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.

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.

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.

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.

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.

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.

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.

Prokineticin 1 expression in gastrointestinal tumors.

AIM: We studied prokineticin 1 (PROK1) expression in human gastrointestinal carcinomas by immunohistochemistry. MATERIALS AND METHODS: PROK1 expression was examined in human gastrointestinal cancer cell lines and primary gastrointestinal lesions. In addition the relationship between the number of blood vessels and PROK1 expression in these primary lesions was examined. RESULTS: PROK1 expression was observed in gastrointestinal cancer cell lines. PROK1 expression was not observed in healthy gastrointestinal mucosa, but was observed in the primary lesions in 23 out of 98 (31.6%) patients with colorectal cancer, 19 out of 55 (34.5%) patients with gastric cancer, and 5 of 10 (50%) patients with cancer of the small intestine. PROK1 expression was observed in many patients with advanced gastrointestinal cancer. The number of blood vessels in PROK1-positive primary gastrointestinal lesions was higher than that in PROK1-negative primary lesions. CONCLUSION: PROK1 expression might be related to the extent of malignancy in gastrointestinal cancer.

Guanylate cyclase C deficiency causes severe inflammation in a murine model of spontaneous colitis.

BACKGROUND: Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses. METHODS: We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C(+/+) and GC-C(-/-) mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10(-/-), GC-C(+/+)IL-10(-/-) and GC-C(-/-)IL-10(-/-) mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line. RESULTS: Relative to GC-C(+/+) animals, intraperitoneal lipopolysaccharide injection into GC-C(-/-) mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C(-/-)IL-10(-/-) animals was significantly more severe relative to GC-C(+/+)IL-10(-/-) mice. Unlike GC-C(+/+)IL-10(-/-) controls, colon pathology in GC-C(-/-)IL-10(-/-) animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C(-/-)IL-10(-/-) mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells. CONCLUSIONS: The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.

Effects of Escherichia coli heat-stable enterotoxin and guanylin on the barrier integrity of intestinal epithelial T84 cells.

Tight junctions contribute to the formation and establishment of intestinal epithelial barriers against microbial infections. However, a variety of enteric pathogens have developed strategies to adhere to and invade epithelial cells and disrupt epithelial integrity. The aim of this study was to ascertain if enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) can cause deterioration of epithelial barrier integrity. Since STa shows amino acid similarity to guanylin, we evaluated the effects of both of these molecules on T84 epithelial cells. T84 epithelial monolayers were grown on 24-well Transwell filters and barrier integrity was assayed by measurement of transepithelial electrical resistance (TER). Macromolecular permeability of the monolayers was determined by measuring the paracellular passage of FITC-labeled dextran 4000 from apical to basolateral compartments of the Transwell filter culture. Treatment of T84 monolayers with either ST or guanylin did not increase paracellular permeability to FITC-dextran. However, although guanylin, which is a physiological guanylate cyclase activator, did not change TER in polarized T84 monolayers, ST did elicit a reduction in TER within 2h, at concentrations above 4µM. These data suggest that STa causes not only induction of water secretion but also intestinal barrier dysfunction.

Endocrine gland-derived vascular endothelial growth factor strengthens cell invasion ability via prokineticin receptor 2 in colon cancer cell lines.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) has recently been identified as one of the vascular endothelial growth factors, and it is considered that the overexpression of EG-VEGF in colon cancer is related to hepatic metastasis. In this study, we report our recent novel findings of the involvement of EG-VEGF in cell invasion of colon cancer cells. Colon cancer cell lines (DLD-1 and HCT116) with high expression of prokineticin receptor (PK-R) 1 and 2 were stimulated with the EG-VEGF protein. Furthermore, Matrigel cell invasion assay was performed to examine the changes in cancer cell invasion. In addition, we investigated the mRNA expression of matrix metalloproteinase (MMP)-2, -7 and -9 in cancer cells. Finally, the EG-VEGF receptor on the colon cancer cell membrane was blocked by anti-PK-R1 and -PK-R2 antibodies to study whether cell invasion ability would be altered. In colon cancer cell lines where the expression of PK-R1 and 2 was confirmed, stimulation with EG-VEGF increased cell invasion a maximum of ~3-5 times. Furthermore, an increase in the mRNA and protein expression of MMP-2, -7 and -9 was observed. We also observed that the cell invasion rate decreased only after exposure to the anti-PK-R2 antibody. The study showed that the EG-VEGF protein may act on MMP-2, -7 and -9 via PK-R2 to strengthen cell invasion ability in colon cancer cell lines.

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.

IL-4 regulates susceptibility to intestinal inflammation in murine food allergy.

