In vitro mesenchymal stem cell differentiation after mechanical stimulation.

OBJECTIVES: Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into adipocytic, chondrocytic and osteocytic lineages on suitable stimulation. We have hypothesized that mechanical loading may influence MSC differentiation and alter their phenotype accordingly. MATERIALS AND METHODS: Mouse bone marrow-derived MSC were established in vitro by differential adherence to plastic culture plates and grown in low glucose medium with 10% foetal calf serum and growth factors. Cells grew out and were subcultured up to 20 times. Differentiation protocols were followed for several cell lineages. Clones with trilineage potential were seeded in type I collagen gels and incubated in a tensioning force bioreactor and real-time cell-derived forces were recorded. Gels were fixed and sectioned for light and electron microscopy. RESULTS: Cell monolayers of parent and cloned mouse bone marrow-derived MSC differentiated into adipocytes, osteocytes and chondrocytes, but not into cardiomyocytes, myotubes or neuronal cells. When cast into type I collagen gels and placed in tensioning bioreactors, MSC differentiated into fibroblast-like cells typical of tissue stroma, and upregulated α-smooth muscle actin, but rarely upregulated desmin. Electron microscopy showed collagen and elastin fibre synthesis into the matrix. CONCLUSIONS: These experiments confirmed that MSC cell fate choice depends on minute, cell-derived forces. Applied force could assist in commercial manufacture of cultured bio-engineered prostheses for regenerative medicine as it mimics tissue stresses and constitutes a good model for development of tissue substitutes.

Intestinal metaplasia in liver of rats after partial hepatectomy and treatment with acetylaminofluorene.

OBJECTIVES: The liver is widely recognized for its ability to self-regenerate after damage. Hepatocyte replication is the primary source of liver restoration, although hepatic stem cells (of one kind or another) may be a secondary font, only brought into effect when primary regeneration is severely compromised. MATERIALS AND METHODS: In experiments using small rodents, such an injury can be inflicted by surgically removing a large portion of the liver followed by treatment with hepatotoxin 2-acetylaminofluorene. Regeneration by hepatocyte replication is blocked and thus, stem cell involvement is promoted. However, other responses may be stimulated and this study describes the presence of mucinous glandular structures in the healing liver after two-thirds of its volume was removed via hepatectomy followed by treatment with 2-acetylaminofluorene. RESULTS: Unique observation of intestinal metaplastic cells was seen under alcian blue/periodic acid-Schiff staining. CONCLUSION: The existence of this phenotype (along with oval cells and small hepatocyte-like cells) is evidence of multipotency of progenitors involved in the hepatic healing response.

Cell proliferation rates in an artificial tissue-engineered environment.

Worldwide, and particularly in Europe, Japan and the USA, cardiovascular disease is a major killer. It can be treated using tissue or organ transplant surgery, but donor organs may be scarce. Tissue engineering is the integration of engineering principles and biology to produce satisfactory synthetic replacement body parts, using viable cells in a suitable matrix, for regenerative medicine. The aim of this study was to measure and compare cell proliferation kinetics after different time intervals of myofibroblasts in a synthetic matrix, thus to be able to deduce the period that a transplanted-cell population can be expected to survive in a tissue-engineered environment. Porcine aortic wall cells were grown in a porous sponge scaffold, that later could be fashioned into aortic or heart valve substitutes. Freshly acquired cells were seeded on identical sponges and were grown under normal culture conditions for a period of 4 weeks. Seeding concentration was a million cells per sponge. Cells progressively populated the sponges, both covering the surface and infiltrating the depth of the matrix, via sponge pores. Samples were taken at 1 week and at 4 weeks, and the rate of cell proliferation was determined by the metaphase arrest technique. Specimens were also taken for light and electron microscopy to determine whether these transplanted cells were capable of synthesizing their own extracellular matrix.

Heart valve and arterial tissue engineering.

