The "Bald Disease" of the Sea Urchin Paracentrotus lividus: Pathogenicity, Molecular Identification of the Causative Agent and Therapeutic Approach.

In recent decades, various species of Mediterranean sea urchins, including Paracentrotus lividus, have been subject to widespread seasonal episodes of mass mortality whose causative agents are still unclear. In particular, P. lividus is subject to late winter events of mortality, due to a disease manifested by a massive loss of spines and the presence of greenish amorphous material on the tests (i.e., the sea urchin skeleton consisting of spongeous calcite). Documented mortality events show a seasonal epidemic diffusion and might produce economic losses also in aquaculture facilities, besides the environmental constraints to its diffusion. We collected individuals showing conspicuous lesions on the body surface and reared them in recirculated aquaria. Samples of external mucous were collected along with coelomic liquids and cultured to isolate bacterial and fungal strains, further submitted to molecular identification through the amplification of prokaryotic 16S rDNA. In addition, pools of infected sea urchins were reared in recirculated tanks after short baths in a formulated therapeutic compound and their survival rates were compared to non-treated individuals for variable periods. Here, we aimed at a redescription of the etiopathogenetic nature of the parasites and tested the efficacy of a possible treatment, to be proposed for aquaculture purposes.

Leishmania spp.-Infected Dogs Have Circulating Anti-Skeletal Muscle Autoantibodies Recognizing SERCA1.

Leishmania spp. infection is associated with an inflammatory myopathy (IM) in dogs. The pathomechanism underlying this disorder is still elusive, however, the pattern of cellular infiltration and MHC I and II upregulation indicate an immune-mediated myositis. This study aimed to investigate the presence of autoantibodies targeting the skeletal muscle in sera of leishmania-infected dogs and individuate the major autoantigen. We tested sera from 35 leishmania-infected dogs and sera from 10 negative controls for the presence of circulating autoantibodies with indirect immunofluorescence. Immunoblot and mass spectrometry were used to identify the main target autoantigen. Immunocolocalization and immunoblot on immunoprecipitated muscle proteins were performed to confirm the individuated major autoantigen. We identified circulating autoantibodies that recognize skeletal muscle antigen(s) in sera of leishmania-infected dogs. The major antigen was identified as the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1). We also found that canine SERCA1 presents several identical traits to the calcium-translocating P-type ATPase of Leishmania infantum. In the present study, we defined circulating anti-SERCA1 autoantibodies as part of the pathogenesis of the leishmania-associated IM in dogs. Based on our data, we hypothesize that antigen mimicry is the mechanism underlying the production of these autoantibodies in leishmania-infected dogs.

Correlation Between Serum Activity of Muscle Enzymes and Stage of the Estrous Cycle in Italian Standardbred Horses Susceptible to Exertional Rhabdomyolysis.

Equine exertional rhabdomyolysis (ER) is a well-recognized clinical syndrome affecting racehorses. Prevalence analysis of ER showed that female sex was a significant risk factor. The aim of this research was to evaluate the differences and correlations in the serum activity of muscle enzymes and the stage of the estrous cycle in ER-susceptible and control (C) mares. Serum muscle enzyme activity before and after exercise and sex hormones were analyzed in the two groups of mares. Ten cyclic ER and 10 cyclic C mares were examined weekly for 4 weeks. During diestrus, ER horses had significantly higher resting and postexercise aspartate aminotransferase (AST) activity, but not creatine kinase (CK) activity, compared with controls; only postexercise AST activity was significantly higher during estrus compared with activity levels in controls. During estrus, 17ß-estradiol and AST activity were significantly negatively correlated in the control but not ER mares. Based on our results, further studies should be performed to characterize the presumptive different roles played by sexual hormones in horses susceptible to ER compared with healthy mares.

Organization Science improves management effectiveness of Marine Protected Areas.

The ecological management effectiveness (EME) of Marine Protected Areas (MPAs) is the degree to which MPAs reach their ecological goals. The significant variability of EME among MPAs has been partly explained by MPA design, management and implementation features (e.g. surface area, enforcement, age of protection). We investigated EME variability by employing, for the first time, Organization Science. Eight Mediterranean MPAs were taken into account as case studies to explore the relationships between EME and MPA features, such as: 1) organizational size (i.e. the ratio between the number of full-time employees and the total MPA surface area), 2) management performance (i.e. the level of effort exerted to enhance and sustain the MPA management, including enforcement), 3) total surface area, and 4) MPA age. The log-response ratios of fish biomass and density in protected vs unprotected (control) areas were used as a proxy of EME. Management performance, organizational size and, to a lesser extent, MPA age were positively correlated with the log-response ratio of fish biomass, whereas total surface area did not display a significant role. None of the four features considered was significantly correlated with the log-response ratio of fish density. Based on our findings, we argue that the employment of Organization Science in the management effectiveness assessment can assist MPA managers to reach MPAs goals more effectively, with a more efficient use of available resources.