Allergies involve a state of immediate hypersensitivity to antigens, including food proteins. The mechanism underlying the initiation and development of allergic responses involves IL-4 that directly induces the differentiation of committed effector Th2 lymphocytes. Although it is clear that Th2 responses play a pivotal role in the development of allergic responses, it remains unclear which mechanisms are involved in the development of the intestinal damages observed in food allergy. Accordingly, this work aimed to study the role of Th2/IL-4-dependent responses in the development of food allergy and intestinal pathology. C57BL/6 wild-type (WT) and IL-4-/- mice were sensitized with peanut proteins, challenged with peanut seeds, and followed for the development of food allergy and intestinal inflammation. Results demonstrated that exposure to peanut seeds led to weight loss in WT but not in IL-4-/- mice that preserved gut integrity with no signs of mucosal inflammation. These animals presented increased levels of IgG2a in sera, suggesting a role for allergic antibodies in the pathogenesis of WT animals. Most importantly, results also showed that lack of IL-4 modulated gut mucosal response in food allergy through diminished expression of TNF-alpha mRNA, increased Th1 IFN-gamma, IL-12p40, regulatory cytokines, and Foxp3, demonstrating their relevance in the control of allergic inflammatory processes, especially in the intestine. Finally, this study highlighted some of the complex mechanisms involved in the pathogenesis of allergic responses to food antigens in the gut, thereby providing valuable tools for directing novel therapeutic or preventive strategies to the control of allergic enteropathy.

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.

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.

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.

Peptide YY attenuates transcription factor activity in tumor necrosis factor-alpha-induced pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is a disease characterized by inflammation. Nuclear factor (NF)-kappaB, Smad proteins, and the steroid hormone family peroxisome proliferator-activated receptors (PPARs) are involved in regulation of gene transcription during the disease process. Peptide YY (PYY), a gastrointestinal hormone, inhibits NF-kappaB translocation to acinar nuclei in tumor necrosis factor (TNF)-alpha-induced AP. We investigated TNF-alpha induction of Smad proteins, PPARalpha/gamma, and NF-kappaB by TNF-alpha, and hypothesized that PYY would attenuate this effect. STUDY DESIGN: Rat acinar cells were treated with recombinant TNF-alpha (200 ng/mL). PYY (3 to 36) was added at 500 pM at 30 minutes after TNF-alpha treatment until cell harvest at 2 hours. Western blot analysis and intracellular staining of the p65 subunit of NF-kappaB were performed. NF-kappaB, Smad3/4, and PPARalpha/gamma binding activities were determined by protein/DNA array analysis and verified by electrophoretic-mobility shift assay and densitometry. RESULTS: Cellular localization of NF-kappaB p65 showed nuclear staining within 2 hours, with controls stained in the cytoplasm. With PYY, p65 stained in the cytoplasm. Nuclear p65 was increased significantly (p < 0.05) by TNF-alpha at 2 hours and PYY reduced it. Array analysis revealed upregulation of NF-kappaB, PPARalpha/gamma, and Smad3/4 with TNF-alpha. TNF-alpha stimulated NF-kappaB activation sevenfold, and binding was enhanced (p < 0.05). PYY reduced NF-kappaB binding to control levels. PPAR binding increased 51% after TNF-alpha treatment and was reduced to 33% with PYY. Smad3/4 binding was increased (p < 0.05) above controls with TNF-alpha and PYY reduced it by 40%. CONCLUSIONS: TNF-alpha increases early nuclear translocation of the p65 subunit of NF-kappaB in acinar cells. Exposure to TNF-alpha activates transcription factors NF-kappaB, Smad3/4, and PPARalpha/gamma. PYY reduces this activation. Treatment with PYY may have therapeutic potential in improving AP.

Screening of gastrointestinal hormone release in patients with lung cancer.

The aim of this study was the screening of gastrointestinal (GI) hormones release as possible sensitive tumor markers. The subjects were eight patients with carcinoma of the lung compared with nine healthy controls. Six kinds of GI hormones were measured by the specific radioimmunoassay. It was evaluated by fasting level (F), increased integrated responses (IIR60, IIR180 pmol/l/min) and total integrated responses (TIR60, TIR180 pmol/l/min) after a meal. The data were analyzed by the Student's t-test. The F of pancreatic polypeptide (PP) was significantly higher in patients than that in healthy controls (24.4 vs. 12.3 pmol/l, p = 0.002). Consequently, the sensitivity was 75% (6 out of 8) and specificity was 100% (9 out of 9). Almost all parameters of IIR60, IIR180, TIR60 and TIR180 in PP and peptide YY were significantly higher in patients than in healthy controls (p value: 0.00020-0.021). The other four kinds of hormones showed similar results to the healthy controls. These results seem to indicate that PP and PYY would be useful tumor markers for lung cancer in clinical management.