Altered pericyte-endothelial relations in the rat retina during aging: Implications for vessel stability

Mural cells (smooth muscle cells and pericytes) regulate blood flow and contribute to vessel stability. We examined whether mural cell changes accompany age-related alterations in the microvasculature of the central nervous system. The retinas of young adult and aged Wistar rats were subjected to immunohistofluorescence analysis of a-smooth muscle actin (SMA), caldesmon, calponin, desmin, and NG2 to identify mural cells. The vasculature was visualized by lectin histochemistry or perfusion of horse-radish peroxidase, and vessel walls were examined by electron microscopy. The early stage of aging was characterized by changes in peripheral retinal capillaries, including vessel broadening, thickening of the basement membrane, an altered length and orientation of desmin filaments in pericytes, a more widespread SMA distribution and changes in a subset of pre-arteriolar sphincters. In the later stages of aging, loss of capillary patency, aneurysms, distorted vessels, and foci of angiogenesis were apparent, especially in the peripheral deep vascular plexus. The capillary changes are consistent with impaired vascular autoregulation and may result in reduced pericyte-endothelial cell contact, destabilizing the capillaries and rendering them susceptible to angiogenic stimuli and endothelial cell loss as well as impairing the exchange of metabolites required for optimal neuronal function. This metabolic uncoupling leads to reactivation of

RNA expression analysis from formalin fixed paraffin embedded tissues

Formalin fixation and paraffin embedding (FFPE) is the most commonly used method worldwide for tissue storage. This method preserves the tissue integrity but causes extensive damage to nucleic acids stored within the tissue. As methods for measuring gene expression such as RT-PCR and microarray are adopted into clinical practice there is an increasing necessity to access the wealth of information locked in the Formalin fixation and paraffin embedding archives. This paper reviews the progress in this field and discusses the unique opportunities that exist for the application of these techniques in the development of personalized medicine.

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine Ib) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylsae (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.

Cholinergic, serotoninergic and peptidergic components of the nervous system of Discocotyle sagittata (Monogenea: Polyopisthocotylea)

Cholinergic, serotoninergic (5-HT) and peptidergic neuronal pathways have been demonstrated in both central and peripheral nervous systems of adult Discocotyle sagittata, using enzyme histochemistry and indirect immunocytochemistry in conjunction with confocal scanning laser microscopy. Antisera to 2 native flatworm neuropeptides, neuropeptide F and the fMRFamide-related peptide (FaRP), GNFFRFamide, were employed to detect peptide immunoreactivity. The CNS is composed of paired cerebral ganglia and connecting dorsal commissure, together with several paired longitudinal nerve cords. The main longitudinal nerve cords (lateral, ventral and dorsal) are interconnected at intervals by a series of annular cross-connectives, producing a ladder-like arrangement typical of the platyhelminth nervous system. At the lever of the haptor, the ventral cords provide nerve roots which innervate each of the 8 clamps. Cholinergic and peptidergic neuronal organisation was similar, but distinct from that of the serotoninergic components. The PNS and reproductive system are predominantly innervated by peptidergic neurones. Copyright (C) 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd.

A CYTOCHEMICAL STUDY OF THE NERVOUS-SYSTEM OF THE PROTEOCEPHALIDEAN CESTODE, PROTEOCEPHALUS-POLLANICOLA

The localization and distribution of cholinergic, serotoninergic and peptidergic nerve elements in the proteocephalidean tapeworm, Proteocephalus pollanicola, have been investigated by enzyme histochemistry, and by an indirect immunofluorescence technique interfaced with confocal scanning laser microscopy. Cholinesterase (ChE) activity was localized in the major components of the central nervous system (CNS) and the peripheral nervous system (PNS), including the innervation of the reproductive structures of the worm. Serotoninergic (5-HT) nerves were found in the paired cerebral ganglia, transverse commissure and in the 10 longitudinal nerve cords. Antisera to 17 mammalian regulatory peptides and the invertebrate peptide FMRFamide have been used to explore the peptidergic nervous system of the worm. The most extensive immunostaining occurred with antisera raised to members of the neuropeptide Y superfamily, namely neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP). In all cases, intense immunoreactivity was found in numerous cell bodies and fibres of both the CNS and PNS, including the innervation of the reproductive apparatus. FMRFamide antisera stained the same structures to a comparable degree as those raised to the NPY superfamily. Cholinergic and peptidergic elements were much more prevalent within the CNS, while the serotoninergic nerve fibres tended to dominate in the PNS. The overlap obtained in staining patterns for the peptidergic and cholinergic components suggests that there may be a certain amount of co-localization of peptides with small-molecule transmitter substances in the same neurone. Weak staining for the tachykinin, substance P and for calcitonin gene-related peptide(CGRP) was confined to the major longitudinal nerve cords.

