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Introduction Pregnancy rhinitis is a common sex hormone-related otorhinolaryngological disorder. There are some epidemiological and physiological studies on pregnancy rhinitis, but histopathological and biomolecular changes have not been studied thoroughly. Objectives The receptors VPAC1 and VPAC2 are known for their roles in allergic rhinitis. On the other hand, activation of subclinical allergy has been suggested in the pathophysiology of pregnancy rhinitis. Therefore, we aimed to compare the physiological and gestational pattern of VPAC1 and VPAC2 expression in rat nasal mucosa. Methods Twenty adult Wister albino female rats were enrolled into the study. Two groups constituted as 10 control (group A) and 10 pregnant (group B) rats. They were fed ad libitum and sheltered at room temperature (22°±2 °C). The rats were sacrificed at the 20th day of gestation by intraperitoneal injection of 400 mg/kg Na-pentobarbitone. Then, 10 − 15 mL of blood was taken, and samples were reserved for the detection of serum estradiol and progesterone levels by ELISA test. The nasal septum was resected and divided in half for immunohistochemical analyses and real time polymerase chain reaction testing of VPAC1 and VPAC2. Results VPAC1 and VPAC2 were found to be in all layers of septal specimens, but the immunostaining of surface epithelium was more distinct in specimens of both groups. We demonstrated higher overall staining intensity in the pregnant group. PCR revealed significant increase in expression of VPAC1 (p = 0.023) and VPAC2 (p = 0.021) in pregnant group when compared with control group. In addition, we demonstrated upregulatory effect of estradiol and progesterone on the vasoactive intestinal peptide receptor expression. Conclusions Gestational up-regulation of nasal VPAC1 and VPAC2 was shown both by PCR and immunohistochemical analysis. These findings support the hypothesis that PR is caused by the activation of subclinical allergy that is present before pregnancy.

Constipation is a condition with a high prevalence rate worldwide and may occur in men and women of any age. Bifidobacterium bifidum has been shown to have a relieving effect on constipation, but the underlying mechanism is still unknown. This study explored the effects of gavage of three strains of B. bifidum (CCFM668, FHNFQ25M12 and FXJCJ32M2) from different sources in mice with loperamide-induced constipation. After 38 days of intervention, B. bifidum CCFM668, FHNFQ25M12 and FXJCJ32M2 showed the ability to modify the levels of gastrointestinal active peptides and promote the expression of 5-hydroxytryptamine (5-HT or serotonin) receptor 4 (5-HT4R), thereby promoting small intestinal peristalsis. The strains could also effectively increase the thickness of the colonic mucosa. However, what was different from previous studies was that these results were independent of the levels of short-chain fatty acids (SCFAs) and 5-HT. Further analysis of the intestinal flora revealed that the relative abundances of the genera Faecalibaculum and Ruminococcaceae_UCG_014 in the constipated mice increased significantly, whereas that of Erysipelatoclostridium decreased. A correlation analysis between the intestinal flora and evaluated gastrointestinal indicators demonstrated that the relative abundances of the genera Anaerotruncus, Angelakisella, Erysipelatoclostridium and Ruminococcaceae_UCG_014 were negatively correlated with the levels of gastrointestinal active peptides. B. bifidum FXJCJ32M2 can increase the relative abundances of Turicibacter and Dubosiella, and this was positively correlated with the expression of aquaporin 8 and vasoactive intestinal peptide receptor 1 but could not effectively alleviate faecal dryness or promote colonic motility. These findings suggest that B. bifidum shows significant intraspecific differences in the remission mechanism and provides a theoretical basis for subsequent population experiments and personalised treatment for constipation.

