Effects of urogastrone (epidermal growth factor) on the intestinal epithelium.

Recombinant urogastrone (human epidermal growth factor) was infused into rats in which intestinal cell proliferation was reduced to a steady state basal level by feeding them intravenously. Urogastrone elevated the augmented metaphase index and weight of all sections of the gastrointestinal tract in a dose-related manner. Infusion of urogastrone at a dose which has a minimal effect on gastric acid secretion significantly increased crypt cell production and tissue weights throughout the gastrointestinal tract. Intravenous urogastrone was also effective in restoring cell proliferation after the intestine had become hypoproliferative. Urogastrone administered luminally had no significant effect on either intestinal weight, crypt cell production rate, or metaphase collection.

Intravenous but not intragastric urogastrone-EGF is trophic to the intestine of parenterally fed rats.

The effects of beta-urogastrone/human epidermal growth factor (URO-EGF) on intestinal epithelial cell proliferation were studied in rats in which intestinal cell proliferation had been reduced to a steady state basal level, by maintaining the rats on total parenteral nutrition. The accumulation of arrested metaphases over a two hour time period was determined in a dose response study. Increasing doses of URO-EGF progressively raised the two hour collection of metaphases and intestinal weights. Intravenous infusion of URO-EGF was also effective in restoring cell proliferation when it was infused after the intestine had become hypoproliferative. beta-urogastrone/human epidermal growth factor administered through an intragastric cannulae thrice daily had no significant effect on intestinal weight or crypt cell production rate or metaphase collection. It is proposed that one of the in vivo actions of urogastrone-epidermal growth factor is the maintenance of gastrointestinal growth and that this occurs through a systemic rather than a luminal mechanism.

Stimulation of neomucosal growth by systemic urogastrone.

Patching small intestinal defects with colon serosa results in the growth of functional neomucosa. However, the rate of neomucosal growth is slow and the defect contracts markedly. The aim of this study was to determine if systemic urogastrone would enhance neomucosal growth. Twenty-two New Zealand white rabbits had two 2 X 5-cm ileal defects patched with colon serosa and osmotic pumps placed subcutaneously. Eleven animals had saline infused at a rate of 5 microliter/hr. The other eleven rabbits had urogastrone (1 mg/ml saline) infused at the same rate. One animal died in this group. There was a modest increase in neomucosal growth after 2 weeks of urogastrone infusion. Three weeks after patching, defect coverage was significantly greater in the urogastrone group (99.8 +/- 0.1 vs 96.0 +/- 1.2%, P less than 0.005) and more defects were completely covered by neomucosa (6/10 vs 1/12, P less than 0.05). Less contraction occurred in the urogastrone group (46 +/- 2 vs 35 +/- 3% initial defect, P less than 0.005) and resultant neomucosal surface area was greater (361 +/- 12 vs 266 +/- 20 mm2, P less than 0.0005). In vitro glucose uptake was significantly greater in the urogastrone group but disaccharidase and diamine oxidase activity were similar. Crypt cell production rate was significantly greater 2 weeks after operation compared to 3 weeks in both groups and was greater in the urogastrone group compared to the saline group at 2 weeks (24.9 +/- 1.1 vs 20.1 +/- 1.1, P less than 0.02). Systemic urogastrone enhances neomucosal growth by increasing the rate of growth and diminishing contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferative effects of urogastrone-EGF on the intestinal epithelium.

The effects of B-urogastrone/human epidermal growth factor on intestinal epithelial cell proliferation were studied in rats in which intestinal cell proliferation was reduced to a steady state basal level (by maintaining the rats on total parenteral nutrition). Increasing doses of urogastrone progressively raised the two hour collection of metaphases and intestinal weights. The crypt cell production rate was measured in animals maintained parenterally with or without urogastrone, and in rats fed a standard laboratory ration. Continuous infusion of 15 micrograms per rat per day of recombinant beta urogastrone (a dose which has a minimal effect on gastric acid secretion) significantly increased cell proliferation and intestinal tissue weights throughout the gastrointestinal tract. Intravenous infusion of urogastrone was also effective in restoring cell proliferation when it was infused after the intestine had become hypoproliferative. Urogastrone administered through an intragastric cannula thrice daily had no significant effect on either intestinal weight, crypt cell production rate, or metaphase collection.

