An immunogenomic exome landscape of triple positive primary antiphospholipid patients.

Primary antiphospholipid syndrome is characterized by thrombosis and autoantibodies directed against phospholipids or associated proteins. The genetic etiology of PAPS remains unknown. We enrolled 21 patients with thromboembolic events associated to lupus anticoagulant, anticardiolipin and anti ß2 glycoprotein1 autoantibodies. We performed whole exome sequencing and a systematic variant-based analysis in genes associated with thrombosis, in candidate genes previously associated with APS or inborn errors of immunity. Data were compared to public databases and to a control cohort of 873 non-autoimmune patients. Variants were identified following a state-of-the-art pipeline. Enrichment analysis was performed by comparing with the control cohort. We found an absence of significant HLA bias and genetic heterogeneity in these patients, including when testing combinations of rare variants in genes encoding for proteins involved in thrombosis and of variants in genes linked with inborn errors of immunity. These results provide evidence of genetic heterogeneity in PAPS, even in a homogenous series of triple positive patients. At the individual scale, a combination of variants may participate to the breakdown of B cell tolerance and to the vessel damage.

Polymorphisms in EGFR and IL28B are associated with spontaneous clearance in an HCV-infected Iranian population.

Although most hepatitis C virus (HCV)-infected individuals develop chronic infection, about 25% of them are able to clear the virus spontaneously without any therapeutic intervention. The aim of the present study was to identify genes associated with spontaneous HCV clearance in a population of Iranian patients. We genotyped 110 single-nucleotide polymorphisms (SNPs) in 59 selected--candidate--genes in a cohort of 107 HCV-infected participants who spontaneously cleared the infection and 176 participants whose infection persisted. Three out of the 110 SNPs were found to be associated with HCV outcome (P-values<0.03). rs11506105 in EGFR (epidermal growth factor receptor gene), and rs11881222 and rs12979860 in IL28B (interferon-λ3 gene). Multivariate logistic regression of the three markers showed that the A/A genotypes in both rs11506105 (EFGR) and rs11881222 (IL28B), and the C/C genotype in rs12979860 (IL28B) are associated with HCV clearance (recessive model: odds ratio (OR)=2.06, 95% confidence interval (95% CI)=1.09-3.88, P=0.025; OR=2.09, 95% CI=1.23-3.60, P=0.007; and OR=1.95, 95% CI=1.15-3.35, P=0.014 for rs11506105, rs12979860 and rs11881222, respectively). In conclusion, EGFR and IL28B SNPs are strong independent predictive markers of spontaneous viral clearance.

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 parasite's 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 acanthocephalan's 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.

Piscidins in the intestine of European perch, Perca fluviatilis, naturally infected with an enteric worm.

This study set out to determine how an enteric parasite, the thorny-headed worm Acanthocephalus lucii, affected the expression of antimicrobial peptides (piscidins) in its host population, the European perch (Perca fluviatilis) collected from Lake Piediluco in Central Italy. A total of 87 perch were examined; 44 (50.5%) were infected with A. lucii (1-18 worms fish(-1)). Pathological changes and immune response were assessed using histological, ultrastructural and immunohistochemical techniques. The acanthocephalans only penetrated the surficial zone of the intestinal wall and induced only slight inflammation. The main damage was destruction of the mucosal epithelium covering the villi adjacent to the parasite's attachment site, and included necrosis and degeneration. Infected intestine had numerous mast cells (MCs), often in close proximity to, and within, the capillaries, and were associated with fibroblasts of the submucosal layer. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bounded granules. Immunostaining of intestine with antibodies against the antimicrobial peptides piscidin 3 and piscidin 4 showed subpopulations of MCs that were positive. Piscidin-positive MCs were mainly observed among the epithelial cells of the intestine, but also within the submucosa. In both uninfected and parasite-infected perch, the number of MCs positive for piscidin 4 was higher than those immunoreactive with piscidin 3 (p < 0.05). For both piscidins, there was no significant difference in the number of positive MCs between parasite-infected and uninfected intestine (p > 0.05). However, uninfected fish showed higher immunostaining intensity for piscidin 3 than infected conspecifics (p < 0.05).

Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream.

Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (<15 parasites per fish) and heavily infected fish (>15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (MCs) were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P<0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections.

A novel heterophilic antibody interaction involves IgG4.

