Comparative Therapeutic Effects of Natural Compounds Against Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae)

The fish parasites Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae) cause important losses in freshwater and marine aquaculture industry, respectively. The possible adverse effects of compounds used to control these parasites in aquaculture resulted in increased interest on the search for natural products with antiparasitic activity. In this work, eighteen plant-derived compounds (2′,4′-Dihydroxychalcone; 7-Hydroxyflavone; Artemisinin; Camphor (1R); Diallyl sulfide; Esculetin; Eucalyptol; Garlicin 80%; Harmalol hydrochloride dihydrate; Palmatine chloride; Piperine; Plumbagin; Resveratrol; Rosmarinic acid; Sclareolide; Tomatine, Umbelliferone, and Usnic Acid) have been tested in vitro. Sixteen of these were used to determine their effects on the gill cell line G1B (ATCC®CRL-2536™) and on the motility of viable dinospores of Amyloodinium ocellatum, and thirteen were screened for inhibitory activity against Saprolegnia spp. The cytotoxicity results on G1B cells determined that only two compounds (2′,4′-Dihydroxychalcone and Tomatine) exhibited dose-dependent toxic effects. The highest surveyed concentrations (0.1 and 0.01 mM) reduced cell viability by 80%. Upon lowering the compound concentration the percentage of dead cells was lower than 20%. The same two compounds revealed to be potential antiparasitics by reducing in a dose-dependent manner the motility of A. ocellatum dinospores up to 100%. With respect to Saprolegnia, a Minimum Inhibitory Concentration was found for Tomatine (0.1 mM), Piperine and Plumbagin (0.25 mM), while 2′,4′-Dihydroxychalcone considerably slowed down mycelial growth for 24 h at a concentration of 0.1 mM. Therefore, this research allowed to identify two compounds, Tomatine and 2′,4′-Dihydroxychalcone, effective against both parasites. These compounds could represent promising candidates for the treatment of amyloodiniosis and saprolegniosis in aquaculture. Nevertheless, further in vitro and in vivo tests are required in order to determine concentrations that are effective against the considered pathogens but at the same time safe for hosts, environment and consumers

Red mark syndrome of trout (Oncorhynchus mykiss; Walbaum, 1792): Histopathological scoring and correlation with gross lesions

Red mark syndrome (RMS) is a skin disorder affecting rainbow trout (Oncorhynchus mykiss). The present work aimed to correlate the gross skin lesions affecting 46 fish sampled from farms surveyed for RMS with their microscopic features, identifying histological parameters that may be suggestive of disease progression. Skin lesions were grossly included in one of three categories (types I, II and III) according to the progressive degree of severity. Histological parameters and anti-proliferating cell nuclear antigen (PCNA) tissue immunoreactivity were semi-quantitatively assessed. In the dermis, PCNA-positive lymphocytes, fibroblasts and endothelial cells were indicative of active phlogosis. A significant increase in PCNA-immunoreactive lymphocytes, from gross type I to type III cases, was found only in the hypodermis. The histological parameters significantly associated with the gross lesion severity were progressive loss of the epithelium and scales, recruitment of inflammatory cells in the stratum compactum, loss of architecture of the stratum compactum, perivascular and perineural granulomatous inflammation and increase in lymphocyte infiltration of the muscular layer. In the type II and type III categories, inflammation in the hypodermis and muscle displayed a granulomatous pattern, reinforcing the hypothesis of an immunopathological mechanism. The morphological diagnosis of “deep chronic dermatitis associated to panniculitis and myositis, characterised by lympho-histiocytic and granulomatous reaction” is suggested

Egg-laying and locomotory screens with C. elegans yield a nematode-selective small molecule stimulator of neurotransmitter release

Nematode parasites of humans, livestock and crops dramatically impact human health and welfare. Alarmingly, parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides that protect crops are facing increasing restrictions because of poor phylogenetic selectivity. Here, we exploit multiple motor outputs of the model nematode C. elegans towards nematicide discovery. This work yielded multiple compounds that selectively kill and/or immobilize diverse nematode parasites. We focus on one compound that induces violent convulsions and paralysis that we call nementin. We find that nementin stimulates neuronal dense core vesicle release, which in turn enhances cholinergic signaling. Consequently, nementin synergistically enhances the potency of widely-used non-selective acetylcholinesterase (AChE) inhibitors, but in a nematode-selective manner. Nementin therefore has the potential to reduce the environmental impact of toxic AChE inhibitors that are used to control nematode infections and infestations

