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Biomedical subjects

M R Alison

Publications and source records attributed to M R Alison.

At least 55 records · Page 3Linked to original sources

Assessing cellular proliferation: what's worth measuring?

The assessment of cellular proliferation is, in the main, carried out on histological material. Ideally, such methods should be applicable to routinely processed tissues, they should be relatively inexpensive and the results easily quantified and interpreted. A knowledge of what is 'normal' within the tissue from both a temporal and spatial point of view is essential. There is a burgeoning literature on novel markers of cell proliferation, usually these are immunohistochemically based, each method with its own unique set of conditions for optimal tissue processing. Some would argue this is simply a process of 'reinventing the wheel' since mitotic counting is a perfectly adequate indicator of proliferation, but we could justly counter that a labelling index (S-phase or growth fraction) is more likely to produce an accurate assessment when the sample size is small. In tumours, tissue heterogeneity is likely to render any single measurement meaningless. To date, there are no reliable validated immunohistochemical markers of the labelling index in animal tissues which visualise naturally occurring proliferation-associated molecules, though this may change shortly.

Animals↗

TGF-alpha can act as morphogen and/or mitogen in a colon-cancer cell line.

Transforming growth factor alpha (TGF-alpha) has multifunctional biological effects on a variety of mesenchymal and epithelial cells. It is a potent mitogen for a number of normal and transformed cell types, regulates extracellular matrix (ECM) production and promotes breast, kidney and lung morphogenesis. To clarify the role of ECM proteins in the morphogenetic and mitogenic effects of TGF-alpha, we have used a human colon carcinoma cell line (SW1222) which expresses EGF receptor. Here we show that TGF-alpha at 1 ng/ml increases the proliferation of SW1222 cells, but only when they are cultured on plastic rather than collagen-coated plates. Higher concentrations of TGF-alpha (10 ng/ml) did not increase cell proliferation but significantly enhanced the crypt-like glandular differentiation when cells were grown in 3-dimensional collagen gel (p = 0.027). These effects were accompanied by increased expression of alpha 2 beta 1 and alpha 3 beta 1 integrin molecules, which are receptors for extracellular matrix proteins, and by a statistically significant increase in binding of SW1222 cells to type-1 collagen. The effects of TGF-alpha both on binding to type-1 collagen and on morphological differentiation in 3-dimensional collagen gel were inhibited by monoclonal antibodies recognizing the alpha 2 beta 1 integrin. These data indicate that the morphogenetic or mitogenic activities of TGF-alpha are critically dependent on cellular interactions with extracellular matrix proteins and are primarily mediated by the alpha 2 beta 1 integrin receptor. Inappropriate expression of this growth factor, seen in tumours whose cell-matrix interactions are greatly impaired, could have deleterious effects on the maintenance of normal tissue architecture and growth control.

Antibodies, Monoclonal↗

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.

Animals↗

Squamous cell carcinoma of the feline thymus with widespread apoptosis.

The present report documents a case of squamous cell carcinoma originating from the thymus of a 12-year-old short-haired male cat. The tumour had metastasised to the sternal lymphoid tissue and to the left lung. To the authors' knowledge, thymic carcinoma has not been reported in domestic animals. Furthermore, extensive apoptosis was observed in the tumour mass.

Animals↗

Divergent effects of epidermal growth factor and calcipotriol on human rectal cell proliferation.

Vitamin D may protect against colorectal cancer by reducing cell proliferation and inducing differentiation. By contrast, epidermal growth factor (EGF) stimulates cell proliferation and may encourage gastrointestinal mucosal healing. This study investigated the effect of a synthetic vitamin D analogue, calcipotriol, and EGF on human rectal epithelial cell proliferation in patients with familial adenomatous polyposis (FAP). In addition, a new technique to measure the cell cycle time is described. Sigmoidoscopic biopsy specimens were obtained from 14 patients with FAP. Tissue was established in organ culture, with or without the addition of EGF (n = 8), or calcipotriol (n = 6). Proliferation was determined using (a) metaphase arrest to measure the crypt cell production rate, (b) native mitotic index, and (c) the growth fraction using PC10 antibody. EGF receptor expression was shown using a polyclonal antibody AP12E. Calcipotriol reduced crypt cell production rate by 52% from mean (SEM) 5.29 (1.18) to 2.56 (0.80) cells/crypt/hour (p < 0.01) and EGF increased crypt cell production rate by 102% from 3.62 (0.59) to 7.33 (0.90) cells/crypt/hour (p < 0.05), and this tissue expressed the EGF receptor. The growth fraction was 48.40 (4.0)%, and the native mitotic index 1.08 (0.14)%. The cell cycle time was estimated as 94.5 hours and the time for mitosis as one hour. Thus, calcipotriol and EGF have divergent effects on human rectal mucosal proliferation.