BackgroundPaucity of T cells in the immune privileged tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is a major reason that PDAC is refractory to immune checkpoint blockade.1 In this study, we show that human PDAC tumors over-express vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, that inhibits effector T cell responses and regulates chemokine receptor expression on activated T cells.2 3 We thus hypothesized that pharmacological inhibition of VIP receptor signaling could enhance anti-tumor responses in PDAC.MethodsVIP levels in plasma were determined via VIP-specific enzyme immunoassay and confirmed with immunohistochemistry (IHC) of tissue sections. VIP receptor (VIP-R) signaling in C57BL/6 immunocompetent murine models of KPC, MT5 or Panc02 pancreatic cancer was inhibited by daily sub-cutaneous treatment with ANT008 or ANT308, two novel VIP-R antagonists with predicted high binding affinities to VIP receptors.4–7 Multiplex IHC or flow cytometry detected frequencies and phenotypes of intra-tumoral T cells across treatment groups.ResultsHuman PDAC tumors expressed VIP by immunohistochemistry, and PDAC patients had significantly elevated plasma VIP levels when compared to healthy volunteers (pConclusionsVIP-R antagonists represent a novel approach to treat PDAC. VIP and VIP-R sequences are highly conserved between humans and mice,8 and human T cells are activated in vitro following treatment with VIP-R antagonists. Thus, we predict comparable anti-tumor activity of the combination of VIP-R antagonist and anti-PD-1 MoAb in human PDAC patients. Further clinical development of this novel concept will require appropriate pre-clinical pharmacokinetic and toxicology studies.AcknowledgementsThe authors thank healthy volunteers and patients for blood and/or tissue samples. The authors also thank the shared resources at Emory University, namely the Emory Integrated Genomics Core (EIGC), Emory Flow Cytometry Core (EFCC), Cancer Animal Models Shared Resource (CAMS), Cancer Tissue Pathology Core (CTP), Biostatistics Shared Resource (BSR) and Integrated Cellular Imaging Core (ICI), that provided services or instruments at subsidized cost to conduct some of the reported experiments. BioRender was used to make figure 4A and 5C. This work was supported in part by Katz Foundation funding and Emory School of Medicine Dean's Imagine, Innovate and Impact (I3) venture award to Edmund K. Waller and NIH R01 CA207619 awarded to Susan N. Thomas. Part of the cost for the immunohistochemistry staining of tissues was covered by Winship Cancer Institute Development Discovery and Therapeutic Program Pilot funding to Sruthi Ravindranathan.Abstract 748 Figure 1VIP is over-expressed by PDAC. (A) VIP mRNA expression levels in various solid malignancies, as obtained from TCGA. (B) Representative images of human PDAC tumor stained with antibodies to VIP or CK19, showing VIP co-expression in islets (black arrow) and cancer epithelial cells (red arrow). Levels of VIP in (C) culture supernatants collected from murine and human PDAC cell lines cultured for 24 hours (n=3 per cell line) were compared to culture supernatants from B16F10 and D4M melanoma cells; (D) plasma of mice bearing melanoma or PDAC tumors (n=5) compared to plasma of non-tumor-bearing mice; (E) plasma of PDAC patients (n=19) compared to that from healthy volunteers (n=26). Statistical differences in C and D were performed by ANOVA followed by Dunnett's post-test and in E were performed by student's t-test. Error bars show mean ± SEM. *pAbstract 748 Figure 2VIP-R antagonists improve responses to anti-PD-1. KPC.Luc, MT5 or Panc02 cells were subcutaneously implanted in immunocompetent C57BL/6 mice. About one week after tumor implantation, when the tumors were palpable, mice were randomized into treatment groups and treated with VIP-R antagonist and/or anti-PD-1 as described in methods. (A) KPC.Luc, MT5 and Panc02 tumor volumes as measured by Vernier calipers on day 22 after subcutaneous tumor implantation. (B) Kaplan-Meier survival plots of C57BL/6 mice with subcutaneously implanted KPC.Luc, MT5 or Panc02 tumors stratified by treatment. Kaplan-Meier survival plots of (C) C57BL/6 mice receiving monoclonal CD4 and/or CD8 monoclonal antibodies (D) CD4KO or (E) CD8KO mice compared to wild-type CD57BL/6 mice with subcutaneously implanted KPC.Luc tumors, stratified by treatment. Statistical differences in A were calculated by ANOVA followed by Dunnett's post-test. Solid line shows mean with in each treatment group. Statistical differences in B-E are calculated via Log-rank test. *pAbstract 748 Figure 3Enhanced T cell response with combination therapy. mRNA expression in T cells isolated from subcutaneous KPC.Luc tumors in C57BL/6 mice treated with ANT008 and/or anti-PD-1 (n=3 per treatment group), were analyzed via Nanostring metabolism panel. Volcano plot showing differential expression of genes in T cells from (A) ANT008+ isotype IgG (IgG) vs scrambled peptide (Scram) + isotype IgG, (B) scrambled peptide +anti-PD-1 vs scrambled peptide + isotype IgG and (C) ANT008+anti-PD-1 vs scrambled peptide + isotype IgG (n=3 mice per treatment group). Genes that are associated with TCR activation and co-stimulation and are at levels significantly higher when compared to Scram+ isotype IgG (FDRAbstract 748 Figure 4Increased T cell density with combination therapy. KPC.Luc cells were orthotopically implanted in the tail of the pancreas of C57BL/6 mice and treated with ANT008 and/or anti-PD-1 with n=9, 10, 8 and 11 in scrambled+IgG, ANT008+IgG, scrambled+anti-PD-1 and ANT008+anti-PD-1, respectively. (A) Schematic showing orthotopic implantation of KPC.Luc cells and treatment strategy with ANT008 and/or anti-PD-1. (B) Waterfall plot showing % change in tumor flux on day 22 relative to day 7 prior to start of treatment. (C) Total flux as measured by IVIS bioluminescent imaging in the different treatment groups. Cross symbol represents mice that were euthanized before day 25 due to ulceration of the tumor and circle symbol represent mouse that were imaged on day 26 via MRI imaging shown in supplementary figure S5. (D) Bar graph showing weight of pancreas on day 25 when the mice were euthanized. 'Star' shaped data points indicate tumor free mice and dotted horizontal line represents the average weight of healthy pancreas from naïve mice. (E) Representative multiplex IHC images (right) showing pancreatic tumors stained for DAPI (blue), CD4 (yellow), CD8 (red) and Ki67 (cyan) and trichrome staining (left) with black arrows showing blue collagen stain in the tissue. XY plot showing the correlation between number of (F) CD4+ or (G) CD8+ T cells/mm2; and (H) Ki67+ CD4+ or (I) Ki67+ CD8+ T cells/mm2 with weight of the pancreas with n=4 to 6 mice per group. P values in panel D were calculated using student ANOVA followed by Dunnett's post hoc test (comparing each treatment group with Scram+IgG). Error bars show mean ± SEM. *pAbstract 748 Figure 5Increased T cell homing with combination therapy. KPC.Luc tumors were subcutaneously implanted in C57BL/6 mice and treated with VIP-R antagonist and/or anti-PD-1 checkpoint therapy for 10 days after the tumors were palpable. Tumor draining lymph nodes were then analyzed for percentage of (A) CXCR4+CD69+ and (B) CXCR4+Ki67+ cells in CD4+ (left) and CD8+ (right) subsets of T cells. In a separate experiment, on day 15 after subcutaneous implantation of KPC.Luc tumors, GFP+ T cells from enhanced GFP transgenic mice (C57BL/6 background) were adoptively transferred (via tail vein injections) and treated with ANT308± aPD-1 for 3 days. (C) Schematic showing GFP+ T cell transfer and treatment strategy in mice with subcutaneous KPC.Luc tumors. (D) Representative Hoescht (blue for nucleus) stained tumor tissues from tumors of each treatment group. Two regions of interest (ROI) in ANT308+aPD-1 treated tumors are shown at higher magnification. Statistical differences in A and B were determined via repeated measures ANOVA and Dunnett's post-test with n=4–5 mice per group. *pReferencesSahin IH, et al. Immunotherapy in pancreatic ductal adenocarcinoma: an emerging entity? Ann Oncol 2017;28(12):2950–2961.Gonzalez-Rey E, Anderson P, Delgado M. Emerging roles of vasoactive intestinal peptide: a new approach for autoimmune therapy. Ann Rheum Dis 2007;66(Suppl 3):p. iii70–6.Anderson P, Gonzalez-Rey E. Vasoactive intestinal peptide induces cell cycle arrest and regulatory functions in human T cells at multiple levels. Mol Cell Biol 2010;30(10):2537–51.Li JM, et al. VIPhyb, an antagonist of vasoactive intestinal peptide receptor, enhances cellular antiviral immunity in murine cytomegalovirus infected mice. PLoS One 2013;8(5):e63381.Moody TW, et al., VIP receptor antagonists and chemotherapeutic drugs inhibit the growth of breast cancer cells. Breast Cancer Res Treat 2001;68(1):55–64.Moody TW, et al. A vasoactive-Intestinal-Peptide antagonist inhibits nonsmall cell lung-cancer growth. Proceedings of the National Academy of Sciences of the United States of America 1993;90(10):4345–4349.Zia H, et al. Breast cancer growth is inhibited by vasoactive intestinal peptide (VIP) hybrid, a synthetic VIP receptor antagonist. Cancer Res 1996;56(15):3486–9.Sena M, et al. High conservation of upstream regulatory sequences on the human and mouse vasoactive intestinal peptide (VIP) genes. DNA Seq 1994;5(1):25–9.Ethics ApprovalAll experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at Emory University. De-identified blood samples from consented patients with PDAC (IRB 00087397) or healthy volunteers (IRB 00046063) were obtained with approval from Institutional Review Boards.

Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.

Worldwide, more than 20 million people suffer from schizophrenia, but effective and definitive new therapeutic drugs/treatments have not been established. Vasoactive intestinal peptide receptor 2 (VIPR2) might be an attractive drug target for the treatment of schizophrenia because both preclinical and clinical studies have demonstrated a strong link between high expression/overactivation of VIPR2 and schizophrenia. Nevertheless, VIPR2-targeting drugs are not yet available. VIPR2 is a class-B G protein-coupled receptor that possesses high structural homology to its subtypes, vasoactive intestinal peptide receptor 1 (VIPR1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). These biological and structural properties have made it difficult to discover small molecule drugs against VIPR2. In 2018, cyclic peptide VIpep-3, a VIPR2-selective antagonist, was reported. The aim of this study was to generate a VIpep-3 derivative for in vivo experiments. After amino acid substitution and structure optimization, we successfully generated KS-133 with 1) a VIPR2-selective and potent antagonistic activity, 2) at least 24 h of stability in plasma, and 3) in vivo pharmacological efficacies in a mouse model of psychiatric disorders through early postnatal activation of VIPR2. To the best of our knowledge, this is the first report of a VIPR2-selective antagonistic peptide that counteracts cognitive decline, a central feature of schizophrenia. KS-133 may contribute to studies and development of novel schizophrenia therapeutic drugs that target VIPR2.

Gestational diabetes mellitus (GDM) is associated with immune metabolic changes that increase women's risk of developing metabolic disorders later in life. Nutritional intervention is a crucial component in reducing the burden of these pathological features. We examined whether protocatechuic acid (PCA), a major metabolite of anthocyanins abundant in plant food, is able to exert insulin-mimetic activity and modulate inflammation in the visceral adipose tissue (VAT) obtained at delivery, from pregnant women with GDM or normal glucose tolerance (NGT). PCA stimulated glucose uptake in the VAT from both GDM and NGT women. This capability was associated with increased phosphorylation of p38 mitogen-activated protein kinase (p38MAPK), as further demonstrated by the inhibitory effect of SB203580, a p38MAPK inhibitor, on PCA-induced glucose uptake. The GDM-VAT expressed lower adiponectin levels and PCA stimulated adiponectin release in the NGT-VAT and, albeit to a lower extent, in the GDM-VAT. Higher levels of IL6 and TNFα were secreted by the GDM-VAT compared with the NGT one, and PCA had no effects on them. PCA reduced the overexpression of vasoactive intestinal peptide receptor 2 (VPAC2) in the GDM-VAT. Further studies are needed to establish whether and how anthocyanins and food rich in these compounds may contribute to prevent or delay metabolic disorders in women with GDM.

Introduction Female sexual arousal disorder (FSAD) is a common issue causing physical and psychological pain, but it has no standard diagnostic criteria or treatment. So its pathogenesis desiderates to be explored. Aim To investigate the specific function of miR-122-5p in FSAD. Methods 18 subjects were grouped into FSAD and normal control groups according to the Chinese version of the Female Sexual Function Index, and the expression levels of miR-122-5p and vasoactive intestinal peptide receptor 1 (VIPR1) protein in their tissue were verified through real-time quantitative polymerase chain reaction (qRT-PCR) and western blot (WB) analysis. Then in vitro experiment, miR-122-5p was overexpressed or inhibited in rat vaginal smooth muscle cells (SMCs). The relaxation of rat vaginal SMCs was reflected by the cell morphology, intracellular free cytosolic calcium ion (Ca2+) levels, cell proliferation and apoptosis, together with the cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activities. Additionally, the expression levels of relaxation-related proteins, including VIPR1, stimulatory G protein (Gs), adenylate cyclase (AC), and PKA, were detected based on WB analysis. Furthermore, a rescue experiment that simultaneously overexpressed or silenced miR-122-5p and VIPR1 was conducted, and all the indicators were evaluated. Main Outcomes Measure The expression level of VIPR1 and downstream proteins, cell morphology, cell proliferation and apoptosis, and intracellular free Ca2+ levels were examined. Results We verified that women with FSAD had higher miR-122-5p and lower VIPR1 protein. Then overexpressing miR-122-5p decreased relaxation of rat vaginal SMCs, which was manifested as a contractile morphology of cells, an increased intracellular free Ca2+ concentration, and lower cAMP concentration and PKA activity. Moreover, by rescue experiments, we inferred that VIPR1 was the target of miR-122-5p and affected the relaxation function of vaginal SMCs. Conclusion miR-122-5p regulates the relaxation of vaginal SMCs in FSAD by targeting VIPR1, ulteriorly providing an underlying diagnostic and therapeutic target for FSAD. Cong S, Gui T, Shi Q, et al. Overexpressing miR-122-5p Inhibits the Relaxation of Vaginal Smooth Muscle in Female Sexual Arousal Disorder by Targeting Vasoactive Intestinal Peptide Receptor 1. Sex Med 2021;9:100390.

