Mucosal Receptors Research Articles

Repression of Peroxisome Proliferator-Activated Receptor γ by Mucosal Ribotoxic Insult-Activated CCAAT/Enhancer-Binding Protein Homologous Protein

CCAAT/enhancer-binding protein homologous protein (CHOP) is a crucial stress-responsive factor in various mucosal injuries, including cellular translational stress conditions. In this study, chemical ribosome-inactivating stresses were assessed for their effects on stress-inducible CHOP expression and its association with epithelial inflammatory cytokine production. Several representative ribotoxic agents (deoxynivalenol, anisomycin, and 15-acetyldeoxynivalenol) enhanced CHOP expression and its nuclear translocation in human intestinal epithelial cells. Moreover, CHOP was a strong positive regulator of IL-8 production, but CHOP-mediated IL-8 production was inversely associated with expression of the mucosal regulatory factor peroxisome proliferator-activated receptor γ (PPARγ). Based on our recent report that PPARγ is a negative regulator of mRNA stability of IL-8, PPARγ was linked to a notable mRNA stabilizing protein, HuR, since ribotoxin-induced IL-8 mRNA is stabilized by HuR protein. Expression of exogenous PPARγ suppressed ribotoxin-triggered cytoplasmic translocation of HuR. In contrast, PPARγ-regulating CHOP was a positive modulator of HuR protein export from nuclei. Taken together, the results indicate that ribotoxin-induced CHOP protein is positively associated with production of proinflammatory cytokine IL-8, but it downregulates PPARγ action, subsequently allowing the cytosolic translocation of HuR protein and stabilization of IL-8 mRNA in gut epithelial cells. CHOP and PPARγ may represent critical mechanistic links between ribotoxic stress and proinflammatory cytokine production, and they may have a broader functional significance with regard to gastrointestinal stresses by toxic mucosal insults.

Spontaneous secretion of interleukin-17 and -22 by human cervical cells in Chlamydia trachomatis infection

To investigate whether IL-17A (IL-17) and IL-22 are produced in response to Chlamydia trachomatis infection, the cervical washes of 27 women with C. trachomatis infection and 17 C. trachomatis negative controls were collected. The levels of cytokines were determined in the cervical wash and in the supernatant of cervical and systemic cell cultures upon C. trachomatis antigen stimulation. C. trachomatis infection appeared to activate local IL-17 and IL-22 production more efficiently than IFN-γ production. In the cervical wash of infected women, median concentrations of IL-17 and -22 were 5- and 3-fold higher, respectively, than in negative controls. The spontaneous intracellular expression of these cytokines was analysed by flow cytometry in blood and cervical cells and 26% of cervical mononuclear cells from infected women were shown to produce IL-22 and 12% to coproduce IL-17 and IL-22. In addition, it was demonstrated that 20–25% of IL-22 producing and IL-17-IL-22 coproducing cervical CD4+ T cells expressed the mucosal homing receptor CCR6. These results suggest that CCR6 is involved in the migration of these cells to the cervix and that IL-17 and IL-22 might play a role in the immune response at the site of C. trachomatis infection.

Toll-like receptor 2 induces mucosal homing receptor expression and IgA production by human B cells

There is a need for developing vaccines that elicit mucosal immunity. Although oral or nasal vaccination methods would be ideal, current strategies have yielded mixed success. Toll-like receptor 2 (TLR2) ligands are effective adjuvants and are currently used in the Haemophilus influenzae type B vaccine. Induction of humoral immunity in the mucosa is critical for effective vaccination; thus, we sought to determine the effects of TLR2 ligands on human mucosal B cell differentiation. We demonstrate that TLR2 ligands induce CCR9 and CCR10 expression by circulating B cells and increased chemotaxis to cognate chemokines CCL25 and CCL28 suggesting that TLR2 induces B cell homing to the gastrointestinal tract. TLR2 stimulation of B cells also induced J chain and IgA production demonstrating the induction of mucosal-like antibody secreting cells. These observations suggest that vaccines containing TLR2-ligands as adjuvants could induce mucosal B cell immunity even when delivered in a non-mucosal manner.

