Palmitic acid induces guanylin gene expression through the Toll-like receptor 4/nuclear factor-κB pathway in rat macrophages.

Recently, we showed that double-transgenic rats overexpressing guanylin (Gn), a bioactive peptide, and its receptor, guanylyl cyclase-C (GC-C), specifically in macrophages demonstrate an antiobesity phenotype and low-expression levels of proinflammatory cytokines in the mesenteric fat even when fed a high-fat diet. Here, we examined the levels and mechanism of Gn and GC-C transcription following saturated fatty acid and lipopolysaccharide (LPS), an activator of Toll-like receptor 4 (TLR4), exposure by using the NR8383 macrophage cell line. In addition, the levels of guanylin and cGMP were increased by addition of either palmitic acid or LPS. Next, we investigated the interaction of the gene transcription and nuclear factor-κB (NF-κB) by using an NF-κB inhibitor and chromatin immunoprecipitation assay. We showed that palmitic acid induced Gn gene expression via TLR4 and NF-κB. Moreover, we demonstrated that NF-κB binding to the Gn promoter was responsible for the induction of gene transcription by palmitic acid or LPS. Our results indicate that saturated fatty acids such as palmitic acid activate Gn gene expression via the NF-κB pathway, raising the possibility that the activated Gn-GC-C system may contribute to the inhibition of high-fat diet-induced proinflammatory cytokines in macrophages.

Immunizations with Enterotoxigenic Escherichia coli Heat-Stable Toxin Conjugates Engender Toxin-Neutralizing Antibodies in Mice That Also Cross-React with Guanylin and Uroguanylin.

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.

Neutralizing Anti-Heat-Stable Toxin (STa) Antibodies Derived from Enterotoxigenic Escherichia coli Toxoid Fusions with STa Proteins Containing N12S, L9A/N12S, or N12S/A14T Mutations Show Little Cross-Reactivity with Guanylin or Uroguanylin.

Heat-stable toxin (STa)-producing enterotoxigenic Escherichia coli (ETEC) strains are a top cause of moderate-to-severe diarrhea in children from developing countries and a common cause of travelers' diarrhea. Recent progress in using STa toxoids and toxoid fusions to induce neutralizing anti-STa antibodies has accelerated ETEC vaccine development. However, concern remains regarding whether the derived anti-STa antibodies cross-react with STa-like guanylin and uroguanylin, two guanylate cyclase C (GC-C) ligands regulating fluid and electrolyte transportation in human intestinal and renal epithelial cells. To further divert STa from guanylin and uroguanylin structurally and antigenically and to eliminate anti-STa antibody cross-reactivity with guanylin and uroguanylin, we mutated STa at the 9th (leucine), 12th (asparagine), and 14th (alanine) residues for the double and triple mutants STaL9A/N12S, STaL9A/A14H, STaN12S/A14T, and STaL9A/N12S/A14H We then fused each STa mutant (three copies) to a monomeric heat-labile toxin (LT) mutant (mnLTR192G/L211A) for the toxoid fusions 3×STaL9A/N12S-mnLTR192G/L211A, 3×STaL9A/A14H-mnLTR192G/L211A, 3×STaN12S/A14T-mnLTR192G/L211A, and 3×STaL9A/N12S/A14H-mnLTR192G/L211A; examined each fusion for anti-STa immunogenicity; and assessed the derived antibodies for in vitro neutralization activity against STa toxicity and for cross-reactivity with guanylin and uroguanylin. Mice subcutaneously immunized with each fusion protein developed anti-STa antibodies, and the antibodies derived from 3×STaN12S-mnLTR192G/L211A, 3×STaL9A/N12S-mnLTR192G/L211A, or 3×STaN12S/A14T-mnLTR192G/L211A prevented STa from the stimulation of intracellular cGMP in T-84 cells. Competitive enzyme-linked immunosorbent assays (ELISAs) showed that guanylin and uroguanylin hardly blocked the binding of anti-STa antibodies to the coated STa-ovalbumin conjugate. These results indicated that antibodies derived from 3×STaN12S-mnLTR192G/L211A, 3×STaL9A/N12S-mnLTR192G/L211A, or 3×STaN12S/A14T-mnLTR192G/L211A neutralized STa and had little cross-reactivity with guanylin and uroguanylin, suggesting that these toxoid fusions are suitable antigens for ETEC vaccines.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Currently, there is no licensed vaccine against ETEC diarrhea. One key challenge is to identify safe antigens to induce antibodies neutralizing the key STa without cross-reacting with guanylin and uroguanylin, two important ligands controlling homeostasis in human intestinal and renal epithelial cells. In this study, we generated nontoxic fusion antigens that induced antibodies that neutralize STa enterotoxicity in vitro and do not cross-react with guanylin or uroguanylin. These fusions have become the preferred antigens for the development of ETEC vaccines to potentially prevent the deaths of hundreds of thousands of young children and hundreds of millions of diarrheal cases each year.