Biomaterials for Personalized Cell Therapy

Cell therapy has already had an important impact on healthcare and provided new treatments for previously intractable diseases. Notable examples include mesenchymal stem cells for tissue regeneration, islet transplantation for diabetes treatment, and T cell delivery for cancer immunotherapy. Biomaterials have the potential to extend the therapeutic impact of cell therapies by serving as carriers that provide 3D organization and support cell viability and function. With the growing emphasis on personalized medicine, cell therapies hold great potential for their ability to sense and respond to the biology of an individual patient. These therapies can be further personalized through the use of patient-specific cells or with precision biomaterials to guide cellular activity in response to the needs of each patient. Here, the role of biomaterials for applications in tissue regeneration, therapeutic protein delivery, and cancer immunotherapy is reviewed, with a focus on progress in engineering material properties and functionalities for personalized cell therapies.Juvenile Diabetes Research Foundation (Grant 2-SRA-2019-714-S-B)Leona M. and Harry B. Helmsley Charitable Trust (Grant 2017PG-T1D027

Contribution on nflammation/cell proliferation markers to the study of RMS pathogenesis

Red mark syndrome (RMS) is a chronic non-lethal skin disease affecting rainbow trout (O. mykiss). It was first noticed in Scotland in 2003, spread rapidly throughout the U.K. and has also been reported in Austria, Germany, Italy, Serbia and U.S.A. Single/multiple skin lesions are macroscopically detectable on the flanks and ventral/dorsal body surfaces, ranging from small pink spots to large bright red areas. Histology shows a lymphocytic/macrophage infiltration in the dermis, enlargement of scale pockets and necrosis of the surrounding connective tissue. Advanced stages are characterised by osteoclastic resorption of scales, and lymphocytes infiltration into the subcutaneous adipose tissue reaching the underling muscle bundles. In the present study, samples of skin and internal organs from naturally infected trouts farmed in northern Italy were evaluated macroscopically and histologically; an immunohistochemical approach was also carried out aiming to study inflammatory cells recruitment and proliferation rate. The evaluation was performed considering the severity of the skin lesion according to Galeotti et al., 2011. The following markers were performed on 4 \ub5m-thick sections from formalin/Bouin\u2019s fixed paraffin embedded specimens: rabbit anti human CD3 (A-0452, Dako); rabbit anti rainbow trout IgT (Prof. O. Sunyer); rabbit anti salmonid HSP70 (AS05061A, Agrisera); rabbit anti human GM-CSFR\u3b1 (sc-690, Santa Cruz Biotech.); rabbit anti histamine (H7403, Sigma-Aldrich); rabbit anti-IL-1\uf062 (Santa Cruz Biotech.); mouse anti PCNA (2586, Cell Signaling Technology); mouse anti AE1/AE3 Cytokeratin (M3515, Dako); mouse anti E Cadherin (M3612, Dako). Anti CD3 and IL-1\uf062 did not cross-react. Anti trout IgT marked a limited number of scattered cells in the dermis, and numerous cells at the base of the intestinal mucosae. HSP70 positive cells were detectable within scale pockets involved in inflammatory changes. Numerous GM-CSFR\u3b1 positive macrophage like-cells and some positive fibroblasts were scattered in the spongiosum derma, especially surrounding the scales. Anti-Cytokeratin and E Cadherin marked the epithelial cells. PCNA positive cells have been semiquantitatively scored (0=absence of staining; 1=up to 25% or 2=25-50% or 3=50-75% or 4=>75% positive cells) separately in epidermis, dermis and hypodermis. In the two latter mesenchymal areas PCNA positive cells were apart scored in infiltrating lymphocytes, vascular endothelium and stromal fibroblasts. Data were compared by the nonparametric Spearman correlation test among lesions macroscopically graded as initial, intermediate and severe. Any difference in proliferation was found in epidermis and in the 3 dermal components (3 compared groups). In hypodermis any reaction was apparent in endothelium and in fibroblasts but only lymphocytes were PCNA immuno-reactive. Statistic revealed, only for these latter, an increasing percentage of proliferating lymphocytes from initials to severe lesions

