Mucosal modulation of contractility in bladder strips from normal and overactive rat models and the effect of botulinum toxin A on overactive bladder strips

AIMS: To investigate the local, regulatory role of the mucosa on bladder strip contractility from normal and overactive bladders and to examine the effect of botulinum toxin A (BoNT-A).

METHODS: Bladder strips from spontaneously hyperactive rat (SHR) or normal rats (Sprague Dawley, SD) were dissected for myography as intact or mucosa-free preparations. Spontaneous, neurogenic and agonist-evoked contractions were investigated. SHR strips were incubated in BoNT-A (3 h) to assess effects on contractility.

RESULTS: Spontaneous contraction amplitude, force-integral or frequency were not significantly different in SHR mucosa-free strips compared with intacts. In contrast, spontaneous contraction amplitude and force-integral were smaller in SD mucosa-free strips than in intacts; frequency was not affected by the mucosa. Frequency of spontaneous contractions in SHR strips was significantly greater than in SD strips. Neurogenic contractions in mucosa-free SHR and SD strips at higher frequencies were smaller than in intact strips. The mucosa did not affect carbachol-evoked contractions in intact versus mucosa-free strips from SHR or SD bladders. BoNT-A reduced spontaneous contractions in SHR intact strips; this trend was also observed in mucosa-free strips but was not significant. Neurogenic and carbachol-evoked contractions were reduced by BoNT-A in mucosa-free but not intact strips. Depolarisation-induced contractions were smaller in BoNT-A-treated mucosa-free strips.

CONCLUSIONS: The mucosal layer positively modulates spontaneous contractions in strips from normal SD but not overactive SHR bladder strips. The novel finding of BoNT-A reduction of contractions in SHR mucosa-free strips indicates actions on the detrusor, independent of its classical action on neuronal SNARE complexes.

Dynamics and Function of Langerhans Cells In Vivo: Dermal Dendritic Cells Colonize Lymph Node Areas Distinct from Slower Migrating Langerhans Cells

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.

Langerhans cells--revisiting the paradigm using genetically engineered mice.

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity and tolerance is poorly defined. 'Knockin' mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene were recently developed in order to discriminate epidermal LCs from other DC subsets and at the same time to track their dynamics under steady-state or inflammatory conditions in vivo. Additional knockin mice expressing a diphtheria toxin receptor fused to EGFP were used to conditionally ablate LCs and assess their role in triggering hapten-specific T cell effectors through skin immunization. We review the insights that have been provided by these various knockin mice and discuss gaps in our knowledge of LCs that need to be filled.

Dermatoxin and phylloxin from the waxy monkey frog, Phyllomedusa sauvagei: cloning of precursor cDNAs and structural characterization from lyophilized skin secretion

Amphibian skin is a morphologically, biochemically and physiologically complex organ that performs the wide range of functions necessary for amphibian survival. Here we describe the primary structures of representatives of two novel classes of amphibian skin antimicrobials, dermatoxin and phylloxin, from the skin secretion of Phyllomedusa sauvagei, deduced from their respective precursor encoding cDNAs cloned from a lyophilized skin secretion library. A degenerate primer, designed to a highly conserved domain in the 5'-untranslated region of analogous peptide precursor cDNAs from Phyllomedusa bicolor, was employed in a 3'-RACE reaction. Peptides with molecular masses coincident with precursor-deduced mature toxin peptides were identified in LC/MS fractions of skin secretion and primary structures were confirmed by MS/MS fragmentation. This integrated experimental approach can thus rapidly expedite the primary structural characterization of amphibian skin peptides in a manner that circumvents specimen sacrifice whilst preserving robustness of scientific data.

Molecular cloning of a novel putative potassium channel-blocking neurotoxin from the venom of the North African scorpion, Androctonus amoreuxi

Scorpion venoms are a particularly rich source of neurotoxic proteins/peptides that interact in a highly specific fashion with discrete subtypes of ion channels in excitable and non-excitable cells. Here we have employed a recently developed technique to effect molecular cloning and structural characterization of a novel putative potassium channel-blocking toxin from the same sample of venom from the North African scorpion, Androctonus amoreuxi. The deduced precursor open-reading frame is composed of 59 amino acid residues that consists of a signal peptide of approximately 22 amino acid residues followed by a mature toxin of 37 amino acid residues. The mature toxin contains two functionally important residues (Lys27 and Tyr36), constituting a functional dyad motif that may be critical for potassium channel-blocking activity that can be affirmed from structural homologs as occurring in the venoms from other species of Androctonus scorpions. Parallel proteomic/transcriptomic studies can thus be performed on the same scorpion venom sample without sacrifice of the donor animal.

Isolation of scorpion (Androctonus amoreuxi) alpha-neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60–70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.

Mature DC from skin and skin-draining LN retain the ability to acquire and efficiently present targeted antigen.

