Giampaolo Bosi

Localization of IGF-I, IGF-I receptor, and IGFBP-2 in developing Umbrina cirrosa (Pisces: Osteichthyes).

In this study, the distribution of IGF-I, IGF type I receptor (IGF-IR), and IGF-binding protein 2 (IGFBP-2) was investigated during larval and post-larval developmental stages of the shi drum (Umbrina cirrosa) by immunohistochemistry using antisera raised against Sparus aurata IGF-I and IGF-IR, and against mouse IGFBP-2. Immunoreactivity of the mitogenic marker PCNA (proliferating cell nuclear antigen) was used for assessment of cellular proliferation. Distribution of IGF-I mRNA was studied by in situ hybridization. IGF-I immunoreactivity was detected in liver and developing intestine already in 1-5 day post-hatching larvae. From day 11, immunostaining in the intestine was evident in the enterocytes of the anterior intestine and in the apical zone of the epithelium of developing posterior intestine. Positive reaction with IGF-I antibody was also detected in chondrocytes, in the epithelium of the skin, gills and in the central nervous system (CNS), and lateral muscle. At hatching IGF-IR immunoreactivity was already detectable in developing CNS, notochord, and skin. From day 6 immunostaining was evident in the olfactory epithelium, in eyes and from day 11 in the developing olfactory bulbs and CNS. Positive reaction with IGF-IR antibody was also detected in chondrocytes, in the epithelium of the skin, gills, heart, and in the lateral muscle. Immunoreactive IGFBP-2, as detected by anti-mouse IGFBP-2 antiserum, exhibited generally a similar distribution pattern to that of IGF-I and IGF-IR. In situ hybridization, which has been performed by using riboprobes from S. aurata cDNA, revealed IGF-I mRNA in skeletal musculature, liver, and CNS. These data strongly suggest a role for the IGF system during development and growth of U. cirrosa.

The chemical code of porcine enteric neurons and the number of enteric glial cells are altered by dietary probiotics

Background  The enteric nervous system (ENS) contains chemically coded populations of neurons that serve specific functions for the control of the gastrointestinal tract. The ability of neurons to modify their chemical code in response to luminal changes has recently been discovered. It is possible that enteric neuronal plasticity may sustain the adaptability of the gut to changes in intestinal activity or injury, and that gut neurons may respond to an altered intestinal environment by changing their neuropeptide expression.

Can nutraceuticals affect the structure of intestinal mucosa? Qualitative and quantitative microanatomy in L-glutamine diet-supplemented weaning piglets

Weaning piglets were fed an L -glutamine-supplemented diet with the aim of monitoring the effects on gut mucosal turnover and barrier function, to elucidate the potential preventive or therapeutic roles of glutamine as a nutraceutical or ‘functional food’. Sixteen female weaning piglets were divided into two groups, which were fed a control diet (Ctr group: n = 8) or a Ctr + 0.5% L -glutamine diet (G group: n = 8) for 28 days. In the ileum of group G piglets the villus height (V) and crypt depth (C) were increased, and the V:C ratio was decreased (p < 0.01). The PCNA and TUNEL immunoreactivities were also tested. The number of mitotic mucosal cells (M) was increased, and that of mucosal cells with apoptotic nuclei (A) decreased in such a way that the A:M index diminished (p < 0.01). The A:M index also decreased at the level of some components of the gut-associated lymphatic tissue (GALT), thus indicating a positive effect on the gut barrier function. This trial showed that L -glutamine supplementation influenced some morphofunctional characteristics of piglet ileal mucosa. These data corroborate the nutraceutical role of glutamine as a trophic agent for mucosal repair, improvement of barrier function and gut adaptation in the swine per se and as an animal model.

Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse

BackgroundThe identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI).MethodsCompact and expanded-cumulus horse oocytes were matured in medium containing different concentrations (1, 10, 100, 1000 ng/ml) of recombinant human leptin and the effects on maturation, fertilization and embryo cleavage were evaluated. Furthermore, early developmental expression of Ob and leptin receptor (Ob-R) was investigated by immunocytochemical staining.ResultsIn expanded-cumulus oocytes, the addition of leptin in IVM medium improved maturation (74% vs 44%, for 100 ng/ml leptin-treated and control groups, respectively; P < 0.05) and fertilization after ICSI (56% vs 23% for 10 ng/ml leptin-treated and control groups, respectively; P < 0.05). However, the developmental rate and quality of 8-cell stage embryos derived from leptin-treated oocytes (100 ng/ml) was significantly reduced, in contrast to previous data in other species where leptin increased embryo cleavage. Ob and Ob-R proteins were detected up to the 8-cell stage with cortical and cytoplasmic granule-like distribution pattern in each blastomere.ConclusionLeptin plays a cumulus cell-mediated role in the regulation of oocyte maturation in the mare. Species-specific differences may exist in oocyte sensitivity to leptin.

