Impairment of intestinal barrier and secretory function as well as egg excretion during intestinal schistosomiasis occur independently of mouse mast cell protease-1.

Deposition of Schistosoma mansoni eggs in the intestinal mucosa is associated with recruitment of mucosal mast cells (MMC) expressing mouse mast cell protease-1 (mMCP-1). We investigated the involvement of mMCP-1 in intestinal barrier disruption and egg excretion by examining BALB/c mice lacking mMCP-1 (Mcpt-1(-/-)). Tissue and faecal egg counts from 6 weeks until 12 weeks post-infection (w p.i.) revealed no differences between wild type (WT) and Mcpt-1(-/-)mice. Using chamber experiments on ileal tissue revealed that at 8 w p.i., the epithelial barrier and secretory capacity were severely impaired, whereas no difference was found between WT and Mcpt-1(-/-)mice in this respect. However, a fragmented distribution of the tight junction (TJ) protein occludin, but not of claudin-3 or ZO-1, was observed in WT mice at 8 w p.i., while no changes in TJ integrity were seen in Mcpt-1(-/-)mice. Therefore, we conclude that in contrast to the situation in Trichinella spiralis-infected mice, in schistosomiasis, mMCP-1 is not a key mediator in egg excretion or impairment of the intestinal barrier. The marked decrease in ileal secretory capacity during S. mansoni egg excretion suggests that the mechanisms facilitating the passage of schistosoma eggs through the gut wall are directed more particularly at the epithelial cells.

TRPV1 receptors on unmyelinated C-fibres mediate colitis-induced sensitization of pelvic afferent nerve fibres in rats.

Patients with inflammatory bowel disease often suffer from gastrointestinal motility and sensitivity disorders. The aim of the current study was to investigate the role of transient receptor potential of the vanilloid type 1 (TRPV1) receptors in the pathophysiology of colitis-induced pelvic afferent nerve sensitization. Trinitrobenzene sulphate (TNBS) colitis (7.5 mg, 30% ethanol) was induced in Wistar rats 72 h prior to the experiment. Single-fibre recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root. Fibres responding to colorectal distension (CRD) were identified in controls and rats with TNBS colitis. The effect of the TRPV1 antagonist N-(4-tertiarybutylphenyl)-4-(3-chlorophyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide (BCTC; 0.25-5 mg kg(-1)) or its vehicle (hydroxypropyl-beta-cyclodextrin) was tested on the afferent response to repetitive distensions (60 mmHg). Immunocytochemical staining of TRPV1 and NF200, a marker for A-fibre neurons, was performed in the dorsal root ganglia L6-S1. TNBS colitis significantly increased the response to colorectal distension of pelvic afferent C-fibres. BCTC did not significantly affect the C-fibre response in controls, but normalized the sensitized response in rats with colitis. TNBS colitis increased the spontaneous activity of C-fibres, an effect which was insensitive to administration of BCTC. TNBS colitis had no effect on Adelta-fibres, nor was their activity modulated by BCTC. TNBS colitis caused an immunocytochemical up-regulation of TRPV1 receptors in the cell bodies of pelvic afferent NF200 negative neurons. TRPV1 signalling mediates the colitis-induced sensitization of pelvic afferent C-fibres to CRD, while Adelta-fibres are neither sensitized by colitis nor affected by TRPV1 inhibition.

Inhibitory purinergic P2 receptor characterisation in rat distal colon.