Adenomatous Polyposis Coli↗

Expression of hepatocyte growth factor mRNA during oval cell activation in the rat liver.

The customary wave of hepatocyte regeneration which occurs in the rat liver after two-thirds partial hepatectomy can be abolished by oral administration of the carcinogen 2-acetylaminofluorene. Instead, regeneration is achieved through the proliferation and differentiation of potential stem cells (oval cells) which appear to emanate from the portal space. Ultrastructural studies have illustrated the undifferentiated nature of these cells in the first 3 days after resection, but very rapidly they acquire features of small hepatocytes or biliary epithelia. Oval cell progeny can form either cohesive columns of cells within sinusoids which may later differentiate into new hepatic plates, or single cells that can insinuate within existing plates. Using a 35S antisense riboprobe to hepatocyte growth factor (HGF) mRNA, the synthesis of HGF mRNA was observed in sinusoid-lining cells. There were few HGF mRNA-expressing cells in the liver removed at resection, but numbers steadily increased in the remnant over the next 7 days. In particular, an almost nine-fold increase in the density of HGF mRNA-producing cells occurred in the periportal areas, resulting in approximately double the density present within the centrilobular parenchyma. The superabundance of HGF-producing cells in the immediate vicinity of oval cell proliferation and differentiation strongly suggests that this growth factor is involved in all aspects of stem cell behaviour--proliferation, migration, and differentiation, through a paracrine mechanism.

Animals↗

Bromodeoxyuridine-labelled apoptosis after treatment with antimetabolites in two murine tumours and in small intestinal crypts.

Antimetabolites are S-phase specific anticancer drugs. Administration of bromodeoxyuridine (BrdUrd) to tumour bearing mice was followed by treatment with cytosine arabinoside or hydroxyurea. Anti-BrdUrd immunocytochemistry visualised susceptible tumour and intestinal crypt cells at electron microscope level, showing unequivocally that cells that were in S-phase at the time of administration of the drugs subsequently died by apoptosis.

Animals↗

Severe subcellular damage in the rat exocrine pancreas after pancreatobiliary diversion.

Cholecystokinin (CCK) may contribute to the genesis of both pancreatic carcinoma and acute pancreatitis. By transposing a long segment of jejunum to lie between the pylorus and the duodenal papilla, pancreatobiliary diversion (PBD) causes a persistent increase in circulating CCK levels, as the normal feedback inhibition of jejunal CCK release by pancreatic juice is evaded. A number of morphological, physiological, and acinar cytokinetic changes ensue. This investigation has examined the ultrastructural changes in pancreatic acinar cells after PBD in the presence and absence of CR1409 (lorglumide), a CCK receptor antagonist. After 14 days there was degranulation and vacuolation of acinar cells with involvement of the enzyme acid phosphatase. The presence of morphologically distinct extracisternal acid phosphatase indisputably predisposed acinar cells to severe damage. Treatment with CR1409 largely prevented degranulation after PBD, but vacuolation of acinar cells still occurred, indicating a possible toxic effect of the receptor antagonist. This is the first report of CCK itself, rather than one of its analogues, causing in vivo pancreatic damage that is generally considered as a forerunner to acute pancreatitis. This is of fundamental importance to the understanding of the earliest stages of the disease.

Acid Phosphatase↗

Transforming growth factor-alpha immunoreactivity in a variety of epithelial tissues.