Monoclonal antibodies to human beta-interferon produced by adoptive transfer in irradiated mice.

Three murine anti-human beta-interferon (IFN-beta) monoclonal antibodies have been isolated following adoptive transfer of immune spleen cells. Adoptive transfer was used to increase the specific efficiency of the fusion. These antibodies have been used to define two epitopes on IFN-beta; the antiviral, antiproliferative, and immunomodulatory effects of IFN-beta are associated with one of these epitopes.

Effect of calcium and calcium antagonists on [3H]-Paf-acether binding to washed human platelets.

[3H]-Platelet activating factor (Paf-acether, 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine) binds to washed human platelets in a specific, dose-dependent, and saturable manner. Scatchard analysis reveals a high affinity site with a KD value of 0.25 +/- 0.033 nM (245 +/- 30 sites per platelet), and a second low affinity site with a KD value of 9.22 +/- 1.17 nM (1616 +/- 165 sites per platelet). Binding to the high affinity site is independent of buffer calcium concentration, inhibited on an equimolar basis by unlabelled 1-O-octadecyl-Paf-acether, but remains unchanged in the presence of 1-O-octadecyl-lyso-Paf-acether. The relative inhibitory effect of four calcium antagonists on [3H]-Paf-acether high affinity binding correlates closely with their respective anti-aggregatory activity against Paf-acether induced responses in human PRP; order of potency being (+)-cis diltiazem greater than (+/-)-verapamil greater than (-)-cis diltiazem greater than nifedipine. In the case of (+)-cis diltiazem, the effect is competitive, stereo-specific and progressively reversed by addition of calcium (1.0 mM and 5.0 mM). A close spatial relationship may thus exist between the Paf-acether receptor and membrane calcium channels in the human platelet.

Influences of gamma interferon on synovial fibroblast-like cells. Ia induction and inhibition of collagen synthesis.

The shape and function of adherent cells cultured from rheumatoid synovial membranes are influenced by immune cells, and their products. The synovial cells produce collagenase and prostaglandin E2 (PGE2), the levels of which are increased when the cells are incubated with the monokine, mononuclear cell factor/interleukin 1. The majority of adherent synovial cells are fibroblastlike in appearance and synthesize collagens and fibronectin; the synthesis of collagens and fibronectins are also increased by a monocyte factor. In the present study we found that the fibroblastlike cells expressed major histocompatibility complex class II (Ia-like) antigens after initial dispersion from the synovial membrane. Monocyte lineage antigens were detected on some round cells in early passage, but no T lymphocytes were identified in established cultures. There was loss of Ia expression on the fibroblastlike cells with age and passage in culture. The addition of the lymphokine, gamma interferon (recombinant), induced class II antigen (DR and DS/DQ) expression in early or late passage cells in a time- and dose-dependent manner and required protein synthesis. Furthermore, the adherent synovial fibroblastlike cells continued to be Ia-positive when examined as long as 10 d after the removal of gamma interferon. Ia expression was also induced by gamma interferon in normal skin fibroblasts. Synovial cells that could be induced to express Ia also bound a monoclonal antibody to type III collagen (a fibroblast marker). Gamma interferon, while inducing Ia expression, decreased the binding of type III collagen antibody on unstimulated as well as monokine-stimulated cells. Analysis of [3H]proline-labeled medium by SDS polyacrylamide gel electrophoresis showed that gamma interferon decreased the synthesis of type I and III collagens and fibronectin by adherent synovial cells in a dose-dependent manner. These findings suggest that Ia expression by synovial tissue cells is not cell-specific, but reflects one or several related events, such as the degree of T lymphocyte infiltration, the presence of factors that stimulate gamma interferon release, or an increased sensitivity of the cells to gamma interferon. Whereas the synthesis of class II antigens is enhanced by the lymphokine gamma interferon, and a monocyte factor(s) stimulates collagen, collagenase and PGE2 synthesis by the same cells, gamma interferon inhibits basal and monokine-induced collagen synthesis. Thus, lymphokines and monokines may influence the extent of fibrosis as contrasted to matrix destruction at various stages of the rheumatoid lesion by affecting the function of fibroblastlike synovial cells.