Characterization of immunological cross-reactivity between enterotoxigenic Escherichia coli heat-stable toxin and human guanylin and uroguanylin.

Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh.

Anti-prokineticin1 (PROK1) monoclonal antibody suppresses angiogenesis and tumor growth in colorectal cancer.

BACKGROUND: The prokineticin1 (PROK1) gene has been cloned as an angiogenic growth factor from endocrine gland cells. However, we have not known about potentials of anti-PROK1 monoclonal antibody in human cancers. Here we investigated how the anti-PROK1 monoclonal antibody (mAb; established by our department) would affect the high-PROK1-expressing colorectal cancer (CRC) cells in vitro and vivo. METHODS: We confirmed PROK1 protein expression in the CRC cells by performing immunohistochemical staining and measured the amount of soluble PROK1 protein. Next, we mixed the CRC cell culture fluid with the anti-PROK1mAb to examine angiogenic activity in vitro and in vivo. Additionally, we investigated whether the anti-PROK1mAb would affect the tumor-forming capability of high PROK1-expressing CRC cells implanted into mice. RESULTS: PROK1 protein expression was confirmed in 3 CRC cell lines, and soluble PROK1 protein was also confirmed in the CRC cell culture fluid. The culture fluid increased angiogenesis in vitro and vivo, whereas the anti-PROK1mAb suppressed angiogenesis. Subcutaneous tumor formation and tumor angiogenesis in mice were suppressed by the anti-PROK1mAb treatment. The anti-PROK1mAb significantly suppressed the number of CD31 stained cells in mice. CONCLUSIONS: The in vitro and vivo experimental system indicated that the anti-PROK1mAb could suppress angiogenesis and tumor growth in the CRC strains.

Bv8 regulates myeloid-cell-dependent tumour angiogenesis.

Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.

G-CSF-initiated myeloid cell mobilization and angiogenesis mediate tumor refractoriness to anti-VEGF therapy in mouse models.

Recent studies suggest that tumor-associated CD11b(+)Gr1(+) myeloid cells contribute to refractoriness to antiangiogenic therapy with an anti-VEGF-A antibody. However, the mechanisms of peripheral mobilization and tumor-homing of CD11b(+)Gr1(+) cells are unclear. Here, we show that, compared with other cytokines [granulocyte-macrophage colony stimulating factor (GM-CSF), stromal derived factor 1alpha, and placenta growth factor], G-CSF and the G-CSF-induced Bv8 protein have preferential expression in refractory tumors. Treatment of refractory tumors with the combination of anti-VEGF and anti-G-CSF (or anti-Bv8) reduced tumor growth compared with anti-VEGF-A monotherapy. Anti-G-CSF treatment dramatically suppressed circulating or tumor-associated CD11b(+)Gr1(+) cells, reduced Bv8 levels, and affected the tumor vasculature. Conversely, G-CSF delivery to animals bearing anti-VEGF sensitive tumors resulted in reduced responsiveness to anti-VEGF-A treatment through induction of Bv8-dependent angiogenesis. We conclude that, at least in the models examined, G-CSF expression by tumor or stromal cells is a determinant of refractoriness to anti-VEGF-A treatment.

Role of Bv8 in neutrophil-dependent angiogenesis in a transgenic model of cancer progression.

