Use of a comprehensive diagnostic algorithm for Anisakis allergy in a high seroprevalence Mediterranean setting.

Summary: Background.Diagnosis of anisakis allergy (AA) is based on the skin prick test (SPT) and specific IgE (sIgE) determination. Anyway, false positivity cases are due to cross reactivity with numerous allergens. The aim of the study was to evaluate the reliability of a comprehensive diagnostic algorithm for the AA. Methods.An observational study was conducted on a sample of consecutive subjects accessing the allergology outpatient ambulatories of two hospitals located in Western Sicily. All the recruited outpatients were tested by Skin Prick Test performed using anisakis extracts by ALK-Abellò (Madrid, Spain). Specific IgE dosage for anisakis extracts was then performed by using ImmunoCAP250 (Immunodiagnostics Uppsala, Sweden). Consequently, outpatients who tested positive to first line tests underwent sIgE testing for ascaris and tropomyosin. Lastly, outpatients positive to the first line were invited to be further tested by basophil activation test (BAT) by using Flow CAST kit and anisakis commercial extract (Bühlmann Laboratories AG, Schönenbuch, Switzerland), as confirmatory analysis. Results.One hundred and eleven outpatients with an anamnesis suggestive of sensitization to anisakis (AS) and 466 subjects with chronic urticaria (CU) were recruited in the study. Of these, 22 with AS and 41 with CU showed a sensitization to anisakis allergens. The diagnostic algorithm revealed that 8.8% of outpatients who tested positive to sIgE determination were affected by CU, while 82.5% of all the sIgE positivity was related to cross-reactivity. Overall, a genuine anisakis seroprevalence of 2.3% was documented. Within a sub-sample of 15 subjects with clinical symptoms related to AA, n. 8 showed a real positivity after BAT. A greater response to A. pegreffii allergens as compared to A. simplex was reported. Conclusions.Our preliminary findings support the high clinical specificity of BAT for AA diagnosis, suggesting implementing this method in a comprehensive diagnostic algorithm.

Vitamin D3 affects innate immune status of European sea bass (Dicentrarchus labrax L.).

The effects of vitamin D3 dietary administration on certain innate immune parameters on the expression of immune-related genes in head-kidney (HK) and gut were investigated in European sea bass Dicentrarchus labrax. Vitamin D3 (vD3) was orally administered to fish in a commercial pellet food supplemented with 0 (control); 3750; 18,750; or 37,500 U kg-1. Furthermore, gut histology was considered. This study showed a modulation in the activities examined in fish fed with the addition of vD3. After just 2 weeks of administration, diet supplementation with the vitamin resulted in increased phagocytic ability, while serum peroxidase content was increased in fish fed with all experimental diets after 4 weeks, no significant differences were observed in protease, anti-protease, natural haemolytic complement activities and total IgM level. At gene level, fbl and rbl transcripts were up-regulated in HK in fish fed with the highest concentration of vD3-supplemented diets after 4 weeks, while in the gut, an up-regulation of hep gene was observed in fish fed with the different doses of vD3. These results suggest that vD3 may be of great interest for immunostimulatory purposes in fish farms.

Tramadol hydrochloride: pharmacokinetics, pharmacodynamics, adverse side effects, co-administration of drugs and new drug delivery systems.

Tramadol hydrochloride (TrHC) is a synthetic analgesic drug exhibiting opioid and non-opioid properties, acting mainly on the central nervous system. It has been mostly used to treat pain, although its use to treat anxiety and depression has also been documented. These properties arise from the fact that they inhibit serotonin (5-HT) reuptake augmenting 5-HT concentration on the synaptic cleft. Despite this, TrHC has also been described to have several side effects which are mainly due to its fast metabolization and excretion which in turn requires multiple doses per day. To surpass this limitation, new pharmaceutical formulations are being developed intending the protection, target and sustained delivery as well as a reduction on daily dose aiming a reduction on the side effects. In the present work we have revised the efficacy, safety, biological and adverse effects of TrHC, and the added value of developing a novel drug delivery system for topical administration.

