Marì Regoli

Alterations in the expression of the a3b1 integrin in certain membrane domains of the glomerular epithelial cells (podocytes) in diabetes mellitus

Summary In view of the major alterations which take place at the level of the extracellular matrix of the glomerular wall in diabetes mellitus and the key roles played by β1 integrins in cell-to-matrix interactions, it is imperative to understand the role played by integrins in the development of diabetic glomerulosclerosis. In the present study, we revealed by immunocytochemistry the ultrastructural distribution of the α3β1 at the level of the plasma membrane of the different renal glomerular cells from short- and long-term diabetic rats. For the endothelial cells, the labelling present on both the luminal and abluminal plasma membranes was low. For the podocyte epithelial cells, the labelling was present on both the luminal and basal plasma membranes, the former being concentrated at points of contact between podocyte foot processes. The labelling on the basal plasma membrane was more significant and similar in domains facing either the glomerular basement membrane or the mesangial matrix. The plasma membrane of mesangial cells also exhibited α3β1. The labelling was recorded under diabetic conditions, at the same sites, with similar intensities, alongside that of the basal plasma membrane of podocytes facing the glomerular basement membrane, the density of which decreased significantly. This decrease in labelling was similar in renal tissues from short- and long-term diabetic animals. These results demonstrate that α3β1 present at the podocyte basal plasma membrane facing the glomerular basement membrane, which undergoes important alterations in diabetes, could be involved in the major dysfunctions of the glomerular wall characteristic of diabetic glomerulosclerosis. Since the changes in integrin were found to occur as early as after 1 month of hyperglycaemia, when morphological alterations of the glomerular basement membrane are not yet established, we propose that they constitute an early event which precedes the onset of diabetic nephropathy. [Diabetologia (1997) 40: 15–22]

Salmonella Induces Flagellin- and MyD88-Dependent Migration of Bacteria-Capturing Dendritic Cells Into the Gut Lumen

BACKGROUND & AIMS Intestinal dendritic cells (DCs) sample bacteria, such as Salmonella, by extending cellular processes into the lumen to capture bacteria and shuttle them across the epithelium; however, direct evidence of bacteria-loaded DCs travelling back into the tissue is lacking. We hypothesized that sampling is paralleled by migration of DCs into the lumen prior to or following the internalization of Salmonella. METHODS The small intestine and the colon of BALB/c and C57BL/6 mice were challenged with noninvasive Salmonella enterica serovar Typhimurium SL1344-DeltaSalmonella pathogenicity island (SPI) 1 or Escherichia coli DH5alpha by using isolated loops or oral administration by gavage. Transepithelial migration of DCs was documented by immunohistochemistry, microscopy, and flow cytometry. The role of flagellin was determined by using flagellin (DeltafliC DeltafljB)- and SPI1-SPI2 (DeltaSPI1 DeltassrA)-deficient Salmonella, flagellated E coli K12, and MyD88 mice. RESULTS Salmonella DeltaSPI1 induced migration of CD11c(+)CX(3)CR1(+)MHCII(+)CD11b(-)CD8alpha(-) DCs into the small intestine, whereas flagellin- and SPI1-SPI2-deficient Salmonella, soluble flagellin, and E coli DH5alpha or flagellated K12, failed to do so. DC migration did not occur in the colon; it was not observed in MyD88 mice, and intraluminal DCs internalized Salmonella but did not cross the epithelium to return into tissues. Finally, DC migration was not linked to Salmonella-induced damage of the epithelium. CONCLUSIONS DC-mediated sampling of Salmonella is accompanied by flagellin- and MyD88-dependent migration of Salmonella-capturing DCs into the intestinal lumen. We suggest that the rapid intraluminal migration of Salmonella-capturing DCs may play a role in the protection of the intestinal mucosa against bacterial infection.

Modifications of the follicle-associated epithelium by short-term exposure to a non-intestinal bacterium

This study was undertaken in order to study the effects of short‐term exposure of the follicle‐associated epithelium (FAE) of rabbit Peyers patches to a non‐intestinal, Gram‐positive bacterium. Isolated ileal loops, each containing one Peyers patch (PP), were stimulated for short periods of time (30 and 60 min) with Streptococcus pneumoniae R36a, a micro‐organism normally not present in the intestinal area. Samples from antigen‐stimulated and control Peyers patches were analysed by light (LM), transmission electron (TEM), and scanning electron microscopy (SEM). Stimulation with living pneumococci induced dramatic changes in FAE architecture and morphology. A massive passage of cells from lymphoid tissue to the FAE was rapidly detectable, accompanied by alterations of the FAE surface, with a marked increase of M‐cell area. Furthermore, TEM analysis revealed that M cells were able to internalize living pneumococci. S. pneumoniae R36a is a valid experimental model for the further study of the unique antigen sampling function which characterizes the highly specialized FAE in Peyers patches.