Ligand arsenic complexation and immunoperoxidase detection of metallothionein in the earthworm Lumbricus rubellus inhabiting arsenic-rich soil

Although earthworms have been found to inhabit arsenic-rich soils in the U.K., the mode of arsenic detoxification is currently unknown. Biochemical analyses and subcellular localization studies have indicated that As3+-thiol complexes may be involved; however, it is not known whether arsenic is capable of inducing the expression of metallothionein (MT) in earthworms. The specific aims of this paper were (a) to detect and gain an atomic characterization of ligand complexing by X-ray absorption spectrometry (XAS), and (b) to employ a polyclonal antibody raised against an earthworm MT isoform (w-MT2) to detect and localize the metalloprotein by immunoperoxidase histochemistry in the tissues of earthworms sampled from arsenic-rich soil. Data suggested that the proportion of arsenate to sulfur-bound species varies within specific earthworm tissues. Although some arsenic appeared to be in the form of arsenobetaine, the arsenic within the chlorogogenous tissue was predominantly coordinated with S in the form of -SH groups. This suggests the presence of an As::MT complex. Indeed, MT was detectable with a distinctly localized tissue and cellular distribution. While MT was not detectable in the surface epithelium or in the body wall musculature, immunoperoxidase histochemistry identified the presence of MT in chloragocytes around blood vessels, within the typhlosolar fold, and in the peri-intestinal region. Focal immunostaining was also detectable in a cohort of cells in the intestinal wall. The results of this study support the hypothesis that arsenic induces MT expression and is sequestered by the metalloprotein in certain target cells and tissues.

Pro-neural transcription factors as cancer markers

BACKGROUND: The aberrant transcription in cancer of genes normally associated with embryonic tissue differentiation at various organ sites may be a hallmark of tumour progression. For example, neuroendocrine differentiation is found more commonly in cancers destined to progress, including prostate and lung. We sought to identify proteins which are involved in neuroendocrine differentiation and differentially expressed in aggressive/metastatic tumours.

RESULTS: Expression arrays were used to identify up-regulated transcripts in a neuroendocrine (NE) transgenic mouse model of prostate cancer. Amongst these were several genes normally expressed in neural tissues, including the pro-neural transcription factors Ascl1 and Hes6. Using quantitative RT-PCR and immuno-histochemistry we showed that these same genes were highly expressed in castrate resistant, metastatic LNCaP cell-lines. Finally we performed a meta-analysis on expression array datasets from human clinical material. The expression of these pro-neural transcripts effectively segregates metastatic from localised prostate cancer and benign tissue as well as sub-clustering a variety of other human cancers.

CONCLUSION: By focussing on transcription factors known to drive normal tissue development and comparing expression signatures for normal and malignant mouse tissues we have identified two transcription factors, Ascl1 and Hes6, which appear effective markers for an aggressive phenotype in all prostate models and tissues examined. We suggest that the aberrant initiation of differentiation programs may confer a selective advantage on cells in all contexts and this approach to identify biomarkers therefore has the potential to uncover proteins equally applicable to pre-clinical and clinical cancer biology.

Abnormal Glycogen Storage by Retinal Neurons in Diabetes

PURPOSE: It is widely held that neurons of the central nervous system do not store glycogen and that accumulation of the polysaccharide may cause neurodegeneration. Since primary neural injury occurs in diabetic retinopathy, we examined neuronal glycogen status in the retina of streptozotocin-induced diabetic and control rats.

METHODS: Glycogen was localized in eyes of streptozotocin-induced diabetic and control rats using light microscopic histochemistry and electron microscopy, and correlated with immunohistochemical staining for glycogen phosphorylase and phosphorylated glycogen synthase (pGS).

RESULTS: Electron microscopy of 2-month-old diabetic rats (n = 6) showed massive accumulations of glycogen in the perinuclear cytoplasm of many amacrine neurons. In 4-month-old diabetic rats (n = 11), quantification of glycogen-engorged amacrine cells showed a mean of 26 cells/mm of central retina (SD ± 5), compared to 0.5 (SD ± 0.2) in controls (n = 8). Immunohistochemical staining for glycogen phosphorylase revealed strong expression in amacrine and ganglion cells of control retina, and increased staining in cell processes of the inner plexiform layer in diabetic retina. In control retina, the inactive pGS was consistently sequestered within the cell nuclei of all retinal neurons and the retinal pigment epithelium (RPE), but in diabetics nuclear pGS was reduced or lost in all classes of retinal cell except the ganglion cells and cone photoreceptors.

CONCLUSIONS: The present study identifies a large population of retinal neurons that normally utilize glycogen metabolism but show pathologic storage of the polysaccharide during uncontrolled diabetes.

Fasciola hepatica surface tegument: glycoproteins at the interface of parasite and host

Fasciola hepatica, commonly known as liver fluke, is a trematode which causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterisation of FhTeg glycosylation using lectin microarrays to characterise carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. While some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components which could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.