Background Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer related death in the U.S, has a 5-year survival rate of only 10%.1 The paucity of T cells in the immune privileged tumor microenvironment (TME) is a major limitation in developing an effective immunotherapy against PDAC.2The cancer genome atlas (TCGA) shows that human PDAC tumors express high levels of vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide (figure 1A), that inhibits effector T cell responses.3 4 We hypothesized that paracrine secretion of VIP in the TME is a targetable mediator of immune paralysis in PDAC, and that pharmacological inhibition of VIP receptor signaling could enhance anti-tumor responses in PDAC. Methods VIP levels in plasma or cell culture supernatant was determined via VIP-specific enzyme immunoassay. Luciferase transfected KPC (KPC.luc) cells were injected subcutaneously or orthotopically into the pancreas of C57BL/6, CD4KO, or CD8KO mice from Jackson Laboratories. C57BL/6 mice T cell subsets in were depleted post tumor implantation with anti-CD4 and/or anti-CD8 antibodies. Tumor-bearing mice were treated daily with ANT008, a novel VIP receptor antagonist peptide, and/or anti-PD1 monoclonal antibody (MoAb) for 10 days, starting 7-10 days after implantation. T cells isolated from peripheral blood of PDAC patients were expanded 9 days ex vivo in anti-CD3 MoAb coated plates with 30U/ml IL-2 and either control peptide (scrambled VIP sequence) or ANT008. Survival by VIP and VIP receptor expression from the TCGA was clinically correlated. Results Increased human and mouse PDAC expression correlates with elevated blood levels (figure 1). While the PDAC cancer cell lines express VIP receptors, ANT008 does not have direct cytotoxic effect on cell growth in vitro (figure 2). However, in orthotopic KPC model, treatment with ANT008 & anti-PD-1 significantly decreased tumor growth rate and burden (figure 3) while increasing the intratumoral levels of CD4 and CD8 proliferating T cells (figure 4) via a T cell dependent mechanism (figure 5). Additionally, in ex vivo cultures of T cells isolated from PDAC patients, ANT008 improved the effector properties of T cells via decreasing expression levels of co-inhibitory molecules and decreasing frequency of regulatory T cells (figure 6). Clinically, VIPR1 receptor expression, but not VIP, provides a survival benefit (figure 7). Conclusions VIP is a targetable mechanism of immune escape in PDAC. Inhibiting VIP receptor signaling improves effector properties of T cells and synergistically improves the anti-tumor response to checkpoint inhibitors in mouse PDAC models. References Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin2020;70(1):7-30. Sahin IH, et al. Immunotherapy in pancreatic ductal adenocarcinoma: an emerging entity?Ann Oncol 2017;28(12):2950-2961. Gonzalez-Rey E, Anderson P, Delgado M. Emerging roles of vasoactive intestinal peptide: a new approach for autoimmune therapy. Ann Rheum Dis 2007;66(Suppl 3):iii70-6. Anderson P, Gonzalez-Rey E. Vasoactive intestinal peptide induces cell cycle arrest and regulatory functions in human T cells at multiple levels. Mol Cell Biol 2010;30(10):2537-51.

Genetically engineered mouse models through gene deletion are useful tools for analyzing gene function. To delete a gene in a certain tissue temporally, tissue-specific and tamoxifen-inducible Cre transgenic mice are generally used. Here, we generated transgenic mouse with cardiac-specific expression of Cre recombinase fused to a mutant estrogen ligand-binding domain (ERT2) on both N-terminal and C-terminal under the regulatory region of human vasoactive intestinal peptide receptor 2 (VIPR2) intron and Hsp68 promoter (VIPR2-ERT2CreERT2). In VIPR2-ERT2CreERT2 transgenic mice, mRNA for Cre gene was highly expressed in the heart. To further reveal heart-specific Cre expression, VIPR2-ERT2CreERT2 mice mated with ROSA26-lacZ reporter mice were examined by X-gal staining. Results of X-gal staining revealed that Cre-dependent recombination occurred only in the heart after treatment with tamoxifen. Taken together, these results demonstrate that VIPR2-ERT2CreERT2 transgenic mouse is a useful model to unveil a specific gene function in the heart.

Synbiotic dietary supplements, as an effective means of regulating the gut microbiota, may have a beneficial effect on constipation. This study evaluated the effects of synbiotic yogurt containing konjac mannan oligosaccharides (KMOS) and Bifidobacterium animalis ssp. lactis BB12 (BB12) on constipated Kunming mice (the model group). Following administration of yogurt containing 2.0% KMOS and BB12 (YBK2.0), black fecal weight and number and gastrointestinal transit rate increased by 97.5, 106.3, and 55.7%, respectively, compared with the model group. Serum levels of excitability neurotransmitters (motilin, substance P, and acetylcholine) in the YBK2.0 group were increased by 139.7, 120.4, and 91.8%, respectively, and serum levels of inhibitory neurotransmitters (vasoactive intestinal peptide, nitric oxide, and acetylcholine) were decreased. Moreover, synbiotic yogurt supplementation significantly downregulated the expression of vasoactive intestinal peptide receptor 1 (VIPR1) and upregulated the expression of serotonin receptor 4 (5-HT4) in the colon, and enhanced the expression of the stem cell factor (SCF)/c-Kit pathway. Additionally, YBK2.0 treatment significantly regulated the community composition and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of gut microbiota, which were positively correlated with physiological parameters of constipation. Thus, supplementation with synbiotic yogurt composed of KMOS and BB12 could facilitate fecal excretion by regulating related pathways and the gut microbiota. These findings demonstrated that the synbiotic yogurt can be considered a functional food for alleviating constipation.

Migraine is a debilitating disease that affects almost 15% of the population worldwide and is the first cause of disability in people under 50 years of age, yet its etiology and pathophysiology remain incompletely understood. Recently, small molecules and therapeutic antibodies that block the calcitonin gene-related peptide (CGRP) signaling pathway have reduced migraine occurrence and aborted acute attacks of migraine in clinical trials and provided prevention in patients with episodic and chronic migraine. Heterogeneity is present within each diagnosis and patient's response to treatment, suggesting migraine as a final common pathway potentially activated by multiple mechanisms, e.g., not all migraine attacks respond to or are prevented by anti-CGRP pharmacological interventions. Consequently, other unique mechanisms central to migraine pathogenesis may present new targets for drug development. Pituitary adenylate cyclase-activating peptide (PACAP) is an attractive novel target for treatment of migraines. We generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1910) with reactivity to both PACAP38 and PACAP27. In vitro, ALD1910 effectively antagonizes PACAP38 signaling through the pituitary adenylate cyclase-activating peptide type I receptor, vasoactive intestinal peptide receptor 1, and vasoactive intestinal peptide receptor 2. ALD1910 recognizes a nonlinear epitope within PACAP and blocks its binding to the cell surface. To test ALD1910 antagonistic properties directed against endogenous PACAP, we developed an umbellulone-induced rat model of neurogenic vasodilation and parasympathetic lacrimation. In vivo, this model demonstrates that the antagonistic activity of ALD1910 is dose-dependent, retaining efficacy at doses as low as 0.3 mg/kg. These results indicate that ALD1910 represents a potential therapeutic antibody to address PACAP-mediated migraine.