Polymeric IgA-secreting and mucosal homing pre-plasma cells in normal human peripheral blood

Clear identification of recently activated mucosal B cells in human blood would greatly facilitate study of mucosal vaccines, immune response to infection and the ongoing mucosal IgA response. We examined blood lymphocytes from normal, healthy individuals to identify IgA-secreting pre-plasma cells' (PPC) functional and phenotypic relevance to mucosal antibody production, in the absence of infection, disease or recent vaccination. PPC are the most recently activated B lymphocytes in blood and are considered in transit between lymphoid tissue and effector tissues, where they terminally differentiate into plasma cells. We observed that all IgA-secreting PPC expressed surface IgA (sIgA) and intracellular IgA (icIgA) and secreted primarily polymeric IgA (pIgA), as determined by flow cytometry, ELISPOT and size exclusion chromatography. A large sub-population of PPC in blood expresses the mucosal chemokine receptor CCR10 and contains the largest fraction of sIgA and icIgA PPC that secrete pIgA. The majority of CCR10(+) PPC expresses high levels of Ki67, indicative of recently activated blasts. In contrast, most CCR10(-) PPC secrete IgG, but a small population secretes pIgA and stains for icIgA. The mucosal integrin alpha(4)beta(7) was detected on a subset of PPC, but this subset did not account for all CCR10(+) PPC or all PPC with sIgA expression. Our data clearly demonstrate that PPC defined by surface expression of CD19, CD27(hi), IgA and CCR10 secrete only pIgA and are the dominant mucosal PPC subset in human blood. These mucosal PPC can now be investigated routinely as indicators of recent human mucosal IgA responses.

Translational research at acute HIV Infection

The initial interaction between HIV and its host is crucial in determining disease progression and uniquely provides the ability to explore the determinants of HIV acquisition. Upon this first encounter, the virus will induce an innate and adaptive immune response that will determine the severity of symptoms and the level of T cell activation. To rationally design the optimal treatment strategy, whether it immunotherapies or ART, this brief time period is of the utmost importance. The challenge here lies in the conflict between the drive for scientific knowledge and supporting the patient though this emotional stage. However, only a delicate balance between these necessities will produce successful translational research. In addition, integrating virological, immunological and behavioural factors has become the sole valid approach of properly addressing both HIV pathogenesis and patient needs. After two decades of HIV research, mucosa immunity has emerged as a cornerstone of future research. Several studies in both SIV and HIV infection have emphasized the importance of depletion of mucosal CD4 T cells and high levels of T cell activation immediately following infection. The contribution of mucosal immunity has shed light on the importance α4β7 mucosal homing receptors, massive loss of memory CD4 T cells, microbial translocation, and T cell activation. Recently, dendritic cells, monocytes, NK cells as well as the interleukin-7 and interleukin-21 signalling pathways have been shown to contribute to the early immune dysfunction observed after infection. The timing of ART initiation plays a major role on the quality of immune recovery. In addition, the establishment of viral reservoir occurs within days after infection and its size is largely determined by the CD4 memory/effector ratio and CD4 cell nadir. New frontiers in acute HIV infection research need to include blood sampling by leukapheresis to collect large quantities of immune cells, mucosa biopsies, and neurocognitive evaluation to pave the route toward HIV eradication and rational design of vaccines and immunotherapies.

M1875b Do Mesenchymal Stem Cells Affect EMG Potentials After Anal Sphincter Injury in an Animal Model?