In the industrialized world, cardiovascular disease alone is responsible for almost half of all deaths. Many of the conditions can be treated successfully with surgery, often using transplantation techniques; however, autologous vessels or human-donated organs are in short supply. Tissue engineering aims to create specific, matching grafts by growing cells on appropriate matrices, but there are many steps between the research laboratory and the operating theatre. Neo-tissues must be effective, durable, non-thrombogenic and non-immunogenic. Scaffolds should be bio-compatible, porous (to allow cell/cell communication) and amenable to surgery. In the early days of cardiovascular tissue engineering, autologous or allogenic cells were grown on inert matrices, but patency and thrombogenicity of grafts were disappointing. The current ethos is toward appropriate cell types grown in (most often) a polymeric matrix that degrades at a rate compatible with the cells' production of their own extracellular matrical proteins, thus gradually replacing the graft with a living counterpart. The geometry is crucial. Computer models have been made of valves, and these are used as three-dimensional patterns for mass-production of implant scaffolds. Vessel walls have integral connective tissue architecture, and application of physiological level mechanical forces conditions bio-engineered components to align in precise orientation. This article reviews the concepts involved and successes achieved to date.

E1B-deleted adenovirus (dl1520) gene therapy for patients with primary and secondary liver tumors.

Clinical studies were performed with a recombinant mutant adenovirus with an E1B 55-kDa deletion, dl1520, to assess its toxicity and efficacy in patients with irresectable primary and secondary liver tumors. A phase I study showed that dl1520 was well tolerated when administered directly intratumorally, intraarterially, or intravenously up to a dose of 3 x 10(11) PFU. Ultrastructural examination of tissue showed the presence of adenovirus in cell cytoplasm around the nucleus and revealed two dissimilar end points of cell death after virus infection: a preapoptotic sequence and necrosis. A phase II study showed that the combination of dl1520 and 5-fluorouracil (5-FU), when infused into the hepatic artery, was well tolerated. Further improvement in the recombinant vector design will be needed in order to achieve better clinical response.

Detection of adenovirus and initiation of apoptosis in hepatocellular carcinoma cells after Ad-p53 treatment.

Transcription of the p53 gene can regulate progression of apoptosis in a wide variety of tissues. Three categories of human hepatocyte culture have been used to show the initiation of apoptosis after treatment with p53-bearing adenovirus. Chang liver cells are derived from normal liver tissue and express native p53, whereas hepatocellular carcinoma (HCC)-derived cell lines were Hep3B (p53-deleted) and PLC/PRF/5 (p53-mutant). Cultures were infected with Ad-p53 (15 particles per cell; 36 hours), and after treatment, morphological changes in all cell categories were observed by electron microscopy. Infection was evident in the cytoplasm of all treated cell types: after entry across the plasma membrane viruses translocated and came to rest surrounding and adjacent to nuclei, cytoplasm proximal to nuclear membranes became dense with virus- and membrane-derived debris, but intact viruses did not enter nuclei. Apoptosis, recognized morphologically by characteristic chromatin and cytoplasmic condensation, occurred more frequently in HCC-derived cells, and the ultimate fate of apoptotic bodies was phagocytosis and degradation by neighboring cells.

Immunolabeling for electron microscopy.

The protocols in this chapter concern postembedding immunolabeling for transmission electron microscopy; other schedules, such as pre-embedding methods, frozen tissue processes, and procedures for scanning electron microscopy, can be found elsewhere (1). In principle, immunolabeling at the electron microscope (EM) level follows the same precepts as immunolabeling at the light microscope level; in tissues or cells, the location of an antigen of interest is identified by a specific antibody, and must be visualized appropriately for investigation. Electron microscopy permits us to distinguish subcellu-lar organelles, and therefore ultrastructural localization of antigen position. At the EM level, however, the "visualizing step" needs to be provided by an electron-dense entity, most often a heavy metal, which reflects incident electrons; this is in contrast to the final step of light microscope level techniques, in which the final reaction product is sought to be colored (and where there is an element of choice of which color to use). Tissue processing for EM is considerably more severe than that for light microscopy, and thus maintenance of antigenicity in tissue is more taxing. Before immunolabeling for electron microscopy can be fruitful, the first step is to ensure that the antigen of interest is present (or is still present) in the tissue; this is done by performing a thorough procedure at the light microscope level on wax-embedded sections. Once a positive result has been obtained, studies can progress to the ultrastructural level. If the presence of an antigen cannot be demonstrated in a wax-embedded block, it will not be demonstrable in a resin-embedded EM block of the same tissue. In such a case, pre-embedding and frozen tissue techniques can be of use at both light and electron microscope levels.