Radiolabeled heterobivalent peptidic ligands (HBPLs), being able to address different receptors, are highly interesting tumor imaging agents as they can offer multiple advantages over monovalent peptide receptor ligands. However, few examples of radiolabeled HBPLs have been described so far. One promising approach is the combination of gastrin-releasing peptide receptor (GRPR)- and vasoactive intestinal peptide receptor subtype 1 (VPAC1R)-targeting peptides into one single radioligand since gastrinomas, prostate and breast cancer have been shown to concomitantly or complementarily overexpress both receptors. Here we report the design and synthesis of different HBPLs, comprising a GRPR-binding (BBN7-14) and a VPAC1R-targeting (PACAP-27) peptide. The heterodimers were varied with regard to the distance between the peptide binders and the steric rigidity of the systems. We radiolabeled the HBPLs 19–23 as well as their monomeric reference standards 26 and 27 with 68Ga, achieving radiochemical yields and purities of 95–99% and non-optimized molar activities of 25–61 GBq/μmol. We tested the stability of the radioligands and further evaluated them in vitro regarding their uptake in different prostate carcinoma cell lines (PC-3, DU-145 and VCaP cells). We found that the heterobivalent substances [68Ga]19 − [68Ga]23 showed comparable uptakes into the tumor cells to those of the respective monomers [68Ga]26 and [68Ga]27, indicating that both peptides are still able to address their target receptors. Furthermore, the obtained results indicate that in case of overall low receptor densities, heterobivalent peptides surpass peptide monomers in tumor cell uptake. Most importantly, it could be shown by blocking studies that both peptide parts of the HBPL [68Ga]19 contributed to tumor cell uptake in VCaP cells, expressing both receptor types. Thus, we describe here the first examples of HBPLs being able to address the GRPR as well as the VPAC1R and have the potential to − by several mechanisms − improve tumor targeting for several malignancies compared to monospecific peptides.

Pancreatic ductal adenocarcinoma (PDAC) is unresponsive to immune checkpoint therapy largely due to a paucity of T cells within the tumor microenvironment (TME) and abundant immunosuppressive signaling pathways. In this study we show that human PDAC tumors over-express vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide that suppresses T cell effector properties and promotes the generation of regulatory T cells (Tregs). Therefore, we treated tumor-bearing mice with VIP receptor peptide antagonists and measured T cell homing, activation, and anti-tumor responses in preclinical murine models of PDAC. Pharmacological inhibition of VIP receptor (VIP-R) signaling using daily subcutaneous injections of peptide antagonists had no discernable toxicity in healthy and tumor-bearing mice. Treatment with VIP-R antagonists in combination with anti-PD-1 checkpoint blockade significantly decreased tumor burden, and improved survival in subcutaneous and orthotopic murine PDAC models. Combination therapy significantly enhanced T cell activation and proliferation and decreased frequencies of Tregs within the TME. Anti-tumor responses were T cell dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. Furthermore, combination therapy significantly increased frequencies of tumor specific T cells (measured with a tetramer reagent) and provided protective immunity against tumor rechallenge. Combination therapy led to significant increases in the infiltration of adoptively transferred GFP+ T cells into PDAC tumors and decreased CXCR4 expression levels on T cells. Encouragingly, peptide-based VIP-R antagonists enhanced the in vitro activation of human T cells isolated from peripheral blood of PDAC patients. Human T cells cultured with VIP-R antagonists had increased proliferation, activation, and decreased proportions of T regs and exhausted T cells co-expressing PD-1, Tim-3 and Lag-3. Taken together, our findings show that VIP is a targetable mechanism of immune escape in PDAC. Inhibiting VIP receptor signaling improves T cell effector properties and synergistically improves anti-tumor responses to checkpoint inhibitors in mouse PDAC models. Additionally, as the VIP sequence is identical between human and mice, and since VIP-R antagonists have similar effects on human and murine T cells in culture, clinical translation is highly feasible. Citation Format: Sruthi Ravindranathan, Passang Tenzin, Jian Ming Li, Rohan Dhamsania, Michael Ware, Mohammad Zaidi, Shuhua Wang, Jingru Zhu, Maria Cardenas, Yuan Liu, Gaurav Joshi, Sanjeev Gumber, Brian Robinson, Anish Sen-Majumdar, Shanmuganathan Chandrakasan, Haydn Kissick, Alan Frey, Susan Thomas, Bassel El-Rayes, Gregory Lesinski, Edmund K. Waller. Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-072.

Receptor-specific peptides labeled with positron emitters play an important role in the clinical imaging of several malignancies by positron emission tomography (PET). Radiolabeled heterobivalent bispecific peptidic ligands (HBPLs) can target more than one receptor type and by this - besides exhibiting other advantages - increase tumor imaging sensitivity. In the present study, we show the initial in vivo evaluation of the most potent heterobivalent gastrin-releasing peptide receptor (GRPR)- and vasoactive intestinal peptide receptor subtype 1 (VPAC1R)-bispecific radiotracer and determined its tumor visualization potential via PET/CT imaging. For this purpose, the most potent described HBPL was synthesized together with its partly scrambled heterobivalent monospecific homologs and its monovalent counterparts. The agents were efficiently labeled with 68Ga3+ and evaluated in an initial PET/CT tumor imaging study in a human prostate carcinoma (PCa) xenograft rat tumor model established for this purpose. None of the three 68Ga-HBPLs enabled a clear tumor visualization and a considerably higher involvement in receptor-mediated uptake was found for the GRPR-binding part of the molecule than for the VPAC1R-binding one. Of the monovalent radiotracers, only [68Ga]Ga-NODA-GA-PESIN could efficiently delineate the tumor, confirming the results. Thus, this work sets the direction for future developments in the field of GRPR- and VPAC1R-bispecific radioligands, which should be based on other VPAC1R-specific peptides than PACAP-27.

Aim The mechanisms underlying the inhibitory effects of oxytocin (OT) on colon tone are not totally understood. We explore the mechanisms of OT on spontaneous contractility in rat distal colon, and identify the mediators involved in this action. Methods In rat distal colon strips, mechanical activity was analyzed and the production of nitric oxide (NO) in tissue loaded with the fluorochrome DAF-FM was visualized by confocal microscopy. OT receptor (OTR) expression was determined by western blotting and immunofluorescence. Results In rat distal colon, OT produced a concentration-dependent reduction of the spontaneous contraction, which was abolished by the OTR antagonist atosiban, the neural blocker tetrodotoxin and the inhibitor of neuronal nitric oxide synthase (nNOS) NPLA. The inhibitory effects of OT were not affected by propranolol, atropine, the nicotinic cholinoceptor blocker hexamethonium, the vasoactive intestinal peptide receptor antagonist VIPHyb, the P2 purinoceptor antagonist PPADS, the adenosine A1 receptors antagonist DPCPX and the prostacyclin receptor antagonist Ro1138452. The soluble guanylyl cyclase (sGC) inhibitor ODQ and the small conductance Ca2+-activated K+ (CaK+) channels blocker apamin significantly reduced the relaxation induced by OT, nicotine, sodium nitroprusside and the sGC activator BAY 41-2272. The neural release of NO elicited by OT was prevented by NPLA, tetrodotoxin, and atosiban. The presence of the OTR and its co-localization with nNOS was detected by immunohistochemistry and western blotting experiments. Conclusion These results demonstrate the NO release from enteric neurons induced by activation of OTR mediates distal colon relaxation. sGC and small conductance CaK+ channels are involved in this relaxation. This article is protected by copyright. All rights reserved.

