Characterization of the immune cells response and ultrastructural study of dendritic cell Golgi Apparatus role in ORF virus infection

Contagious Ecthyma is an acute skin anthropozoonosis caused by orf virus (ORFV), which affects sheep and goat. The infectious agent is an epitheliotropic, double- stranded DNA poxvirus. Infection happens via the hurt skin, and causes a localized virus production in the epidermal cells and keratinocytes. This paper characterize the cellular immune response by cytochemistry in ORFV infection and studies the role of Golgi Apparatus (GA) of keratinocytes by transmission electron microscopy (TEM) and 3D models. Twenty cutaneous biopsies in sheep from ORFV infected lesions were fixed in 10% formalin and embedded in paraffin for light microscopy. Paraffin sections were immunocytochemically stained (DAKO LSAB+/HRP) to identify major histocompatibility complex (MHC) II, CD1, CD79a, CD3, TCRγδ, and CD21. Furthermore, a portion from the biopsies was fixed with 1% glutaraldehyde/0.1M Na-cacodylate buffer, post-fixed in cacodylate buffer/1% osmium tetroxide and embedded in Durcupan for TEM. Ultrathin sections were observed and marked for MHC II with 10 nm gold particles. Three-dimensional computer-generated GA models were reconstructed from serial ultrathin sections. Light microscopy images showed keratinocites positive for MHC II and negative for CD1, supporting the idea that Langerhans cells start the skin immune response. Positive cells for TCRγδ/CD3, CD21 and CD79a suggested the prevailing attraction for dendritic cells instead of T and B cells. Transmission electron micrographs revealed poxvirus-like particles. The images of GA showed the presence of large structures with bud profiles in the process of fusing with a Golgi stack and appeared emptying its ORFV content into it. ORFV induced swelling of Golgi vesicles, and perforation and fragmentation of GA, leading to a disorganized structure. Lateral vesicles close the Golgi stack were positive for MHC II, but in heavy infected cells the Golgi structures positive for MHC II were dispersed into the cytoplasm. An increased distance between GA and cell nucleus in infected cells was detected. Computer 3D reconstructions clarify the dynamics of GA in ORFV infected cell. Overall, these results suggest that ORFV targets the vesicular export machinery and the structure and function of the GA might aid to escape cellular immune recognition

Anisakis pegrefii and Anisakis simplex sensu strictu in Mediterranean sea

BACKGROUND: Anisakiasis is a parasitic zoonosis caused by ingestion of nematode larvae belonging to Anisakidae family, following consumption of raw, undercooked or improperly processed fish. Mediterranean sea represent an aquatic ecosystem particularly suitable for the development of Anisakid larvae. The aim of this work is to found intra-species and inter-species nucleotide differences by phylogenetic analysis in this geographical area. METHODS: In the period from January to November 2013, 584 fish from Mediterranean sea were screened, and they were found parasitised of 6318 type I anisakid larvae. Fish were eviscerated and observed by stereo microscope to collect larvae relived in the viscera, organs and muscles. After genus identification, the larvae were subjected to molecular analysis by extraction of DNA, amplification of ITS gene and restriction enzyme. PCR products were sequenced and the sequences were analyzed and aligned to examine the relationship of nucleotides. RESULTS: The anisakid species we have identified were Anisakis pegreffii and Anisakis simplex s.s.. Phylogenetic analysis detects nucleotide differences between the two species. In the positions 251 and 267 was found a Cytosine in Anisakis pegreffii and a Thymine in Anisakis simplex s.s., respectively. No difference was found in Anisakis pegreffii specie becoming from different fish of different areas. CONCLUSIONS:. The parasite DNA were amplified and sequenced to identify any nucleotide differences between the different species as well as within the same species.No intra-species sequence differences were found in Anisakis pegreffii. Two inter-species differences were found between Anisakis pegreffii and Anisakis simplex. Further studies will be conducted to confirm nucleotide differences in other target genes

Characterization of the immune cells response and ultrastructural study of dendritic cell Golgi Apparatus role in ORF virus infection

