Pathogenic bacteria of the species Yersinia, including Yersinia pestis, block phagocytosis by macrophages. This process involves the YopE protein, which induces disruption of the host cell actin microfilament structure. Here, we show that the contact between the pathogen and the mammalian cell induces expression and then polarized transfer of YopE into the eukaryotic cell. While the bacteria remain at the surface of the target cell, the YopE cytotoxin is transferred through the host cell plasma membrane and YopE is only recovered within the cytosol of the target cell. The results suggest that the pathogen senses cell structures and focuses the transfer of YopE to occur solely at the interaction zone between the bacterium and the eukaryotic cell. The regulation of this process is shown to involve surface‐located YopN sensor protein of the bacterium.

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 beta-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. beta-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.

Nonopsonized as well as immunoglobulin G (IgG)-opsonized Yersinia pseudotuberculosis resists phagocytic uptake by the macrophage-like cell line J774 by a mechanism involving the plasmid-encoded proteins Yops. The tyrosine phosphatase YopH was of great importance for the antiphagocytic effect of the bacteria. YopHnegative mutants did not induce antiphagocytosis; instead, they were readily ingested, almost to the same extent as that of the translocation mutants YopB and YopD and the plasmid-cured strain. The bacterial determinant invasin was demonstrated to mediate phagocytosis of nonopsonized bacteria by these cells. In addition to inhibiting uptake of itself, Y. pseudotuberculosis also interfered with the phagocytic uptake of other types of prey: J774 cells that had been exposed to virulent Y. pseudotuberculosis exhibited a reduced capacity to ingest IgG-opsonized yeast particles. This effect was impaired when the bacterium-phagocyte interaction occurred in the presence of gentamicin, indicating a requirement for in situ bacterial protein synthesis. The Yersinia-mediated antiphagocytic effect on J774 cells was reversible: after 18 h in the presence of gentamicin, the phagocytic capacity of Yersinia-exposed J774 cells was completely restored. Inhibition of the uptake of IgG-opsonized yeast particles was dependent on the Yops in a manner similar to that seen for blockage of Yersinia phagocytosis. This similarity suggests that the pathogen affected a general phagocytic mechanism. Despite a marked reduction in the capacity to ingest IgG-opsonized yeast particles, no effect was observed on the binding of the prey. Taken together, these results demonstrate that Yop-mediated antiphagocytosis by Y. pseudotuberculosis affects regulatory functions downstream of the phagocytic receptor and thereby extends to other types of phagocytosis.

The intestinal mucosa plays a fundamental role as the site for absorption ofnutrients, and as an important barrier from potentially harmful agents in the intestinal lumen. Little is known of the permeability properties of the intestinal mucosa in uraemic patients. The intestinal permeability to differently sized polyethylene glycols (PEGs; range 326-1254 daltons) was studied in nine patients with chronic renal failure (24 hour endogenous creatinine clearance 5-27 ml/minute). We found an over all reduction in intestinal permeability of PEGs but a relatively increased permeability to larger PEG molecules daltons) in patients with chronic uraemia. The permeability profiles of the uraemic patients and normal subjects were also compared with the results of computer simulations of a multicompartment model, focussing on the effects of reduced renal excretion capacity. Patients and methods PATIENTS

All previously published papers were reproduced with permission from the publisher. ABSTRACT AimThe over-arching aim of this thesis was to study some metabolic functions of the gut microflora in children with known or screening detected celiac disease (CD) and their first-degree relatives. Materials Study I. A number of 36 untreated CD children, 47 after at least 3 months on glutenfree diet (GFD) and 42 healthy controls (HC). Study II. A number of 76 first-degree relatives to CD children and 93 healthy controls (HC). Study III. A number of 17 screening detected CD children were included to be compared with the untreated children and controls from study I; with exchange of one child in the untreated group, due to low age. Study IV. A comparative study regarding correlation between iso-forms of short chain fatty acids (SCFAs) in humans as well as in animals. Methods Faecal short chain fatty acids were measured in all four studies. Additionally faecal tryptic activity (FTA) was measured in study II. Major findingsAll groups of CD children demonstrated a similar SCFAs profile, i.e. significantly more total SCFAs and acetic acid and a strong tendency to more iso-butyric and iso-valeric acids compared with HC. The first-degree relatives demonstrated another SCFAs profile, i.e. significantly less total SCFAs and acetic acid and significantly more FTA than HC. Conclusions and future outlookBased upon the strong similarities between all groups of CD children we are allowing ourselves hypothesising that CD children have a "celiacogenic" flora compared with healthy controls. In a similar way it can be said that the first-degree relatives are harbouring a "celiacprotective" microflora. Our findings open up for challenging new diagnostic, therapeutic and prognostic possibilities.