The secreted Bv8 protein has been recently characterized as a regulator of myeloid cell mobilization and a neutrophil-derived mediator of tumor angiogenesis in several xenografts, but its role in tumor progression in an endogenous setting was unknown. The rat insulin promoter (RIP)-T-antigen (Tag) is a well characterized transgenic mouse model of multistage pancreatic beta-cell tumorigenesis. Also, the role of neutrophils in RIP-Tag angiogenic switching, as assessed by systemic ablation using anti-Gr1 antibodies at different stages of tumor progression, has been recently described. Here, we show that early treatment of RIP-Tag mice with anti-Bv8 antibodies resulted in a significant reduction in the number of angiogenic islets relative to control antibody-treated mice, implicating Bv8 in the angiogenic switch during neoplasia. Histological analysis showed a significant reduction in vascular surface areas in hyperplastic and angiogenic lesions in pancreatic islets from anti-Bv8-treated mice. Anti-Bv8 treatment also inhibited the mobilization and homing of CD11b+Gr1+ cells to the peripheral blood and the emerging neoplastic lesions. However, anti-Bv8 treatment had no effect on tumor vascularization or burden when initiated at later stages of tumor progression. The stage-dependent efficacy of anti-Bv8 treatment appears remarkably similar to that reported after neutrophil ablation, suggesting that Bv8 is an important mediator of neutrophil-dependent angiogenesis in this transgenic model. In summary, our studies verify a role for Bv8 in the mobilization and recruitment of myeloid cells and in the induction of tumor angiogenesis in the early stages of neoplastic progression.

Virus-like particle-display of the enterotoxigenic Escherichia coli heat-stable toxoid STh-A14T elicits neutralizing antibodies in mice.

Enterotoxigenic Escherichia coli (ETEC) causes diarrhoea by secreting enterotoxins into the small intestine. Human ETEC strains may secrete any combination of three enterotoxins: the heat-labile toxin (LT) and the heat-stable toxins (ST), of which there are two variants, called human ST (STh) and porcine ST (STp). Strains expressing STh, either alone or in combination with LT and/or STp, are among the four most important diarrhoea-causing pathogens affecting children in low- and middle-income countries. ST is therefore an attractive target for ETEC vaccine development. To produce a safe ST-based vaccine, several challenges must be solved. ST must be rendered immunogenic and non-toxic, and antibodies elicited by an ST vaccine should neutralize ST but not cross-react with the endogenous ligands uroguanylin and guanylin. Virus-like particles (VLPs) tend to be highly immunogenic and are increasingly being used as carriers for presenting heterologous antigens in new vaccines. In this study, we have coupled native STh and the STh-A14T toxoid to the coat protein of Acinetobacter phage AP205 by using the SpyCatcher system and immunized mice with these VLPs without the use of adjuvants. We found that both STs were efficiently coupled to the VLP, that both the STh and STh-A14T VLPs were immunogenic in mice, and that the resulting serum antibodies could completely neutralize the toxic activities of native STh. The serum antibodies showed a high degree of immunological cross-reaction to STp, while there was little or no unwanted cross-reaction to uroguanylin and guanylin. Moreover, compared to native STh, the STh-A14T mutation did not seem to negatively impact the immunogenicity of the construct or the neutralizing ability of the resulting sera. Taken together, these findings demonstrate that VLPs are suitable carriers for making STs immunogenic, and that the STh-A14T-coupled AP205 VLP represents a promising ETEC vaccine candidate.

Activation of the NLRP3 Inflammasome Pathway by Prokineticin 2 in Testicular Macrophages of Uropathogenic Escherichia coli- Induced Orchitis.

Infections of the reproductive tract are known to contribute to testicular inflammatory impairment, leading to an increase of pro-inflammatory cytokines such as IL-1ß, and a decline in sperm quality. Prokineticin 2 (PK2), a secretory protein, is closely associated with the secretion of pro-inflammatory cytokines in inflamed tissue. It was reported that increased PK2 is related to the upregulation of IL-1ß, but the underlying mechanism remains elusive. Here, we illustrated that PK2 was upregulated in testicular macrophages (TM) in a rat model of uropathogenic Escherichia coli (UPEC) infection, which induced the activation of the NLRP3 inflammasome pathway to boost IL-1ß secretion. Administration of PK2 inhibitor alleviated the inflammatory damage and suppressed IL-1ß secretion. Moreover, PK2 promoted NLRP3 expression and the release of cleaved IL-1ß from TM to the supernatants after the challenge with UPEC in vitro. IL-1ß in the supernatants affected Leydig cells by suppressing the expression of genes encoding for the enzymes P450scc and P450c17, which are involved in testosterone production. Overall, we revealed that increased PK2 levels in TM in UPEC-induced orchitis may impair testosterone synthesis via the activation of the NLRP3 pathway. Our study provides a new insight into the mechanisms underlying inflammation-associated male infertility and suggests an anti-inflammatory therapeutic target for male infertility.

