Veronika Jancsik

Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs

Maternal immunity is mediated exclusively by colostral immunoglobulins in ruminants. As the neonatal Fc receptor (FcRn) is suggested to be involved in the transport of immunoglobulin G (IgG) in the mammary gland, we cloned this receptor from sheep and analysed its expression in the mammary gland around the time of parturition and also in the small intestine from the newborn lamb. FcRn heavy‐chain mRNA was detected (by using in situ hybridization) exclusively in the acinar and ductal epithelial cells in mammary gland biopsies both before and after parturition. Immunohistochemistry revealed that the cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition. A remarkable difference was observed in the pattern after lambing, where the apical side of the cells was strongly stained. The presence of the FcRn in the acinar and ductal epithelial cells of the mammary gland, and the obvious change in distribution before and after parturition, indicate that the FcRn plays an important role in the transport of IgG during colostrum formation in ruminants. Immunohistochemical analysis detected a strong apical and a weak basal FcRn signal in the duodenal crypt cells of a neonatal lamb, which have been previously demonstrated to secrete IgG1 in newborn ruminants. The FcRn was not detected in the duodenal enterocytes, which absorb intact IgG from the colostrum in a non‐specific manner. These data suggest that FcRn is involved in IgG1 secretion in ruminant epithelial cells.

Localization of the sheep FcRn in the mammary gland

Among the multiple functions, which have been identified for the neonatal Fc receptor (FcRn), we study its role in the IgG transport in the mammary gland during the colostrum formation. For this reason, we have obtained several mammary gland biopsies from a pregnant sheep around parturition. The presence of the FcRn heavy chain mRNA was detected exclusively in the acinar and ductal epithelial cell by in situ hybridization (ISH). We detected strong signal in samples harvested 24 and 10 days prepartum; however, in samples we collected postpartum was barely detectable. Immunohistochemistry confirmed our ISH data. The cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition, although a remarkable difference was observed in the pattern after lambing. The signal indicated uneven distribution of the FcRn alpha chain in the epithelial cells 1 and 5 days postpartum, since the apical sides of the epithelial cells were highlighted. The presence of the FcRn in the acinar and ductal epithelial cells and the obvious change of its distribution before and after parturition suggest that FcRn plays an important role in the IgG transport during colostrum formation. FcRn expression was also found in the lamb duodenal crypt epithelial cells, which have been previously demonstrated to secrete IgG1 in newborn ruminants, suggesting secretory role of the FcRn in ruminant epithelial cells.

Immunofluorescence mapping of dystrophin in the rat brain: astrocytes contain the splice variant Dp71f, but this is confined to subpopulations

Dystrophins are membrane-associated actin-binding proteins, recognized at first in muscular dystrophies. In the brain the full-length Dp427 has been detected, as well as Dp140 and Dp71 of the shorter variants. Dp71 seems to be their major representative in the brain, and it occurs as splice variants, Dp71f and Dp71d. Dystrophins have been demonstrated mainly in neurons. In tissue cultures, the glial data, mainly in situ, are still insufficient. The present mapping study reveals the astroglial localization of the splice variant Dp71f, using a monoclonal antibody (5F3, developed by D. Mornet) specific for its additional 31 last amino acids. In parallel, another monoclonal antibody was used (Dys2, Novocastra) that detects the Dp71d, Dp427, as well as Dp140 and other short variants. Rats were overdosed with ether and perfused transcardially with 4% phosphate-buffered paraformaldehyde solution. Floating Vibratome sections were processed for immunohistochemical labeling with fluorescent secondary antibodies. In some animals the reactive glia were investigated following stab wounds in ketamine-xylazine anesthesia. Only the 5F3 antibody labeled astrocytes, however, not in general but in special localizations, mainly along the glia limitans of the pial surface, below the ependyma and in the reactive glia. Perivascular astrocytes were consistently labeled only where the vessels entered the brain, and in some circumventricular organs. The 5F3 antibody also labeled the ependyma and the residual subventricular zone. In contrast to the astrocytes, neurons were labeled throughout the brain. Dys2 antibody (to Dp71d and longer isoforms) labeled neurons in a distribution similar to that of 5F3, but rarely labeled astroglia and only in perivascular rings. Dp71f positivity seems to occur in those astrocyte populations that proved to be immunopositive to glial fibrillary acidic protein (GFAP) and produced laminin in former studies.

Restenosis and Therapy

The vascular disease involves imbalanced function of the blood vessels. Risk factors playing a role in development of impaired vessel functions will be briefly discussed. In ischemia/reperfusion (I/R), ischemic hypoxia is one of the cardinal risk factors of restenosis. Various insults are shown to initiate the phenotype switch of VSMCs. The pathological process, leading to activated inflammatory process, complement activation, and release of growth factors, initiate the proliferation of VSMCs in the media and cause luminal narrowing and impaired vascular function. The review summarizes the alteration process and demonstrates some of the clinical genetic background showing the role of complement and the genotypes of mannose-binding lectin (MBL2). Those could be useful markers of carotid restenosis after stent implantation. Gene therapy and therapeutic angiogenesis is proposed for therapy in restenosis. We suggest a drug candidate (iroxanadine), which ensures a noninvasive treatment by reverse regulation of the highly proliferating VSMCs and the disturbed function of ECs.