IgG4 has been implicated in a diverse set of complex pathologies - e.g. autoimmune pancreatitis (AIP), idiopathic membranous nephropathy - and carries unique features including lack of activation of the classical complement pathway and a dynamic Fab-arm exchange. We recently showed that the rheumatoid factor (RF)-like activity of IgG4 is achieved through a hitherto unknown, Fc-Fc (and not Fab-Fc as is the case in classical RF; CRF) interaction; hence the name, novel RF (NRF). Here, we further explore the resemblance/difference between CRF and NRF. As heterophilic interactions of human IgM RF (CRF) are well known, we checked whether this is the case for IgG4. Human IgG4 showed variable reactivity to animal IgGs: reacting intensely with rabbit and mouse IgGs, but weakly with others. The binding to rabbit IgG was not through the Fab (as in CRF) but via the Fc piece, as was recently shown for human IgG (NRF). This binding correlates with the IgG4 concentration per se and could therefore be of diagnostic usage and incidentally explain some observed interferences in biological assays. In conclusion, here is defined a novel heterophilic antibody interaction and is established the universality of the unique Fc-Fc binding, both involving IgG4.

Immunocytochemical localization of piscidin in mast cells of infected seabass gill.

Annual losses of approximately 5-10% of the juvenile stock of European seabass, Dicentrarchus labrax (L.) in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans. Immunocytochemical, light and ultrastructural studies were carried out on seabass naturally parasitized with this monogenean. The site of the worm's attachment was marked by the common presence of haemorrhages and white mucoid exudate. In histological sections, infected gills showed hyperplasia, as well as proliferation of mucous cells and rodlet cells. Disruption and fusion of the secondary lamellae were common in all infected seabass, with several specimens also showing marked inflammation and erosion of the primary and secondary lamellar epithelium. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of mast cells that were positive. Mast cells were both within and outside the blood vessels of the primary and secondary lamellae, and often made intimate contact with vascular endothelial cells. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Our data provide evidence showing the presence of piscidin 3 in the cytoplasmic granules of an important group of fish inflammatory cells, the mast cells resident in seabass gill tissue. There was no significant difference in the number of HAGR-positive mast cells between infected and uninfected fish (ANOVA, p > 0.05). However, mast cells in parasitized gills usually showed much stronger immunostaining intensity compared to those in unparasitized gills. These data are the first to document a response of piscidins or any other antimicrobial peptide of fish to parasite infection and suggest that mast cells may play a role in certain inflammatory responses without a detectable increase in their numbers.

Ultrastructural study on the body surface of the acanthocephalan parasite Dentitruncus truttae in brown trout.

Scanning electron microscope (SEM) investigations on the holdfast elements, proboscis hooks, and trunk spines of Dentitruncus truttae (Acanthocephala, Palaeacanthocephala), an endoparasite of Salmo trutta (brown trout), provide more data about the surface of these taxonomic relevant structures. In both acanthocephalan sexes, the fully everted cylindrical proboscis possessed 18 longitudinal rows of hooks with 18 hooks per row (rarely 19-20). Hook length varied according to position on the proboscis; apical hooks were 40-52 microm long, middle hooks were 31.7-36.6 microm, and basal hooks were 38.1-40 microm. Starting from the anterior end of the metasoma, numerous cuticular spines (26.7-30 microm in length) were visible and their number progressively decreased posteriorly. SEM observations of D. truttae hooks and spines revealed the presence of many surface striations on each proboscis hook. These surface striations were absent from trunk spines. From the base of the hook, the striations ran parallel toward the point of convergence. Additionally, survey of longitudinal and transversal sections of the hook using transmission electron microscope confirmed that the hook surface was not smooth. SEM comparison with the hooks of several palaeacanthocephalan species, as well as with the hooks of species belonging to Eoacanthocephala and Polyacanthocephala, indicated that the striations are currently exclusive to D. truttae proboscis hooks.

Ultrastructure of microtriches on the scolex of Cyathocephalus truncatus (Cestoda: Spathebothriidea).

The scolex surface of the mature spathebothriidean Cyathocephalus truncatus (Pallas, 1781), a parasite of the brown trout Salmo trutta fario L., was studied using scanning and transmission electron microscopy. A particular attention was paid to microtriches, unique structure on the surface of the Cestoda. The scolex of C. truncatus is covered with two types of filiform microtriches (filitriches): aciculate (approximately 3 microm long) and capillate (approximately 10 microm long). Capillate microtriches, which have never been reported in any other spathebothriideans, are described for the first time using transmission electron microscopy. The tegument covered with filiform microtriches only (no spiniform microtriches are present) is typical of cestode groups supposed to be the most basal, e.g., Gyrocotylidea, Spathebothriidea, and Caryophyllidea.