CONTRIBUTION OF CELL MARKERS TO THE STUDY OF RMS PATHOGENESIS

Red mark syndrome (RMS) is a chronic non-lethal skin disease affecting farmed rainbow trout (O. mykiss) in U.K., Austria, Germany, Italy (Galeotti et al., 2011), Serbia and U.S.A. Histology shows a lymphocyte/macrophage infiltration in scale pockets, dermis and ipodermis. Aiming at the full comprehension of RMS aetio-pathogenesis, we focused on the mechanisms of cell recruitment/activation in the skin lesions, in order to elucidate if and how an hypothetical microbial agent might trigger the host inflammatory response. Samples of skin from infected fish were evaluated by histology, immunohistochemistry and electron microscopy. The following markers were used: rabbit to human CD3 (A-0452, Dako); rabbit to rainbow trout IgT/IgM (Prof. Sunyer); rabbit to salmonid HSP70 (AS05061A, Agrisera); rabbit to human GM-CSFR\u3b1 (sc-690, Santa Cruz Biotech.); mouse to PCNA (2586, Cell Signaling Technology); mouse to AE1/AE3 Cytokeratin (M3515, Dako); mouse to E Cadherin (M3612, Dako). Anti trout IgT and IgM marked a limited number of scattered cells in the dermis and ipodermis. Anti CD3 marked a relevant number of cells composing the skin infiltrate. HSP70 marked monocyte/macrophages, dendritic like-cells and endothelial cells, within the scale pockets involved by inflammation. GM-CSFR\u3b1 positive monocyte-macrophage were scattered in the derma, surrounding the scales. Anti-Cytokeratin and E Cadherin marked the epithelial cells. PCNA positive cells have been detected in epidermis, dermis and hypodermis, as well among infiltrating lymphocytes, stromal fibroblasts and vascular endothelial cells. HSP70 are considered not only as acute phase proteins, but also as molecules able to mediate immunity and inflammation (Pockley, 2003); they can be released by several cell populations in response to various stimuli. Briefly HSP70 could act as an \u201cantigen\u201d inducing a severe T lymphocyte response, leading to an auto-immune like reaction. Dendritic cells and APCs are stimulated by HSP70 to release TNF-\u3b1, IL1-\uf062and GM-csf. We might speculate that a microbial agent promotes HSP70 expression by macrophages/endothelial cells, within scale pockets. HSP70 might be also internalized by skin APCs. The pro-inflammatory cytokines released could then trigger the local inflammatory process. The GM-csf stimulates the development of osteoclasts. Skin APCs could express HSP70 and therefore stimulate T cell proliferation. These findings might justify the severe cell infiltration detectable in the skin of RMS affected fish

Dioxin-like Compounds in Lake Fish Species: Evaluation by DR-CALUX Bioassay

Fish consumption is the principal source of intake of organochlorinated compounds in humans. Compared with other types of foods of animal origin, \ufb01sh contain the highest levels of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans, all of which are classi\ufb01ed as highly toxic organochlorine compounds. Currently, lakes and \ufb01sh farms in northern Italy are not regularly monitored for PCBs and dioxins in areas contaminated by industrial sources, partially because of the high costs of traditional analytical methods that limit the number of samples to be analyzed. The DR-CALUX cell bioassay is based on the uptake of the cellular aryl hydrocarbon receptor (AhR) for dioxins and dioxin-like compounds. The aim of this study was to assess the levels of dioxins and dioxin-like PCB contamination in Lake Maggiore and Lake Como, two lakes in northwestern Italy, and in nearby areas. The levels were quanti\ufb01ed using the cell bioassay DR-CALUX and reference controls in two wild \ufb01sh species, perch (Perca \ufb02uviatilis) and roach (Rutilus rutilus), and in a farmed species, rainbow trout (Oncorhynchus mykiss). Tissue samples collected from the farmed rainbow trout were also submitted to immunohistochemical analysis of CYP1A expression as a marker for environmental pollutant-induced liver damage. The levels of dioxins, furans, and dioxin-like PCBs were all below the maximum levels and action limits set by European Union Regulation, suggesting no risk for human health associated with the consumption of the \ufb01sh species caught or farmed in these areas