Contagious Ecthyma is an acute skin anthropozoonosis caused by orf virus (ORFV), which affects sheep and goat. The infectious agent is an epitheliotropic, double- stranded DNA poxvirus. Infection happens via the hurt skin, and causes a localized virus production in the epidermal cells and keratinocytes. This paper characterize the cellular immune response by cytochemistry in ORFV infection and studies the role of Golgi Apparatus (GA) of keratinocytes by transmission electron microscopy (TEM) and 3D models. Twenty cutaneous biopsies in sheep from ORFV infected lesions were fixed in 10% formalin and embedded in paraffin for light microscopy. Paraffin sections were immunocytochemically stained (DAKO LSAB+/HRP) to identify major histocompatibility complex (MHC) II, CD1, CD79a, CD3, TCRγδ, and CD21. Furthermore, a portion from the biopsies was fixed with 1% glutaraldehyde/0.1M Na-cacodylate buffer, post-fixed in cacodylate buffer/1% osmium tetroxide and embedded in Durcupan for TEM. Ultrathin sections were observed and marked for MHC II with 10 nm gold particles. Three-dimensional computer-generated GA models were reconstructed from serial ultrathin sections. Light microscopy images showed keratinocites positive for MHC II and negative for CD1, supporting the idea that Langerhans cells start the skin immune response. Positive cells for TCRγδ/CD3, CD21 and CD79a suggested the prevailing attraction for dendritic cells instead of T and B cells. Transmission electron micrographs revealed poxvirus-like particles. The images of GA showed the presence of large structures with bud profiles in the process of fusing with a Golgi stack and appeared emptying its ORFV content into it. ORFV induced swelling of Golgi vesicles, and perforation and fragmentation of GA, leading to a disorganized structure. Lateral vesicles close the Golgi stack were positive for MHC II, but in heavy infected cells the Golgi structures positive for MHC II were dispersed into the cytoplasm. An increased distance between GA and cell nucleus in infected cells was detected. Computer 3D reconstructions clarify the dynamics of GA in ORFV infected cell. Overall, these results suggest that ORFV targets the vesicular export machinery and the structure and function of the GA might aid to escape cellular immune recognition

Analysis of Body Perception, Preworkout Meal Habits and Bone Resorption in Child Gymnasts

The beneficial effects of physical activity on body image perception and bone are debated among artistic gymnasts. Gymnasts seem to be at greater risk of developing body dissatisfaction, eating disorders and osteoporosis due to inadequate nutrition and attention to the appearance of the body. The objective of this work was to investigate the association between the artistic gymnast and a more favorable body image compared to their sedentary peers and if a preworkout high-carbohydrate meal (HCM; 300 kcal, 88% carbohydrates, 9% protein, 3% fat) or high-protein meal (HPM; 300 kcal, 55% carbohydrates, 31% protein, 13% fat) is able to attenuate bone resorption in young rhythmic gymnasts. Twenty-eight preadolescent female gymnasts were examined. Self-esteem tests were used to analyze body image perception. Preworkout eating habits were examined by short food frequency questions (FFQ) validated for children. The biomarker of the bone resorption C-terminal telopeptide region of collagen type 1 (CTX) was measured in the urine (fasting, postmeal and postworkout). Gymnasts reported higher satisfaction with their body appearance compared to sedentary peers. Of the gymnasts, 30% did not have a preworkout meal regularly, and the timing of the consumption was variable. Bone resorption was decreased by the HCM, consumed 90 min before the training, with respect to the HPM. The study suggests that playing artistic gymnastics is associated with a positive body self-perception in a child. The variability in preworkout meal frequency and timing need attention to prevent inadequate eating habits in light of the ability of the HCM to reduce acute bone resorption

Changes in eggshell ultrastructure of Falco naumanni and Tyto alba exposed to pesticides and polychlorinated biphenyls (PCBs)

Changes in the quality of eggs of birds exposed to environmental contaminants have been described, but few reports concern eggshell ultrastructure. In this study, infertile or addled Lesser Kestrels (Falco naumanni) and Barn owls (Tyto alba) eggs were collected from the polluted area of Gela plain (Sicily) during 2007, and compared in terms of organophosphate and organochlorine pesticides, and PCBs levels, and eggshell ultrastructure as determined by scanning electron microscopy. Pesticide and PCB residues in eggs were determined by Gas chromatography/ mass spectrometry (GC/MS) [GC Agilent 7890A/MS Agilent 5975C (Agilent technologies) using a DB-5 capillary column in the selected ion monitoring mode]. The GC/MS analysis revealed that eggs contained measurable amounts of some pesticides and PCBs. There was a low detection of organophosphate pesticides while the most abundant organochlorine residues detected were p,p’ DDT, p,p’ DDE, and Hexachlorobenzene. While, the most abundant PCBs detected congeners were PCB 138, 153, 170, 180, and 187. Although the general structure of the eggshell layers was maintained, the results showed ultrastructural differences in mammillary and palisade eggshell layers between high level and low level contaminated eggs in Tyto alba. Furthermore, mammillary cores of the eggshell had an increased distance between themselves with respect to well organized structures present in uncontaminated egg. In this paper we verify the presence of environmental contaminants in the eggs and document structural changes in bird of prey eggshells. The data could suggest that some contaminants can contribute to reduced reproductive performance (infertile or addled egg) by structural changes in the eggshell. The alteration in morphological disposition of mammillary cores could also suggest an impairment of gas exchange

