Olive Mill Waste-Water Extract Enriched in Hydroxytyrosol and Tyrosol Modulates Host-Pathogen Interaction in IPEC-J2 Cells.

The dietary supplementation of olive oil by-products, including olive mill waste-water (OMWW) in animal diets, is a novel application that allows for their re-utilization and recycling and could potentially decrease the use of antibiotics, antimicrobial resistance risk in livestock species, and the occurrence of intestinal diseases. Salmonella serovar typhimurium is one of the most widespread intestinal pathogens in the world, causing enterocolitis in pigs. The aim of this study was to investigate the effect of an OMWW extract enriched in polyphenols (hydroxytyrosol and tyrosol) in the immune response of an intestinal porcine epithelial cell line (IPEC-J2) following S. typhimurium infection. Cells were pre-treated with OMWW-extract polyphenols (OMWW-EP, 0.35 and 1.4 µg) for 24 h and then infected with S. typhimurium for 1 h. We evaluated bacterial invasiveness and assayed IPEC-J2 gene expression with RT-qPCR and cytokine release with an ELISA test. The obtained results showed that OMWW-EP (1.4 µg) significantly reduced S. typhimurium invasiveness; 0.35 µg decreased the IPEC-J2 gene expression of IL1B, MYD88, DEFB1 and DEFB4A, while 1.4 µg down-regulated IL1B and DEFB4A and increased TGFB1. The cytokine content was unchanged in infected cells. This is the first study demonstrating the in vitro immunomodulatory and antimicrobial activity of OMWW extracts enriched in polyphenols, suggesting a protective role of OMWW polyphenols on the pig intestine and their potential application as feed supplements in farm animals such as pigs.

Vulvo-vaginal epithelial tumors in mares: A preliminary investigation on epithelial-mesenchymal transition and tumor-immune microenvironment.

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and ß-catenin expression as well as nuclear ß-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and ß-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/ß-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions.

Bovine Papillomavirus Type 1 Infection in an Equine Congenital Papilloma.

Papillomas are benign epithelial lesions protruding on the epithelial surfaces as finger-like or warty projections. These lesions are often caused by papillomavirus (PV) infection. Congenital papillomas have been reported in foals. However, to date, no evidence of PV infection has been provided. In the present paper, we describe the main clinical-pathological features of a congenital papilloma observed in a foal. In addition, biomolecular tests demonstrated BPV1 infection in the case under study. Such data stimulate further investigations, even on archived samples, aiming to clarifying the etiology of equine congenital papilloma and the clinical relevance, if any, of BPV1 vertical transmission in horses.

Epithelial-mesenchymal transition in an EcPV2-positive vulvar squamous cell carcinoma of a mare.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) has been recently associated with Equus caballus papillomavirus type 2 (EcPV2) infection. Still, few reports concerning this disease are present in the literature. OBJECTIVE: To describe a case of naturally occurring EcPV2-induced VSCC, by investigating tumour ability in undergoing the epithelial-to-mesenchymal transition (EMT). STUDY DESIGN: Case report. METHODS: A 13-year-old Haflinger mare was referred for a rapidly growing vulvar mass. After surgical excision, the mass was submitted to histopathology and molecular analysis. Histopathological diagnosis was consistent with a VSCC. Real-time qPCR, real-time reverse transcriptase (RT)-qPCR and RNAscope were carried out to detect EcPV2 infection and to evaluate E6/E7 oncogenes expression. To highlight the EMT, immunohistochemistry (IHC) was performed. Expression of EMT-related and innate immunity-related genes was investigated through RT-qPCR. RESULTS: Real-time qPCR, RT-qPCR and RNAscope confirmed EcPV2 DNA presence and expression of EcPV2 oncoproteins (E6 and E7) within the neoplastic vulvar lesion. IHC highlighted a cadherin switch together with the expression of the EMT-related transcription factor HIF1α. With RT-qPCR, significantly increased gene expression of EBI3 (45.0 ± 1.62, p < 0.01), CDH2 (2445.3 ± 0.39, p < 0.001), CXCL8 (288.7 ± 0.40, p < 0.001) and decreased gene expression of CDH1 (0.3 ± 0.57, p < 0.05), IL12A (0.04 ± 1.06, p < 0.01) and IL17 (0.2 ± 0.64, p < 0.05) were detected. MAIN LIMITATIONS: Lack of ability to generalise and danger of over-interpretation. CONCLUSION: The results obtained were suggestive of an EMT event occurring within the neoplastic lesion.