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.

Pericardial substitution after cardiopulmonary bypass surgery: a trial of an absorbable patch.

Primary closure of the pericardium affords some protection against adhesion formation and the consequent hazards of resternotomy. However, its completion may be impractical and hazardous, and therefore the pursuit of an ideal pericardial substitute has prompted much research. Twenty calves were divided into 3 groups for the study. All animals underwent right posterolateral thoracotomy. The test group (group X), consisting of 6 animals, received a poly-beta-hydroxybutyrate patch (PHB) to close the pericardium following cardiopulmonary bypass (CPB). In group Y (9 animals) the pericardium was left open following CPB. Group Z (5 animals) also had their pericardium left open but did not undergo CPB (non-CPB). The plasminogen activating activity (PAA) of homogenates of pericardial tissue samples were measured in 5 animals in group X, and 5 in group Z. Samples were taken at three time points from the time of pericardiotomy, and at reoperation 4 weeks later. In group X (CPB) there was a significant reduction in the PAA during the operation with some recovery at reoperation. The reduction in the pericardial PAA of group Z (non-CPB) animals did not reach significance. For both group X and group Z the progress of mesothelial damage, compared with that at zero time, showed a significant increase. In addition, their pericardial inflammatory features became more apparent in the later samples but more significantly in group Z. This study demonstrated no significant short-term differences in adhesion formation or postoperative coronary anatomy visibility between any of the groups. At reoperation the patch material contained pronounced macrophage activity but no regenerative mesothelium. There were no infective episodes in any of the animals studied. Furthermore, this study suggests that CPB in comparison to non-CPB has a significant affect on pericardial PAA.

Hepatic extramedullary granulopoiesis after treatment with liver-stimulatory xenobiotics, in lpr mice.

This study demonstrates that extramedullary hematopoiesis occurs in livers of adult lpr mice and, after treatment with each of three xenobiotic compounds--phenobarbital, cyproterone acetate, and nafenopin--it includes granulopoiesis. lpr mice are used as a model of the human disease systemic lupus erythematosus (SLE). The develop a syndrome very similar to that of human sufferers. In untreated lpr mice, mononuclear white blood cells were discernible in hepatic sinusoidal foci; T and B lymphocytes were distinguished from each other by immunocytochemistry at light microscope level. After treatment with any of the xenobiotic compounds, immunolabeling demonstrated the additional presence of granulocytes in foci, and, at electron microscope level neutrophils, eosinophils and their precursors were clearly recognizable.

Wild-type p53 induces apoptosis in Hep3B through up-regulation of bax expression.

We demonstrated that introduction and expression of wild-type p53 gene in the human hepatocellular carcinoma cell line, Hep3B, resulted in up-regulation of both p21WAF1/CIP1 and bax gene expression and apoptosis. This cell line contains integrated hepatitis B virus sequences and lacks the expression of both p53 and retinoblastoma tumor suppressor genes because of deletions. Our results suggest that whereas an increased level of bax expression mediates apoptosis, an increased level of p21WAF1/CIP1 expression does not induce arrest of cell growth, presumably because of the deletion of the retinoblastoma gene. This study also confirms reported observations that p53 is a tumor suppressor gene, which induces apoptosis in malignant cells that lack normal p53 activity because of mutation, deletion, or inactivation of the gene by the presence of oncogenic viral proteins.

Reversal of phenobarbital-induced hyperplasia and hypertrophy in the livers of lpr mice.