Dystroglycan is involved in laminin-1-stimulated motility of Müller glial cells: combined velocity and directionality analysis.

We investigate the role of dystroglycan, a major laminin‐1 receptor and central member of the dystrophin–glycoprotein complex, in the laminin‐1 induced motility of cultured Müller glial cells. Binding of laminin‐1 to dystroglycan was prevented by IIH6, a function‐blocking monoclonal antibody against α‐dystroglycan. As an alternative means of inhibition, we used heparin to mask the dystroglycan binding site of the laminin‐1, known to overlap with heparin binding sites. Cell motility was characterized in a two‐dimensional motility assay based on computer‐controlled videomicroscopy and statistical analysis of cellular trajectories. We obtained data on both the cell velocity and the diffusion index, a measure of direction‐changing frequency. Both means of inhibition of dystroglycan function led to a significant decrease in the ability of laminin‐1 to stimulate cell migration. At the same time, dystroglycan function does not appear to be involved in laminin‐1‐dependent increase in process dynamism and direction‐changing activity.

Expression of α-dystrobrevin in blood-tissue barriers: sub-cellular localisation and molecular characterisation in normal and dystrophic mice

The α- and β-dystrobrevins (DBs) belong to a family of dystrophin-related and dystrophin-associated proteins that are members of the dystrophin-associated protein complex (DAPC). This complex provides a link between the cytoskeleton and the extracellular matrix or other cells. However, specific functions of the two dystrobrevins remain largely unknown, with α-DB being believed to have a role mainly in skeletal muscle. Here, we describe previously unknown expression patterns and the localisation and molecular characteristics of α-DB isoforms in non-muscle mouse tissues. We demonstrate a highly specific sub-cellular distribution of α-DB in organs forming blood-tissue barriers. We show α-DB expression and localisation in testicular Sertoli cells, stomach and respiratory epithelia and provide electron-microscopic evidence for its immunolocalisation in these cells and in the central nervous system. Moreover, we present the molecular characterisation of α-DB transcript in these tissues and provide evidence for a distinct heterogeneity of associations between α-DB and dystrophins and utrophin in normal and dystrophic non-muscle tissues. Together, our results indicate that α-DB, in addition to its role in skeletal muscle, may also be required for the proper function of specific non-muscle tissues and that disruption of DAPC might lead to tissue-blood barrier abnormalities.

Synaptic alpha-dystrobrevin: localization of a short alpha-dystrobrevin isoform in melanin-concentrating hormone neurons of the hypothalamus

The expression of the two members of the dystrobrevin (DB) family in the adult brain was thought to be highly specific for the two main cell types: alpha-dystrobrevin (alpha-DB) and beta-dystrobrevin (beta-DB) has been identified as glial and neuronal proteins, respectively. In the present work we show that a subset of neurons in the hypothalamus contains alpha-DB. Comparative immunohistochemical studies with two alpha-DB antibodies of different specificity indicate that the neurons contain short alpha-DB isoform(s) alpha-DB-2 and/or alpha-DB-4. Immunoreactive multipolar or spindle-shaped neurons form clusters with bilateral symmetry, localized predominantly in the lateral hypothalamic area, with extensions into the zona incerta and the dorso-medial and ventro-medial hypothalamic region. alpha-DB immunoreactivity was localized in cell processes and at postsynaptic densities, furthermore in the endoplasmic reticulum within the perikarya. alpha-DB-positive neurons are beta-dystrobrevin immunoreactive, but alpha- and beta-DB do not co-localize with their usual molecular anchors like dystrophins or high molecular weight forms of utrophin. Colocalization with nNOS was also not observed. The pattern of alpha-DB immunoreactive neurons gave a perfect colocalization with melanin-concentrating hormone (MCH) neurons throughout the whole region studied. We propose that alpha-DB plays a role in a structure or regulation mechanism unique to MCH-expressing neurons.

Three-dimensional visualization of the distribution of melanin-concentrating hormone producing neurons in the mouse hypothalamus.

We present here a new procedure to represent the 3D distribution of neuronal cell bodies within the brain, using exclusively softwares free for research purposes. Our technique is based on digitalized photos of brain slices processed by immunohistochemical technique, and the 3D Slicer software. The technique presented enables transposition of immunohistochemical or in situ hybridization data to the stereotaxic mouse brain atlas (e.g. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates. second ed. Academic Press, San Diego). By exporting the finalized models into a popular 3D design software (3DS Max) arbitrary environment and motion simulation can be created to improve the visual understanding of the area studied. Application of this technique provides the possibility to store, analyze and compare data - e.g. on the hypothalamic neuropeptides - across experimental techniques and laboratories. The method is exemplified by visualizing the distribution of immunohistochemically identified melanin-concetrating hormone (MCH) containing perikarya within the mouse hypothalamus.