Skin-draining LN contain several phenotypically distinguishable DC populations, which may be immature or mature. Mature DC are generally considered to have lost the capacity to acquire and present newly encountered Ag. Using antibody-opsonized liposomes as Ag carriers, we show that mature DC purified from skin explants are able to efficiently capture liposomes, process Ag encapsulated within them and activate Ag-specific CD4(+) T cells. Explant DC from mice with Langerhans cells (LC) expressing the primate diphtheria toxin receptor that were exposed to diphtheria toxin in vivo presented Ag as well as explant DC from wild-type mice, indicating that LC are not required and dermal DC are probably responsible for this presentation. We further show that all DC subtypes from LN that capture opsonized Ag are capable of cross-presenting it to CD8(+) T cells. Induction of additional maturation in vivo by LPS or treatment with double-stranded RNA did not alter the Ag presentation capacity of the skin or LN DC subtypes. These results suggest that mature DC present in skin-draining LN may play an important role in the induction of primary and/or secondary immune responses against Ag delivered to the LN that they take up by receptor-mediated endocytosis.

A switch in the cellular localization of macrophage migration inhibitory factor in the rat testis after ethane dimethane sulfonate treatment.

Macrophage migration inhibitory factor (MIF), one of the first cytokines to be discovered, has recently been localized to the Leydig cells in adult rat testes. In the following study, the response of MIF to Leydig cell ablation by the Leydig cell-specific toxin ethane dimethane sulfonate (EDS) was examined in adult male rats. Testicular MIF mRNA and protein in testicular interstitial fluid measured by ELISA and western blot were only marginally reduced by EDS treatment, in spite of the fact that the Leydig cells were completely destroyed within 7 days. Immunohistochemistry using an affinity-purified anti-mouse MIF antibody localized MIF exclusively to the Leydig cells in control testes. At 7 days post-EDS treatment, there were no MIF immunopositive Leydig cells in the interstitium, although distinct MIF immunostaining was observed in the seminiferous tubules, principally in Sertoli cells and residual cytoplasm, and some spermatogonia. A few peritubular and perivascular cells were also labelled at this time, which possibly represented mesenchymal Leydig cell precursors. At 14 and 21 days, Sertoli cell MIF immunoreactivity was observed in only a few tubule cross-sections, while some peritubular and perivascular mesenchymal cells and the re-populating immature Leydig cells were intensely labeled. At 28 days after EDS-treatment, the MIF immunostaining pattern was identical to that of untreated and control testes. The switch in the compartmentalization of MIF protein at 7 days after EDS-treatment was confirmed by western blot analysis of interstitial tissue and seminiferous tubules separated by mechanical dissection. These data establish that Leydig cell-depleted testes continue to produce MIF, and suggest the existence of a mechanism of compensatory cytokine production involving the Sertoli cells. This represents the first demonstration of a hitherto unsuspected pattern of cellular interaction between the Leydig cells and the seminiferous tubules which is consistent with an essential role for MIF in male testicular function.

Epidermal Langerhans Cells are Dispensable for Humoral and Cell-Mediated Immunity Elicited by Gene Gun Immunization.

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8(+) T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4(+) Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4(+) and CD8(+) T cells, IFN-gamma secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity.

Paralytic shellfish poisoning detection by surface plasmon resonance-based biosensors in shellfish matrixes.

The detection of paralytic shellfish poisoning (PSP) toxins in contaminated shellfish is essential for human health preservation. Ethical and technical reasons have prompted the search for new detection procedures as an alternative to the mouse bioassay. On the basis of the detection of molecular interactions by surface plasmon resonance (SPR) biosensors, an inhibition assay was developed using an anti-GTX2/3 antibody (GT13-A) and a saxitoxin-CM5 chip. This assay allowed for quantification of saxitoxin (STX), decarbamoyl saxitoxin (dcSTX), gonyautoxin 2,3 (GTX2/3), decarbamoyl gonyautoxin 2,3 (dcGTX2/3), gonyautoxin 5 (GTX5), and C 1,2 (C1/2) at concentrations from 2 to 50 ng/mL. The interference of five shellfish matrixes with the inhibition assay was analyzed. Mussels, clams, cockles, scallops, and oysters were extracted with five published methods. Ethanol extracts and acetic acid/heat extracts (AOAC Lawrence method) performed adequately in terms of surface regeneration and baseline interference, did not inhibit antibody binding to the chip surface significantly, and presented STX calibration curves similar to buffer controls in all matrixes tested. Hydrochloric acid/heat extracts (AOAC mouse bioassay method) presented surface regeneration problems, and although ethanol-acetic acid/dichloromethane extracts performed well, they were considered too laborious for routine sample testing. Overall the best results were obtained with the ethanol extraction method with calibration curves prepared in blank matrix extracts. STX recovery rate with the ethanol extraction method was 60.52 ± 3.72%, with variations among species. The performance of this biosensor assay in natural samples, compared to two AOAC methods for PSP toxin quantification (mouse bioassay and HPLC), suggests that this technology can be useful as a PSP screening assay. In summary, the GT13-A-STX chip inhibition assay is capable of PSP toxin detection in ethanol shellfish extracts, with sufficient sensitivity to quantify the toxin in the range of the European regulatory limit of 80 g/100 g of shellfish meat.