Fas is a cell surface receptor that mediates apoptosis, and Fas mRNA has been demonstrated in hepatocytes. MRL/MP-lpr/lpr mice carry the mutated lymphoproliferation-associated gene, lpr, that codes for truncated Fas protein, resulting in reduced apoptotic potential in some circumstances. Phenobarbital treatment of experimental animals induces cytochrome P450 enzymes, and thus acts as a growth stimulus to the liver with both hyperplasia and hypertrophy; cessation results in reversion of liver to normal size with apoptosis playing a role. This study has determined the respective contributions of atrophy and apoptosis to this involution in Fas-defective and normal-FAs bearing animals. Between the first day and the fifth day after phenobarbital cessation, the weights of both Fas-defective (lpr/lpr) livers and control (lpr/+) livers reduced. Hepatocyte hypertrophy gradually reverted in both categories of mouse and this was the greater contribution to reduction in liver size. In lpr/lpr animals, there was a consistent level of apoptosis which remained relatively constant, while numbers of apoptotic cells in control livers increased over the period. This investigation has shown that in liver, a mechanism to execute apoptosis is operative even in Fas-defective mice, but it is not sensitive to signals activated by the removal of the growth stimulus. This is in contrast to mice which can mount a Fas-mediated response; thus a separate apoptotic pathway is indicated.

Liver stem cells: when the going gets tough they get going.

The ability of the liver to regenerate is widely acknowledged, and this is usually accomplished by the entry of normally proliferatively quiescent hepatocytes into the cell cycle. However, when hepatocyte regeneration is impaired, small bile ducts proliferate and invade into the adjacent hepatocyte parenchyma. In humans and experimental animals these ductal cells are referred to as oval cells, and their association with defective regeneration has led to the belief that they are the progeny of facultative stem cells. Oval cells are of great biological interest since they may represent a target population for hepatic carcinogens, and they may also be useful vehicles for ex vivo gene therapy for the correction of inborn errors of metabolism. The ability of oval cells to differentiate into hepatocytes has been demonstrated unequivocally. However, this process only occurs when the regenerative capacity of hepatocytes is overwhelmed, and thus, unlike the intestinal epithelium, the liver is not behaving as a classical continually renewing stem cell-fed lineage.

Effect of total hepatic vascular exclusion during liver resection on hepatic ultrastructure.

The aim of this investigation was to observe ultrastructural changes in the liver in response to warm ischemia during liver surgery. In 11 noncirrhotic patients, hepatic resection was performed under total vascular exclusion (TVE). The mean duration of warm ischemia was 28 minutes (range 16-48 minutes). Three specimens were taken from each patient: before clamping, at the end of TVE, and after reperfusion. Biopsy specimens were studied by light microscopy and by transmission electron microscopy (EM). At the end of the ischemic phase sinusoids were collapsed with resultant loss of normal hepatic architecture. Morphological changes to hepatocytes included focal chromatin condensation at the nuclear margins, distended nuclear envelope, and swelling of both mitochondria and endoplasmic reticulum. After reperfusion these changes reversed. The phenomenon of sinusoidal and hepatocellular recovery after TVE was seen in all the cases of this study, irrespective of age, sex, disease, type and severity of surgical intervention, and duration of TVE. It can be concluded that TVE over a period of 48 minutes has no irreversible deleterious effects on the ultrastructure of the noncirrhotic liver.

An analysis of apoptosis in lymphoid organs and lupus disease in murine systemic lupus erythematosus (SLE).

Apoptosis is a programmed cell death process that helps to regulate both T cell and B cell development. In this study, we have investigated the levels of apoptotic death in cells of the thymuses and spleens (white matter) of autoimmune MRL-lpr/lpr mice with progressive lymphadenopathy and SLE disease activity; we also examined the renal pathology in these animals. Fas is a cell surface receptor, which when activated initiates the sequence of events that lead to apoptosis. In MRL-lpr/lpr mice Fas is defective, so the competency for apoptosis may be reduced. In young animals of advancing age the thymuses enlarged until in 5-month-old females the average weight was three times that at 1 month, and spleen and kidney weights also increased in size disproportionately. At light microscope level apoptotic cells in tissue sections were counted using both routine eosin and haematoxylin staining (to identify them by their morphology) and in situ end-labelling of cells with DNA strand breaks; their presence was further confirmed by electron microscopy. As the mice aged, the numbers of apoptotic cells in thymic cortex, thymic medulla and spleen white pulp areas reduced significantly (P < 0.01-0.001), whereas in BALB/c normal controls they increased significantly (P < 0.05). These changes were coincident with the development of severe lupus, whose activity was assessed by measuring serum anti-ssDNA and anti-dsDNA antibody titres and urinary protein (albumin) level which were elevated significantly by 5 months of age (P < 0.001 for both ssDNA and dsDNA and P < 0.01 for urine albumin) compared with their younger counterparts. Thus, lymphoid organ enlargement, decrease in apoptotic indices, elevated serum anti-ssDNA and anti-dsDNA antibody levels, and impaired renal function coincided with the onset and severity of lupus disease in lpr mice. It seems likely that there is a causal relationship between defective deletion of autoreactive lymphoid cells, imperfect Fas-mediated apoptosis and development of murine SLE.