The immunohistochemical expression of transforming growth factor-alpha (TGF alpha) has been examined in a range of normal adult epithelial tissues from both man and rat using an anti-hTGF alpha 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 TGF alpha 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 TGF alpha 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 TGF alpha immunoreactivity.

Adult↗

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.

Animals↗

The nature of cytotoxic drug-induced cell death in murine intestinal crypts.

The nature of cell death in murine small intestinal crypts caused by potentially lethal doses of four classes of cancer chemotherapeutic agents was studied. The drugs used were cytosine arabinoside, vincristine, adriamycin and nitrogen mustard. The compounds readily induced massive cell death in the proliferating compartment of the crypt. In each case, cell death was apparent within an hour, and the incidence of dead cells peaked during the following 4-8 h. By 24 h, little damage was discernible in the crypt systems. Remarkably, dead cells or dead cell fragments were phagocytosed rapidly (within about 1 h) by neighbouring healthy enterocytes. When examined by light microscopy, transmission electron microscopy and scanning electron microscopy, the dead cells showed the characteristic features of having succumbed to an apoptotic mode of cell death without any trace of cell and organelle oedema characteristic of necrosis. The study suggests that cell death by apoptosis operates even when the cells are exposed to severe pathological perturbation and that the phenomenon is not solely a process which operates in response to either physiological stimuli or to mild physical or chemical trauma.

Animals↗

WEB 2086 inhibits neutrophil dependent increases in coronary resistance in blood perfused rabbit heart.

OBJECTIVE: The aim was to investigate the mechanism of the pressor action of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine in the blood perfused Langendorff preparation of the isolated rabbit heart; and in particular to establish whether the response was dependent on the presence of neutrophils and whether the release of platelet activating factor contributed to the pressor effect. METHODS: An isolated rabbit heart was perfused with blood from an anesthetised support rabbit. Formyl-methionyl-leucyl-phenylalanine was injected intra-arterially proximal to the isolated heart and measures of cardiac performance recorded. In some experiments the support animal was depleted of neutrophils by pretreatment with mechlorethamine while in others the specific platelet activating factor receptor antagonist WEB 2086 was given to the support animal before formyl-methionyl-leucyl-phenylalanine. Differential blood cell counts were determined throughout the course of each experiment. Each heart was examined histologically after the experiment. RESULTS: Formyl-methionyl-leucyl-phenylalanine caused a significant rise in perfusion pressure which was virtually abolished by leucocyte depletion of the support animal. The response could also be reduced by about 80% with intravenous WEB 2086. Histological examination of the perfused hearts showed that the number of accumulated neutrophils was very variable and not correlated with the rise in perfusion pressure. There was no significant difference between control hearts and those receiving WEB 2086. CONCLUSIONS: The results confirm previous reports that the response to formyl-methionyl-leucyl-phenylalanine is neutrophil dependent and show that this model of a blood perfused heart can be used successfully to examine the response to a leucocyte dependent stimulus. The results also suggest that the response to formyl-methionyl-leucyl-phenylalanine may not only be due to physical obstruction of the coronary circulation or "neutrophil plugging", but may also be due to the release of platelet activating factor.

Animals↗

Transforming growth factor alpha expression in normal gastric mucosa, intestinal metaplasia, dysplasia and gastric carcinoma--an immunohistochemical study.

Using a monoclonal antibody (GF10) and a standard immunohistochemical technique, immuno-expression of transforming growth factor alpha (TGF-alpha) was found consistently within the differentiated compartment of normal adult human gastric mucosa. In 70% of mucosal samples exhibiting intestinal metaplasia there was more or less uniform TGF-alpha immunoreactivity throughout the intestinalized mucosa. Similarly, 60% of cases of dysplasia and 60% of gastric carcinomas showed strong immunoreactivity in most of the cells. However, whereas 93% of intestinal-type cancers showed strong immunoreactivity only 30% of the diffuse type were stained and then only weakly. Transforming growth factor alpha expression is thus a fairly regular feature of several types of differentiated gastric epithelia (normal, metaplastic, dysplastic and intestinal-type carcinoma), while its relative absence may be a factor in the histogenesis of the diffuse type of gastric carcinoma.