Inflammatory Myopathy in Horses With Chronic Piroplasmosis.

Horses affected by chronic piroplasmosis may develop poor performance and muscle atrophy. Here we investigate the pathological and immunopathological aspects of myopathy occurring in chronic equine piroplasmosis. The study included 16 horses serologically positive for equine piroplasms presenting with clinical signs and supporting serum biochemical evidence of a myopathy. Skeletal muscle was evaluated by histopathology, immunohistochemistry, indirect immunofluorescence, and molecular detection of piroplasms and inflammatory cytokines in skeletal muscle. Histologic lesions included muscle fiber atrophy (100% of cases), degenerative changes (13/16, 81%), and perivascular perimysial and endomysial lymphocytic infiltrates (81% of cases). In 15 cases (94%), muscle fibers had strong immunostaining for major histocompatibility complex classes I and II. T lymphocyte populations were mainly CD3+, CD8+, and CD4+ in equal proportions, with a lower number of CD79α+ cells. The serum from affected horses was tested by indirect immunofluorescence for binding of IgG, IgM, or IgA to sections of normal equine muscle to detect circulating autoantibodies against muscle antigen(s). In all cases, distinct sarcolemmal staining was detected in sections incubated with serum from affected horses, in contrast to sections incubated with phosphate-buffered saline or equine control sera. Reverse transcription polymerase chain reaction (RT-PCR) testing of muscles from affected animals revealed a significant increase of interferon-γ, interleukin-12, and tumor necrosis factor-α gene expression compared to healthy controls. Theileria equi or Babesia caballi was not detected in samples of affected muscle by RT-PCR. Thus, inflammatory myopathy associated with equine piroplasmosis may involve an autoimmune pathogenesis with upregulation of inflammatory cytokines that may cause myofiber atrophy and degeneration.

Periprostatic Perineurioma, Description of a Case With Unusual Localization.

Perineurioma is a rare entity, it is a benign peripheral nerve sheath tumor entirely composed of perineurial cells. A 62-year-old male patient was admitted to our hospital, suffering from scrotal and pelvic pain combined with a severe and continuous pain in his right thigh. A transrectal ultrasound revealed a periprostatic oval lesion of about 5 cm in maximum diameter. A sovrapubic laparotomy was performed with a complete tumor excision. The morphological and immunohistochemical data were most consistent with the diagnosis of perineurioma.

Immunogenicity of two oat varieties, in relation to their safety for celiac patients.

OBJECTIVE: Most of the recent studies suggest that oats are well tolerated by celiac disease (CD) patients. However, it is still possible that different oat cultivars may display different biological properties relevant for CD pathogenesis. We aimed to investigate biological and immunological properties of two oat varieties, Avena genziana and Avena potenza, in relation to their safety for CD patients. MATERIAL AND METHODS: Phosphorylation of extracellular signal-regulated kinase (ERK) and trans-epithelial electrical resistance (TEER) were evaluated in CaCo-2 cells treated with peptic-tryptic (PT) digests from the two oats and from gliadin (PTG). With the same PT-digests, duodenal biopsies from 22 CD patients were treated in vitro for 24 h and density of CD25+ cells in lamina propria and of intraepithelial CD3+ T cells was measured, as well as crypt cell proliferation and epithelial expression of interleukin 15. Finally, interferon γ (IFN-γ) production was measured as evidence of gliadin-specific T-cell activation by PT-digests. RESULTS: In contrast to PTG, oats PT-digests were not able to induce significant increase in ERK phosphorylation and decrease in TEER in CaCo-2 cells. In the organ culture system, oats PT-digests, unlike PTG, did not induce significant increase in crypt enterocyte proliferation, increase in interleukin 15 expression or in lamina propria CD25+ cells. Nevertheless Avena potenza increased intraepithelial T-cell density, while Avena genziana-induced IFN-γ production in 3/8 CD intestinal T cell lines. CONCLUSIONS: Our data show that Avena genziana and Avena potenza do not display in vitro activities related to CD pathogenesis. Some T-cell reactivity could be below the threshold for clinical relevance.

Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers via EGFR activation.

Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 muM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) abolished the effect. A concentration of 50 muM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment.

Unconjugated bilirubin modulates the intestinal epithelial barrier function in a human-derived in vitro model.