Histopathology and ultrastructure of Platichthys flesus naturally infected with Anisakis simplex s.l. larvae (Nematoda: Anisakidae).

The histopathology, ultrastructure, and immunohistochemistry of the alimentary canal of flounder Platichthys flesus (L.), naturally infected with the nematode Anisakis simplex s.l. (Rudolphi 1809) from the River Forth (Scotland), were investigated and described. Eight of the 16 flounders were infected with A. simplex s.l. larvae (L3); parasites were encapsulated by serosa on the external surface of the host's digestive tract (intensity of infection 1-8 parasites per host), although nematode larvae were found encysted under the peritoneal visceral serosa of the host spleen and liver and, occasionally, in the liver parenchyma (intensity of infection 3-10 parasites per host). In all sites, different structural elements were recognized within the capsule surrounding larvae. Among the epithelial cells of the intestine of 5 flounders with larvae encysted on external surface of the gut, the presence of several rodlet cells (RCs) was observed. Furthermore, often the occurrence of macrophage aggregates (MAs) was noticed in infected liver and spleen, mainly around the parasite larvae. Eight neuropeptide antisera were tested with immunohistochemistry methods on gut sections of 4 P. flesus infected with extraintestinal nematodes. Sections from the gut of 5 uninfected flounder were used for comparative purposes. In the tunica mucosa of parasitized P. flesus, several endocrine epithelial cells were immunoreactive to anti-CCK-39 (cholecystokinin 39) and -NPY (neuropeptide Y) sera; furthermore, in the myenteric plexus, a high number of neurons were immunoreactive to antibombesin, -galanin, and several to -NPY and -PHI (peptide histidine isoleucine) sera.

Spermiogenesis and sperm ultrastructure of Cyathocephalus truncatus (Pallas, 1781) Kessler, 1868 (Cestoda: Spathebothriidea).

Spermiogenesis and sperm ultrastructure of adult Cyathocephalus truncatus, a member of presumably basal group of "true" cestodes (Eucestoda), have been examined for the first time by using transmission electron microscopy. The process of sperm formation corresponds in basic pattern to that of the Pseudophyllidea. In addition, the 2 pairs of electron-dense attachment zones are present in median cytoplasmic process of C. truncatus. However, mature spermatozoa of C. truncatus differ significantly from those of the pseudophyllideans, especially in the morphology of the proximal and distal spermatozoon extremities. The proximal extremity of the mature spermatozoon lacks a crested body, which is present in more derived cestodes and some pseudophyllideans. The distal end of the mature spermatozoa exhibits different morphology in the gametes from testes and those from receptaculum seminis. New for the Eucestoda is a finding that a lateral cytoplasmic extension creates the distal end of the spermatozoa from testes, resembling sperm of some Monogenea and Digenea. In contrast, the distal extremity of the spermatozoa from receptaculum seminis contains only a nucleus. Despite the above-mentioned peculiarities, the ultrastructural data on sperm/spermiogenesis suggest close relationships of the Spathebothriidea and Pseudophyllidea.

[The bronchial response to inhaled formaldehyde]. / Le formaldéhyde inhalé et la réponse bronchique.

INTRODUCTION: Formaldehyde is an ubiquitous indoor chemical polutant. Occupational exposure to high concentrations has revealed its irritant and allergenic potential. Nevertheless, domestic exposure to low concentrations may also have an effect on respiratory health in a non-specific way, just as has been found for other pollutants. STATE OF KNOWLEDGE: Potentiation of the response to allergens has been observed in animals and children. This effect has also been found on respiratory symptoms, with a 39% increase in the risk of asthma for a domiciliary exposure of more than 60 microgrammes.m(-3). We have recently been able to show, in a study with asthmatics sensitised to house dust mite, that the response to allergen provocation was increased following a 30 minutes exposure to 100 microgrammes.m(-3) formaldehyde. VIEWPOINT AND CONCLUSIONS: All the data show that mild exposure to formaldehyde in the home is sufficient to provoke sensitisation and also an aggrevation of symptoms in patients with allergic asthma. Taking into account the published evidence it is advisable that the concentrations of formaldehyde in domestic products should be made known in order to improve domiciliary air quality.