// Rongjie Yu 1, 2 , Hongyu Liu 1, 2 , Xinhe Peng 1, 2 , Yue Cui 1, 2 , Suqin Song 1, 2 , Like Wang 1, 2 , Huahua Zhang 3 , An Hong 1, 2 and Tianhong Zhou 4 1 Institute of Biomedicine, School of Life Science and Technology, Jinan University, Guangzhou, Guangdong, China 2 National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, Guangdong, China 3 Department of Medical Genetics, Guangdong Medical University, Dongguan, Guangdong, China 4 Department of Bioengineering, School of Life Science and Technology, Jinan University, Guangzhou, Guangdong, China Correspondence to: Rongjie Yu, email: rongjie_yu1123@163.com Keywords: vasoactive intestinal peptide receptor 1 (VPAC1), cysteine (Cys), palmitoylation, nuclear translocation, anti-apoptotic activity Received: March 06, 2017 Accepted: April 14, 2017 Published: April 27, 2017 ABSTRACT VPAC1 is class B G protein-coupled receptors (GPCR) shared by pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). The first cysteine (Cys37) in the N-terminal extracellular domain of mature VPAC1 is a free Cys not involved in the formation of conserved intramolecular disulfide bonds. In order to investigate the biological role of this Cys37 in VPAC1, the wild-type VPAC1 and Cys37/Ala mutant (VPAC1-C37/A) were expressed stably as fusion proteins with enhanced yellow fluorescent protein (EYFP) respectively in Chinese hamster ovary (CHO) cells. Both VPAC1-EYFP and VPAC1-C37/A-EYFP trafficked to the plasma membrane normally, and CHO cells expressing VPAC1-EYFP displayed higher anti-apoptotic activity against camptothecin (CPT) induced apoptosis than the cells expressing VPAC1-C37/A-EYFP, while VPAC1-C37/A-CHO cells showed higher proliferative activity than VPAC1-CHO cells. Confocal microscopic analysis, western blotting and fluorescence quantification assay showed VPAC1-EYFP displayed significant nuclear translocation while VPAC1-C37/A-EYFP did not transfer into nucleus under the stimulation of VIP (0.1 nM). Acyl-biotin exchange assay and click chemistry-based palmitoylation assay confirmed for the first time the palmitoylation of Cys37, which has been predicted by bioinformatics analysis. And the palmitoylation inhibitor 2-bromopalmitate significantly inhibited the nuclear translocation of VPAC1-EYFP and its anti-apoptotic activity synchronously. These results indicated the palmitoylation of the Cys37 in the N-terminal extracellular domain of VPAC1 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity. These findings reveal for the first time the lipidation-mediating nuclear translocation of VPAC1 produces a novel anti-apoptotic signal pathway, which may help to promote new drug development strategy targeting VPAC1.

Innervation of the intestinal mucosa has gained more attention with demonstrations of tuft and enteroendocrine cell innervation. However, the role(s) these fibers play in maintaining the epithelial and mucus barriers are still poorly understood. This study therefore examines the proximity of mouse ileal goblet cells to neuronal fibers, and the regulation of goblet cell production by vasoactive intestinal peptide (VIP). An organotypic intestinal slice model that maintains the cellular diversity of the intestinal wall ex vivo was used. An ex vivo copper‐free click‐reaction to label glycosaminoglycans was used to identify goblet cells. Pharmacological treatment of slices was used to assess the influence of VIP receptor antagonism on goblet cell production and neuronal fiber proximity. Goblet cells were counted and shown to have at least one peripherin immunoreactive fiber within 3 µm of the cell, 51% of the time. Treatment with a VIP receptor type I and II antagonist (VPACa) resulted in an increase in the percentage of goblet cells with peripherin fibers. Pharmacological treatments altered goblet cell counts in intestinal crypts and villi, with tetrodotoxin and VPACa substantially decreasing goblet cell counts. When cultured with 5‐Ethynyl‐2’‐deoxyuridine (EdU) as an indicator of cell proliferation, colocalization of labeled goblet cells and EdU in ileal crypts was decreased by 77% when treated with VPACa. This study demonstrates a close relationship of intestinal goblet cells to neuronal fibers. By using organotypic slices from mouse ileum, vasoactive intestinal peptide receptor regulation of gut wall goblet cell production was revealed., A close relationship between neuronal fibers and enteric goblet cells was demonstrated, with more neuronal fibers being in close proximity to goblet cells in vasoactive intestinal peptide (VIP) receptor antagonized ileum slices. Further, antagonizing VIP receptors ex vivo was shown to regulate goblet cell production in the ileal crypt.

A paradigm shift has emerged in Parkinson’s disease (PD) highlighting the prominent role of CD4+ Tregs in pathogenesis and treatment. Bench to bedside research, conducted by others and our own laboratories, advanced a neuroprotective role for Tregs making pharmacologic transformation of immediate need. Herein, a vasoactive intestinal peptide receptor-2 (VIPR2) peptide agonist, LBT-3627, was developed as a neuroprotectant for PD-associated dopaminergic neurodegeneration. Employing both 6-hydroxydopamine (6-OHDA) and α-synuclein (α-Syn) overexpression models in rats, the sequential administration of LBT-3627 increased Treg activity without altering cell numbers both in naïve animals and during progressive nigrostriatal degeneration. LBT-3627 administration was linked to reductions of inflammatory microglia, increased survival of dopaminergic neurons, and improved striatal densities. While α-Syn overexpression resulted in reduced Treg activity, LBT-3627 rescued these functional deficits. This occurred in a dose-dependent manner closely mimicking neuroprotection. Taken together, these data provide the basis for the use of VIPR2 agonists as potent therapeutic immune modulating agents to restore Treg activity, attenuate neuroinflammation, and interdict dopaminergic neurodegeneration in PD. The data underscore an important role of immunity in PD pathogenesis.

