Graham Robinson

Electron microscope studies of Fasciola hepatica. XII. The fine structure of the gastrodermis.

Abstract The gastrodermis of Fasciola hepatica comprises a single continuous layer of epithelial cells which show considerable variation in fine structure. These differences in structure, however, reflect only the different functional states of a single cell type. Using ultrastructural morphology as a basic criterion, the cells can be classified into one of three groups, arbitrarily termed Groups I, II, and III. Group I cells are considered to be predominantly secretory in function and Group II cells predominantly absorptive. Each cell shows a cyclical transformation between the secretory and absorptive forms, but since all the cells in any particular area of the diverticula are not all in the same state, secretion, absorption and digestion are occurring simultaneously and continuously. This situation has been observed only in the cells of the lateral diverticula. The main caeca, lined by Group III cells, appear to be more concerned with the movement of material back and forth within the lumen of the gut system, although they must also both secrete and absorb.

Pre‐clinical evaluation of the Gastrimmune immunogen alone and in combination with 5‐fluorouracil/leucovorin in a rat colorectal cancer model

Mature and post‐translational precursor gastrin forms are growth factors for colorectal tumours. The immunogen Gastrimmune is composed of the amino terminus of gastrin‐17 linked to diphtheria toxoid and raises antibodies in situ which neutralise amidated and glycine‐extended gastrin‐17. The aim of the study was to determine the effect of treatment with 5‐fluorouracil(5‐FU)/leucovorin on the antibody titres induced by Gastrimmune and the effect of combination therapy on the growth of the rat colon tumour DHDK12. Gastrimmune was administered to rats s.c. at 3 weekly intervals. The rat colon tumour line DHDK12 was injected into the abdominal wall of BDIX rats. Combinations of 5‐FU/leucovorin were injected i.v. on days 1, 3 and 5, with the cycle repeated every 4 weeks. Antibody titres were measured by an ELISA technique. Antibody titres were followed for 40 weeks after Gastrimmune (500 μg · ml−1) immunization, with titres peaking between 10 and 20 weeks after a single immunisation and falling by week 30. At termination, no effect was observed on either the histological appearance of the gastro‐intestinal tract or the proliferation of the colonic mucosa. Pre‐ and post‐treatment with 5‐FU/leucovorin (30 mg · kg−1) had no effect on the kinetics and level of antibody response to Gastrimmune. Gastrimmune (200 μg · ml−1) and 5‐FU/leucovorin combinations (12.5 and 20 mg · kg−1) increased the therapeutic effects on the in vivo growth of DHDK12 tumors when compared to the agents given singly. Gastrimmune immunisation may be a therapeutic option for the treatment of colorectal cancer in combination with 5‐FU/leucovorin. Int. J. Cancer 75:873–877, 1998.

Immunocytochemical localization of angiotensin II receptor subtypes 1 and 2 in the porcine fetal, prepubertal and postpubertal ovary.

There is considerable evidence for a mammalian ovarian renin–angiotensin system, which may influence ovulation, angiogenesis and steroidogenesis via the autocrine and/or paracrine actions of the biologically active product of the cascade, angiotensin II (AngII). There are two characterized AngII receptors – type 1, AT1 and type 2, AT2. We report the localization of these receptor subtypes within porcine fetal, prepubertal and postpubertal ovaries. Positive staining for AT1 and AT2 receptors was observed in egg nests in all fetal ovaries studied, as well as in a defined two‐cell layer at the ovarian periphery. In prepubertal tissue, positive AT1 and AT2 staining was localized to granulosa cells adjacent to the basement membrane of pre‐antral and antral follicles, with no staining in the thecal layer. There was immunostaining for both receptors in prepubertal oocytes and zona pellucida. In postpubertal tissue, positive AT1 and AT2 immunostaining was localized to areas of putative neovascularization, the zona pellucida and the oocyte. Further AT1 staining was located to the postpubertal antral follicle granulosa cells. The results indicate that there are higher densities of AT1 receptors than AT2 receptors in the porcine fetal, prepubertal and postpubertal ovary, and this has profound implications for the role of AngII in ovarian development.