Back to the future: bacteriophages as promising therapeutic tools.

Bacteriophages (phages), natural predators of bacteria, are becoming increasingly attractive in medical and pharmaceutical applications. After their discovery almost a century ago, they have been particularly instrumental in the comprehension of basic molecular biology and genetics processes. The more recent emergence of multi-drug-resistant bacteria requires novel therapeutic strategies, and phages are being (re)considered as promising potential antibacterial tools. Furthermore, phages are also used for other purposes, e.g. vaccine production, gene/drug carriers, bacterial detection and typing. These new alternative approaches using phages are of major interest and have allowed unexpected developments, from the decipherment of fundamental biological processes to potential clinical applications.

Common carp Cyprinus carpio responses to sub-chronic exposure to perfluorooctanoic acid.

Perfluorooctanoic acid (PFOA) is an important and diffuse perfluorinated alkylated substance, but knowledge of the toxicological effects of this endocrine disrupter in fish is limited. Adult common carp Cyprinus carpio, L. were exposed to 200 ng/l (a concentration reported in impacted aquatic ecosystems) and 2 mg/l PFOA solutions in a flow-through system for 56 days to determine tissue accumulation and histological alterations of the primary target organs. PFOA was extracted from blood, gill, liver, muscle, kidney, gonad, and brain by an ion-pairing liquid extraction procedure and quantified using high performance liquid chromatography with electrospray ionization tandem mass spectrometry. The limit of detection (LOD) was 0.4 ng/g wet weight (ww). PFOA was not detectable in unexposed fish or in fish exposed to 200 ng/l, but was >LOD in most samples of carp exposed to 2 mg/l. Mean PFOA concentration ranged from 0.5 to 65 ng/g ww, depending on the tissue, with highest levels in the blood and liver. There were no significant differences in condition factor, hepato-somatic index, or gonado-somatic index among the fish of the three groups. Histological, histochemical, and immunohistochemical staining was performed on sections of liver and gonad. Occurrence of atretic oocytes and a paucity of spermatozoa were documented in carp treated with 2 mg/l PFOA. Exposed fish did not show gross hepatic anomalies, but there was enhancement of hepatocytes in proliferation (positive to anti-PCNA antibody) compared to controls.

Fine structure of the male reproductive ducts, vagina and seminal receptacle of Cyathocephalus truncatus (Cestoda: Spathebothriidea).

Fine structure of the vas efferens, vas deferens, ejaculatory duct with accessory glands and vagina with seminal receptacle is described in the spathebothriidean tapeworm, Cyathocephalus truncatus (Pallas, 1781) Kessler, 1868. The numerous well-developed prostate glands are characterised by having secretory granules with an electron-dense core surrounded by a matrix of lower electron density. Coalescence of the outer part of the granules with each other takes place in the terminal end of the secretory ducts. The position of prostate glands around the proximal part of the cirrus pouch and terminating in the ejaculatory duct is a characteristic feature of the Spathebothriidea. Up to 20 closely arranged muscle layers make up the muscular cirrus pouch wall with 4 well-developed muscular layers in the ejaculatory duct and cirrus. Both the cirrus and the vagina are covered with the same uniform cone-shaped microtriches. The vagina has an extensive seminal receptacle. All of these structures are well-adapted to insure successful sperm transfer involving ejaculation and storage, probably for both self- and cross-insemination. Cyathocephalus truncatus has a cirrus similar to that of the monozoic, progenetic caryophyllidean, Archigetes sieboldi and well-developed prostate glands like those of the polyzoic pseudophyllidean, Diphyllobothrium latum. The ultrastructural aspects of the male and female reproductive system of C. truncatus are compared with those of other tapeworms.

Histopathology and the inflammatory response of European perch, Perca fluviatilis muscle infected with Eustrongylides sp. (Nematoda).