Methods: 30 SD rats underwent placement of a tunneled catheter into the proximal colon. 22 underwent parasympathetic denervation (bilateral vagotomy and transection of pelvic nerves) while the others received a sham operation. To measure colonic transit, 51Cr was injected into the proximal colon at post op days 1, 3, and 7 and the geometric center (GC) of distribution of Cr was calculated. 10 SD rats were divided into 2 groups, sham operation and parasympathetic denervation. At post op day 3, mucosa/submucosal layers of the proximal, middle and distal colon were harvested. 5-HT4 receptor expression was evaluated by Western blot and real time PCR. Results: At post op day 1, colonic parasympathetic denervation caused a significant decrease in GC (4.4, p<0.01, n=6), compared to sham operation (6.3, n=8). Between post op days 3 and 7, there was a significant trend of increasing GC [5.2 at day 3, (n=8) and 6.0, p<0.05 (n=8)] in the denervation group. 5-HT4 receptor protein expression was significantly increased in the distal colon in the denervation group (n=5, P<0.01), compared to sham operation group (n=5). A trend toward significance was seen in 5-HT4 receptor mRNA expression in the denervation group (n=5, P=0.06), compared to sham operation group (n=5). Conclusions: Mucosal colonic 5-HT4 receptors are upregulated following parasympathetic denervation of the distal colon in rats. Upregulation of 5HT4 receptors of intrinsic origin may be a contributing factor in the mechanism involved in restoring normal colorectal motility after parasympathetic denervation.

M1875a Mucosal 5-HT4 Receptor Expression is Altered Following Parasympathetic Denervation of the Rat Colon

M1875a Mucosal 5-HT4 Receptor Expression is Altered Following Parasympathetic Denervation of the Rat Colon

Adenovirus 5 serotype vector-specific immunity and HIV-1 infection: a tale of T cells and antibodies

Adenovirus 5 serotype vector-specific immunity and HIV-1 infection: a tale of T cells and antibodies

Effects of short‐term food deprivation on orexin‐A‐induced intestinal bicarbonate secretion in comparison with related secretagogues

Studies of gastrointestinal physiology in humans and intact animals are usually conducted after overnight fast. We compared the effects of orexin-A, vasoactive intestinal polypeptide (VIP), melatonin, serotonin, uroguanylin, ghrelin and prostaglandin E(2) (PGE(2)) on duodenal bicarbonate secretion in fed and overnight fasted animals. This review is a summary of our findings. Secretagogues were administered by intra-arterial infusion or luminally (PGE(2)). Enterocyte intracellular calcium ([Ca(2+)](i)) signalling was studied by fluorescence imaging. Total RNA was extracted, reverse transcripted to cDNA and expression of orexin receptors measured by quantitative real-time PCR. Orexin-A stimulates the duodenal secretion in continuously fed animals but not in food-deprived animals. Similarly, short-term fasting causes a 100-fold decrease in the amount of the muscarinic agonist bethanechol required for stimulation of secretion. In contrast, fasting does not affect secretory responses to intra-arterial VIP, melatonin, serotonin, uroguanylin and ghrelin, or that to luminal PGE(2). Orexin-A induces [Ca(2+)](i) signalling in enterocytes from fed rats but no significant [Ca(2+)](i) responses occur in enterocytes from fasted animals. In addition, overnight fasting decreases the expression of mucosal orexin receptors. Short-term food deprivation thus decreases duodenal expression of orexin receptors and abolishes the secretory response to orexin-A as well as orexin-A-induced [Ca(2+)](i) signalling. Fasting, furthermore, decreases mucosal sensitivity to bethanechol. The absence of declines in secretory responses to other secretagogues tested strongly suggests that short-term fasting does not affect the secretory capacity of the duodenal mucosa in general. Studies of intestinal secretion require particular evaluation with respect to feeding status.

The mouth: a gateway or a trap for HIV?