Activation of the ppp(A2'p)nA system in interferon-treated, herpes simplex virus-infected cells and evidence for novel inhibitors of the ppp(A2'p)nA-dependent RNase.

High doses (100-1000 reference units/ml) of alpha or beta interferons are required to inhibit the growth of herpes simplex virus types I and II (HSV-I and HSV-II) in human Chang cells. In contrast, much lower doses (10-100 reference units/ml) of interferon inhibit replication of encephalomyocarditis virus (EMCV) in these cells. In the HSV-infected cells these high doses did not prevent the virus-induced shut off of host protein synthesis. The interferons were more effective in reducing the virus yield of HSV-I than of HSV-II. At the above concentrations they inhibited HSV-I protein synthesis but had little apparent effect on that of HSV-II. Similar amounts of (2'-5')oligo(adenylate)s were synthesised in response to HSV-I, HSV-II and EMCV infection of Chang cells after treatment with alpha or beta interferons. No (i.e. less than 1 nM) (2'-5')oligo(adenylate)s were found in control cells or on virus infection alone. Only low levels of ppp(A2'p)nA-specific rRNA cleavage were observed in the interferon-treated HSV-infected cells. In contrast, high levels were found in response to EMCV, despite the fact that ppp(A2'p)nA accumulated to similar levels with each of the three viruses in these cells. High-performance liquid chromatographic analysis of material from interferon-treated Chang cells 18 h after infection with HSV-I or HSV-II, combined with radiobinding, radioimmune and rRNA cleavage assays, confirmed the presence of ppp(A2'p)2A and ppp(A2'p)3A at greater than nanomolar concentration. In addition, apparently equivalent amounts of two other putative (2'-5')oligo(adenylate) derivatives which compete in the radiobinding and radioimmune assays, were present. These compounds were only weak activators of the ppp(A2'p)nA-dependent RNase and under appropriate conditions were capable of inhibiting the activation of this RNase by authentic ppp(A2'p)nA. The presence of these potentially inhibitory compounds provides a possible explanation for the relatively low levels of activation of the ppp(A2'p)nA-dependent RNase in interferon-treated, HSV-infected Chang cells.

Studies on the pathogenesis of equine tendonitis following collagenase injury.

The histological and morphological characteristics of the scar tissue formed during healing after traumatic equine tendon injury have been compared with those of scar tissue produced in response to an enzyme-induced pony tendon injury. Several techniques are currently in use in the treatment of equine tendon sprain and this work formed part of a study of their respective efficacy. It was concluded that the enzyme-induced lesion is resolved by a reparative process very similar in its prognosis and extent to that following a naturally occurring tendon sprain. It may therefore justifiably be used as a model of naturally occurring tendon sprain in the comparative study of treatment methods.

The distribution of types I and III collagen and fibronectin in the healing equine tendon.

During tissue response to injury the glycoproteins fibronectin and Type III collagen are synthesized in increased amounts. We have studied the distribution of these molecules in the healing tendon at various times after injury by comparison with that of the major constituent of normal tendon, Type I collagen. Immunofluorescent localization demonstrated the presence of fibronectin throughout the tendon within one week after injury. Staining was found in the matrix, both around capillaries and around fibroblast-like cells. Fibronectin was still apparent in the healing tendon at one month after injury, but after a further two months was no longer detectable. Type III collagen was present both in pericellular and matrix locations until three months after injury, and matrix staining was apparent during the entire fourteen-month period under study. Type III collagen was also found throughout the matrix of the contralateral superficial flexor tendon during this period.

