal imaging. PKC activation caused a reorganization of TJ, which was parallel to increased localization of a-DB to TJ areas, most pronounced in MDCK I cells. Moreover, actin and ZO-1 co-immunoprecipitated with a-DB, as displayed with immunoblotting. Our findings suggest that a-dystrobrevin specifically is associated with the tight junctions during their reorganization. © Springer Science+Business Media, Inc. 2005.

Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgC1₂-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.

We show here, that activation of protein kinase C by the phorbol ester PMA improves barrier function in colon carcinoma (HT 29) cells. By contrast, in canine kidney (MDCK I) cells it caused increased permeability and opening of tight junctions; the latter has also been noticed in other studies. Thus, with PMA confluent HT 29 cells responded with a reduced passage of 330 kDa sodium fluorescein, increased transepithelial electrical resistance, and a change in the cell shape of the HT 29 cells from an irregular to a regular, hexagonal form. Confocal imaging revealed parallel distinct changes in the staining of occludin and caludin-1, viz. a translocation from cytoplasmic clusters to apical cell–cell contacts. Interestingly, in both cell lines protein kinase A activation caused a decreased in the threonine phosphorylation of occludin that correlated with tight junction assembly in HT 29 cells and tight junction disassembly in MDCK I cells. We conclude that protein kinase C regulation of the epithelial barrier involves specific molecular mechanisms and achieves distinct effects at different developmental stages.

The development of new conjugated-polymer tools for the study of the biological realm, and for use in a clinical setting, is reviewed in this article. Conjugated-polymer actuators, based on the changes of volume of the active conjugated polymer during redox transformation, can be used in electrolytes employed in cell-culture media and in biological fluids such as blood, plasma, and urine. Actuators ranging in size from 10 μm to 100 μm suitable for building structures to manipulate single cells are produced with photolithographic techniques. Larger actuators may be used for the manipulation of blood vessels and biological tissue.

We present the development of a cell clinic. This is a micromachined cavity, or microvial, that can be closed with a lid. The lid is activated by two polypyrrole/Au microactuators. Inside the microvials two Au electrodes have been placed in order to perform impedance studies on single or a small number of cells. We report on impedance measurements on Xenopus leavis melanophores. We could measure a change in the impedance upon cell spreading and identify intracellular events such as the aggregation of pigment granules. The electrical data is correlated to optical microscopy.

Background & Aims: Crohn's disease (CD) is associated with a disturbed intestinal barrier. Permeability studies have focused on inert molecules, but little is known about transepithelial transport of macromolecules with antigenic potential in humans. The aim of this study was to quantify permeation and to characterize passage routes for macromolecules in ileal mucosa in CD.

Methods: Noninflamed and inflamed ileal mucosa specimens from patients with CD (n = 12) and ileal specimens from patients with colon cancer (n = 7) were studied regarding transmucosal permeation of ovalbumin, dextran (mol wt, 40,000), and ⁵¹Cr-EDTA for 90 minutes in vitro in Ussing chambers. Transepithelial passage routes for fluorescent ovalbumin and dextran 40,000 were investigated by confocal microscopy.

Results: Noninflamed ileum from CD patients showed increased permeation of ovalbumin compared with ileum from colon cancer patients (P < 0.05). Dextran permeation was equal in the three groups, whereas ⁵¹Cr-EDTA permeability was increased in inflamed ileum. Ovalbumin passed both transcellularly and paracellularly, but dextran followed a strictly paracellular route. Both markers were subsequently endocytosed by cells of the lamina propria.

Conclusions: Noninflamed ileal mucosa from patients with CD shows increased epithelial permeability to ovalbumin, probably by augmented transcytosis. This increase in antigen load to the lamina propria could be an initiating pathogenic event in CD.

The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with β-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. β-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.

Epithelial cells of the human body form a physical barrier to harmful agents and potential invading microorganisms. In the small intestine they must also produce enzymes for digestion of food and be able to absorb nutrients.

The aim of this work was to study properties of epithelial cells in model systems using cell lines and toad bladder cells, and to study the effect of gluten intolerance (celiac disease, CD), viz. a pathological condition, on human small-intestine epithelial cells, enterocytes. Fluores-cence microscopy techniques, and primarily confocallaser scanning microscopy (CLSM), have beeen used as the major methods in the investigation. Part of the work has also been to develop, and apply, tools for measurements in confocal rnicroscopy images to obtain semi-quantitative information on structures.

Fluorescence recovery after photobleaching (FRAP) was used to study the effect of maturation of small intestine-like epithelial cells. Lateral diffusion of membrane components was measured to reveal alterations in membrane fluidity induced by differentiation. No general effects on the lateral mobility of membrane components was observed, rather distinct effects were noticed on protein diffusion.