Abundance, diversity and seasonal succession of dytiscid and noterid beetles (coleoptera: Adephaga) in two marshes of the Eastern Po Plain (Italy)

The dytiscid (plus Noteridae) communities of two neighbouring marshes (A and B) in the Eastern plain of the Po river (Italy) was investigated. The two marshes showed similar surface area, plant species and abundance, quantity of detritus, exposure and depth variations. Physical and chemical variables of the two habitats were similar. Phenology curves based on larval and adult water beetles showed two peaks, in spring and autumn. However, the number of specimens and the variety of species were significatively higher in marsh A than in marsh B. The occurrence and the abundance of the main vertebrate and macroinvertebrate taxa in the two habitats were measured using a relative method. There was little difference in densities of prey animals (tadpoles, newt larvae, crustaceans, ephemeropteran nymphs and dipteran larvae) in the two marshes. Instead, a higher abundance of odonate larvae and hemipteran nymphs and adults were found in marsh B compared to marsh A. Difference in the number of species of aquatic beetles and their abundance could be partially due to the stability of marsh B compared to marsh A. Probably, competition and predation by hemipterans and odonate larvae on dytiscid larval stages could be implicated in determining water beetle communities structure in the two neighbouring marshes; thus, this phenomenon is discussed here.

Fish innate immunity against intestinal helminths

Most individual fish in farmed and wild populations are infected with parasites. Upon dissection of fish, helminths from gut are often easily visible. Enteric helminths include several species of digeneans, cestodes, acanthocephalans and nematodes. Some insights into biology, morphology and histopathological effects of the main fish enteric helminths taxa will be described here. The immune system of fish, as that of other vertebrates, can be subdivided into specific and aspecific types, which in vivo act in concert with each other and indeed are interdependent in many ways. Beyond the small number of well-described models that exist, research focusing on innate immunity in fish against parasitic infections is lacking. Enteric helminths frequently cause inflammation of the digestive tract, resulting in a series of chemical and morphological changes in the affected tissues and inducing leukocyte migration to the site of infection. This review provides an overview on the aspecific defence mechanisms of fish intestine against helminths. Emphasis will be placed on the immune cellular response involving mast cells, neutrophils, macrophages, rodlet cells and mucous cells against enteric helminths. Given the relative importance of innate immunity in fish, and the magnitude of economic loss in aquaculture as a consequence of disease, this area deserves considerable attention and support.

Enteric neuromodulators and mucus discharge in a fish infected with the intestinal helminth Pomphorhynchus laevis

BackgroundIn vertebrates, the presence of enteric worms can induce structural changes to the alimentary canal impacting on the neuroendocrine system, altering the proper functioning of the gastrointestinal tract and affecting the occurrence and relative density of endocrine cells (ECs). This account represents the first immunohistochemistry and ultrastructure-based study which documents the intimate relationship between the intestinal mucous cells and ECs in a fish-helminth system, investigating the potential effects of enteric neuromodulators on gut mucus secretion/discharge.MethodsA modified dual immunohisto- and histochemical staining technique was applied on intestinal sections from both infected and uninfected fish. Sections were incubated in antisera to a range of neuromodulators (i.e. leu-enkephalin, met-enkephalin, galanin and serotonin) and the glycoconjugate histochemistry of the mucous cells was determined using a subsequent alcian blue – periodic acid Schiff staining step. Dual fluorescent staining on sections prepared for confocal laser scanning microscopy and transmission electron microscopy were also used to document the relationship between ECs and mucous cells.ResultsFrom a total of 26 specimens of Squalius cephalus sampled from the River Paglia, 16 (i.e. 62 %) specimens were found to harbour an infection of the acanthocephalan Pomphorhynchus laevis (average intensity of infection 9.2 ± 0.8 parasites host−1, mean ± standard error). When acanthocephalans were present, the numbers of mucous cells (most notably those containing acidic or mixed glycoconjugates) and ECs secreting leu-enkephalin, met-enkephalin, galanin, serotonin were significantly higher than those seen on sections from uninfected fish. The relationship between met-enkephalin-like or serotonin-like ECs and lectin DBA positive mucous cells was demonstrated through a dual fluorescent staining. The presence of tight connections and desmosomes between mucous and ECs in transmission electron micrographs provides further evidence of this intimate relationship.ConclusionsThe presence of P. laevis induces an increase in the number of enteric ECs that are immunoreactive to leu- and met-enkephalin, galanin, and serotonin anti-sera. The mucous cells hyperplasia and enhanced mucus secretion in the helminth-infected intestines could be elicited by the increase in the number of ECs which release these regulatory substances.