A histological study of eosinophilic granuloma in mice following infestation with Anisakis larvae

Anisakidosis is a parasitic anthropozoonosis caused by the nematode larvae of the Anisakidae family. Anisakid nematodes belonging to the Anisakis pegreffii are highly prevalent in several fish species living in the Mediterranean sea. The larvae can accidentally infect humans following the ingestion of infected raw or undercooked sea fish. A migrating larva causes a clinical disease when it invades the stomach or intestinal wall and peritoneum, mimicking an eosinophilic gastroenteritis or an ulcer. In this preliminary experiment, the histopathology of the newly-formed parasitic granulomas in mice infested with third stage Anisakis pegreffii larvae, was studied and described. The larvae were morphologically identified as Anisakis type I by the presence of a boring tooth and a mucron, without ventriculus and caecum. The larval DNA was extracted and amplified by polymerase chain reaction (PCR). After PCR, the samples were processed to undergo restriction fragment length polymorphism analysis (RFLP). This was done to scan the restriction profiles for genetic identification. PCR products were purified and sequenced. The sequences were analysed to detect the relationship between nucleotides and perform a phylogenetic analysis. The paraffin-embedded granuloma samples showed worms having a diameter of 0.55 mm x 0.37 mm, polymyarian muscle cells and a circular intestine. The histological profiles showed a primary lesion at the site of anisakid penetration marked by oedema and neutrophil and eosinophil infiltration. The presence of histiocytes or epithelioid histiocytes, lymphocytes, monocytes, and plasma cells was also possible. Fibrinous exudation, hemorrhage, or vascular damage were detected within the first week of the acute intestine infection with a massive eosinophilic infiltration. Two weeks after the infestation, the infiltrating host cells formed a granuloma in the tissue surrounding the penetrated worm mainly consisting of eosinophils, a large number of fibroblasts and a varying number of admixed multinucleated giant cells. In order to explain the origin of the eosinophilic granulomas, a study into the produced substance attracting eosinophils and other host white blood cells to the area will carry out

Chronic administration of green tea extract to TRAMP mice induces the collapse of Golgi apparatus in prostate secretory cells and results in alterations of protein post-translational processing.

Considering its long latency, prostate cancer (PCa) represents an ideal target for chemoprevention strategies. Green tea extract (GTE) has been proved to be one of the most promising natural substances capable of inhibiting PCa progression in animal models (transgenic adenocarcinoma of mouse prostate), as well as in humans. However, the cellular targets of the GTE action are mostly unknown. The main objective of this work was to investigate whether the endoplasmic reticulum (ER) and the Golgi apparatus (GA), known to be actively involved in sensing stress stimuli and initiating and propagating cell death signalling, may represent the subcellular targets of GTE action. To this end, 42 TRAMP mice were divided into four experimental groups: groups II and IV, received GTE in tap water (0.3 g/100 ml solution) starting at 8 weeks of age and up to the time of sacrifice. Groups I and III were respective age-matched water-fed controls. The animals were sacrificed after 4 weeks (groups I and II) or 40 weeks of treatment (groups II and IV). We also treated TRAMP-C2 cells with GTE (20 µg/ml for 7 days) to check the expression profile of clusterin (CLU), a protein involved in prostate tumourigenesis, extensively processed through ER-GA before being secreted through the plasma membrane. In vivo we found that chronic administration of GTE in TRAMP mice results in collapse of ER and GA in prostate epithelial cells. Consistently, in vitro we found that the mature, fully processed form of CLU, sCLU, is strongly reduced by GTE treatment in TRAMP-C2 cells. Taking into account the sCLU biogenesis dependence on the ER-GA integrity and the proposed anti-apoptotic role of sCLU, the possibility for GTE to counteract PCa progression by interfering with sCLU biogenesis is suggested

In vivo Micro-computed tomography imaging of adipose tissue of mice fed Sicilian pistachio