The aim of this study was to characterise the P2 receptors involved in purinergic relaxant responses in rat distal colon circular muscle. Concentration-response curves for purinergic agonists were constructed on methacholine-precontracted circular muscle strips of rat distal colon in the absence and presence of the nerve blocker TTX and the ecto-nucleotidase inhibitor ARL67156. The effects of the P2 receptor antagonists RB2, PPADS, suramin, MRS2179 and NF279, the NO-synthase inhibitor L-NAME and the small conductance K(+) channel blocker apamin were investigated. The localisation of the different P2 receptors was examined immunocytochemically. Immunocytochemistry demonstrated the expression of P2Y(1), P2Y(6) and P2X(1) receptors on smooth muscle cells and P2Y(2), P2Y(12), P2X(2) and P2X(3) receptors in the myenteric plexus; almost a quarter of the P2Y(2)-immunopositive neurons co-expressed nNOS. The P2X-selective agonist alphabetameATP and the P2Y-selective agonist ADPbetaS were the most potent relaxants; their effects were abolished by apamin. The effect of ADPbetaS was antagonised by the P2Y(1)-selective antagonist MRS2179 pointing to interaction with the muscular P2Y(1)-receptors. The relaxant effect of alphabetameATP was partially reduced by TTX and concentration-dependently antagonised by PPADS, suramin, RB2 and the P2X(1)-selective antagonist NF279; this correlates with an interaction with neuronal P2X(3) and muscular P2X(1) receptors. UTP was the least potent agonist; its effect was markedly increased by ARL67156, nearly abolished by TTX and reduced by L-NAME. This points to interaction with the neuronal P2Y(2)-receptors inducing relaxation, at least partially, by NO release.

Effect of intestinal inflammation on the cell-specific expression of somatostatin receptor subtypes in the murine ileum.

Despite our knowledge of somatostatin (SOM) in gastrointestinal functions, little information is available on the SOM receptors (SSTRs) mediating these effects. This study focussed on the expression of SSTRs in non-inflamed and Schistosoma mansoni-infected murine ileum using immunocytochemistry, reverse transcriptase (RT)-PCR and quantitative real time RT-PCR (qPCR). In the non-inflamed ileum, SSTRs showed a widespread, cell-type specific expression pattern. For instance, SSTR2A immunoreactivity was detected in a minor population of submucous but not myenteric glial cells. In the inflamed ileum, significant changes in the expression pattern of SSTRs occurred, with SSTR1 and SSTR3 expression on mucosal mast cells (MMCs) and mucosal nerve fibres. SSTR4-immunoreactive nerve fibres were detected in granulomas and the lamina propria. qPCR experiments indicated significantly increased mRNA levels for SOM, SSTR1 and SSTR3 in inflamed ileum. This study reveals that SSTRs are expressed in specific cell types in murine ileum. Expression of SSTR1 and SSTR3 on MMCs and increased density of SOM-expressing nerve fibres in the lamina propria during inflammation, support the hypothesis that SOM is implicated in the physiological control of MMCs during intestinal inflammation. Evidence is provided that in mouse mainly SSTR1, SSTR3 and SSTR4 are involved in the somatostatinergic inflammatory effects during intestinal schistosomiasis.

The zebrafish mutant lessen: an experimental model for congenital enteric neuropathies.

BACKGROUND: Congenital enteric neuropathies of the distal intestine (CEN) are characterized by the partial or complete absence of enteric neurons. Over the last decade, zebrafish has emerged as a leading model organism in experimental research. Our aim was to demonstrate that the mutant zebrafish, lessen, expressing CEN characteristics, is an equally valuable animal model alongside mammalian models for CEN, by studying its enteric phenotype. METHODS: The effect of the lessen mutation on the development of the enteric nervous system (ENS), interstitial cells of Cajal (ICC), and intestinal motility in each intestinal region of mutant and wild-type (wt) zebrafish embryos at 3-6 dpf, was analyzed by immunofluorescent detection of neurochemical markers and motility assays. KEY RESULTS: Development of intestinal motility in the mutant was delayed and the majority of the observed contractions were disturbed. A significant disturbance in ENS development resulted in a distal intestine that was almost free of neuronal elements, in reduced neuronal density in the proximal and mid-intestine, and in a defect in the expression of neurochemical markers. Furthermore, markedly disturbed development of ICC gave rise to a less dense network of ICC. CONCLUSIONS & INFERENCES: The observed alterations in intestinal motility, intrinsic innervation and ICC network of the mutant in comparison with the wt zebrafish, are similar to those seen in the oligo- and aganglionic regions of the intestine of CEN patients. It is concluded that the zebrafish mutant lessen is an appropriate animal model to investigate CEN.