Association between islets of Langerhans and pancreatic ductal system in adult rat. Where endocrine and exocrine meet together

Aims/hypothesis. Studies on the functional and morphological relations between exocrine and endocrine pancreas have been conducted mainly to disclose the influence of islets of Langerhans on acinar parenchyma. Less attention has been paid to the relations occurring between islets and pancreatic ducts. Methods. A series of consecutive sections of normal adult rat pancreas were double stained with islet (hormones) and duct (cytokeratin 20) markers. Electron microscopy was conducted to investigate the ultrastructural features of duct-islet relations and anti-insulin immunogold labelling was carried out to reveal the presence of insulin in the pancreatic duct system. Results. Consecutive double-stained sections demonstrated that 73.60 ± 2.97 % of the islets were attached to the ducts. For each series, 93.48 ± 5.43 % of the islets contacting the duct tree were associated with small-sized ducts or centroacinar cells. Electron microscopy revealed that some insulin and somatostatin cells do face the duct lumen. Insulin was detected within the duct lumen and in the endosomal compartment of the duct cells. Conclusions/interpretation. The finding that most islets are connected with the duct system in the adult pancreas is discussed in terms of hormone secretion into the ducts, islet histogenesis and the relation among the three tissue components of the pancreas, the endocrine, the exocrine and the duct system. [Diabetologia (2001) 44: 575–584]

Nestin Expression in Adult and Developing Human Kidney

Nestin is considered a marker of neurogenic and myogenic precursor cells. Its arrangement is regulated by cyclin-dependent kinase 5 (CDK5), which is expressed in murine podocytes. We investigated nestin expression in human adult and fetal kidney as well as CDK5 presence in adult human podocytes. Confocal microscopy demonstrated that adult glomeruli display nestin immunoreactivity in vimentin-expressing cells with the podocyte morphology and not in cells bearing the endothelial marker CD31. Glomerular nestin-positive cells were CDK5 immunoreactive as well. Western blotting of the intermediate filament-enriched cytoskeletal fraction and coimmunoprecipitation of nestin with anti-CDK5 antibodies confirmed these results. Nestin was also detected in developing glomeruli within immature podocytes and a few other cells. Confocal microscopy of experiments conducted with antibodies against nestin and endothelial markers demonstrated that endothelial cells belonging to capillaries invading the lower cleft of S-shaped bodies and the immature glomeruli were nestin immunoreactive. Similar experiments carried out with antibodies raised against nestin and α-smooth muscle actin showed that the first mesangial cells that populate the developing glomeruli expressed nestin. In conclusion, nestin is expressed in the human kidney from the first steps of glomerulogenesis within podocytes, mesangial, and endothelial cells. This expression, restricted to podocytes in mature glomeruli, appears associated with CDK5.

Macrophage Migration Inhibitory Factor Plays a Role in the Regulation of Microfold (M) Cell-Mediated Transport in the Gut

It has been shown previously that certain bacteria rapidly (3 h) up-regulated in vivo microfold cell (M cell)-mediated transport of Ag across the follicle-associated epithelium of intestinal Peyer’s patch. Our aim was to determine whether soluble mediators secreted following host-bacteria interaction were involved in this event. A combination of proteomics and immunohistochemical analyses was used to identify molecules produced in the gut in response to bacterial challenge in vivo; their effects were then tested on human intestinal epithelial cells in vitro. Macrophage migration inhibitory factor (MIF) was the only cytokine produced rapidly after in vivo bacterial challenge by CD11c+ cells located beneath the M cell-rich area of the follicle-associated epithelium of the Peyer’s patch. Subsequently, in vitro experiments conducted using human Caco-2 cells showed that, within hours, MIF induced the appearance of cells that showed temperature-dependent transport of microparticles and M cell-specific bacterium Vibrio cholerae, and acquired biochemical features of M cells. Furthermore, using an established in vitro human M cell model, we showed that anti-MIF Ab blocked Raji B cell-mediated conversion of Caco-2 cells into Ag-sampling cells. Finally, we report that MIF−/− mice, in contrast to wild-type mice, failed to show increased M cell-mediated transport following in vivo bacterial challenge. These data show that MIF plays a role in M cell-mediated transport, and cross-talk between bacteria, gut epithelium, and immune system is instrumental in regulating key functions of the gut, including M cell-mediated Ag sampling.

Adoptive transfer of dendritic cells from allergic mice induces specific immunoglobulin E antibody in naïve recipients in absence of antigen challenge without altering the T helper 1/T helper 2 balance.