Toll-like Receptors and Cytokine Modulation by Goat Milk Extracellular Vesicles in a Model of Intestinal Inflammation.

Extracellular vesicles (EVs) are nanometric spherical structures, enclosed in a lipid bilayer membrane and secreted by multiple cell types under specific physiologic and pathologic conditions. Their complex cargo modulates immune cells within an inflammatory microenvironment. Milk is one of the most promising sources of EVs in terms of massive recovery, and milk extracellular vesicles (mEVs) have immunomodulatory and anti-inflammatory effects. The aim of this study was to characterize goat mEVs' immunomodulating activities on Toll-like receptors (TLRs) and related immune genes, including cytokines, using a porcine intestinal epithelial cell line (IPEC-J2) after the establishment of a pro-inflammatory environment. IPEC-J2 was exposed for 2 h to pro-inflammatory stimuli as a model of inflammatory bowel disease (IBD), namely LPS for Crohn's disease (CD) and H2O2 for ulcerative colitis (UC); then, cells were treated with goat mEVs for 48 h. RT-qPCR and ELISA data showed that cell exposure to LPS or H2O2 caused a pro-inflammatory response, with increased gene expression of CXCL8, TNFA, NOS2 and the release of pro-inflammatory cytokines. In the LPS model, the treatment with mEVs after LPS determined the down-regulation of NOS2, MMP9, TLR5, TGFB1, IFNB, IL18 and IL12A gene expressions, as well as lower release of IL-18 in culture supernatants. At the same time, we observed the increased expression of TLR1, TLR2, TLR8 and EBI3. On the contrary, the treatment with mEVs after H2O2 exposure, the model of UC, determined the increased expression of MMP9 alongside the decrease in TGFB1, TLR8 and DEFB1, with a lower release of IL-1Ra in culture supernatants. Overall, our data showed that a 48 h treatment with mEVs after a pro-inflammatory stimulus significantly modulated the expression of several TLRs and cytokines in swine intestinal cells, in association with a decreased inflammation. These results further highlight the immunomodulatory potential of these nanosized structures and suggest their potential application in vivo.

Corrigendum: Goat milk extracellular vesicles: immuno-modulation effects on porcine monocyte-derived macrophages in vitro.

[This corrects the article DOI: 10.3389/fimmu.2023.1209898.].

Goat milk extracellular vesicles: immuno-modulation effects on porcine monocyte-derived macrophages in vitro.

Introduction: Extracellular vesicles (EVs) are nanometric-membrane-bound sub-cellular structures, which can be recovered from milk. Milk EVs have drawn increasing interest due to their potential biomedical applications, therefore it is important to investigate their impact on key immune cells, such as macrophages. Methods: In this work, the immunomodulatory effects of goat milk EVs on untreated (moMФ) and classically activated (moM1) porcine monocyte-derived macrophages were investigated using flow cytometry, ELISA, and gene expression assays. Results: These particles were efficiently internalized by macrophages and high doses (60 mg protein weight) triggered the upregulation of MHC I and MHC II DR on moMФ, but not on moM1. In moMФ, exposure to low doses (0.6 mg) of mEVs enhanced the gene expression of IL10, EBI3, and IFNB, whereas high doses up-regulated several pro-inflammatory cytokines. These nanosized structures slightly modulated cytokine gene expression on moM1. Accordingly, the cytokine (protein) contents in culture supernatants of moMФ were mildly affected by exposure to low doses of mEVs, whereas high doses promoted the increased release of TNF, IL-8, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, and IL-12. The cytokines content in moM1 supernatants was not critically affected. Discussion: Overall, our data support a clinical application of these molecules: they polarized macrophages toward an M1-like phenotype, but this activation seemed to be controlled, to prevent potentially pathological over-reaction to stressors.

Heterogeneity of Phenotypic and Functional Changes to Porcine Monocyte-Derived Macrophages Triggered by Diverse Polarizing Factors In Vitro.

