Catherine E. Sarraf

Trefoil peptide gene expression in gastrointestinal epithelial cells in inflammatory bowel disease

BACKGROUNDnThis work expands on recent observations that the trefoil peptides pS2 and human spasmolytic polypeptide (hSP) are expressed in the ulceration-associated cell lineage (UACL) glands developing in chronic ulcerative conditions.nnnMETHODSnTrefoil peptide expression in small intestinal Crohns disease was examined by in situ hybridization to reveal sites of expression of the messenger RNAs encoding pS2 and hSP and by immunohistochemistry and immunoelectron microscopy to localize the peptides in the UACL and adjacent goblet and neuroendocrine cells.nnnRESULTSnGoblet cells near the UACL expressed pS2 messenger RNA and peptide; ultrastructural immunolocalization revealed pS2 copackaged within mucous cell granules. Neuroendocrine cell hyperplasia was marked in crypts near the UACL; pS2 was copackaged with the neuroendocrine granules.nnnCONCLUSIONSnCopackaging of a secretory protein, pS2, in both mucous and neuroendocrine granules, which have different functions, is unusual and indicates an important role for pS2 in the secretory process itself or as a ligand delivered to its receptors via different routes. It is concluded that trefoil peptides are of considerable potential functional importance in inflammatory bowel disease.

Pluripotential liver stem cells: facultative stem cells located in the biliary tree

Abstract. The ability of the liver to regenerate after parenchymal damage is usually accomplished by the ephemeral entry of normally proliferatively quiescent (G0) hepatocytes into the cell cycle. However, when hepatocyte regeneration is defective, arborizing ductules which are continuous with the biliary tree, proliferate and migrate into the surrounding parenchyma. In man these biliary cells have variously been referred to as ductular structures, neoductules and neocholangioles, and have been observed in many forms of chronic liver disease, including cancer. In experimental animals similar ductal cells are usually called oval cells, and their association with defective regeneration has led to the belief that these cells represent a progenitor cell population. Oval cells are thought to take over the burden of regenerative growth after substantial hepatocyte loss, suggesting that they are the progeny of facultative stem cells. The liver is not, however, generally considered as a stem cellfed hierarchy, although this is disputed by others. Despite this, the subject of oval cells has aroused intense interest as these cells may represent a target population for hepatic carcinogens, and they may be useful vehicles for ex vivo gene therapy.

Review : Apoptosis: regulation and relevance to toxicology

1 Apopotosis is a remarkably stereotyped morphological event across all tissues in response to a vast array of dam aging agents. 2 Our very existence depends upon a willing exchange of old life for new: apoptotic cell death is our guardian and saviour from genetic damage. 3 There is a close link between cell proliferation and apoptosis: When a cell picks up the machinery to prolifer ate it also acquires an abort pathway - better dead than wrong. 4 A wide variety of highly conserved genes have been implicated in triggering apoptosis. 5 The release of DNA loops from the nuclear scaffold is a more crucial intracellular event than DNA laddering in apoptotic cells. 6 The manipulation of apoptotic rates in many of the common diseases in man will be a major therapeutic strategy in the future.

The role of growth factors in gastrointestinal cell proliferation.

The epithelial lining of the gastrointestinal (GI) tract is in a state of continuous cell renewal, and the proliferating and differentiating/differentiated cell populations are spatially clearly demarcated. Members of the epidermal growth factor (EGF) family of peptides, the trefoil peptides and enteroglucagon appear to be the most important enterotrophic molecules for both normal cell renewal and healing after cell damage. Transforming growth factor‐a (TGF‐a) appears to be the primary physiological ligand for the EGF receptor (EGFR), promoting normal cell renewal, and TGF‐a/EGFR are part of an autocrine loop in many intestinal cancers. In response to damage, a differentiating cell lineage arises from adjacent epithelium secreting EGF, TGF‐a and trefoil peptides; this may be viewed as part of a ‘repair kit’ in damaged endodermally‐derived tissue.