Microgel encapsulated nanoparticles for glucose-responsive insulin delivery

An insulin delivery system that self-regulates blood glucose levels has the potential to limit hypoglycemic events and improve glycemic control. Glucose-responsive insulin delivery systems have been developed by coupling glucose oxidase with a stimuli-responsive biomaterial. However, the challenge of achieving desirable release kinetics (i.e., insulin release within minutes after glucose elevation and duration of release on the order of weeks) still remains. Here, we develop a glucose-responsive delivery system using encapsulated glucose-responsive, acetalated-dextran nanoparticles in porous alginate microgels. The nanoparticles respond rapidly to changes in glucose concentrations while the microgels provide them with protection and stability, allowing for extended glucose-responsive insulin release. This system reduces blood sugar in a diabetic mouse model at a rate similar to naked insulin and responds to a glucose challenge 3 days after administration similarly to a healthy animal. With 2 doses of microgels containing 60 IU/kg insulin each, we are able to achieve extended glycemic control in diabetic mice for 22 days.National Cancer Institute (Grant P30-CA14051

In field study on immune-genes expression during a lactococcosis outbreak in rainbow trout (Oncorhynchus mykiss)

Italian rainbow trout (Oncorhynchus mykiss) farms are periodically affected by outbreaks of lactococcosis caused by Lactococcus garvieae, and rearing basins reveal simultaneously asymptomatic and symptomatic fish. The present study, as part of an “in field” sanitary survey, was designed to describe the expression of immune related genes in infected versus healthy fish, as well as to explore a Near Infrared Spectroscopy (NIR) as a novel approach to discriminate between them. Measurements/sampling were performed in a farm of Northern Italy and included two groups of fish (150–300 g), sharing the same basin: asymptomatic (n = 10) and symptomatic (n = 9). Fingerling (n = 5, 15 g) were also sampled from the farm internal hatchery and considered as L. garvieae-free reference group. NIR spectra were collected from the abdominal region of the fish through the SCiO molecular device. Spleen and head kidney were sampled for bacterial detection (PCR). Head kidney was also subjected to gene expression analysis (qPCR). Seven out of nine (7/9) symptomatic trout were L.garvieae positive (bacterial DNA) while all the asymptomatic (10/10) and fingerling (2/2) were L. garvieae negative. SCiO scanning provided two different spectral populations, almost coinciding with the two fish groups considered (asymptomatic and symptomatic). Among cytokines, IL-1β showed significant up-regulation in symptomatic group compared to asymptomatic group (P < 0.05) while IL-8 showed very high (P < 0.01) and high significant (P < 0.05) differences in its up-regulation in symptomatic group compared to asymptomatic group and fingerling, respectively. IL-10 showed a tendency of significant over-expressed in symptomatic fish compared to fingerling (P < 0.1). TCR-β showed significant (P < 0.05) down-regulation in symptomatic fish compared to asymptomatic ones and MHC-II resulted in down-regulation in a very significant (P < 0.01) manner in symptomatic trout compared to fingerling and asymptomatic. Concerning immunoglobulin genes expression, the level of mRNA transcript of IgT was significantly higher in symptomatic trout compared to fingerling (P < 0.05) and tended to be higher in asymptomatic trout compared to fingerling (P < 0.1). No significant differences were observed for IL-6, TNF-α, TLR5, MHC-I and IgM. In this study, a NIR based approach was used for the first time in the field of fish pathology/immunology with the aim of differentiating between healthy and diseased fish. Moreover, our results on immune related genes modulation highlighted how the outcome of a L. garvieae infection might be influenced by a specific gene expression pattern, which is crucial in the mode of action of trout immune system against L. garvieae