Mucosal sites represent the primary routes of HIV transmission, yet the oral mucosa appears uniquely resistant to HIV-1 infection. Since the oral cavity is not conducive to either infection or transmission of HIV, characterizing the responsible resistance factors, particularly components of innate immunity, and their mechanisms of action may identify new opportunities for interference with viral acquisition at other mucosal sites. Although many of the innate immune factors described to date exhibit both antibacterial and antiviral activities, molecules that inhibit HIV-1 are largely HIV-1 specific. Nonetheless, if these oral innate factors can be identified, characterized, and harnessed, they may be useful in protection and/or intervention in acute HIV-1 infection. Endogenous molecules acting independently or synergistically, given their lack of substantive side effects, may represent an alternative novel approach for reducing HIV risk and suppressing infection. While HIV infection via breast milk does involve oral trafficking and is an important area of study, it will only be briefly discussed here. The major emphasis of this article is on the oral cavity and salivary substances that can influence HIV infection Epidemiology of oral transmission of HIV More than twenty-five years into the global HIV/AIDS epidemic, it is well established that in addition to blood, the reproductive and/or rectal mucosae represent the primary portals of HIV entry in adults. There is less definitive evidence regarding whether other mucosal sites serve as viral conduits and in particular, uncertainty remains as to the possibility of transmission of HIV-1 via the oral entrance to the gastrointestinal (GI) tract. This is not a trivial concern, since despite the successes of combination therapy for HIV/AIDS, infection rates remain high, compounded by persistence of latent viral reservoirs, antiretroviral drug resistance, and the lack of an effective vaccine or potential cure. Although it is often accepted that oral transmission is of little or no consequence, we have failed to capitalize on that information to identify the mechanism(s) of protection at the oral mucosal surface. These mechanisms could provide clues for novel strategies of prevention/intervention at other more vulnerable viral entry sites. Early in the epidemic, evidence of the presence of HIV-1 in the oral cavity suggested the possibility of transmissibility via this route [1, 2]. Whereas in some early studies [3–7], oral transmission could not be established, a few studies implicated this route as a potential, even likely mode of transmission (e.g., [8]). In larger cohorts of individuals and in well-designed studies to monitor oral transmission, the rarity of HIV-1 infection by this route was substantiated [9]. Nonetheless, potential shortcomings in these studies, including accurately documenting the definitive route of infection, underscore our lack of certainty regarding the relative risk of oral transmission. (Reviews: [10, 11]). The low frequency of documented oral transmission of HIV-1 could reflect low titers of infectious virus in saliva. While high levels of HIV-1 RNA and antigens can routinely be measured in oral fluids, infectious virus was not detected in early studies attempting to isolate infectious virus from saliva [12, 13] or more recently in large cohort studies, despite plasma viremia [14, 15].

Muscarinic Receptor Antagonists, the Overactive Bladder and Efficacy against Urinary Urgency

The overactive bladder (OAB) is a debilitating condition in which patients suffer from urinary urgency, frequency and nocturia with or without urge urinary incontinence. The mainstay of pharmacotherapy for OAB is muscarinic receptor antagonists, which have been shown to be effective treatments for the symptoms of OAB. The mechanism underlying the efficacy of antimuscarinic agents against the symptoms of OAB is not completely understood. This review explores the role of bladder mucosal muscarinic receptors in the signaling pathways that are activated in response to bladder filling. The cholinergic system is seen to be involved in bladder afferent signaling at many levels and as such muscarinic receptor antagonists may affect bladder signaling via numerous pathways including release of mediators from the bladder urothelium and activation of suburothelial myofibroblasts and afferent nerves. Therefore the mucosal cholinergic system may represent another target for the antimuscarinic agents used to treat OAB.

The integrin α 4 β 7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1

Both activated and resting CD4(+) T cells in mucosal tissues play important roles in the earliest phases of infection after sexual transmission of HIV-1, a process that is inefficient. HIV-1 gp120 binds to integrin alpha(4)beta(7) (alpha(4)beta(7)), the gut mucosal homing receptor. We find that alpha(4)beta(7)(high) CD4(+) T cells are more susceptible to productive infection than are alpha(4)beta(7)(low-neg) CD4(+) T cells in part because this cellular subset is enriched with metabolically active CD4(+) T cells. alpha(4)beta(7)(high) CD4(+) T cells are CCR5(high) and CXCR4(low); on these cells, alpha(4)beta(7) appears in a complex with CD4. The specific affinity of gp120 for alpha(4)beta(7) provides a mechanism for HIV-1 to target activated cells that are critical for efficient virus propagation and dissemination following sexual transmission.