Role of oxidative stress in the pathogenesis of septic ileus in mice.

We investigated the role of oxidative stress in the pathogenesis of septic ileus. Sepsis was induced by intraperitoneal (i.p.) injection of lipopolysaccharides (LPS, 20 mg kg(-1)) in mice. The effect of two i.p. injections of superoxide dismutase [polyethylene glycol (PEG)-SOD, 4000 U kg(-1)] and catalase (PEG-CAT, 15,000 U kg(-1)) was investigated on gastric emptying, intestinal transit and total nitrite plasma concentrations. We also performed immunohistochemical experiments on gastric and ileal tissue. LPS significantly delayed gastric emptying and intestinal transit while plasma nitrite levels increased. Polyethylene glycol (PEG)-SOD reversed the endotoxin-induced delay in gastric emptying and improved the delay in intestinal transit without effect on plasma nitrite levels. PEG-CAT slightly improved the delay in gastric emptying without effect on intestinal transit. Immunohistochemistry showed the presence of nitrotyrosine (NT) and 4-hydroxy-2-nonenal (HNE) in the gastric and ileal mucosa of LPS-treated mice. Treatment with PEG-SOD or PEG-CAT of LPS mice diminished the presence of NT or HNE in both tissues. In addition, LPS induced a significant increase in inducible nitric oxide synthase (iNOS)-positive residential macrophages in the external musculature of stomach and ileum, which significantly decreased after PEG-SOD or PEG-CAT treatment. The present results support a role for oxidative and nitrosative stress in the pathogenesis of septic ileus in mice.

Effect of schistosomiasis on CX3CR1-expressing mononuclear phagocytes in the ileum and mesenteric lymph nodes of the mouse.

BACKGROUND: Intestinal dendritic cells (DCs) maintain immune homeostasis, only initiating an active immune response against invading pathogens. However, little information is available on the reaction of mononuclear phagocytes (MNP) to intestinal trematode infection, a reaction equally important in helminth-based therapies. The CD11c(+)  CX3CR1(+)  F4/80(-) DCs in the ileal lamina propria (LP) of the mouse were proven to migrate to the mesenteric lymph nodes (MLNs). We analyzed all MNP subsets present in the mouse LP and MLNs, under steady-state conditions and during acute Schistosoma mansoni-induced inflammation. Furthermore, we studied the uptake of schistosomal antigens by MNP in vivo in the LP and MLNs. METHODS: Using a combination of immunohistochemistry and multiparametric flow cytometry, we investigated distributional changes of the MNP during acute intestinal schistosomiasis. Next, S. mansoni-derived products, i.e., S. mansoni soluble worm proteins (SmSWP) and S. mansoni soluble egg antigens (SmSEA) were intraperitoneally injected into CX3CR1(+/) (GFP) C57BL/6 mice and antigen uptake was analyzed using confocal microscopy. KEY RESULTS: The CD11c(+)  CX3CR1(+)  F4/80(-) DCs significantly increased during intestinal schistosomiasis in the LP and MLNs. Only CX3CR1-expressing DC and MФ subsets, but not other LP DCs, are involved in both SmSWP and SmSEA antigen uptake and processing. CONCLUSIONS & INFERENCES: The significant upregulation of CD11c(+)  CX3CR1(+)  F4/80(-) DCs during intestinal schistosomiasis and the restriction of phagocytosis of parasitic antigens to CX3CR1-expresssing MNP indicate a crucial role for this immune cell niche in response to trematodiasis. These findings add insight into the functional specialization of LP immune cells and add to the understanding of cellular mechanisms behind helminth-based therapies.

Localization of apoptotic cells by direct immunogold detection of digoxigenin-labeled genomic DNA in semithin sections.

In this study, which correlates apoptosis with avian ovarian physiology, we modified an in situ DNA nick end-labeling method using immunogold reagents to detect apoptotic cells in semithin sections of quail ovaries embedded in glycol methacrylate resin. Special attention was paid to the prevention of background staining. The results are comparable with those of the ApopTag peroxidase kit in paraffin-embedded ovaries.