Swine are attracting increasing attention as a biomedical model, due to many immunological similarities with humans. However, porcine macrophage polarization has not been extensively analyzed. Therefore, we investigated porcine monocyte-derived macrophages (moMΦ) triggered by either IFN-γ + LPS (classical activation) or by diverse "M2-related" polarizing factors: IL-4, IL-10, TGF-ß, and dexamethasone. IFN-γ and LPS polarized moMΦ toward a proinflammatory phenotype, although a significant IL-1Ra response was observed. Exposure to IL-4, IL-10, TGF-ß, and dexamethasone gave rise to four distinct phenotypes, all antithetic to IFN-γ and LPS. Some peculiarities were observed: IL-4 and IL-10 both enhanced expression of IL-18, and none of the "M2-related" stimuli induced IL-10 expression. Exposures to TGF-ß and dexamethasone were characterized by enhanced levels of TGF-ß2, whereas stimulation with dexamethasone, but not TGF-ß2, triggered CD163 upregulation and induction of CCL23. Macrophages stimulated with IL-10, TGF-ß, or dexamethasone presented decreased abilities to release proinflammatory cytokines in response to TLR2 or TLR3 ligands: IL-10 showed a powerful inhibitory activity for CXCL8 and TNF release, whereas TGF-ß provided a strong inhibitory signal for IL-6 production. While our results emphasized porcine macrophage plasticity broadly comparable to human and murine macrophages, they also highlighted some peculiarities in this species.

Genetic Characterization of a Novel Equus caballus Papillomavirus Isolated from a Thoroughbred Mare.

Papillomaviruses (PVs) are small, non-enveloped viruses, ubiquitous across the animal kingdom. PVs induce diverse forms of infection, such as cutaneous papillomas, genital papillomatosis, and carcinomas. During a survey on the fertility status of a mare, a novel Equus caballus PV (EcPV) has been identified using Next Generation Sequencing, and it was further confirmed with genome-walking PCR and Sanger sequencing. The complete circular genome 7607 bp long shares 67% average percentage of identity with EcPV9, EcPV2, EcPV1, and EcPV6, justifying a new classification as Equus caballus PV 10 (EcPV10). All EcPV genes are conserved in EcPV10, and phylogenetic analysis indicates that EcPV10 is closely related to EcPV9 and EcPV2, genus Dyoiota 1. A preliminary EcPV10 genoprevalence study, carried out on 216 horses using Real Time PCRs, suggested a low incidence of this isolate (3.7%) compared to EcPVs of the same genus such as EcPV2 and EcPV9 in the same horse population. We hypothesize a transmission mechanism different from the one observed in the closely related EcPV9 and EcPV2 that particularly infect Thoroughbreds. This horse breed is usually submitted to natural mating, thus indicating a possible sexual diffusion. No differences were detected for breeds in terms of susceptibility to EcPV10. Further studies are needed to investigate the molecular mechanisms behind the host and EcPV10 infection to explain the reduced viral spread.

Antimicrobial and Immunomodulatory Potential of Cow Colostrum Extracellular Vesicles (ColosEVs) in an Intestinal In Vitro Model.

Extracellular Vesicles (EVs) are nano-sized double-lipid-membrane-bound structures, acting mainly as signalling mediators between distant cells and, in particular, modulating the immune response and inflammation of targeted cells. Milk and colostrum contain high amounts of EVs that could be exploited as alternative natural systems in antimicrobial fighting. The aim of this study is to evaluate cow colostrum-derived EVs (colosEVs) for their antimicrobial, anti-inflammatory and immunomodulating effects in vitro to assess their suitability as natural antimicrobial agents as a strategy to cope with the drug resistance problem. ColosEVs were evaluated on a model of neonatal calf diarrhoea caused by Escherichia coli infection, a livestock disease where antibiotic therapy often has poor results. Colostrum from Piedmontese cows was collected within 24 h of calving and colosEVs were immediately isolated. IPEC-J2 cell line was pre-treated with colosEVs for 48 h and then infected with EPEC/NTEC field strains for 2 h. Bacterial adherence and IPEC-J2 gene expression analysis (RT-qPCR) of CXCL8, DEFB1, DEFB4A, TLR4, TLR5, NFKB1, MYD88, CGAS, RIGI and STING were evaluated. The colosEVs pre-treatment significantly reduced the ability of EPEC/NTEC strains to adhere to cell surfaces (p = 0.006), suggesting a role of ColosEVs in modulating host−pathogen interactions. Moreover, our results showed a significant decrease in TLR5 (p < 0.05), CGAS (p < 0.05) and STING (p < 0.01) gene expression in cells that were pre-treated with ColosEVs and then infected, thus highlighting a potential antimicrobial activity of ColosEVs. This is the first preliminarily study investigating ColosEV immunomodulatory and anti-inflammatory effects on an in vitro model of neonatal calf diarrhoea, showing its potential as a therapeutic and prophylactic tool.

Molecular Characterization of CF33 Canine Cell Line and Evaluation of Its Ability to Respond against Infective Stressors in Sight of Anticancer Approaches.