Measurement of gut hormones in plasma.

The gastrointestinal tract is the largest endocrine organ and holds a special place in endocrinology since the concept of blood-borne communication between cells was first established through experiments on the gut. Gut peptide hormones and neurotransmitters regulate the complex processes of digestion, motility, epithelial growth, and integrity. Investigation of this complex endocrine organ has depended on the development of sensitive and specific radioimmunoassay. Radioimmunoassays have also increased our understanding of pathophysiological processes affecting the gut, including rare gastroenteropancreatic neuroendocrine tumours. The object of this chapter is to describe the techniques used in the radioimmunoassay of common gastrointestinal hormones.

Sequence analysis of porcine gut GLI-1.

A protein from porcine gut with 100 amino acid residues (porcine gut GLI-1) and having glucagon-like immunoreactivity has been characterized by partial sequences. The sequence of the C-terminal amino acid residues is -Met-Asn-Thr-Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala and includes the C-terminal amino acid residue sequence (-Met-Asn-Thr) of porcine glucagon. Evidence is presented that the glucagon sequence -Thr-Ser-Asp-Tyr-Ser-Lys-Tyr- is found in the gut GLI-1 as well. The data support the theory that gut GLI-1 contains the full glucagon sequence and that gut GLI-1 and glucagon are formed from a common precursor.

The anti-tumor effect is enhanced by simultaneously targeting VEGF and PROK1 in colorectal cancer.

Hematogenous metastasis, mainly hepatic metastasis, is a frequent metastatic mode in colorectal cancer involving angiogenic growth factors. Two angiogenic growth factors, in particular, Vascular endothelial growth factor (VEGF) and Prokineticin1(PROK1), are considered to have an important role in hematogenous metastasis of colorectal cancer. Accordingly, we report our findings on the importance of the anti-tumor efffect by inhibiting these two factors in human colorectal cancer.When the culture fluid of Colorectal cancer cell lines(DLD-1, HCT116, and LoVo) with high levels of VEGF/PROK1 expression was injected subcutaneously into mice, the culture fluid increased subcutaneous angiogenesis. But when both anti-PROK1 and anti-VEGF antibodies were present in the culture fluid, the length and size of the blood vessels were reduced compared with those seen in the fluid-only, anti-PROK1, and anti-VEGF controls. Also, tumor masses were produced in mice by subcutaneously embedding colorectal cancer cells with high levels VEGF/PROK1 expression. When both anti-PROK1 and anti-VEGF antibodies were simultaneously applied, tumor formation and peritumoral angiogenesis were strongly suppressed, compared with when either anti-PROK1 antibody or anti-VEGF antibody was applied alone.Simultaneous targeting of both angiogenic growth factors (VEGF/PROK1) may prove more useful in colorectal cancer.

Glicentin immunoreactive cells: their relationship to glucagon-producing cells.

The cellular and subcellular localization of one of the gut glucagon-like immunoreactants (GLI-1 or glicentin) and the relative distribution of glicentin- and glucagon-containing cells were investigated by immunocytochemistry. By immunofluorescence, the antiglicentin serum, which does not react with glucagon, revealed positive cells in the islets of Langerhans and in the gut mucosa, particularly in the terminal ileum and colon. In the intestinal mucosa, it was proven ultrastructurally that the glicentin immunoreactive cells correspond to the L cell and that the secretory granules represent the storage compartment of the immunoreactive material. In pancreatic islets, consecutive semithin sections treated with antiglicentin and specific antiglucagon sera showed that the same A cell population reacted with both sera, while immunoperoxidase staining on thin sections revealed that the immunoreactive material was confined to the secretory granules. The same results were obtained on dog oxyntic mucosa, where the glicentin- and glucagon-containing cells were identified as the gastric A cell. The immunocytochemical demonstration of a common glicentin-like material in the A and L cells together with the known presence of a common immunoreactant in glicentin and glucagon strongly support the idea that the A and L cells are ontogenetically related and synthesize their secretory product via a glicentin-like precursor which, by specific cleavage, could yield glucagon and gut glucagon-like immunoreactants.