Vasoactive intestinal peptide receptor 1 (VPAC1) is a member of a secretin-like subfamily of G protein-coupled receptors. Its endogenous neuropeptide (VIP), secreted by neurons and immune cells, modulates various physiological functions such as exocrine and endocrine secretions, immune response, smooth muscles relaxation, vasodilation, and fetal development. As a drug target, VPAC1 has been selected for therapy of inflammatory diseases but drug discovery is still hampered by lack of its crystal structure. In this study we presented the homology model of this receptor constructed with the well-known web service GPCRM. The VPAC1 model is composed of extracellular and transmembrane domains that form a complex with an endogenous hormone VIP. Using the homology model of VPAC1 the mechanism of action of potential drug candidates for VPAC1 was described. Only two series of small-molecule antagonists of confirmed biological activity for VPAC1 have been described thus far. Molecular docking and a series of molecular dynamics simulations were performed to elucidate their binding to VPAC1 and resulting antagonist effect. The presented work provides the basis for the possible binding mode of VPAC1 antagonists and determinants of their molecular recognition in the context of other class B GPCRs. Until the crystal structure of VPAC1 will be released, the presented homology model of VPAC1 can serve as a scaffold for drug discovery studies and is available from the author upon request., info:eu-repo/semantics/published

Xenin-25 is a neurotensin-like peptide that is secreted by enteroendocrine cells in the small intestine. Xenin-8 is reported to augment duodenal anion secretion by activating afferent neural pathways. The intrinsic neuronal circuits mediating the xenin-25-induced anion secretion were characterized using the Ussing-chambered, mucosa-submucosa preparation from the rat ileum. Serosal application of xenin-25 increased the short-circuit current in a concentration-dependent manner. The responses were abolished by the combination of Cl−-free and [Formula: see text]-free solutions. The responses were almost completely blocked by TTX (10−6M) but not by atropine (10−5M) or hexamethonium (10−4M). The selective antagonists for neurotensin receptor 1 (NTSR1), neurokinin 1 (NK1), vasoactive intestinal polypeptide (VIP) receptors 1 and 2 (VPAC1 and VPAC2, respectively), and capsaicin, but not 5-hydroxyltryptamine receptors 3 and 4 (5-HT3and 5-HT4), NTSR2, and A803467, inhibited the responses to xenin-25. The expression of VIP receptors ( Vipr) in rat ileum was examined using RT-PCR. The Vipr1 PCR products were detected in the submucosal plexus and mucosa. Immunohistochemical staining showed the colocalization of NTSR1 and NK1 with substance P (SP)- and calbindin-immunoreactive neurons in the submucosal plexus, respectively. In addition, NK1 was colocalized with noncholinergic VIP secretomotor neurons. Based on the results from the present study, xenin-25-induced Cl−/[Formula: see text] secretion is involved in NTSR1 activation on intrinsic and extrinsic afferent neurons, followed by the release of SP and subsequent activation of NK1 expressed on noncholinergic VIP secretomotor neurons. Finally, the secreted VIP may activate VPAC1 on epithelial cells to induce Cl−/[Formula: see text] secretion in the rat ileum. Activation of noncholinergic VIP secretomotor neurons by intrinsic primary afferent neurons and extrinsic afferent neurons by postprandially released xenin-25 may account for most of the neurogenic secretory response induced by xenin-25.NEW & NOTEWORTHY This study is the first to investigate the intrinsic neuronal circuit responsible for xenin-25-induced anion secretion in the rat small intestine. We have found that nutrient-stimulated xenin-25 release may activate noncholinergic vasoactive intestinal polypeptide (VIP) secretomotor neurons to promote Cl−/[Formula: see text] secretion through the activation of VIP receptor 1 on epithelial cells. Moreover, the xenin-25-induced secretory responses are mainly linked with intrinsic primary afferent neurons, which are involved in the activation of neurotensin receptor 1 and neurokinin 1 receptor.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse effects in the nervous system. The present study investigated whether stimulation of PACAP receptors (PACAPRs) induces responses in neurons and satellite cells of the superior cervical ganglia (SCG), with special reference to intracellular Ca2+ ([Ca2+]i) changes. The expression of PACAPRs in SCG was detected by reverse transcription-PCR. PACAP type 1 receptor (PAC1R), vasoactive intestinal peptide receptor type (VPAC)1R, and VPAC2R transcripts were expressed in SCG, with PAC1R showing the highest levels. Confocal microscopy analysis revealed that PACAP38 and PACAP27 induced an increase in [Ca2+]i in SCG, first in satellite cells and subsequently in neurons. Neither extracellular Ca2+ removal nor Ca2+ channel blockade affected the PACAP38-induced increase in [Ca2+]i in satellite cells; however, this was partly inhibited in neurons. U73122 or xestospongin C treatment completely and partly abrogated [Ca2+]i changes in satellite cells and in neurons, respectively, whereas VPAC1R and VPAC2R agonists increased [Ca2+]i in satellite cells only. This is the first report demonstrating the expression of PACAPRs specifically, VPAC1 and VPAC2 in SCG and providing evidence for PACAP38-induced [Ca2+]i changes in both satellite cells and neurons via Ca2+ mobilization.

Neuroprotective immunity is defined by transformation of T-cell polarity for therapeutic gain. For neurodegenerative disorders and specifically for Parkinson's disease (PD), granulocyte-macrophage colony stimulating factor or vasoactive intestinal peptide receptor 2 (VIPR2) agonists elicit robust anti-inflammatory microglial responses leading to neuronal sparing in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. While neurotherapeutic potential was demonstrated for PD, there remain inherent limitations in translating these inventions from the laboratory to patients. One obstacle in translating such novel neurotherapeutics centers on the availability of suitable noninvasive methods to track disease progression and therapeutic efficacy. To this end, we developed manganese-enhanced magnetic resonance imaging (MEMRI) assays as a way to track a linkage between glial activation and VIPR2 agonist (LBT-3627)-induced neuroprotective immunity for MPTP-induced nigrostriatal degeneration. Notably, LBT-3627-treated, MPTP-intoxicated mice show reduced MEMRI brain signal intensities. These changes paralleled reduced astrogliosis and resulted in sparing of nigral tyrosine hydroxylase neurons. Most importantly, the data suggest that MEMRI can be developed as a biomarker tool to monitor neurotherapeutic responses that are relevant to common neurodegenerative disorders used to improve disease outcomes.

Vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2) is a class B G-protein coupled receptor (GPCR) and plays important roles in the physiology of central nervous system (CNS) by interaction with natural ligands; vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Because it has been reported that high-expression and/or overactivation of VIPR2 link to schizophrenic symptoms, VIPR2 antagonists could be good drug candidates for schizophrenia therapeutics. In this study, we discovered several artificial peptides that antagonize both human and rodent VIPR2 with selectivities against receptor subtypes VIPR1 (also known as VPAC1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). Of them, the representative 16-mer cyclic peptide VIpep-3 (Ac-CPPYLPRRLCTLLLRS-OH) exhibited strong binding affinity with KD value of 41 nM to extracellular domain of human VIPR2 in SPR analysis and showed potent antagonist activity with IC50 values of 47 nM (human), 180 nM (mouse), and 44 nM (rat) against VIP–VIPR2 signal in cell-based Ca influx assay. This is not only the first report on artificial VIPR2-selective antagonist peptides but also good example of the effective approach to discover novel antagonist against class B GPCR. Our peptides will contribute to study and development of the novel CNS drugs targeting to VIPR2.