Spontaneous mammary tumors are the most frequent neoplasms in bitches and show similarities with human breast cancer in risk factors, clinical course, and histopathology. The poor prognosis of some cancer subtypes, both in human and dog, demands more effective therapeutic approaches. A possible strategy is the new anticancer therapy based on immune response modulation through bacteria or their derivatives on canine mammary carcinoma cell lines. The aim of the present study was to analyze the CF33 cell line in terms of basal expression of immune innate genes, CXCR4 expression, and interaction with infectious stressors. Our results highlight that CF33 maintains gene expression parameters typical of mammary cancer, and provides the basal gene expression of CF33, which is characterized by overexpression of CXCR4, CD44, RAD51, LY96, and a non-continuous expression of TP53 and PTEN. No mutations appeared in the CXCR4 gene until the 58th passage; this may represent important information for studying the CXCR4 pathway as a therapeutic target. Moreover, the CF33 cell line was shown to be able to interact with Salmonella Typhimurium (ST) (an infective stressor), indicating that these cells could be used as an in vitro model for developing innovative therapeutic approaches involving bacteria.

Vesicular Glutamate Release from Feeder-FreehiPSC-Derived Neurons.

Human-induced pluripotent stem cells (hiPSCs) represent one of the main and powerful tools for the in vitro modeling of neurological diseases. Standard hiPSC-based protocols make use of animal-derived feeder systems to better support the neuronal differentiation process. Despite their efficiency, such protocols may not be appropriate to dissect neuronal specific properties or to avoid interspecies contaminations, hindering their future translation into clinical and drug discovery approaches. In this work, we focused on the optimization of a reproducible protocol in feeder-free conditions able to generate functional glutamatergic neurons. This protocol is based on a generation of neuroprecursor cells differentiated into human neurons with the administration in the culture medium of specific neurotrophins in a Geltrex-coated substrate. We confirmed the efficiency of this protocol through molecular analysis (upregulation of neuronal markers and neurotransmitter receptors assessed by gene expression profiling and expression of the neuronal markers at the protein level), morphological analysis, and immunfluorescence detection of pre-synaptic and post-synaptic markers at synaptic boutons. The hiPSC-derived neurons acquired Ca2+-dependent glutamate release properties as a hallmark of neuronal maturation. In conclusion, our study describes a new methodological approach to achieve feeder-free neuronal differentiation from hiPSC and adds a new tool for functional characterization of hiPSC-derived neurons.

In vitro evaluation of immunomodulatory activities of goat milk Extracellular Vesicles (mEVs) in a model of gut inflammation.

Gut represents a major immunological defense barrier with mucosal immune system and intestinal epithelial cells (IECs). In all intestinal diseases, in particular inflammatory bowel disease (IBD), both the absorption and the local immune system are compromised and alternative effective therapies are sought after. Extracellular Vesicles (EVs) have the capability to regulate immune cells within the inflammatory microenvironment, by dampening inflammation and restoring intestinal barrier integrity. Recently, the immune-modulatory role of EVs has also been confirmed for milk EVs (mEVs), notable for their easy production, high sample volumes, cost-effective scalable production and non-toxic and non-immunogenic behavior. In this context, the aim of this study was to evaluate goat mEV anti-inflammatory and immuno-modulating effects on an in vitro model (IPEC-J2) of intestinal inflammation through gene expression evaluation with RT-qPCR and cytokine release dosage with ELISA test. After the establishment of a pro-inflammatory environment due to LPS stimuli, IL6, CXCL8, IL12p35, IL12p40, IFNB, IL18, TLR7 and NOS2 resulted significantly up-regulated in stimulated IPEC-J2 cells compared to those of the basal culture. After 48 h of mEV treatment in inflamed IPEC-J2 a partial restoration of initial conditions was detected, with the IL18 and IL12p40 significant down-regulation, and IL12p35, EBI3, TLR7, BD1 and BD3 up-regulation. IL-18 reduced protein production was also detected in supernatants. Moreover, a decrease of MMP9 and NOS2 together with a strong up-regulation of MUC2 indicated a recovery of cellular homeostasis and, therefore, potential beneficial effects on the intestinal mucosa. Nevertheless, 48 h post-treatment, an increased gene expression and protein release of IL-8 was observed. This paper is one of the firsts to assess the effect of goat mEVs and the first one, in particular, of doing this on an in vitro model of gut inflammation. The obtained results show a potential capability of goat mEVs to modulate inflammation and to play beneficial effects on the intestinal mucosa.