Identification of a novel population of Langerin+ dendritic cells.

Langerhans cells (LCs) are antigen-presenting cells that reside in the epidermis of the skin and traffic to lymph nodes (LNs). The general role of these cells in skin immune responses is not clear because distinct models of LC depletion resulted in opposite conclusions about their role in contact hypersensitivity (CHS) responses. While comparing these models, we discovered a novel population of LCs that resides in the dermis and does not represent migrating epidermal LCs, as previously thought. Unlike epidermal LCs, dermal Langerin(+) dendritic cells (DCs) were radiosensitive and displayed a distinct cell surface phenotype. Dermal Langerin(+) DCs migrate from the skin to the LNs after inflammation and in the steady state, and represent the majority of Langerin(+) DCs in skin draining LNs. Both epidermal and dermal Langerin(+) DCs were depleted by treatment with diphtheria toxin in Lang-DTREGFP knock-in mice. In contrast, transgenic hLang-DTA mice lack epidermal LCs, but have normal numbers of dermal Langerin(+) DCs. CHS responses were abrogated upon depletion of both epidermal and dermal LCs, but were unaffected in the absence of only epidermal LCs. This suggests that dermal LCs can mediate CHS and provides an explanation for previous differences observed in the two-model systems.

The dermis contains langerin+ dendritic cells that develop and function independently of epidermal Langerhans cells.

Langerhans cells (LCs) constitute a subset of dendritic cells (DCs) that express the lectin langerin and that reside in their immature state in epidermis. Paradoxically, in mice permitting diphtheria toxin (DT)-mediated ablation of LCs, epidermal LCs reappeared with kinetics that lagged behind that of their putative progeny found in lymph nodes (LNs). Using bone marrow (BM) chimeras, we showed that a major fraction of the langerin(+), skin-derived DCs found in LNs originates from a developmental pathway that is independent from that of epidermal LCs. This pathway, the existence of which was unexpected, originates in the dermis and gives rise to langerin(+) dermal DCs (DDCs) that should not be confused with epidermal LCs en route to LNs. It explains that after DT treatment, some langerin(+), skin-derived DCs reappear in LNs long before LC-derived DCs. Using CD45 expression and BrdU-labeling kinetics, both LCs and langerin(+) DDCs were found to coexist in wild-type mice. Moreover, DT-mediated ablation of epidermal LCs opened otherwise filled niches and permitted repopulation of adult noninflammatory epidermis with BM-derived LCs. Our results stress that the langerin(+) DC network is more complex than originally thought and have implications for the development of transcutaneous vaccines and the improvement of humanized mouse models.

Langerin expressing cells promote skin immune responses under defined conditions.

There are conflicting data in the literature regarding the role of epidermal Langerhans cells (LC) in promoting skin immune responses. On one hand, LC can be extremely potent APCs in vitro, and are thought to be involved in contact hypersensitivity (CHS). On the other hand, it seems counterintuitive that a cell type continually exposed to pathogens at the organism\'s barrier surfaces should readily trigger potent T cell responses. Indeed, LC depletion in one model led to enhanced contact hypersensitivity, suggesting they play a negative regulatory role. However, apparently similar LC depletion models did not show enhanced CHS, and in one case showed reduced CHS. In this study we found that acute depletion of mouse LC reduced CHS, but the timing of toxin administration was critical: toxin administration 3 days before priming did not impair CHS, whereas toxin administration 1 day before priming did. We also show that LC elimination reduced the T cell response to epicutaneous immunization with OVA protein Ag. However, this reduction was only observed when OVA was applied on the flank skin, and not on the ear. Additionally, peptide immunization was not blocked by depletion, regardless of the site. Finally we show that conditions which eliminate epidermal LC but spare other Langerin(+) DC do not impair the epicutaneous immunization response to OVA. Overall, our results reconcile previous conflicting data in the literature, and suggest that Langerin(+) cells do promote T cell responses to skin Ags, but only under defined conditions.

Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Priming of CD8+ and CD4+ T Cells in Experimental Leishmaniasis Is Initiated by Different Dendritic Cell Subtypes

The biological role of Langerin(+) dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin(+) DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of The biological role of Langerin+ dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin+ DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of Langerin+ DCs in vivo. For the first time, infection experiments with DT-treated Lang-DTR mice revealed that proliferation of L. major-specific CD8+ T cells is significantly reduced during the early phase of the immune response following depletion of Langerin+ DCs. Consequently, the total number of activated CD8+ T cells within the draining lymph node and at the site of infection is diminished. Furthermore, we show that the impaired CD8+ T cell response is due to the absence of Langerin+ dDCs and not Langerhans cells. Nevertheless, the CD4+ T cell response is not altered and the infection is cleared as effectively in DT-treated Lang-DTR mice as in control mice. This clearly demonstrates that Langerin+ DCs are, in general, dispensable for an efficient adaptive immune response against L. major parasites. Thus, we propose a novel concept that, in the experimental model of leishmaniasis, priming of CD4+ T cells is mediated by Langerin− dDCs, whereas Langerin+ dDCs are involved in early priming of CD8+ T cells.