Perioperative histologic and ultrastructural changes in the pericardium and adhesions

The presence of pericardial adhesions prolongs the operation time and increases the risk of serious damage to the heart and other major vascular structures during resternotomy. The reported incidence of such damage is 2% to 6%. Pericardial mesothelial cells exhibit fibrinolytic activity, and therefore have an actual or potential role in the breakdown of the fibrinous adhesions that serve as the initial scaffolding for the firm collagenous adhesions seen at reoperation. Ten patients undergoing primary cardiac procedures were studied to assess the morphologic changes that take place within the pericardium and to relate these to accompanying changes in the pericardial plasminogen activating activity. Samples were taken at 0, 75, and 135 minutes after pericardiotomy. Compared with samples obtained at the time of pericardiotomy, those taken at 75 and 135 minutes demonstrated a significant progression in the mesothelial cell damage (p < 0.01), together with increasing evidence of pericardial inflammation (p < 0.01). The findings from electron microscope studies confirmed and supplemented these findings. Furthermore, compared with its initial levels (median, 2.06 IU/cm2; range, 1.28 to 6.48 IU/cm2), the plasminogen activating activity of pericardial biopsy specimens was significantly reduced at 75 minutes (median, 0.64 IU/cm2; range, 0.12 to 2.44 IU/cm2; P < 0.05), with some recovery at 135 minutes (median, 1.45 IU/cm2; range, 0.12 to 4.39 IU/cm2; p = 0.059). This study has revealed that, during cardiac procedures, the pericardium undergoes inflammatory changes with concomitant damage to its mesothelium, together with a reduction in the pericardial mesothelial fibrinolytic potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Trefoil Peptide Gene Expression in Gastrointestinal Epithelial Cells in Inflammatory Bowel Disease

Trefoil peptides are a growing group of proteins with interesting structural and functional properties. We have defined the pattern of trefoil peptide gene expression in the ulceration-associated cell lineage (UACL) and in the nearby mucosa in Crohns disease. In the UACL, human spasmolytic polypeptide (hSP) mRNA is expressed in the acinar and proximal duct cells, while pS2 mRNA and peptide are found in the distal duct cells and in the surface cells. In adjacent mucosa, pS2 mRNA and protein are expressed by goblet cells, with the pS2 peptide concentrated in the area of the Golgi and also in the theca. Ultrastructural immunolocalisation showed the pS2 to be co-packaged in the mucous cell granules before being secreted into the intestinal lumen. In addition, pS2 peptide was demonstrated in local neuroendocrine cells and was also co-packaged with the neuroendocrine granules. The crypts associated with the UACL also showed marked neuroendocrine cell hyperplasia. We conclude that pS2 peptide is secreted locally into the viscoelastic coat covering the intestinal mucosa which surrounds Crohns disease ulcers. In addition, it is clear that intestinal goblet cells, in addition to producing mucins, are a rich source of regulatory peptides. Moreover, pS2 is clearly co-packaged with neurosecretory granules, which are released through basal and lateral membranes so that the contained peptides can act in a paracrine manner. These findings are interpreted in terms of the epidermal growth factor/urogastrone released by the UACL, stimulating pS2 gene expression in surrounding cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Trefoil Peptide gene expression in small intestinal Crohn's disease and dietary adaptation