The origins, pathways and terminations of neurons with VIP-like immunoreactivity in the guinea-pig small intestine.

We have analyzed changes in the distributions of terminals with vasoactive intestinal polypeptide (VIP)-like immunoreactivity, and accumulations in severed processes, that occur after lesions of intrinsic and extrinsic nerve pathways of the guinea-pig small intestine. The observations indicate that enteric vasoactive intestinal polypeptide immunoreactive neurons have the following projections. Nerve cell bodies in the myenteric plexus provide varicose processes to the underlying circular muscle; the majority of these pathways, if they extend at all in the anal or oral directions, do so for distances of less than 1 mm. Nerve cell bodies of the myenteric plexus also project anally to provide terminals to other myenteric ganglia. The lengths of the majority of these projections are between 2 and 10 mm, with an average length of about 6 mm. Processes of myenteric neurons also run anally in the myenteric plexus and then penetrate the circular muscle to provide varicose processes in the submucous ganglia at distances of up to 15 mm, the average length being 9-12 mm. In addition, there is an intestinofugal projection of myenteric neurons whose processes end around nerve cell bodies of the coeliac ganglia. A similar projection from the colon supplies the inferior mesenteric ganglia. The nerve cell bodies in submucous ganglia give rise to a subepithelial network of fibres in the mucosa and also supply terminals to submucous arterioles. It is concluded that vasoactive intestinal polypeptide is contained in neurons of a number of intrinsic nerve pathways, influencing motility, blood flow and mucosal transport. The myenteric neurons that project to prevertebral sympathetic ganglia may be involved in intestino-intestinal reflexes.

Gastric inhibitory peptide-like immunoreactivity in glucagon and glicentin cells: properties and origin. An immunocytochemical study using several antisera.

Although gastric inhibitory peptide (GIP) has never been detected outside the upper small intestine by immunochemical methods, GIP-like immunoreactivity has been demonstrated by immunocytochemistry in the glucagon/glicentin cells of pancreas, and gut. In the present study several GIP antisera (five polyclonal and one monoclonal) were tested on specimens from pancreas and intestines of several mammalian species, including man. Two of the polyclonal antisera and the monoclonal one stained cells in the upper small intestine only, while the other three also stained cells in the pancreas, ileum, and colon. Monoclonal anti-GIP did not stain GIP cells in man. The immunostaining produced could not be abolished by pretreatment of the antisera with glucagon or glicentin in excess, whereas small amounts of synthetic or natural porcine GIP prevented the immunostaining. Thus, three of the antisera are specific for GIP, while the other three recognize not only GIP but also GIP-like peptides. The results suggest that the glucagon/glicentin cells contain peptides distinct from GIP but sharing an immunodeterminant with GIP. The GIP-like immunoreactivity in the glucagon cells of the rat pancreas was not altered by infusion of GIP or by elimination of the bulk of endogenous GIP by resection of the upper small intestine, indicating that the GIP-like peptide is produced in the glucagon cells rather than accumulated from the circulation. The nature of this GIP-like peptide is unknown. Conceivably, it represents the cryptic portion of the glucagon precursor molecule. In some species a proportion of the GIP cells in the proximal small intestine displayed glicentin-like immunoreactivity as well, emphasizing the relationship between GIP cells on the one hand and glucagon/glicentin cells on the other.

Localization of xenin-immunoreactive cells in the duodenal mucosa of humans and various mammals.