BACKGROUND: The European perch, Perca fluviatilis L. is a common paratenic host of dioctophymatid nematodes belonging to the genus Eustrongylides. In this host, once infected oligochaetes, which serve as the first intermediate host, are ingested, Eustrongylides migrates through the intestine and is frequently encountered within the musculature, free within the body cavity, or encapsulated on the viscera. The current study details the first Italian record of Eustrongylides sp. with larvae reported in the muscle of P. fluviatilis. METHODS: Uninfected and nematode-infected muscle tissues of perch were fixed and prepared for histological evaluation and electron microscopy. Some sections were subjected to an indirect immunohistochemical method using anti-PCNA, anti-piscidin 3 and anti-piscidin 4 antibodies. RESULTS: A total of 510 P. fluviatilis (TL range 15-25 cm) from Lake Trasimeno, Perugia were post-mortemed; 31 individuals had encysted nematode larvae within their musculature (1-2 worms fish(-1)). Histologically, larvae were surrounded by a capsule with an evident acute inflammatory reaction. Muscle degeneration and necrosis extending throughout the sarcoplasm, sarcolemmal basal lamina, endomysial connective tissue cells and capillaries was frequently observed. Within the encapsulating reaction, macrophage aggregates (MAs) were seen. Immunohistochemical staining with the proliferating cell nuclear antigen (PCNA) revealed numerous PCNA-positive cells within the thickness of the capsule and in the immediate vicinity surrounding Eustrongylides sp. larvae (i.e. fibroblasts and satellite cells), suggesting a host response had been initiated to repair the nematode-damaged muscle. Mast cells (MCs) staining positively for piscidin 3, were demonstrated for the first time in response to a muscle-infecting nematode. The piscidin 3 positive MC's were seen principally in the periphery of the capsule surrounding the Eustrongylides sp. larva. CONCLUSIONS: A host tissue response to Eustrongylides sp. larvae infecting the musculature of P. fluviatilis was observed. Numerous fibroblasts, MAs and MCs were seen throughout the thick fibroconnectival layer of the capsule enclosing larvae. PCNA positive cells within the capsule suggest that host repair of nematode damaged muscle does occur, while the presence of the antimicrobial peptide piscidin 3 is shown for the first time. This is first report of Eustrongylides sp. in an Italian population of P. fluviatilis.

Triplet repeat polymorphism in the MICA gene in HLA-B27 positive and negative caucasian patients with ankylosing spondylitis.

Previously, we reported a triplet repeat polymorphism in the transmembrane region within the MICA gene closely linked to HLA-B in a limited number of B27-positive Caucasian patients with ankylosing spondylitis (AS) (N = 48). In this study, we enrolled much more patients including some negative for B27, 162 AS subjects consisting of 140 B27-positive, and 22 B27-negative patients. The microsatellite allele consisting of 4 repetitions of (GCT/AGC) (A4 allele) was present at a significantly higher phenotype frequency in the patient group than in the ethnically matched control group (Pc < 0.000001). However, the frequency of the A4 allele was not significantly higher in the B27-positive and B27-negative patient groups, as compared to the B27-positive and B27-negative control groups, respectively. The higher phenotype frequency of the A4 allele in the patient group was supposed to be due to a strong linkage disequilibrium between the MICA and HLA-B genes. Thus, the possibility that the MICA gene is involved in the pathogenesis of AS can be excluded, supporting the hypothesis of a primary association of AS with HLA-B27.

The intestinal parasite Pomphorhynchus laevis (Acanthocephala) interferes with the uptake and accumulation of lead (210Pb) in its fish host chub (Leuciscus cephalus).

Uninfected chub as well as fish experimentally infected with the acanthocephalan parasite Pomphorhynchus laevis were exposed to (210)Pb(2+) for up to 38 days and the uptake and distribution of lead within different fish organs and the parasites was determined at various time points. Highest metal concentrations were detected in the acanthocephalans, followed by intestine, bile, liver, gill and muscle of the fish host. Infected chub had significantly lower (210)Pb levels in the gills on day 17 (P< or =0.01), in the bile on day 24 (P< or =0.05) and in the gills as well as in the intestine on day 38 compared with uninfected fish. A subsequent polynomial regression revealed that lead levels for the infected fish ranged below the levels determined for uninfected fish during most of the exposure period. This is the first proof that P. laevis reduces lead levels in the bile thereby diminishing or even impeding the hepatic intestinal cycling of lead, which may reduce the amount of metals available for the fish organs. This is especially important for ecotoxicological research. For example, organisms used as accumulation indicators may erroneously indicate low levels of pollution if they are infected with parasites which alter their pollutant uptake mechanisms. Additionally, the results gave further experimental evidence for acanthocephalans as accumulation indicators for metals.