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

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 cell-fed 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. This review proposes that the liver does harbour stem cells which are located throughout the biliary epithelium, and that oval cells represent the progeny of these stem cells and function as an amplification compartment for the generation of 'new' hepatocytes. This is a conditional process which only occurs when the regenerative capacity of hepatocytes is overwhelmed and thus, unlike the intestinal epithelium, the liver is not behaving as a classical continually renewing stem cell-fed lineage. We focus on the biliary network, not merely as a conduit for bile, but also as a cell compartment with the potential to proliferate under appropriate conditions and give rise to fully differentiated hepatocytes and other cell types.

Liver damage in the rat induces hepatocyte stem cells from biliary epithelial cells.

BACKGROUND & AIMS: When rat hepatocyte regeneration after partial hepatectomy is blocked by 2-acetylaminofluorene, a proliferation of biliary epithelia sends out ductules into the parenchyma. The ability of these neoductules to act as a significant progenitor compartment for hepatocytes is in dispute. This study aims to resolve this question by varying the amount of 2-acetylaminofluorene administered. METHODS: Rats were fed 2-acetylaminofluorene fr 6 days before and up to 7 days after partial hepatectomy was performed at a dose of either 2.5 (low) or 5 (high) mg/kg(-1)/day(-1). The response was monitored by the immunohistochemical expression of intermediate filaments and cytochrome P450 enzymes. RESULTS: No regeneration by mature hepatocytes occurred with either dose, and new ductules expressed the biliary cytokeratins 7, 8, 18, and 19 and, in addition, vimentin. At the high dose, hepatocytic differentiation was infrequent, whereas apoptosis and intestinal differentiation were common. At the low dose, almost all ductules differentiated into hepatocytes within 14 days of hepatectomy. CONCLUSIONS: Biliary epithelium is an effective and substantiative hepatocyte progenitor compartment under appropriate conditions.

Pericardial trauma and adhesions in relation to reoperative cardiac surgery.

In an attempt to appreciate the changes that favour adhesion formation we compared the morphological and fibrinolytic changes that occur in human primary and reoperative pericardium. Ten patients undergoing primary elective open heart surgery and ten undergoing first time reoperative open heart surgery were studied. Pericardial samples were taken at four time points. At 0 (time A) and 30 (time B) minutes from the time of pericardiotomy (before the commencement of CPB), 30-50 minutes (time C) after the commencement of CPB, and then finally 10 minutes (time D) after the patient had been rewarmed. The fibrinolytic activity, as measured by the plasminogen activating activity (PAA), in the pericardial samples of the ten primary cases was compared with that in 5 of the reoperative cases. For the primary group, the PAA after 30 minutes of exposure (median 6.65 IU/cm2, range 3.85-11.89 IU/cm2, p = 0.14, n = 10) was not significantly reduced when compared to the initial activity (median 8.74 IU/cm2, range 2.22-17.68 IU/cm2, n = 10). After 30-50 minutes CPB the PAA was significantly reduced (median 3.93 IU/cm2, range 1.5-13.24 IU/cm2, p = 0.028, n = 10) and still reduced after rewarming for 10 minutes (median 3.12 IU/cm2, range 0.88-19.93 IU/cm2, p = 0.047, n = 10). The simultaneous plasma tissue-type plasminogen activator activity showed a significant (p < 0.05) increase after 30-50 minutes bypass with a later decline. The changes in the reoperative pericardial PAA were similar. In addition, the degree of PAA in reoperative pericardium was consistently lower than that observed in primary tissue. The extent of primary pericardial mesothelial damage at times B, C, and D compared with that at time A showed a significant (p < 0.01 for times B, C, and D) increase. Similarly there was a significant worsening of the degree of inflammation. Compared with primary pericardium, the reoperative samples showed a significant (p < 0.01 for times A, B, and C) preponderance of damaged mesothelium at the earlier stages of the operation. It appears that, following the initial bypass surgery, the processes that cause pericardial and mesothelial healing with recovery of PAA compete with those leading to pericardial adhesions and fibrosis. The histological and biochemical outcome seen in reoperative pericardium is the result of these competitive actions.