We examined the patterns of trefoil peptide gene expression in the ulcer-associated cell lineage (UACL) and mucosa adjacent to Crohns disease in humans and during gastrointestinal adaptation to enteral feeding in rats. In the UACL, human spasmolytic polypeptide (hSP) mRNA and peptide are present in the acinar and proximal duct cells, whereas pS2 mRNA and peptide are found in the distal duct cells and in the surface cells. In mucosa adjacent to UACL, pS2 mRNA and peptide are expressed ectopically by goblet cells and neuroendocrine cells. Intestinal crypts associated with the UACL showed marked neuroendocrine cell hyperplasia. Ultrastructural immunolocalization showed pS2 to be copackaged in the mucous cell and neuroendocrine granules. The copackaging of a secretory protein in both mucous and neuroendocrine granules, which have different functions, is unusual and indicates an important role for pS2 in the secretory process itself or as a ligand delivered to its receptor via multiple routes. We also cloned the newest trefoil peptide, intestinal trefoil factor (ITF), from human and rat intestinal mucosa. Using in situ hybridization we demonstrated its synthesis by normal rat intestinal goblet cells. RNAse protection analysis revealed that the level of mRNA for rat ITF in small and large intestine was affected by the process of enteral feeding. We conclude that trefoil peptides are widely distributed in the intestine in human inflammatory bowel disease and are of considerable potential functional importance.

Transforming growth factor‐α immunoreactivity in a variety of epithelial tissues

Abstract. The immunohistochemical expression of transforming growth factor‐alpha (TGFα) has been examined in a range of normal adult epithelial tissues from both man and rat using an anti‐hTGFα monoclonal antibody (GF10). No differences in distribution were apparent between man and rat. In the continually renewing epithelium of the gastrointestinal tract, no staining was seen within the proliferative compartments, but strong immunoexpression was noted in various differentiated populations. In the testis, the spermatogonia were unstained, but the more luminally orientated germ cells were strongly positive. In the gastrointestinal tract, at least, any mitogenic action of TGFα must be mediated through a relatively long paracrine loop. In contrast, the differentiated parenchyma of kidney, salivary gland and liver remained unstained apart from collecting ducts in the kidney, striated ducts in salivary glands and bile ducts in the liver. The association of TGFα with tubule formation was reinforced by the very strong staining of newly forming bile ducts in a model of liver oval cell proliferation. Thus, in all the epithelia studied there was a distinct spatial pattern of TGFα immunoreactivity.

Evaluation of a proposed technique to assess unscheduled DNA synthesis and genotoxicity.

Results from a recent, new assay suggest that omeprazole, a potent inhibitor of gastric acid secretion, is genotoxic. The principle of this assay is that the non-proliferating zone of surface gastric epithelial cells can be selectively removed by controlled digestion so that any incorporation of tritiated thymidine into these cells represents unscheduled DNA synthesis. Parietal cells (which are located below the uppermost proliferating cells) and proliferating cells in semiconservative, regular DNA synthesis could always be shown in the digested fraction, and as regular DNA synthesis takes up a thousand fold more thymidine than unscheduled DNA synthesis, any signal from unscheduled synthesis would therefore be swamped. The digestion process was also uneven, as histological analysis showed denuded patches of mucosa, and gland like structures were seen in the digest. Quantification of the number of silver grains over the nuclei showed no increase in low level labelling after omeprazole administration, indicating that there was no unscheduled DNA synthesis. The labelling index of undigested gastric tissue from omeprazole treated rats was not significantly different from that of the control group, despite an increase in the plasma gastrin value.

Electron Microscopic Assessment of Adenovirus-Mediated Transfer

This chapter describes the method of preparing and observing hepatocellular carcinoma (HCC) and surrounding normal liver cells infected with therapeutically p (53)-transfected adenovirus (Ad-p (53)), so that morphology of the cells and viruses, and crucially their relationships to each other, are revealed. In standard practice, ultrastructural analysis of viruses carried in body fluids (e.g., stool or mucus) is sufficient for diagnosis, using the technique of phosphotungstate - dark field staining-of the aqueous extract. That method, however, is not suitable when one needs to examine precise subcellular location of viruses in situ, with tissue and cells intact, for complete pathological assessment; here, we describe our method (1) for transmission electron microscopy of the ultrastructure of virus-infected tumors. Tissue fixation, osmication, embedding, section cutting, and observation of Ad-p (53) infection will be included.