Gastric Mucosa↗

Immunoreactivity of transforming growth factor alpha in the normal adult gastrointestinal tract.

The immunolocalisation of transforming growth factor alpha (TGF alpha) in the normal human oesophagus and the gastrointestinal tract was elucidated using two different antibodies - Ab2, a monoclonal antibody reacting with part of the human TGF alpha molecule, and 26T a rabbit affinity purified polyclonal antibody raised against part of the rat TGF alpha peptide. There have been conflicting reports on the distribution of this growth factor in the gut. The results clearly showed that this peptide regulatory factor is confined to the luminal surface and foveolae in the stomach, restricted to the villous epithelium in the small intestine, and in the colon was seen in the upper one third of the crypt. This pattern indicates that the distribution of this peptide regulatory factor is within the differentiated compartment and indicates a role in differentiation besides its well known proliferative effects.

Adult↗

Role of cholecystokinin in pancreatic adaptation to massive enterectomy.

Since pancreatic adaptation to massive proximal small bowel resection (PSBR) may be modulated through cholecystokinin (CCK) secretion, we tested the effect of the CCK antagonist CR-1409 on this response. Male Wistar rats (n = 72) weighing 220-225 g were randomised to receive either PSBR or transection/resuture followed by saline or CR-1409 (12 mg/kg daily subcutaneously). Rats were killed one, two, and three weeks post-operatively, at which time blood was obtained for CCK assay and the pancreas was assessed for proliferative activity by three parameters: nucleic acid and protein content, bromode-oxyuridine (BrdUrd) labelling index, and proliferating cell nuclear antigen (PCNA) expression. PSBR increased plasma CCK concentration by 83-102% at 1-3 weeks, irrespective of CR-1409 administration. Total pancreatic DNA content per 100 g body weight increased by 34% at two weeks (p less than 0.05) and by 82% at three weeks (p less than 0.05), while RNA content increased by 60% and 178% (p less than 0.001) and protein content by 20% and 57% (p less than 0.05). PSBR increased the BrdUrd labelling index and the percentage of PCNA immunoreactive cells. CR-1409 completely abolished this proliferative response and also prevented the rise in nucleic acid and protein contents. Apart from growth stimulation, PSBR also enhanced pancreatic exocrine function, as shown by ultrastructural evidence of an appreciable decrease in zymogen granules; CR-1409 also inhibited this functional effect of hypercholecystokininaemia. The results confirm the tropic role of CCK after PSBR, and CR-1409 prevents this pancreatic adaptation.

Animals↗

Apoptosis: a gene-directed programme of cell death.

Apoptosis is a particular type of programmed cell death which commonly occurs in the developing embryo, in normal healthy adult tissues and in many pathological settings. In contrast to necrosis, apoptosis is not a passive phenomenon but is gene-directed, usually requiring ongoing protein synthesis. The dying cell is characterised by having a raised level of cytosolic Ca2+; this activates a non-lysosomal Ca(2+)- and Mg(2+)-dependent endonuclease which digests the chromatin into oligonucleosome length fragments. The dying cell may or may not fragment into a number of apoptotic bodies, but in all cases the cell contents are bounded by a membrane which prevents the spillage of harmful substances such as DNA. Apoptotic cells are eliminated through phagocytosis by neighbouring cells and macrophages, and cell surface changes on apoptotic cells aid their recognition and engulfment by the phagocytosing cells. Extrinsic signals can both stimulate and inhibit apoptosis, and even direct damage to the cell can activate the process. Apoptosis is widely involved in organ formation in the embryo, and its occurrence in response to noxious stimuli such as cytotoxic drugs, irradiation and hyperthermia may be viewed as an altruistic suicide. Apoptosis provides a safe disposal mechanism for neutrophils at inflamed sites, and within the immune system it is considered responsible for eliminating self-reactive T-cell clones and for the affinity maturation of antibody producing cells. A failure to undergo apoptosis has been invoked in the pathogenesis of low-grade follicular lymphoma, and the triggering of apoptosis with monoclonal antibodies specifically in tumour cells has been achieved in one or two cases.

Animals↗