A clinical and experimental study of tendon injury, healing and treatment in the horse.

This project was carried out over a five year period (1977 to 1981 inclusive) at the University of Bristol following discussion between the British Veterinary Association and the Royal College of Veterinary Surgeons about the efficacy of, and ethical justification for, the practice of 'firing' (cautery). These discussions had been promoted by parliamentary questions but led to no firm conclusions because previously reported clinical investigations on specific treatments lacked adequate comparisons and controls and thus did not provide scientifically acceptable, statistically valid data. The project was originally conceived as having two main sections, one clinical and the other experimental. The Veterinary Advisory Committee of the Horserace Betting Levy Board felt that primary attention should be given to the clinical investigation and this was done.

Biological properties of human interferon beta 1 synthesized in recombinant bacteria.

Human fibroblast interferon, designated IFN-beta 1, has been produced in E. coli by direct expression of the cloned cDNA coding for the mature polypeptide. Bacterial lysates from recombinant cultures contain a polypeptide with an apparent molecular weight of 17,500 that corresponds in size to the unglycosylated IFN-beta 1 molecule. The latter could be specifically immunoprecipitated by antibodies to purified natural IFN-beta and could inhibit the replication of Herpes simplex virus types 1 and 2 in many different cell lines. Like the natural fibroblast IFN-beta, the bacterial IFN-beta 1 was active in many human cell lines, less active in a monkey cell line and inactive in rabbit and mouse fibroblasts. The antibody titre required to neutralise the anti-herpes activity of both IFN preparations was similar suggesting that they have the same specific activities. Similarly, the bacterial IFN-beta 1 was equally active in inhibiting the proliferation of Daudi cells grown in culture. Bacterial IFN-beta 1 was also capable of enhancing natural killer cell activity and antibody-dependent cellular cytotoxicity in vitro. Thus, IFN-beta 1 produced in recombinant bacteria displays a large range of biological properties ascribed to the natural fibroblast IFN-beta molecule.

Experimental pyelonephritis in the cat: 3. Collagen alterations in renal fibrosis.

Chronic pyelonephritis was induced in young adult cats by the intravenous injection of a human or a feline strain of Escherichia coli after ligation of one ureter for 24 or 48 h. In the 3 cats infected with the feline strain, scarred kidneys from the obstructed side were removed at necropsy 3, 4 and 5 months later. Collagen was extracted from pyelonephritic and normal kidney tissue with dilute acetic acid and limited proteolysis with pepsin. Scarred kidneys gave higher yields of both acid-soluble collagen (normal = 0.57 +/- 0.12 mg per g tissue; scarred = 0.88 +/- 0.10 mg per g tissue) and pepsin-solubilized collagen (normal = 9.69 +/- 1.79 mg per g tissue; scarred = 20.02 +/- 2.84 mg per g tissue). There was no significant increase in the collagen yield from the kidneys of the 2 cats in which mild focal lesions were found 14 and 16 months after infection with the human strain of E. coli. Pepsin released collagens were separated by fractional salt precipitation and identified by agarose gel chromatography and polyacrylamide gel electrophoresis. Normal kidney was shown to contain collagen of Types I, IV and V (AB). The Type IV collagen extracted consisted of a mixture of 4 major pepsin-resistant chains of apparent molecular weights of 150 000, 115 000, 85 000 and 60 000. The collagen extracted from scarred kidneys was predominantly Type I, only trace amounts of Type IV and V components being present. These findings suggest that basement membrane collagens of the kidney are selectively degraded during the atrophy and scarring of chronic feline pyelonephritis and are preferentially replaced by interstitial Type I collagen.

Connective tissue composition of the equine sarcoid.