Validation of an UV spectrophotometric assay for the quantification of polymyxin B in solid lipid nanoparticles.

Polymyxins are efficient antibiotic drugs used for the treatment of Gram-negative bacterial infections. These compounds are not absorbed in the gastrointestinal tract and are responsible for serious toxicological effects. In order to enhance their therapeutic effectiveness, decrease the adverse/toxic side effects and promote a sustained release profile, a derivative--polymyxin B sulphate--has been formulated in solid lipid nanoparticles (SLNs) intended for buccal administration. To quantify polymyxin B in the formulation, UV spectrophotometry analysis was applied, validating the analytical methodology by assessing the selectivity, accuracy, precision, linearity, and repeatability. Analyses were performed at 210 nm keeping the samples at 25 degrees C. Results showed that lipid composition of SLNs did not interfere with the polymyxin B spectra. The linearity showed a correlation coefficient of 0.9977 in the range of 5-90 µg/mL. Quantification of polymyxin B by UV spectrophotometry, at 210 nm in SLN formulations, was approved in all analyzed parameters, validating the methodology proposed in this work.

Seabream (Sparus aurata) long-term dominant-subordinate interplay affects phagocytosis by peritoneal cavity cells.

Fish are sensitive to stressful conditions that affect their innate immune systems and increase their susceptibility to diseases. We examined the social stress of paired gilthead seabream (Sparus aurata). Social hierarchies (dominant/subordinate) were characterised by behavioural changes, such as "aggressiveness" and "feeding order"; hierarchical positions were established within an hour of exposure to social stress and remained unchanged for approximately 1 year. To characterise physiological stress, we measured blood plasma levels of cortisol, glucose, and lactate as well as osmolarity and observed that the levels of these stress markers were higher in subordinate individuals than in dominant ones. The discriminant analysis revealed a separation of the subordinate fish groups, and at 15 days, a significant separation among groups was observed. Moreover, diminished phagocytic and respiratory burst activities revealed that social stress appeared to affect the cellular innate immune response of the subordinate specimens. Finally, to examine the effect of cortisol on phagocytosis, peritoneal cavity cells were treated in vitro, and an inhibitory effect was observed.

A lytic mechanism based on soluble phospholypases A2 (sPLA2) and ß-galactoside specific lectins is exerted by Ciona intestinalis (ascidian) unilocular refractile hemocytes against K562 cell line and mammalian erythrocytes.

Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca²+-dependent cytotoxic activity toward mammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte populations were separated (B1-B6 bands) through a Percoll discontinuous density gradient, the hemocyte cytotoxic activity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS) were assayed. In addition the separated hemocytes were cultured and the cell-free culture medium (CFM) assayed after 3 h culture. Results support that unilocular refractile hemocytes (URGs), enriched in B5, are cytotoxic. The B5-HLS contains lysins and the activity of B5-CFM shows that lysins can be released into a culture medium. The B5 activity was blocked by D-galactose, α-lactose, lactulose, LacNAc, thiodigalactoside (TDG), L-fucose, D-mannose, D-glucose, sphingomyelin (SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain, quinacrine). Accordingly, HLS chemico-physical properties (alkaline medium, high thermostability, Ca²+-dependence, trypsin treatment, protease inhibitors) and SEM observations of the affected targets suggested that sPLA2 could be responsible for changes and large alterations of the target cell membrane. An apoptotic activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very diluted HLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cells respectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme activity.

F-type lectin from the sea bass (Dicentrarchus labrax): purification, cDNA cloning, tissue expression and localization, and opsonic activity.