Effects of Schistosoma mansoni infection on somatostatin and somatostatin receptor 2A expression in mouse ileum.

Intestinal schistosomiasis is accompanied by motility-related dysfunctions but the underlying mechanisms are not well-known. Therefore, the presence and effects on intestinal contractility of somatostatin (SOM) and its receptor, SSTR2A, were investigated in the ileum of normal and infected mice. The distribution of SOM and SSTR2A was visualized using immunocytochemistry. Radioimmunoassay combined with oogram studies was performed to determine SOM levels and contractility measurements were determined in organ bath experiments. Schistosomiasis resulted in a significant decrease in somatostatin-positive endocrine cells, whereas the number of somatostatin-immunoreactive (IR) neuronal cell bodies did not change. From 8 weeks postinfection onwards, an increase was noted in somatostatin-IR nerve fibres in both villi and granulomas. The staining intensity for SSTR2A, expressed in somatostatin-negative myenteric cholinergic neurones, increased during infection suggesting an upregulation of this receptor. SOM levels were negatively correlated with the number of eggs during the acute phase, and were elevated during the chronic phase. Pharmacological experiments revealed that schistosomiasis diminished the inhibitory effect of SOM on neurogenic contractions. We can conclude that schistosomiasis influences the distribution and expression levels of SOM and SSTR2A in the murine ileum, which might explain the changed motility pattern.

Distribution of apoptosis-related proteins in the quail ovary during folliculogenesis: BCL-2, BAX and CPP32.

It is suggested that follicular apoptosis is driven by the status of the BCL-2: BAX rheostat, and that CPP32 is a key effector of granulosa cell death. In the present study, we have immunohistochemically localized two BCL-2 family members, BCL-2 and BAX, and one caspase, CPP32, in the quail ovary during folliculogenesis. BCL-2 was predominantly found in the granulosa cells of developing follicles. BAX was detected in some follicular cells of atretic follicles, and in the nucleus of some prelampbrush oocytes. Expression of CPP32 was detected in leukocytes and in follicular cells of atretic follicles. Immunostaining was also found in interstitial cells, in surface epithelial and vascular endothelial cells, and in some thecal cells of post-ovulatory follicles. In the granulosa cells of non-growing and small prehierarchal follicles, a weak immunostaining was observed. We can conclude that in the avian ovary, BAX and CPP32 are involved in atresia. The present results support the BCL-2: BAX rheostat hypothesis.

Localisation of epidermal growth factor receptor in the quail ovary.

The present study focuses on the distribution of EGFR in the follicular and stromal compartments of the Japanese quail (Coturnix coturnix japonica) ovary, using immunohistochemical methods and a histological ligand binding assay. EGFR was predominantly present in granulosa cells during each stage of the folliculogenesis. Furthermore, EGFR was also detected in thecal cells and in oocytes. In some cells of the vascular endothelium, of the ovarian smooth muscle, and of the surface epithelium, the presence of EGFR was detected as well. The presence of EGFR allows to formulate the hypothesis that EGFR-ligands are involved in the autocrine and/or paracrine regulation of oocyte maturation and of folliculogenesis in the quail, and possibly in all birds.

Smooth muscle cells in the peritubular tissue of the quail testis.

The aim of the present study was to reveal the presence of smooth muscle cells in the quail testis. We used an immunocytochemical and ultrastructural approach. The features of smooth muscle cells were present in the cells of the peritubular tissue and of the tunica albuginea. The peritubular cells formed a pseudo-stratified tissue. We suggest that the smooth muscle cells of the quail testis may be important in testicular contractile processes.

Immunolocalization of smooth muscle-like cells in the quail ovary.