Xenin is a 25-amino-acid peptide extractable from mammalian tissue. This peptide is biologically active. It stimulates exocrine pancreatic secretion and intestinal motility and inhibits gastric secretion of acid and food intake. Xenin circulates in the human plasma after meals. In this study, the cellular origin of xenin in the gastro-entero-pancreatic system of humans, Rhesus monkeys, and dogs was investigated by immunohistochemistry and immunoelectron microscopy. Sequence-specific antibodies against xenin detected specific endocrine cells in the duodenal and jejunal mucosa of all three species. These xenin-immunoreactive cells were distinct from enterochromaffin, somatostatin, motilin, cholecystokinin, neurotensin, and secretin cells, and comprised 8.8% of the chromogranin A-positive cells in the dog duodenum and 4.6% of the chromogranin A-positive cells in human duodenum. In all three species, co-localization of xenin was found with a subpopulation of gastric inhibitory polypeptide (GIP)-immunoreactive cells. Immunoelectron microscopy in the canine duodenal mucosa demonstrated accumulation of gold particles in round, homogeneous, and osmiophilic secretory granules with a closely adhering membrane of 187 +/- 19 nm diameter (mean +/- SEM). This cell type was found to be identical to the previously described canine GIP cell. Immunocytochemical expression of the peptide xenin in a subpopulation of chromogranin A-positive cells as well as the localization of xenin immunoreactivity in ultrastructurally characterized secretory granules permitted the identification of a novel endocrine cell type as the cellular source of circulating xenin.

Duodenal gangliocytic paraganglioma. Clinicopathologic and immunocytochemical study of 11 cases.

The clinicopathologic features of 11 cases (8 in men) of duodenal gangliocytic paraganglioma are presented. The patients averaged 56 years of age; none showed evidence of phakomatosis. Ten tumors occurred in the second portion of the duodenum, and one arose in the third portion. All tumors were polypoid, and half presented with gastrointestinal bleeding. The neoplasms were composed of paraganglioma and carcinoid-like elements, neurons, and Schwann as well as sustentacular cells. All tumors behaved in a benign fashion after local resection or snare polypectomy; long-term follow-up (1-25 years; mean, 8.3 years) showed no recurrence in any case. Immunocytochemical examination demonstrated the presence of somatostatin, serotonin, and human pancreatic polypeptide within endocrine cells and neurons.

Motilin-antimotilin reaction reveals two antigenic determinants in motilin.

The reaction of motilin with four rabbit antimotilin sera raised by immunization with synthetic porcine motilin-bovine serum albumin conjugate was studied with respect to various binding parameters and specificity. All four antisera exhibited an extremely high degree of specificity and high affinity (K greater than 10(11) M-1) for porcine motilin. Studying the various synthetic motilin fragments. These antisera appeared to contain binding sites reacting strongly with the N-terminal sequence in which the first three amino acid residues are essential for high affinity binding. One of the antisera, R-3-6, appeared to contain approximately 20% of its binding sites with high affinity for C-terminus-containing fragments. These results suggest that motilin possesses two antigenic domains along its primary structure; one contains the N-terminal tripeptide and the other contains the C-terminal nanopeptide as essential parts. Thus, in addition to heterogeneity in affinity, a given antibody preparation may be heterogenous with respect to the specificity along the sequence of a peptide. Gel filtration studies of the methanol extracts of human and dog plasma indicated that an immunoreactive motilin-like material with a molecular size similar to natural porcine motilin was measured by our routine radioimmunoassay.

Delta sleep-inducing peptide (DSIP)-like immunoreactivity in gut: coexistence with known peptide hormones.

Delta sleep-inducing peptide-like immunoreactivity (DSIP-LI) has previously been demonstrated in brain neurons and in endocrine cells of the pituitary and the adrenal medulla. By means of three different antisera against synthetic DSIP we now describe the occurrence and distribution of DSIP-LI in several gut endocrine cells. The human gut was the richest source, where DSIP-LI was located in gastrin/CCK, secretin and PYY/glicentin cells. The rat and pig gut harbour a moderate number of immunoreactive cells in the antral mucosa but in the intestines DSIP-LI-containing cells were very few. By radioimmunoassay, the concentration of DSIP-LI was determined in extracts of various gut regions from man, pig and rat. The highest concentrations were found in all human specimens compared with corresponding samples in the pig and rat. In all three species, high-performance liquid chromatography revealed a single peak of DSIP-like material with approximately the same retention time as DSIP 3-9. Taken together, the present results provide evidence for the presence of DSIP-LI in gut endocrine cells in man, pig and rat; the human gut seems to be the richest source of DSIP-like peptides.