Differential Sensitivity of Human Nonpregnant and Pregnant Myometrium to Calcitonin Gene-Related Peptide

Objectives: Calcitonin gene-related peptide (CGRP) is a smooth musicle relaxant with potent vasodilating properties. To investigate its inhibitory effects on human myometrial contractions, we obtained excised human myometria from term pregnant, with and without spontaneous labor, and nonpregnant patients. Methods: Myometrial strips were mounted in tissue baths in which contractile activity was recorded. Spontaneously contracting tissues in vitro were exposed to increasing concentations of CGRP (10-13-10-7 mol/L). Tissues without spontaneous contractions were induced to contract with either oxytocin or KCl before being exposed to CGRP. The IC25 (CGRP ocncentration required to inhibit contracitility by 25%) was used as comparison between groups. Results: Tissues with spontaneous or oxytocin-induced in vitro contractions responded equally to CGRP relaxation. Tissues induced to contract with KCl in vitro required approximately 2000 times more CGRP for equal relaxation. In tissues with spontaneous or oxytocin-induced in vitro contractions, those from pregnant unlabored patients were 60 times more sensitive to CGRP than those from pregnant labored or nonpregnant patients. The latter two groups responded equally to CGRP. Conclusions: The sensitivity of myometrial tissues to CGRP relaxation in vitro is increased from the nonpregnant to the pregnant term state. This increased sensitivity is lost once patients develop spontaneous term labor. The relative ineffectiveness of CGRP in relaxing KCl-induced in vitro contractions probably reflects its known mechanism of action, namely the hyperpolarization of cell membrane potentials via activation of membrane potassium channels.

Immunochemical studies of the endocrine cells of the gastrointestinal tract. I. The use and value of peroxidase-conjugated antibody techniques for the localization of gastrin-containing cells in the human pyloric antrum

SynopsisThis paper describes an immunochemical localization of gastrin-containing endocrine cells in the human pyloric antrum using antibodies conjugated to horseradish peroxidase. With pre-fixed cryostat sections, a distinct clear-cut staining of gastrincontaining cells can be obtained either by direct or indirect single stain procedures, but many cells containing endogenous peroxidase activity also stain. In order to abolish staining due to endogenous peroxidase, sections were pretreated with a number of inhibitors prior to incubation in immune sera, but the inhibitors used appeared to interfere with the antigenicity of the gastrin molecule since subsequent immunochemical localization was impossible. The application of a double-staining technique, however, allowed us to distinguish easily between those cells which contained endogenous peroxidase and those on to which labelled antibody had been adsorbed. No labelled cells were found in post-fixed cryostat sections of fresh-frozen tissue. The technique is of value because preparations are permanent, a fluorescence microscope is not required, and the same technique can be adapted for use with the electron microscope.

Transforming growth factor‐α–mediated growth pathways in human gastro‐intestinal cell lines in relation to the gastrin autocrine pathway

Epidermal growth factor (EGF) and transforming growth factor‐α (TGF‐α) increase transcription of the gastrin gene, and the gastrin peptide may be phosphorylated by EGF‐stimulated tyrosine kinase. Our aims were to compare EGF/TGF‐α interactions in 2 human gastro‐intestinal cell lines: MGLVA1, with a strong gastrin autocrine pathway, and C170HM2, with a weak pathway. Both cell lines expressed the TGF‐α gene. MGLVA1 expressed TGF‐α protein as determined by immuno‐cytochemistry, which was absent in C170HM2. Both cell lines expressed the same level of EGF receptors, as assessed by flow cytometry; however, MGLVA1 did not have enhanced in vitro proliferation in response to EGF or TGF‐α, unlike C170HM2. The basal growth of MGLVA1 was inhibited by anti‐sera against TGF‐α, the EGF receptor and G17. C170HM2 was not inhibited by any of the anti‐sera. Neutralisation of TGF‐α resulted in undetectable cell‐associated progastrin levels in MGLVA1 (untreated had 391.7 fmol/5 × 106 cells). The progastrin level of C170HM2 remained unaffected. Tyrosine kinase activity, as assessed by phosphopeptide concentration, of unstimulated MGLVA1 was 2.6 times higher than that of C170HM2 in the cell membrane fraction (0.097 compared to 0.037 μg/mg protein, p < 0.001) and 4.8 times higher in the cytosolic fraction (0.269 compared to 0.056 μg/mg protein, p < 0.05). Following treatment with EGF, the phosphopeptide concentration increased in both the membrane and cytosolic fractions of both cell lines. Tyrphostin B42, which inhibits autophosphorylation of the EGF receptor, inhibited the basal growth of MGLVA1 (IC50 1.3 μM) and C170HM2 (9.5 μM, p < 0.05 from MGLVA1). Herbimycin, which inhibits pp60c‐src kinase, reduced the basal growth of MGLVA1 (0.67 μM) but not C170HM2. Immunofluorescence studies confirmed the presence of tyrosine‐phosphorylated proteins and pp60c‐src within the cytoplasm of unstimulated MGLVA1 cells. There was no specific immunofluorescence for either parameter in C170HM2 cells until after treatment with EGF. Int. J. Cancer 87:20–28, 2000.