Equus caballus Papillomavirus Type-9 (EcPV9): First Detection in Asymptomatic Italian Horses.

Papillomavirus (PV) infections may be related to anogenital lesions and cancer development in humans and several other animal species. To date, 11 different PVs have been reported in horses. Among them, a newly described PV named Equus caballus Papillomavirus Type9 (EcPV9) was thus far only reported in the semen of a stallion with penile lesions in Australia. This study reports for the first time the presence of EcPV9 in asymptomatic Italian horses. From July 2020 to January 2022, genital brush samples were collected from 209 horses with no apparent signs of neoplastic disease and no PV-associated lesions, clinically examined at the Didactic Veterinary University Hospital (OVUD) of Perugia and at the Veterinary University Hospital (OVU) of Turin. Brushes were submitted to real-time PCR targeting the EcPV9-L1 region. The first amplification targeted a region of ~116 bp, followed by the amplification and sequencing of ~533 bp of the positive samples. EcPV9-L1 DNA was found in eleven horses (5.3%), all female and mainly English Thoroughbred. Co-infection with EcPV2-L1 was found in 7 out of the 11 EcPV9-L1 positive horses (63.6%). This study contributes to the description of the prevalence of exposure or infection of EcPVs in the horse population in Italy, for which data are still limited. In this regard, here we provide a phylogenetic analysis and the completely reconstructed viral genomes of two Italian EcPV type 9 isolates, as well as four EcPV type 2 obtained from co-infected animals.

Equine Gastric Squamous Cell Carcinoma in a Friesian Stallion.

A 7-year-old Friesian stallion with a history of oesophageal stenosis, weight loss, inappetence, and recurrent hyperthermia was referred for gastroscopy. The stomach mucosa surrounding the oesophageal opening showed a large, necrotic, and ulcerated mass. On post-mortem examination, a very large, cauliflower-like neoplasm was seen, affecting non-glandular gastric mucosa. Nodular lesions were observed, scattered on the omentum, the spleen, and the liver. Microscopic findings allowed the diagnosis of gastric squamous cell carcinoma with abdominal metastasis. Biomolecular investigations demonstrated the presence of EcPV-2 genes in neoplastic lesions, thus supporting the role of EcPV-2 in the ethiology of equine gastric cancer.

Analyses of the Impact of Immunosuppressive Cytokines on Porcine Macrophage Responses and Susceptibility to Infection to African Swine Fever Viruses.

African swine fever viruses (ASFV), currently a serious threat to the global pig industry, primarily target porcine macrophages. Macrophages are characterized by their remarkable plasticity, being able to modify their phenotype and functions in response to diverse stimuli. Since IL-10 and TGF-ß polarize macrophages toward an anti-inflammatory phenotype, we analyzed their impact on porcine monocyte-derived macrophages' (moMΦ) susceptibility to infection and their responses to two genotype I ASFV strains, virulent 26544/OG10 and attenuated NH/P68. At a low multiplicity of infection (MOI), NH/P68, but not 26544/OG10, presented a higher ability to infect moM(IL-10) compared to moMΦ and moM(TGF-ß), but no differences were appreciated at a higher MOI. Both strains replicated efficiently in all moMΦ subsets, with no differences at later times post-infection. Both strains downregulated CD14 and CD16 expression on moMΦ, irrespective of the activation status. ASFV's modulation of CD163 and MHC II DR expression and cytokine responses to NH/P68 or 26544/OG10 ASFV were not affected by either IL-10 or TGF-ß pre-treatment. Our results revealed little impact of these anti-inflammatory cytokines on moMΦ interaction with ASFV, which likely reflects the ability of the virus to effectively modulate macrophage responses.

Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide.

Toll-like receptor 2 (TLR2) ligands are attracting increasing attention as prophylactic and immunotherapeutic agents against pathogens and tumors. We previously observed that a synthetic diacylated lipopeptide based on a surface protein of Mycoplasma agalactiae (Mag-Pam2Cys) strongly activated innate immune cells, including porcine monocyte-derived macrophages (moMΦ). In this study, we utilized confocal microscopy, flow cytometry, multiplex cytokine ELISA, and RT-qPCR to conduct a comprehensive analysis of the effects of scalar doses of Mag-Pam2Cys on porcine moMΦ. We observed enhanced expression of activation markers (MHC class I, MHC class II DR, CD25), increased phagocytotic activity, and release of IL-12 and proinflammatory cytokines. Mag-Pam2Cys also upregulated the gene expression of several IFN-α subtypes, p65, NOS2, and molecules with antimicrobial activities (CD14, beta defensin 1). Overall, our data showed that Mag-Pam2Cys polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. However, Mag-Pam2Cys downregulated the expression of IFN-α3, six TLRs (TLR3, -4, -5, -7, -8, -9), and did not interfere with macrophage polarization induced by the immunosuppressive IL-10, suggesting that the inflammatory activity evoked by Mag-Pam2Cys could be regulated to avoid potentially harmful consequences. We hope that our in vitro results will lay the foundation for the further evaluation of this diacylated lipopeptide as an immunopotentiator in vivo.