Oval cell differentiation into hepatocytes in the acetylaminofluorene-treated regenerating rat liver.

When hepatocyte regeneration after a two-thirds partial hepatectomy (PH) in rats is blocked by oral gavage of acetylaminofluorene, a proliferation of ductular cells ensues that results in a profusion of neoductules radiating from each portal tract. To examine the possibility that this population of newly emerging cells harbors cells capable of differentiating into hepatocytes, we have looked in these cells for expression of functional markers of hepatocyte commitment at both the RNA and protein levels. Expression of albumin and alpha-fetoprotein (alpha-FP) messenger RNA (mRNA) transcripts were sought in situ using antisense riboprobes, and the expression of a number of cytochrome P450 enzymes was examined immunohistochemically. Before any signs of differentiation the ductular cells strongly expressed cytokeratins 7, 8, 18, and 19 in the same manner as authentic bile ducts, but unlike the latter also expressed vimentin. In situ hybridization studies showed that small bile ducts close to the limiting plate, as well as the newly formed ducts, expressed albumin and alpha-fetoprotein messenger RNAs, and immunocytochemistry showed that the distribution of the respective proteins was similar. Beginning at 1 week after partial hepatectomy, areas of differentiation could be found in the new ducts, with cells resembling either columnar intestinal-type epithelia or hepatocytes. Intestinal-like cells expressed neither albumin, alpha-FP, nor cytochrome P450 enzymes, whereas ductular cells appearing like hepatocytes with the typical membranous distribution of cytokeratin 8 strongly expressed a variety of cytochrome P450 enzymes normally associated with functional hepatocytes. These observations further support the belief that reactive ductules, sprouted from small ducts, can represent an adaptive response of the liver to replenish lost hepatocytes, although some of the newborn cells appear to differentiate along intestinal lines.

Subcellular distribution of peptides associated with gastric mucosal healing and neoplasia.

The trefoil peptides pS2 and human spasmolytic peptide are putative growth factors, particularly associated with mucus-producing cells of the gastrointestinal tract including those of the stomach. The receptor for transforming growth factor alpha (TGF alpha) takes its name from one of its alternative ligands, epidermal growth factor, and is called the epidermal growth factor receptor. Although there is immunoreactive epidermal growth factor in the stomach, it is TGF alpha and the epidermal growth factor receptor that are abundant. Immunolabelling at electron microscope level allows for subcellular localisation of antigens; pS2 and human spasmolytic peptide co-localise to cytomembranes, including the Golgi apparatus, and thecae of surface/pit mucous cells. TGF alpha is abundant on the membranes of tubulovesicles of parietal cells and is also present in chief cells: in mucous producing cells it can be detected but not in association with mucous. The distribution of the epidermal growth factor receptor mimics that of TGF alpha but with preferential clustering on the basolateral membranes of gastric cells. The trefoil peptides are associated with healing and probably act, together with mucus, to protect the gastric mucosa and maintain a viable environment. TGF alpha, transduced via the epidermal growth factor receptor, inhibits gastric acid secretion, thus aids the trefoils in the maintenance of a gastric microenvironment conducive to healing after damage. TGF alpha, however, is also a potent mitogen; while this property plays a vital part in repairing mucosal defects, if this peptide or indeed its receptor are overexpressed, the result can be neoplasia.