Vasopressin induces the fusion of vesicles containing water channels with the apical membrane of toad bladder epithelial cells. This fusion is known to result in depolymerization of fllamentous actin (F-actin) of the cell. CLSM was used to assess where in the cell the depolymerization occurs. It was demonstrated that the depolymerization is not evenly distributed, but confined only to the apical region of the cells.

In children suffering from celiac disease the mucosa of the small intestine is severely damaged. The damage at the enterocyte level is, however, less investigated. In the present work, CLSM was applied to compare the distributions ofF-actin and of glycoconjugates in enterocytes  from children with CD to enterocytes from children not suffering from the  disease. The results show that in children with active CD the distribution of both structures was altered, but also that compliance to a gluten-free diet results in the return to normal-looking enterocytes.

We have studied the effect of maturation to small intestinal-like epithelial cells of the human colonic calcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21-day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM-Gal. In comparison to control cells grown in DMEM-Glu medium, the more small intestinal-like cells grown in DMEM-Gal displayed no alteration of the lateral mobility of either cholera toxin (B subuni)-labelled ganglioside GM₁ (diffusion coefficient, D [x 10⁸] = 0.8–0.9 cm²s⁻¹; mobile fraction, R = 50-60%) or antibody-stained Class 2 histocompatibility (HLA-DR) antigen (D [x 10⁹] = 2 cm²s⁻¹; R = 60–70%). However, antibody-labelled β₂-microglobulin of HLA Class 1 antigen displayed increased mobility in HT29-Gal cells; D was × 1.4 and R × 1.8 larger in the HT29-Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were × 0.60 and × 0.69 of the values seen in HT29-Glu cells. It is thus concluded that DMEM-Gal-induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobil-ity of different membrane components.

Alpha-dystrobrevin (α -DB) has been described primarily as a cytoplasmic component of the dystrophin-glycoprotein complex (DGC) in skeletal muscle cells. Isoforms of α -DB show different localization in cells and tissues; at basolateral membranes in epithelial cells, dystrobrevins mediate contact with the extracellular matrix, peripheral and transmembrane proteins and the filamentous actin cytoskeleton. Beside their structural role, α -DBs are assumed to be important in cell signalling and cell differentiation. We have assessed the role of a-DB in two epithelial cell lines (MDCK I and HT 29) that are in different developmental stages and exhibit distinct permeability characteristics. Using a polyclonal anti-α-DB antibody, we have investigated its localization and association with tight junction (TJ)-associated proteins before and after protein kinase C (PKC) activation with phorbol myristate acetate (PMA). In both cell lines, there was submembranous localization of α -DB both apically and basolaterally, as assessed with confocal imaging. PKC caused a reorganization of TJ, which was parallel with increased localization of α -DB to TJ areas, especially in MDCK I cells. Moreover, the TJ-associated protein Z0-1 co-immunoprecipitated with α -DB, as displayed with SDS-PAGE and immunoblotting. Thus, α -DBs not only take part in the regulation of the contact between intra- and extracellular proteins at basolateral membranes, but possibly also in the regulation of tight junctions via ZO-1 and PKC-activation.

Melanophores are cells specialized for transport of pigment-filled organelles called melanosomes. Melanosomes are aggregated in the center of a melanophore or dispersed throughout the cytoplasm by motor proteins moving along the actin and microtubule cytoskeleton. In angelfish (Pterophyllum scalare), aggregation of melanosomes (as compared to dispersion) increases the height of the central part of melanophores by 300%. Our objective was to detennine whether such a height increase also occurs in frog (Xenopus laevis) melanophores. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that elevation of the melanophore plasma membrane is due to local swelling caused by influx of water through HgCl₂-sensitive aquaporins and subsequent polymerization of actin. Confocal microscopy revealed a 30% increase in height in X. laevis melanophores during melatonin-induced aggregation. This was not due to actin polymerization, because it also occurred when aggregation was induced by the polymerization inhibitor latrunculin B. The nitric oxide (NO) synthase inhibitor L-NAME induced dispersion and lowered the plasma membrane, which suggests that NO is involved in the upward movement. Furthermore, neither dispersion nor aggregation was affected by inhibition of water flux through HgCl₂ sensitive aquaporins. Together, these observations imply that melanosomes in X. laevis melanophores are driven upwards during aggregation by a mechanism other than actin polymerization, possibly involving microtubules, intermediate filaments, or a motor protein that may be regulated by NO. Furthermore, influx of water through HgCl₂-sensitive aquaporins is probably not necessary for aggregation-induced elevation of the cell membrane, because both aggregation and dispersion can occur in the presence of HgCl₂.