In obesity condition the adipose tissue undergoes molecular and cellular alterations affecting systemic metabolism via the release of different pro-inflammatory mediators. Nut-derived polyphenols and fatty acids have a documented role in the modulation of energy metabolism and antiobesity effect. We investigate the effects of Pistachia vera nuts on body fat mass and its distribution in a mouse models of obesity by Micro-computed tomography (CT). For this purpose, three groups of C57BL/6J male mice were fed for 16 weeks with a standard chow, a high fat diet (HFD) or HFD supplemented with pistachio harvested in plantation of the valley of the Platani river (Sicily). Fatty acids extraction from Pistachia vera nuts was carried out and analysed by a gas-chromatographic technique for contributing 20% of total diet energy. Total body fat mass was estimated and compared between groups by micro-CT (Quantum FX Micro CT scanner). Furthermore, the effects of pistachio consumption on total cholesterol, triglycerides, fat liver accumulation, adipose tissue inflammatory cytokines (TNFα and IL-1β) expression and serum ROS levels were also evaluated. The micro-CT slices and three-dimensional image stacks of control and obese mice showed a reduced body fat deposit and less visceral fat in mice fed HFD supplemented with pistachios. Furthermore, cholesterol and triglycerides levels, hepatic lipid accumulation, pro-inflammatory cytokines and serum ROS levels were significantly reduced in mice fed HFD supplemented with pistachio compared with the HFD mice. Our results suggest that pistachio consumption could have counteracting effects on metabolic dysfunctions in obesity through acting on the fat mass. In particular, it lower the accumulation of visceral and subcutaneous fat that could be responsible of lower level of circulating and hepatic lipids and of the decreased inflammatory condition

Changes in eggshell ultrastructure of Falco naumanni and Tyto alba exposed to pesticides and polychlorinated biphenyls (PCBs)

Changes in the quality of eggs of birds exposed to environmental contaminants have been described, but few reports concern eggshell ultrastructure. In this study, infertile or addled Lesser Kestrels (Falco naumanni) and Barn owls (Tyto alba) eggs were collected from the polluted area of Gela plain (Sicily) during 2007, and compared in terms of organophosphate and organochlorine pesticides, and PCBs levels, and eggshell ultrastructure as determined by scanning electron microscopy. Pesticide and PCB residues in eggs were determined by Gas chromatography/ mass spectrometry (GC/MS) [GC Agilent 7890A/MS Agilent 5975C (Agilent technologies) using a DB-5 capillary column in the selected ion monitoring mode]. The GC/MS analysis revealed that eggs contained measurable amounts of some pesticides and PCBs. There was a low detection of organophosphate pesticides while the most abundant organochlorine residues detected were p,p’ DDT, p,p’ DDE, and Hexachlorobenzene. While, the most abundant PCBs detected congeners were PCB 138, 153, 170, 180, and 187. Although the general structure of the eggshell layers was maintained, the results showed ultrastructural differences in mammillary and palisade eggshell layers between high level and low level contaminated eggs in Tyto alba. Furthermore, mammillary cores of the eggshell had an increased distance between themselves with respect to well organized structures present in uncontaminated egg. In this paper we verify the presence of environmental contaminants in the eggs and document structural changes in bird of prey eggshells. The data could suggest that some contaminants can contribute to reduced reproductive performance (infertile or addled egg) by structural changes in the eggshell. The alteration in morphological disposition of mammillary cores could also suggest an impairment of gas exchange

A novel enzyme blend for efficient tissue dissociation and primary cells isolation

Tissue dissociation/primary cell isolation and cell harvesting are principal appli- cations for enzymes in tissue culture research and cell biology studies. The goal of a cell isolation procedure is to maximize the yield of functionally viable dissoci- ated cells. Among the parameters which affect the outcome of any particular dissociating procedure there are enzyme(s) used and related impurities presents in crude enzyme preparation. ABIEL srl recently produced the recombinant collagenase class I (Col G) and II (Col H) from Clostridium histolyticum (PCT WO 2011/073925 A9). The enzymes were produced in Escherichia coli and purified by affinity chromatography. The method of production adopted allows absolute control of the final composition of these enzymes, as well as their stability, purity, activity, absence of toxicity and higher reproducibility of batches. The two collagenases produced separately have been used in conjunction according to precise proportions to dissociate calvaria, liver, pancreas, retina of the BALB/c mouse; and bovine hoof. The analyses carried out on all isolated cell populations suggest that the cells maintain the structural and functional integrity of specific tissues/organs originating. Recombinant Col G and Col H enzymes produced by ABIEL are promising in the context of the tissue/cells dissociation, with the aim to make innovation in the fields of tissue engineering and transplantation medicine