Multi-membrane spanning proteins, such as G protein-coupled receptors (GPCRs) and ion channels, are extremely difficult to purify as native proteins. Consequently, the generation of antibodies that recognize the native conformation can be challenging. By combining genetic immunization, phage display, and biopanning, we identified a panel of monovalent antibodies (nanobodies) targeting the vasoactive intestinal peptide receptor 1 (VPAC1) receptor. The nine unique nanobodies that were classified into four different families based on their CDR3 amino acid sequence and length, were highly specific for the human receptor and bind VPAC1 with moderate affinity. They all recognize a similar epitope localized in the extracellular N-terminal domain of the receptor and distinct from the orthosteric binding site. In agreement with binding studies, which showed that the nanobodies did not interfere with VIP binding, all nanobodies were devoid of any functional properties. However, we observed that the binding of two nanobodies was slightly increased in the presence of VPAC1 agonists [vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27)], but decreased in the presence of VPAC1 antagonist. As no evidence of allosteric activity was seen in VIP binding studies nor in functional assays, it is, therefore, possible that the two nanobodies may behave as very weak allosteric modulators of VPAC1, detectable only in some sensitive settings, but not in others. We demonstrated that the fluorescently labeled nanobodies detect VPAC1 on the surface of human leukocytes as efficiently as a reference mouse monoclonal antibody. We also developed a protocol allowing efficient detection of VPAC1 by immunohistochemistry in paraffin-embedded human gastrointestinal tissue sections. Thus, these nanobodies constitute new original tools to further investigate the role of VPAC1 in physiological and pathological conditions., SCOPUS: ar.j, info:eu-repo/semantics/published

Background In migraineurs pituitary adenylate cyclase activating peptide1–38 (PACAP1–38) is a potent migraine provoking substance and the accompanying long lasting flushing suggests degranulation of mast cells. Infusion of the closely related vasoactive intestinal peptide (VIP) either induces headache or flushing. This implicates the pituitary adenylate cyclase activating peptide type I receptor (PAC1) to be involved in the pathophysiology of PACAP1–38 provoked headaches. Here we review studies characterizing the effects of mainly PACAP but also of VIP on cerebral and meningeal arteries and mast cells. Discussion PACAP1–38, PACAP1–27 and VIP dilate cerebral and meningeal arteries from several species including man. In rat cerebral and meningeal arteries the dilation seems to be mediated preferably via vasoactive intestinal peptide receptor type 1 (VPAC1) receptors while, in human, middle meningeal artery dilation induced via vasoactive intestinal peptide receptor type 2 (VPAC2) receptors cannot be ruled out. PACAP1–38 is a strong degranulator of peritoneal and dural mast cells while PACAP1–27 and VIP only have weak effects. More detailed characterization studies suggest that mast cell degranulation is not mediated via the known receptors for PACAP1–38 but rather via a still unknown receptor coupled to phospholipase C. Conclusion It is suggested that PACAP1–38 might induce migraine via degranulation of dural mast cells via a yet unknown receptor.

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease that selectively attacks human joints. The common non-targeted treatment approaches lead to obvious side effect and systemtic complication for RA patients. Therefore, targeted drug delivery for treatment of RA has gained much attetntion in the past few years. In this paper, we reviewed the potential targets (folate receptor, angiogenesis, matrix metalloproteases, selectins, vasoactive intestinal peptide receptor andFc-γ receptor) that could be utilised to facilitate the specific delivery of drugs to the inflammed synovium and also presented different drug delivery systems for targeting RA, including the liposomes, various types of nanoparticles, polymeric micelles and the macromolecular prodrugs. The strategies combining nanotechnologies and ligand mediated active targeting for RA would be emphatically illustrated, which was expected to be helpful for identifying technologies and drug delivery methods for targeted treatment of RA.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by salivary and lacrimal gland dysfunction. Clinical observations and results from animal models of SS support the role of aberrant epithelial cell apoptosis and immune homeostasis loss in the glands as triggering factors for the autoimmune response. Vasoactive intestinal peptide (VIP) promotes potent anti-inflammatory effects in several inflammatory and autoimmune disease models, including the non-obese diabetic (NOD) mouse model of SS. With the knowledge that VIP modulates monocyte function through vasoactive intestinal peptide receptors (VPAC) and that immune homeostasis maintenance depends strongly upon a rapid and immunosuppressant apoptotic cell clearance by monocytes/macrophages, in this study we explored VPAC expression on monocytes from primary SS (pSS) patients and the ability of VIP to modulate apoptotic cell phagocytic function and cytokine profile. Monocytes isolated from individual pSS patients showed an increased expression of VPAC2 subtype of VIP receptors, absent in monocytes from control subjects, with no changes in VPAC1 expression. VPAC2 receptor expression could be induced further with lipopolysaccharide (LPS) in pSS monocytes and VIP inhibited the effect. Moreover, monocytes from pSS patients showed an impaired phagocytosis of apoptotic epithelial cells, as evidenced by reduced engulfment ability and the failure to promote an immunosuppressant cytokine profile. However, VIP neither modulated monocyte/macrophage phagocytic function nor did it reverse their inflammatory profile. We conclude that monocytes from pSS patients express high levels of VPAC2 and display a deficient clearance of apoptotic cells that is not modulated by VIP. Fil: Hauk, Vanesa Cintia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Fraccaroli, Laura Virginia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Grasso, Esteban Nicolas. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Eimon, Alicia. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; Argentina Fil: Ramhorst, Rosanna Elizabeth. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hubscher, Osvaldo. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; Argentina Fil: Perez Leiros, Claudia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Agonists of vasoactive intestinal peptide receptor 2 (VPAC2) stimulate glucose-dependent insulin secretion, making them attractive candidates for the treatment of hyperglycaemia and type-II diabetes. Vasoactive intestinal peptide (VIP) is an endogenous peptide hormone that potently agonizes VPAC2. However, VIP has a short serum half-life and poor pharmacokinetics in vivo and is susceptible to proteolytic degradation, making its development as a therapeutic agent challenging. Here, we investigated two peptide cyclization strategies, lactamisation and olefin-metathesis stapling, and their effects on VPAC2 agonism, peptide secondary structure, protease stability, and cell membrane permeability. VIP analogues showing significantly enhanced VPAC2 agonist potency, glucose-dependent insulin secretion activity, and increased helical content were discovered; however, neither cyclization strategy appeared to effect proteolytic stability or cell permeability of the resulting peptides.