Dendritic cells (DCs) are important in the regulation of immune responses and it has been proposed that these cells play an important role in asthma; however, their role in food allergy is still largely unknown. Our aim was to study specific immunoglobulin E (IgE) and immunoglobulin G (IgG) responses in naïve recipients following adoptive transfer of myeloid DCs from allergic and control mice. The phenotypic features and lymphokine production of DCs were also investigated. CD11c+/hi B220− DCs isolated from spleen and Peyers patches (PP) of cows milk (CM) allergic and control mice were transferred intravenously (i.v.) into naïve syngeneic recipients, and IgE‐ and IgG‐specific responses were evaluated. Experiments were also carried out to determine the levels of interferon‐γ (IFN‐γ) and interleukin (IL)‐4 produced by splenocytes from naïve recipients following the adoptive transfer, and CD40 ligand (CD40L)‐mediated IL‐10 production by DCs from allergic and control mice. DCs isolated from spleen and PP of allergic mice, but not control groups, induced CM‐specific IgG and IgE antibody production in naïve recipients in the absence of previous immunization, but did not modify the T helper 1 (Th1) and T helper 2 (Th2) balance. Furthermore, although no difference was observed in the expression of canonical DC surface markers, PP DCs from allergic mice produced less IL‐10 than DCs from controls. We interpret these data as showing that DCs play a pivotal role in allergen‐specific IgE responses and that a Th2‐skewed response may not be involved in the early phase of allergic responses. The identification of the mechanisms underlying these events may help to design novel strategies of therapeutic intervention in food allergy.

Gold nanoparticles uptake and cytotoxicity assessed on rat liver precision-cut slices.

A major obstacle in the field of nanotoxicology is the development of an in vitro model that accurately predicts an in vivo response. To address this concern, rat liver precision-cut slices were used to assess the impact of 5-nm gold nanoparticles (GNPs) coated with polyvinylpyrrolidone (PVP) on the mammalian liver, following exposure to different concentrations and for a duration of up to 24 h. The presence of GNPs inside endocytotic vesicles of hepatocytes was appreciable within 30 min of their addition. After 2 h, GNPs were clearly visualized inside endosome-like vesicles within the slice, not only in hepatocytes but also in endothelial and Kupffer cells located within the first two cellular layers. This uptake did not translate into modifications of either phase I or phase II of 7-ethoxycoumarin metabolism or alter activities of cytochrome P450 toward marker substrates. Furthermore, although the GNPs were rapidly internalized, no overt signs of cytotoxicity, assessed through lactate dehydrogenase release, reduction of methylthiazolyldiphenyl tetrazolium bromide, and glutathione levels, were observed. In conclusion, the use of rat liver slices successfully enhanced nanomaterial screening and determined that PVP-coated 5-nm GNPs were biocompatible with rat liver cells.

Nestin expression in rat adrenal gland

Abstract. The constituents of the intermediate filament network of adrenal gland cells have not been deeply investigated in vivo. Adrenocortical cells have been reported to express cytokeratins and vimentin, but the intermediate filament components of the adrenomedullary cells are still unknown. Nestin is an intermediate filament protein that is mainly expressed in the developing nervous and muscle systems. It has been reported to be unable to form filaments by itself and it co-assembles with vimentin. Using immunocytochemical and biochemical approaches, the present study demonstrates that nestin is expressed in situ either in the cortex or in the medulla of adult rat adrenal glands. Nestin-negative cells prevalently form the zona glomerulosa whereas the zona fasciculata and the zona reticularis are mainly nestin-immunoreactive. Nestin-positive cells always express vimentin-like immunoreactivity but several cells apparently expressing only vimentin are detectable too. Nestin is also expressed by adrenomedullary cells that also display a faint vimentin-like immunoreactivity. We hypothesise that the inconstant detection of nestin in adrenocortical cells depends on their different functional moments. Moreover, even though our data do not allow to confirm vimentin in adrenomedullary cells, in situ detection of nestin in the adrenal medulla indirectly supports in vivo expression of vimentin in chromaffin cells.

Rabbit Tonsil-associated M-cells Express Cytokeratin 20 and Take Up Particulate Antigen

M-cells are believed to play a pivotal role in initiation of the immune response. These cells, located in the epithelia that overlie mucosal lymphoid follicles, are responsible for the active uptake of particulate antigens and for their translocation to the underlying lymphoid tissue. The identification of reliable markers for M-cells is therefore extremely important for the study of the initial steps that lead to the immune response. For this purpose, we studied cytokeratin 20 (CK20) expression in the epithelium of rabbit palatine tonsils by immunofluorescence, confocal microscopy, and Western blotting. CK20+ cells were observed in all rabbit palatine tonsils examined. By Western blotting, one CK20-immunoreactive band was identified at 46 kD on samples of proteins from the intermediate filament-enriched cytoskeletal fraction of tonsil epithelium. Double labeling of CK20+ cells with cell-specific markers confirmed that such cells were actually M-cells. Moreover, CK20+ M-cells displayed a mature phenotype (they formed pockets harboring lymphoid cells) and were functionally competent because they could take up particulate antigens from the pharyngeal lumen. We conclude that CK20 is an M-cell marker for rabbit palatine tonsils. Moreover, we can hypothesize the use of M-cells as a possible site for antigen delivery of particle-based vaccines.