Comparative Phenotypic and Functional Analyses of the Effects of IL-10 or TGF-ß on Porcine Macrophages.

Macrophages are phagocytic cells involved in maintaining tissue homeostasis and defense against pathogens. Macrophages may be polarized into different functionally specialized subsets. M2c macrophages arise following stimulation with IL-10 or TGF-ß and mediate anti-inflammatory and tissue repair functions. M2c macrophages remain poorly characterized in the pig, thus we investigated the impact of these regulatory cytokines on porcine monocyte-derived macrophages (moMΦ). The phenotype and functionality of these cells was characterized though confocal microscopy, flow cytometry, ELISA, and RT-qPCR. Both cytokines induced CD14 and MHC II DR down-regulation and reduced IL-6, TNF-α, and CD14 expression, suggestive of an anti-inflammatory phenotype. Interestingly, neither IL-10 or TGF-ß were able to trigger IL-10 induction or release by moMΦ. Differences between these cytokines were observed: stimulation with IL-10, but not TGF-ß, induced up-regulation of both CD16 and CD163 on moMΦ. In addition, IL-10 down-regulated expression of IL-1ß and IL-12p40 4h post-stimulation and induced a stronger impairment of moMΦ ability to respond to either TLR2 or TLR4 agonists. Overall, our results provide an overview of porcine macrophage polarization by two immunosuppressive cytokines, revealing differences between IL-10 and TGF-ß, and reporting some peculiarity of swine, which should be considered in translational studies.

Distal motor neuropathy associated with novel EMILIN1 mutation.

Elastin microfibril interface-located proteins (EMILINs) are extracellular matrix glycoproteins implicated in elastogenesis and cell proliferation. Recently, a missense mutation in the EMILIN1 gene has been associated with autosomal dominant connective tissue disorder and motor-sensory neuropathy in a single family. We identified by whole exome sequencing a novel heterozygous EMILIN1 mutation c.748C>T [p.R250C] located in the coiled coil forming region of the protein, in four affected members of an autosomal dominant family presenting a distal motor neuropathy phenotype. In affected patient a sensory nerve biopsy showed slight and unspecific changes in the number and morphology of myelinated fibers. Immunofluorescence study of a motor nerve within a muscle biopsy documented the presence of EMILIN-1 in nerve structures. Skin section and skin derived fibroblasts displayed a reduced extracellular deposition of EMILIN-1 protein with a disorganized network of poorly ramified fibers in comparison with controls. Downregulation of emilin1a in zebrafish displayed developmental delay, locomotion defects, and abnormal axonal arborization from spinal cord motor neurons. The phenotype was complemented by wild-type zebrafish emilin1a, and partially the human wild-type EMILIN1 cRNA, but not by the cRNA harboring the novel c.748C>T [p.R250C]. These data suggest a role of EMILIN-1 in the pathogenesis of diseases affecting the peripheral nervous system.

Progress of Induced Pluripotent Stem Cell Technologies to Understand Genetic Epilepsy.

The study of the pathomechanisms by which gene mutations lead to neurological diseases has benefit from several cellular and animal models. Recently, induced Pluripotent Stem Cell (iPSC) technologies have made possible the access to human neurons to study nervous system disease-related mechanisms, and are at the forefront of the research into neurological diseases. In this review, we will focalize upon genetic epilepsy, and summarize the most recent studies in which iPSC-based technologies were used to gain insight on the molecular bases of epilepsies. Moreover, we discuss the latest advancements in epilepsy cell modeling. At the two dimensional (2D) level, single-cell models of iPSC-derived neurons lead to a mature neuronal phenotype, and now allow a reliable investigation of synaptic transmission and plasticity. In addition, functional characterization of cerebral organoids enlightens neuronal network dynamics in a three-dimensional (3D) structure. Finally, we discuss the use of iPSCs as the cutting-edge technology for cell therapy in epilepsy.