An insight into testis and gubernaculum dynamics of INSL3–RXFP2 signalling during testicular descent in the dog

Insulin-like 3 (INSL3) plays a prominent role in male development and is supposed to induce the growth of the gubernaculum testis (g.t.), thus being directly involved in testicular descent in humans and rodents. This happens through activation of the RXFP2 receptor (GREAT or LGR8). The INSL3-RXFP2 complex is reputed to play an additional paracrine role in the testis, possibly acting as part of an autocrine feedback loop. The present work provides evidence of the immunolocalisation of INSL3 in the Leydig cells of canine fetuses and of the expression of RXFP2 receptor in different tissues of the g.t. of the same specimens. RXFP2 was localised at the cell membrane of g.t. muscle and connective cells, as well as in the epithelial cells of the developing excurrent ducts. Notably, RXFP2 immunoreactivity of the g.t. was limited to fetuses at ~35-45 days of gestation, which is also the fetal period when the endocrine compartment of the dog testis is active endocrinologically, as confirmed by the anti-P450c17 and anti-INSL3 immunoreactivities of the fetal Leydig cells, and by anti-Müllerian hormone immunoreactivity of the Sertoli cells. The same immunoreactivities were also evaluated in the testes of cryptorchid dogs of different ages. RXFP2 immunoreactivity was absent from genital tracts of cryptorchid testes and g.t. remnants.

Occurrence of immune cells in the intestinal wall of Squalius cephalus infected with Pomphorhynchus laevis.

A sub-population of 34 specimens of chub, Squalius cephalus, was sampled from the River Brenta (Northern Italy) and examined for ecto- and endo-parasites. Pomphorhynchus laevis (Acanthocephala) was the only enteric helminth encountered. Immunofluorescence and ultrastructural studies were conducted on the intestines of chub. Near the site of parasites attachment, mucous cells, mast cells (MCs), neutrophils and rodlet cells (RCs) were found to co-occur within the intestinal epithelium. The numbers of mucous cells, MCs and neutrophils were significantly higher in infected fish (Mann-Whitney U test, p < 0.05). Dual immunofluorescence staining with the lectin Dolichos Biflorus Agglutinin (DBA) and the macrophage-specific MAC387 monoclonal antibody, with parallel transmission electron microscopy, revealed that epithelial MCs often made intimate contact with the mucous cells. Degranulation of a large number of MCs around the site of the acanthocephalans attachment and in proximity to mucous cells was also documented. MCs and neutrophils were abundant in the submucosa. Immune cells of the intestinal epithelium have been described at the ultrastructural level and their possible functions and interactions are discussed.

Responses of Squalius cephalus intestinal mucous cells to Pomphorhynchus laevis

Intestinal mucous cell numbers and their glycoconjugate composition were investigated by histochemical methods in uninfected chub, Squalius cephalus, and in conspecifics naturally parasitised with the acanthocephalan Pomphorhynchus laevis. A sub-population of 42 chub from the River Tiber (Perugia, Italy) were sampled and screened for ecto and endoparasites. No parasites were found in gills and in other visceral organs of chub and P. laevis appeared to be the only enteric worm encountered. In all infected chub (twenty-eight out of 42) this acanthocephalan was encountered mainly in the mid-gut. In situ, an excessive yellowish mucus or catarrh was observed around each acanthocephalan. Hyperplasia and hypertrophy of the mucous cells were only evident near the site of P. laevis attachment where the total number of mucous cells and the number of those containing acidic, particularly non-sulphated mucins, or mixed glycoconjugates were significantly higher. In intestinal regions of infected fish far away from the point of parasite attachment, there were no statistical differences in the density of mucous cells in comparison to uninfected fish. Interestingly, in parasitised chub, the length of intestinal folds was significantly larger close to the sites at which P. laevis attach when compared to the length of the intestinal folds located further away from the acanthocephalans and/or in uninfected intestines. The effect of P. laevis on intestinal mucous cells of S. cephalus was compared to other parasite-host systems and the role of enhanced mucus production in parasitized intestines was discussed.