Attenuation of Non‐neuronal Adenosin Triphosphate Release from Human Bladder Mucosa by Antimuscarinic Agents

Objectives: To clarify the contribution of mucosal muscarinic receptors to bladder function, we investigated the effects of various antimuscarinic drugs on stretch‐induced non‐neuronal adenosin triphosphate (ATP) release in human bladder.Methods: Human bladders were obtained from 17 patients. Bladder strips with and without mucosa were suspended in organ baths. A microdialysis probe was inserted into the strip, Ringer's solution was perfused into the probe, and dialysate was collected under tetrodotoxin pretreatment. The amount of ATP released in dialysate was measured by luciferin‐luciferase assay. The effects of various antimuscarinic drugs on non‐neuronal ATP release were evaluated.Results: Non‐neuronal ATP release from bladder strips without mucosa was approximately 10% of that from strips with mucosa. Non‐neuronal ATP release was significantly inhibited by pretreatment with nifedipine or in Ca2+‐free medium. Both methoctramine (M2 receptor selective antagonist) and 4‐diphenyl‐acetoxy‐N‐methylpiperidine methiodide (4‐DAMP [M3 receptor selective antagonist]) significantly inhibited release. However, M1 receptor selective antagonist (pirenzepine) did not have a significant effect on release. Oxybutynin, propiverine, tolterodine and solifenacin caused concentration‐dependent inhibition in non‐neuronal ATP release. The rank order of the maximum inhibition rate was propiverine ≥ solifenacin ≥ tolterodine ≥ oxybutynin. Solifenacin showed an inhibitory effect at a lower concentration compared to other drugs.Conclusion: The data suggest that human bladder mucosa is a main source of stretch‐induced non‐neuronal ATP release, and that stimulation of M2 and M3 receptor subtypes of mucosa partly contributes to non‐neuronal ATP release. Various antimuscarinic drugs used for the treatment of overactive bladder may have different inhibitory effects on non‐neuronal ATP release.

P07-01. The gut mucosal homing receptor integrin α4β7 forms a complex with CD4 and defines a T cell subset that is highly susceptible to infection by HIV-1

P07-01. The gut mucosal homing receptor integrin α4β7 forms a complex with CD4 and defines a T cell subset that is highly susceptible to infection by HIV-1

Mechanosensitive Primary Bladder Afferent Activity in Rats With and Without Spinal Cord Transection

Spinal cord injury induces functional and morphological changes in bladder afferent pathways. However, direct evidence for changes in the excitability of afferent nerve activity primarily originating from the bladder has not been clearly demonstrated. Thus, we determined the characteristics of peripheral mechanosensitive bladder afferents in the pelvic nerve and possible afferent changes in Adelta and C fibers after spinal cord injury. Adult female rats were divided into 2 groups, including spinal cord injured and neurologically intact animals. In the spinal cord injury group the spinal cord was transected at Th9 at 4 weeks before functional experiments. For single unit afferent activity monitoring fine filaments were dissected from the L6 dorsal root and bladder afferent fibers were identified. Single unit afferent activity was studied during constant filling with saline. Two afferent patterns were linked to small phasic increases in intravesical pressure during bladder filling, including accelerated and nonaccelerated types. The incidence of the accelerated type was significantly higher in the spinal cord injury group than in the neurologically intact group regarding Adelta and C fibers. However, we found no relationship between conduction velocity and the functional properties of bladder mechanosensitive afferent fibers in neurologically intact or spinal cord injured rats. Results indicate that mechanosensitive bladder afferent activity has several patterns and is facilitated after spinal cord injury, especially in concert with small bladder contractions (micromotions). The functional properties of these individual afferent fibers are not related in an obvious manner to their conduction velocity and, thus, probably the afferent fiber type.