Recently described biochemical and structural aspects of fucose-binding lectins from the European eel (Anguilla anguilla) and striped bass (Morone saxatilis) led to the identification of a novel lectin family ("F-type" lectins) characterized by a unique sequence motif and a characteristic structural fold. The F-type fold is shared not only with other members of this lectin family, but also with apparently unrelated proteins ranging from prokaryotes to vertebrates. Here we describe the purification, biochemical and molecular properties, and the opsonic activity of an F-type lectin (DlFBL) isolated from sea bass (Dicentrarchus labrax) serum. DlFBL exhibits two tandemly arranged carbohydrate-recognition domains that display the F-type sequence motif. In situ hybridization and immunohistochemical analysis revealed that DlFBL is specifically expressed and localized in hepatocytes and intestinal cells. Exposure of formalin-killed Escherichia coli to DlFBL enhanced their phagocytosis by D. labrax peritoneal macrophages relative to the unexposed controls, suggesting that DlFBL may function as an opsonin in plasma and intestinal mucus.

The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis.

Phenoloxidase (PO) activity was examined in the tunic tissue of Ciona intestinalis following lipopolysaccharide (LPS) intratunic injection. Tunic homogenate supernatant (THS), assayed with the Dopa-MBTH reaction, displayed Ca(2+)-independent PO activity that was raised by LPS and further enhanced by proteases. Specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. Assay with soybean trypsin inhibitor showed that, in the tunic, PO activation with trypsin was not significantly inhibited suggesting that proteases diverse from serine proteases were involved. In vivo experiments were carried out by injecting isosmotic medium or LPS, and THS was assayed for its PO activity. Analysis of variance of the time-course profiles showed that LPS was more effective in activating proPO. To disclose the PO response at the injured site, an assay with Dopa-MBTH was performed in vitro. Quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. Microscopic observations and immunohistochemistry with anti-CinPO-2 antibodies showed granulocytes and unilocular refractile granulocytes containing PO, whereas few morula cells were stained. In THS zymograms (SDS-polyacrylamide gel electrophoresis), PO activity linked to 90-kDa and 120-kDa bands was observed as an effect of LPS injection, whereas the density of 170-kDa PO was weak. A third presumptive PO enzyme (CinPO-3) containing the CinPO-2 peptide was identified in the recent Ciona genome version. Presumably, LPS stimulated the production and dimerization (120 kDa) of CinPO-3 (66 kDa). Thus, the activated proPO system includes several POs that are distinguishable by size and that are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars.

Glucocorticoid receptor (DlGR1) is expressed in pre-larval and larval stages of the teleost fish Dicentrarchus labrax.

Glucocorticoid hormone receptors (GR), members of the nuclear hormone receptor superfamily, are ligand-dependent transcription factors expressed in various tissues by binding to specific DNA sequences. Since glucocorticoids have a role in maintaining the homeostatic status in fish, we previously cloned and sequenced a GR (DlGR1) of adult Dicentrarchus labrax; we also showed mRNA expression (in situ hybridization) and tissue immunohistochemical localization of DlGR1 in several organs. This work has now been extended to the examination of the expression, tissue distribution, and cytolocalization of DlGR1 in larval developmental stages by similar methods to those used for the adult organs. The riboprobe included the DlGR1 cDNA transcriptional activation domain (1.0-1,300 nucleotide sequence) showing no significant similarity with a known second GR cDNA sequence of sea bass. The antibody was specific for an opportunely selected peptide sequence of the DlGR1 transcriptional domain. In histological sections of brain, head kidney, gills, liver, anterior intestine, and spleen cells, the riboprobe was mainly located in the cell nucleus. The antibody identified DlGR1 in the head kidney, gills, liver, and anterior intestine, mainly located in the cytosol. These results are in agreement with the receptor location in adult tissues. The greater presence of both the transcript and protein of DlGR1 in the late developmental stages suggests an increasing expression of this receptor. The cytolocalization (nuclear-cytosolic) and presumptive roles of DlGR1-containing tissues are discussed.

Expression of a glucocorticoid receptor (DlGR1) in several tissues of the teleost fish Dicentrarchus labrax.

