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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.

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.

Background: This 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., Methods: Trefoil peptide expression in small intestinal Crohn's 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., Results: Goblet 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., Conclusions: Copackaging 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.

Apoptosis or programmed cell death of senescent neutrophils leading to their uptake by phagocytes is a general mechanism by which neutrophils may be removed from inflamed sites in vivo, promoting resolution rather than persistence of inflammation. We now report morphological evidence of neutrophil apoptosis leading to uptake by glomerular cells in rats with experimental glomerulonephritis. In addition to confirming that inflammatory macrophages take up apoptotic neutrophils, these studies indicated that glomerular mesangial cells can also participate in this mode of neutrophil clearance. Furthermore, human neutrophils which had been "aged" in vitro so as to undergo apoptosis were ingested by 31.5 +/- 1.3% (mean +/- SE) of cultured human mesangial cells, but there was minimal recognition of freshly isolated neutrophils (2.2 +/- 0.1%). Centrifugal elutriation of aged neutrophil populations yielded fractions with varying degrees of apoptosis (from 11.1 to 79.4%). Uptake of these fractions (by 8.2% to 59.8% of mesangial cells) was closely correlated with apoptosis (r = 0.96, P less than 0.0001). This demonstrated that recognition was dependent upon apoptosis, as in previous reports of macrophage recognition of aged neutrophils. However, by contrast, a partial requirement for serum was observed. These data indicate a hitherto unexpected function for the mesangial cell in clearance of senescent neutrophils from the glomerulus which may supplement inflammatory macrophage uptake of leucocytes undergoing apoptosis.

Apoptosis is a process by which cells die in a controlled and programmed manner in response to specific stimuli, often following extrinsic and intrinsic signals which ultimately cause the "switching on" of cell death regulatory genes. Condensation of chromatin and cytoplasm, fragmentation of the cell and formation of membrane-bound bodies containing intact organelles (apoptotic bodies), and phagocytosis of these bodies by resident cells are the major structural changes associated with apoptosis. Biochemically, activation of a nonlysosomal endonuclease is a cardinal feature of this mode of cell death. Several genes have been implicated in the execution of apoptosis. A signal transduction mechanism is suspected to regulate the phenomenon. Although apoptosis is widely considered as an adaptive response to physiological or near physiological stimuli, several noxious agents can initiate the reaction and thus it is often a toxicological response.

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.

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.

Interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) have been reported to stimulate human mesangial cells (HMC) to proliferate and synthesize eicosanoids. We have examined whether they also induce HMC to release cytokines. In this study we show that both IL-1 and TNF stimulate HMC to release IL-6 and IL-8. Cycling and quiescent HMC were stimulated with various concentrations of either recombinant IL-1 beta or TNF for 1 to 24 hours. IL-1 beta at doses as low as 6 pg/ml stimulated mesangial cells to synthesize mRNA for both IL-6 and IL-8 as assessed by Northern analysis; mRNA for tubulin remained constant, which demonstrated a specific increase in mRNA. Secretion of IL-6 and IL-8 into the culture medium increased (4.5 to 18 ng/ml and 4 to 40 ng/ml, respectively) measured by ELISAs. TNF had similar effects but only in high concentrations (greater than 100 ng/ml). IL-1 beta did not stimulate cells to proliferate, as measured by 3H thymidine incorporation. TNF caused proliferation but only in concentrations over 100 ng/ml. We conclude that IL-1 beta is a potent stimulator of human mesangial cell production of IL-6 and IL-8, both of which may influence injury in nephritis. TNF also stimulates mesangial cells but only in pharmacological doses.

Previous studies on the subject of silver-staining nucleolar organizer regions (AgNORs) as indicators of precise proliferative status of tissues have sometimes resulted in ambiguity. The studies, however, have most frequently addressed themselves to the prognosis of neoplasias, with the aim of using AgNORs principally to distinguish between benign and malignant tumours. This investigation was to determine a base-line relationship of AgNOR clusters to proliferation and thus concentrated on normally proliferative tissues and conditionally renewing tissues after appropriate stimulation. Two murine transplantable tumours were also examined as examples of frank malignancy. As an example of the former, variations in AgNOR clusters were noted in the small intestine of man, mouse, and rat. The conditionally renewing systems of liver, prostate, and salivary glands were stimulated into proliferation by two-thirds partial hepatectomy, castration followed by treatment with testosterone, and isoproterenol treatment, respectively, in rat models; the murine sarcoma SaF and carcinoma CaNT provided relatively simple malignant tumours for AgNOR investigation. Proliferation was monitored by noting labelling indices after injection with bromodeoxyuridine (BrdUrd) in vivo followed by immunocytochemical visualization of S-phase cells. In all tissues, an increase in the size of AgNOR clusters rather than their number correlated positively with elevated labelling, particularly with the emergence of silver-staining regions of 2-3 microns visible diameter. Thus, increased AgNOR cluster size (diameter) as representative of AgNOR cluster/nucleolus volume was found to be dependent on proliferative activity in a range of normal and neoplastic tissues.

Proliferating cell nuclear antigen (PCNA), also called DNA polymerase delta-associated protein, is found in the cells of the proliferative compartment of normal tissues and is essential for DNA replication. It can be recognized by many monoclonal antibodies to various epitopes on the molecule. In this investigation one of these, PC10, has been used on formalin-fixed, paraffin-embedded, human and rodent gastro-intestinal epithelial tissues to assess numerically the labelling index of PC10 and to compare it, in the rat liver and gastrointestinal tract, with the S-phase fraction as determined by bromodeoxyuridine (BrdUrd) labelling. The distribution of PC10-labelled cells was recorded with respect to cell position in the intestinal crypts of man. In tissues where both modes of assessment were used, PC10 staining in the well-established proliferative compartments was found to be more extensive than that of BrdUrd. The higher labelling index with PC10 can be explained by its identification of PCNA outside the S phase of the cell cycle and also by the long half-life of PCNA protein in post-proliferative intestinal epithelial cells as they migrate towards the villus. Nevertheless the data suggest PC10 immunostaining in gastro-intestinal epithelia is an operational marker of cell proliferation which is reproducible, quantifiable and can be performed on routinely processed tissues.

The quantitative aspect of apoptosis in experimental tumours is often neglected. In this study, the apoptotic and mitotic indices for a range of tumours have been determined at light microscope level. It has been found that the apoptotic levels fall into a consistent range for all tumour types and agree well with those described by previous workers. It is suggested that these might be basic parameters of tumour expansion, as relevant to growth kinetics as mitotic levels.