Luminal chemosensing and upper gastrointestinal mucosal defenses

The upper gastrointestinal mucosa is exposed to endogenous and exogenous substances, including gastric acid, carbon dioxide, and foodstuffs. Physiologic processes such as secretion, digestion, absorption, and motility occur in the gastrointestinal tract in response to ingested substances, which implies the presence of mucosal sensors. We hypothesize that mucosal acid sensors and tastelike receptors are important components of the mucosal chemosensing system. We have shown that luminal acid/carbon dioxide is sensed via ecto- and cytosolic carbonic anhydrases and ion transporters in the epithelial cells and via acid sensors on the afferent nerves in the duodenum and esophagus. Furthermore, a luminal l-glutamate signal is mediated via mucosal l-glutamate receptors with activation of afferent nerves and cyclooxygenase in the duodenum, which suggests the presence of luminal l-glutamate sensing. These luminal chemosensors help to activate mucosal defense mechanisms to maintain the mucosal integrity and physiologic responses of the upper gastrointestinal tract. Because neural pathways are components of the luminal chemosensory system, investigation of these pathways may help to identify novel molecular targets in the treatment and prevention of mucosal injury and visceral sensation.

Avian Chemoreception

The first detailed physiological evidence for olfactory and trigeminal chemoreception in an avian species is provided by a series of investigations in the chicken (Gallus domesticus). Initial work indicated that the activity of avian olfactory bulb neurones closely resembles that of other vertebrates, exhibiting variable spontaneous temporal firing patterns with mean firing rates between those reported for mammals and reptiles. Application of odors directly to the olfactory epithelium showed that like mammals, avian olfactory bulb neurones respond in the form of inhibition and excitation with accompanying changes in temporal firing pattern. When exposed to a range of concentrations of a single odor, all responsive neurones exhibited an ability to discriminate small step-changes in concentration producing clear stimulus response relationships. Avian trigeminal chemoreception was also investigated by examining the responses of single mucosal receptors in the nasal cavity and palate. Slowly and rapidly adapting nasal mechanoreceptors were identified, some of which exhibited chemical sensitivity when exposed to ammonia gas, acetic acid vapor or carbon dioxide. These results demonstrate that polymodal nociceptors are present in avian nasal mucosa and represent the first attempt in any species to quantify the responses of single trigeminal receptors to a range of concentrations of noxious airborne chemicals. Collectively, the findings demonstrate how an electrophysiological approach can improve our understanding of the underlying sensory physiology relating to avian perception of the chemical environment.

Nitric Oxide as an Endogenous Peripheral Modulator of Visceral Sensory Neuronal Function

Nitric oxide (NO) plays important roles in CNS and smooth muscle function. Here we reveal an additional function in peripheral sensory transmission. We hypothesized that endogenous NO modulates the function of gastrointestinal vagal afferent endings. The nonselective NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride increased responses to tactile mechanical stimuli of mucosal afferent endings in two species, in some cases severalfold. This was mimicked by a neuronal NOS inhibitor but not an endothelial NOS inhibitor. NOS inhibitors did not affect the responsiveness of smooth muscle afferent endings, suggesting that the endogenous source of NO is exclusively accessible to mucosal receptors. The role of the NO-soluble guanylyl cyclase (sGC)-cGMP pathway was confirmed using the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one and the cGMP phosphodiesterase 5' inhibitor sildenafil. The first enhanced and the second inhibited mechanosensory function. Exogenous NO, from the donor S-nitroso-N-acetylpenicillamine, significantly reduced mechanosensitivity of both types of ending. Up to one-third of stomach-projecting afferent neurons in the nodose ganglia expressed neuronal NOS (nNOS). However, anterograde-traced vagal endings were nNOS negative, indicating NOS is not transported peripherally and there are alternative sources of NO for afferent modulation. A subpopulation of enteroendocrine cells in the gut mucosa were nNOS positive, which were found anatomically in close apposition with mucosal vagal afferent endings. These results indicate an inhibitory neuromodulatory role of epithelial NO, which targets a select population of vagal afferents. This interaction is likely to play a role in generation of symptoms and behaviors from the upper gastrointestinal system.

S1249 Substance P and Its Mucosal Receptors - Possible Mediators of Inflammation and Noxious Sensation in Irritable Bowel Syndrome

S1249 Substance P and Its Mucosal Receptors - Possible Mediators of Inflammation and Noxious Sensation in Irritable Bowel Syndrome

Index of Suspicion

Index of Suspicion