We localized alpha-smooth muscle actin (alpha-SMA) in the quail ovary, using the peroxidase-anti-peroxidase technique. Special attention was paid to the influence of fixation on the immunoreactivity of the antigen. The immunostaining of alpha-SMA largely depended on the nature of the fixative. The antigen could most successfully be localized in ovaries fixed in Carnoy's fluid. We also localized alpha-SMA and desmin in semi-thin glycol methacrylate sections of the pre-ovulatory follicle, using the immunogold-silver staining method. The sections were pretreated with Lugol's iodine or sodium metaperiodate to enhance the immunoreactivity. alpha-SMA was demonstrated in the cells of the chordae, the tunica albuginea, and the theca externa of each follicle. These structures were inter-connected, forming an ovarian suspensory apparatus. The thecal cells of prelampbrush follicles also expressed alpha-SMA. In the wall of the pre-ovulatory follicle, desmin was found in the cells of the chordae and the tunica albuginea, and in a few cells of the theca externa. In the theca interna, desmin, and sometimes alpha-SMA, was observed in cells adjacent to the endothelium of sinusoids, which are probably pericytes. Our results support the hypothesis that in birds the ovarian follicles possess a thecal contractile system, that is presumably involved in the ovulatory process.

Presence and function of growth factors in the avian ovary.

It is now evident that a number of growth factors and cytokines are intimately involved in ovarian processes. This review focuses on these autocrine/paracrine modulators in terms of describing their presence and function in avian ovaries.

Persistence after hypothalamectomy of a tamoxifen-like effect in the ovary of quail embryos exposed cyclically in ovo to low incubation temperatures.

Three days old quail embryos were hypothalamectomized in ovo by the decapitation technique according to Callebaut (1993). Half of these embryos was exposed to day cyclically repeated prolonged low subnormal incubation temperatures (25 degrees C). The other half of these decapitated embryos was normally continuously incubated. As is the case for not decapitated embryos the ovaries of the first group presented a tamoxifen like effect: cortical rim atrophy with proportional hypertrophy of the medulla and a pronounced decrease in the number of oogonia and oocytes. By contrast the ovaries of the second group of decapitated embryos presented a normal aspect without sterilization of the ovigerous sex cords. It was concluded that: 1. the hypophysis, the hypothalamus or other prosencephalic tissues play no intermediary role in the establishment of a tamoxifen like effect in the ovary; 2. the early embryonic development of the quail ovary during the considered period takes place without the intervention of the hypothalamus.

Avian gastrulation and neurulation are not impaired by the removal of the marginal zone at the unincubated blastoderm stage.

In the present study, we removed the whole area marginalis, Rauber's sickle and the peripheral part of the area centralis from unincubated chicken blastoderms (st IV, Vakaet, 1962a). By placing a fragment of a quail Rauber's sickle (functioning as early gastrulation organizer: Callebaut and Van Nueten, 1994) at different places and oriented in different directions on the remaining central part of the area centralis, we observed, after in vitro culture, a normal embryonic development. This indicates that the area marginalis itself is not indispensable for gastrulation and neurulation. Our study also indicates that none of the three elementary tissues (Rauber's sickle, endophyll and upper layer) of the avian unincubated blastoderm present an irreversible functional polarity.

Induction of (pre) gastrulation and/or (pre) neurulation by subgerminal ooplasm and Rauber's sickle in cultured anti-sickle regions of avian unincubated blastoderms.

Rauber's sickle fragments from unincubated quail blastoderms, associated or not with chicken central subgerminal ooplasm, were placed on the deep side of the upper layer (UL) of the isolated anti-sickle region of unincubated chicken blastoderms and cultured in vitro. When only a Rauber's sickle fragment was placed, we observed always a pronounced thickening of the UL (pregastrulation) in the immediate neighbourhood. Some times a primitive streak (PS) developed. When the Rauber's sickle fragment was "sandwiched" between the UL and a central subgerminal ooplasmic mass [containing the nucleus of Pander (1817)], always a (pre)neural plate accompanied by endophyll developed, not or well associated with a primitive streak. In the latter case a complete miniature embryo developed. De novo formation of endophyll was observed. As it contained quail nuclei it was derived from Rauber's sickle cells which colonized the subgerminal ooplasm. Our experiments indicate that the uncommitted upper layer (UL) of the anti-sickle of unincubated blastoderms constitutes an excellent reactor tissue for inductions. The thickening induced in the UL of the anti-sickle region by Rauber's sickle initiates (pre)gastrulation and the thickening induced by endophyll initiates (pre)neurulation. In the beginning, pregastrulation and preneurulation seem to be independent phenomena. It is only later, when both become correctly linked at the right place and time, that a normal embryo will develop.