Unconjugated bilirubin promotes intestinal secretion without affecting nutrient digestion or absorption. In the current study, the effects of unconjugated bilirubin (UCB) on the barrier function of the intestinal epithelium were investigated. The apical side of human intestinal cell line Caco-2 monolayers was challenged with purified UCB. Transepithelial electrical resistance and paracellular fluxes of 10 kD Cascade blue conjugate dextran were measured. Cell monolayer viability was studied using LDH release and trypan blue exclusion tests. Redistribution of enterocyte tight junction occludin was studied by confocal microscopy. Bilirubin induced a dose-dependent decrease of transepithelial electrical resistance (TEER). This effect was maximal at 6 h and tended to be reversed at 48 h. Oxidated bilirubin was ineffective. Bilirubin significantly increased fluorescent dextran paracellular passage. Cell viability was not affected by UCB over the 5-200 nmol/L concentration range. Finally, bilirubin triggered a reversible redistribution of tight junctional occludin. UCB increases the permeability of intestinal epithelium. This effect is reversible, dependent on the redox status of the molecule and the rearrangement of the tight junction. These data attribute to bilirubin a novel role of functional modulator of intestinal paracellular permeability in vitro.

Reactive nitrogen species modulate the effects of rhein, an active component of senna laxatives, on human epithelium in vitro.

BACKGROUND: Senna laxatives are used worldwide. However, their misuse can lead to chronic mucosal inflammation with the accumulation of pigment-laden leukocytes and may cause colon cells to undergo apoptosis. This study explores the mechanisms by which rhein, an active component of senna, acts on a human intestinal cell line to induce ion secretion, apoptosis, and indirect chemotaxis of polymorphonuclear leukocytes. METHODS: Human colonic adenocarcinoma (CaCo-2) monolayer cells, in the presence or in the absence of rhein, were used to monitor the production of reactive nitrogen species using the Griess reaction. Modified Ussing chambers were used to study electrolyte secretion. The capacity to recruit human polymorphonuclear leukocytes was evaluated using masked well chemotaxis chambers. Rhein-induced apoptosis was investigated by counting apoptotic nuclei stained with Hoechst 33258 dye. RESULTS: Rhein caused a dose-dependent increase in short-circuit current that was abolished in chloride-free bathing buffer or by preincubating with 100 micromol/L NG-nitro-L-arginine (L-NAME) methyl ester. The concentration that maximally stimulated intestinal secretion, 50 micromol/L rhein, induced nitrate production. Supernatants obtained from CaCo-2 cultures after incubation with 50 micromol/L rhein stimulated a time-dependent polymorphonuclear leukocytes chemotaxis that was significantly decreased with 100 micromol/L L-NAME, whereas rhein per se was not active. Neutralizing antibodies anti-interleukin-8 (IL-8) and anti-ENA78 also inhibited chemotaxis. Overnight rhein incubation produced an increased number of apoptotic cells in the culture supernatant that was significantly decreased by preincubation with 100 micromol/L L-NAME. Light-degraded rhein had no effects on CaCo-2 monolayers. CONCLUSIONS: The integrity of rhein is crucial to generating nitric oxide, which mediates, with different time courses, ion secretion, chemotaxis, and apoptosis of human-derived cells.

Unconjugated bile acids modulate adult and neonatal neutrophil chemotaxis induced in vitro by N-formyl-met-leu-phe-peptide.

In this study, we have investigated the effect of hydrophobic and hydrophilic unconjugated bile acids (UBAs)-ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), lithocholic acid, and colic acid-on chemotaxis in adult and neonatal human polymorphonuclear leukocytes (PMNs). The trypan blue exclusion dye test was preliminarily performed to determine the toxicity of the studied UBAs on PMNs. N-formyl-methionyl-leucyl-phenylalanine (100 nM) was used as a chemoattractant. Chemotaxis (1 x 10(6)cells/mL) was analyzed in the presence or absence of UBAs (10 microM) by blind well chambers. The antioxidants vitamin E and vitamin C were tested for their ability to reduce the inhibitory effect of UBAs. We found that only CDCA was able to induce damage in PMNs in the range of 1-40 microM. Both CDCA and UDCA were able to inhibit chemotaxis in PMNs, whereas lithocholic acid and colic acid were ineffective. The inhibitory effect was reversible inasmuch as PMNs incubated with either CDCA or UDCA and subsequently washed showed normal chemotaxis. Concomitant incubation of PMNs with UBAs and vitamins C or E reversed the inhibition. We did not find substantial differences between PMNs from adults or newborns. In conclusion, CDCA and UDCA are able to reduce, in a specific and reversible fashion, both adult and newborn neutrophil chemotaxis. As concomitant incubation of UBAs and electron scavengers restores PMN chemotaxis to control values, we conclude that free radicals may be involved in the mechanism of inhibition. We speculate that this defect may contribute to the impaired host response described in cholestatic patient.