Since glucocorticoids have a role in maintaining the homeostatic status in fish, in the present paper mRNA expression (in situ hybridization) and tissue immunohistochemical localization of a glucocorticoid receptor (DlGR1) in several Dicentrarchus labrax organs are reported. Riboprobe and specific antibodies were prepared by using the DlGR1 that has been previously cloned and sequenced from peritoneal cavity leukocytes. Both mRNA and receptor were identified in head kidney, spleen, gills, intestine, heart and liver tissues. The functional roles of DlGR1 localization are discussed.

Anaphylatoxin-like molecules generated during complement activation induce a dramatic enhancement of particle uptake in rainbow trout phagocytes.

Here we have identified a serum fraction containing approximately 8-kDa molecules with an unexpected capacity to greatly enhance particle uptake in trout head kidney leukocytes (HKLs). This approximately 8-kDa particle-uptake enhancing fraction (PUEF-8) was purified from complement-activated serum by gel filtration chromatography. Mass spectrometric analysis and reactivity of anti-trout C3-1 and C4 antibodies, indicated the presence of C3a, C4a and C5a molecules in PUEF-8. Using a newly developed flow cytometric assay that measures the capacity of cells to ingest fluorescent beads, we showed that PUEF-8 induced a striking enhancement (344+/-50% higher than the PBS control value) in the number of HKLs ingesting three or more beads. In contrast, the effect of PUEF-8 on peripheral blood leukocytes (PBLs) was almost negligible. Interestingly, PUEF-8 acted as a strong chemoattractant for both HKLs and PBLs. These findings suggest a novel role for the anaphylatoxins generated during complement activation in teleost fish.

A serum fucolectin isolated and characterized from sea bass Dicentrarchus labrax.

A lectin specific for fucose and galactose was isolated by affinity chromatography on Sepharose CL-6B from the serum of Dicentrarchus labrax. The hemagglutinating activity against rabbit erythrocytes was calcium-independent, and reached its maximum at 37 degrees C. Two protein components were found in the hemagglutinating fractions eluted from the Sepharose column. Only the 34 kDa component (DLL2) eluted from the polyacrylamide gels (SDS-PAGE) showed agglutinating activity against rabbit erythrocytes. SDS-PAGE, in non-reducing conditions, revealed a single 66 kDa protein that reacted with antibodies to the 34 kDa component. Therefore, a dimeric structure stabilized by disulfide bonds can be proposed. The Ca(2+)-independent fucose-binding specificity, a significant amino acid sequence homology of the N-terminal trait, and cross-reaction of eel fucolectin with antibodies to DLL2 suggest that this lectin may be included in the recently identified fucolectin family.

Spontaneous cytotoxic activity of eosinophilic granule cells separated from the normal peritoneal cavity of Dicentrarchus labrax.

In this study the spontaneous in vitro cytotoxic activity to tumour cell lines, (K562), by unstimulated sea bass (Dicentrarchus labrax) leukocytes was examined by trypan blue exclusion test and lactate dehydrogenase release assay. A high anti-tumour cell line activity of resident peritoneal leukocytes was found at an effector to target ratio (E:T) of 25:1 after incubation for 2 h at 18 degrees C. Rabbit and sheep erythrocytes were not lysed. A low activity was displayed by head kidney and spleen cell populations whereas blood leukocytes revealed no significant activity. The effect of E:T ratio on cytotoxicity as well as microscopy observations suggested that the cytotoxic reaction required effector-target cell contact. Eosinophilic granule cells, isolated on a Percoll density gradient from a peritoneal wash, appeared to be responsible for the in vitro cytotoxic activity.

[Current trends in the treatment of perforated colonic diverticulitis]. / Attualità nel trattamento della diverticolite perforata del colon.

The authors, after having examined the cases of complicated diverticulitis operated between 1985 and 1993, confirm that the surgical treatment for this particular pathology is strictly dependent on the local or general seriousness. In the light of this they consider an operation for resection and anastomosis 1st stage justifiable only in selected cases; colostomy and drainage in particularly serious cases, preferring Hartmann's method of operation for the 3rd and 4th stages of Chappuis and Chon. The results obtained agree in a satisfactory manner with the data in the literature.