The connective tissue composition and organisation of the "equine sarcoid" was compared with that of normal adult equine skin to determine whether the cells which produce their respective connective tissue matrices show similar biosynthetic characteristics. No major qualitative difference could be found between the collagen compositions of skin and sarcoid material, although the organisation into fibres of Type III collagen in the sarcoid was markedly greater than that of skin.

A placebo-controlled blind trial of cyclosporin-A in prevention of corneal graft rejection in rabbits.

Cyclosporin-A (CyA) administered to rabbits intramuscularly in a dose of 25 mg/kg/day for 14 days following interlamellar corneal grafting had a significant effect in preventing rejection of the corneal graft (p less than 0.05), and the benefit was maintained. Rejection was attended by an initial mild inflammatory reaction and followed by a protracted intense response involving a variety of cell types, with subsequent loss of epithelium. endothelium and keratocytes, and the development of areas of stromal necrosis. CyA suppressed this rejection response.

Immunological studies with human aortic elastin.

Antibodies to human fetal aortic elastin were isolated from sheep immunized with alpha-elastin peptides. In preliminary tests of specificity using passive hemagglutination, partial cross-reactivity was demonstrated with alpha-elastin from other species. However, in a double antibody radio-immunoassay alpha-elastin peptides from other mammalian species failed to compete with 125I-labelled human alpha elastin. These results suggest the existence of at least two different antigenic sites on the elastin molecule. One, a high affinity site, demonstrates species specificity at low antigen/antibody concentrations. The other, a low affinity site, is common to mammalian elastins and is demonstrated at high antibody/antigen concentrations. In the radioimmunoassay the antibodies showed considerably less avidity for adult human alpha-elastin than for the fetal antigen. This implies that the species specific site is age-dependent and probably involves the cross-linking region of the elastin molecule. Using the sheep antiserum immunohistochemical staining of elastic tissue has been developed. This should prove to be a useful technique for studying polymeric elastin in intact tissue by light microscopy and at the ultrastructural level.

The actual cautery -- myth and reality in the art of firing.

The historical development of "firing" as a treatment for various complaints in animals and man is followed from the first surviving written account by Vegetius (approximately AD 500) to the present day. Changing concepts as to the mechanism of action of the actual cautery are examined in relation to physiological understanding at various times and conflicting views and evident fallacies are discussed. It is of particular interest that firing of "sinewes" (tendons and ligaments) was expressly forbidden until the 18th century. It is concluded that "firing" is an outmoded practice for which there is no scientific justification.

Ischaemic myocardial fibrosis and aortic strongylosis in the horse.

The hearts and aortas of 2076 unselected horses of all ages were examined immediately after slaughter. Focal zones of fibrosis, observed in the myocardium of 14.3 per cent of hearts examined, were found in both atria and ventricles and were unrelated to age. Microscopically the majority of lesions involved myocardial fibre lysis and replacement fibrosis, although acute infarction was present in some cases. Intramyocardial arterioles in or adjacent to the lesions exhibited occlusive arteriosclerotic changes whereas those elsewhere remained patent. The evidence strongly suggests that the myocardial lesions were ischaemic in origin and related to the distribution of intramyocardial arteriosclerosis. Nodular fibrous plaques and mural thrombi associated with migrating larvae of Strongylus vulgaris were observed in the thoracic aorta of 9.4 per cent of horses. These intimal lesions were often present in the aortic bulb and proximal 10 cm of the thoracic aorta indicating that larval migration in this zone is common. Statistical analysis revealed a highly significant association between the occurrence of proximal aortic S vulgaris lesions and the presence of focal ischaemic lesions in the myocardium. The association was not the result of direct larval damage but appeared to be caused by microembolisation from parasitic lesions in the proximal aorta, producing obstructive arteriosclerotic lesions in myocardial arterioles.

Newly synthesized elastin is associated with neoplastic epithelial cells in human mammary carcinoma.

Indirect immunofluorescence with a purified antiserum to human foetal elastin has identified newly synthesized elastin on the membranes of neoplastic epithelial cells in human mammary carcinoma.