Map of the Anlage fields in the avian unincubated blastoderm.

By excision at different sites of rectangular fragments from unincubated chicken blastoderms and replacement by isotopic fragments from unincubated quail blastoderms, we could make the first complete map of the Anlage fields in the freshly laid avian blastoderm. All the Anlage fields (Fig. 11) are found in the upper layer (UL) of the caudal half of the area centralis (bordered by the Rauber-Koller's sickle). In the UL of the area marginalis, peripheral to Rauber-Koller's sickle, neither gastrulation nor neurulation phenomena could be observed. Similar heterotopic replacement experiments indicate that before incubation, the different parts of the UL of the area centralis are still uncommitted or reversibly committed. The Anlage fields of chordamesoblast and definitive endoderm (gut endoderm) in unincubated avian blastoderms appeared to be disposed caudally in the caudal half of the area centralis. As far as we know we are the first to demonstrate that the Anlage field of the definitive gut endoderm (which is derived from the upper layer: Hunt, 1937; Vakaet, 1962b) is localized in the most caudal upper layer part of the area centralis just centrally to the Rauber-Koller's sickle. The Anlage field of the neural plate is localized in the upper layer over the more cranial endophyll. The Anlage of the brain is shield-shaped, whilst the other Anlage fields are sickle-shaped, parallel with the Rauber-Koller's sickle. Their general hemicircular disposition and form still seem to reflect (together with the Rauber-Koller's sickle) the original ooplasmic radial symmetry (Callebaut, 1972) combined with the eccentricity of the deep layer components, which was observed during early symmetrization by gravitational orientation of the egg yolk (Callebaut, 1993a,b). The Rauber-Koller's sickle might be homologous with the vegetal dorsalizing cells or centre of Nieuwkoop (1973) in amphibian blastulas.

Endophyll orients and organizes the early head region of the avian embryo.

By placing endophyll on the caudal area marginalis situated behind Rauber's sickle of avian unincubated blastoderms, we observed after using the quail-chick chimera system and culture the development of a (pre)neural plate or a miniature embryo, head-oriented towards this endophyll. A Rauber's sickle fragment placed in the same conditions gives no reaction. If we place endophyll close to Hensen's node (stage 4 Vakaet, 1962) on an isolated anti-sickle region of an avian unincubated blastoderm in vitro, a similar endophyll-oriented development takes place after culture. Under the same conditions, but in the absence of endophyll, a Hensen's node provokes a thickening of the upper layer in the immediate neighbourhood, eventually with formation of a neural axis, oriented according to the original caudocranial direction of the graft. Our study indicates that avian endophyll (from unincubated blastoderms) can induce in the upper layer a (pre)neural plate, with or without neural folds. By interaction with sickle endoblast coming from Rauber's sickle (the early gastrulation organizer: Callebaut and Van Nueten, 1994), or from Hensen's node (a later avian organizer: Waddington, 1932), it can orient or re-orient the head region and the caudocranial direction of an induced miniature embryo. The conclusions from our embryological experiments are in agreement with the results obtained by recent molecular biology studies.

Avian junctional endoblast has strong embryo-inducing and -dominating potencies.

In the present study, we demonstrate that quail junctional endoblast fragments have powerful embryo-inducing potencies when placed on the deep side of the upper layer (UL) of the anti-sickle region of unincubated chicken blastoderms. Moreover, in most cases the inducing potencies of the autochthonous Rauber's sickle-endophyll complex were inhibited at distance. Also, junctional endoblast still in situ in an early streak embryo strongly inhibits the inducing capacities of a Rauber's sickle - endophyll complex apposed on the deep side of the upper layer of the cranial part of the area pellucida. So junctional endoblast, besides embryo inducing potencies also seems to dominate and to inhibit at distance the gastrulation potencies of an ectopically placed or autochthonous Rauber's sickle - endophyll complex.