The mechanism of immune dysregulation caused by porcine reproductive and respiratory syndrome virus (PRRSV).

PRRSV is capable of evading the effective immune response, thus persisting in piglets and throughout the swine herd. We show here that PRRSV invades the thymus and causes depletion of T-cell precursors and alteration of the TCR repertoire. Developing thymocytes are affected during negative selection when they transit from the triple-negative to triple-positive stages at the corticomedullary junction just before entering the medulla. The restriction of repertoire diversification occurs in both helper and cytotoxic αß-T cells. As a result, critical viral epitopes are tolerated, and infection becomes chronic. However, not all viral epitopes are tolerated. Infected piglets develop antibodies capable of recognizing PRRSV, but these are not virus neutralizing. Further analysis showed that the lack of an effective immune response against the critical viral structures results in the absence of a germinal center response, overactivation of T and B cells in the periphery, robust production of useless antibodies of all isotypes, and the inability to eliminate the virus. Overall, the results show how a respiratory virus that primarily infects and destroys myelomonocytic cells has evolved strategies to disrupt the immune system. These mechanisms may be a prototype for how other viruses can similarly modulate the host immune system.

Proteomic Analysis of Human Neural Stem Cell Differentiation by SWATH-MS.

The unique properties of stem cells to self-renew and differentiate hold great promise in disease modelling and regenerative medicine. However, more information about basic stem cell biology and thorough characterization of available stem cell lines is needed. This is especially essential to ensure safety before any possible clinical use of stem cells or partially committed cell lines. As proteins are the key effector molecules in the cell, the proteomic characterization of cell lines, cell compartments or cell secretome and microenvironment is highly beneficial to answer above mentioned questions. Nowadays, method of choice for large-scale discovery-based proteomic analysis is mass spectrometry (MS) with data-independent acquisition (DIA). DIA is a robust, highly reproducible, high-throughput quantitative MS approach that enables relative quantification of thousands of proteins in one sample. In the current protocol, we describe a specific variant of DIA known as SWATH-MS for characterization of neural stem cell differentiation. The protocol covers the whole process from cell culture, sample preparation for MS analysis, the SWATH-MS data acquisition on TTOF 5600, the complete SWATH-MS data processing and quality control using Skyline software and the basic statistical analysis in R and MSstats package. The protocol for SWATH-MS data acquisition and analysis can be easily adapted to other samples amenable to MS-based proteomics.

Dysbiosis of skin microbiome and gut microbiome in melanoma progression.

BACKGROUND: The microbiome alterations are associated with cancer growth and may influence the immune system and response to therapy. Particularly, the gut microbiome has been recently shown to modulate response to melanoma immunotherapy. However, the role of the skin microbiome has not been well explored in the skin tumour microenvironment and the link between the gut microbiome and skin microbiome has not been investigated in melanoma progression. Therefore, the aim of the present study was to examine associations between dysbiosis in the skin and gut microbiome and the melanoma growth using MeLiM porcine model of melanoma progression and spontaneous regression. RESULTS: Parallel analysis of cutaneous microbiota and faecal microbiota of the same individuals was performed in 8 to 12 weeks old MeLiM piglets. The bacterial composition of samples was analysed by high throughput sequencing of the V4-V5 region of the 16S rRNA gene. A significant difference in microbiome diversity and richness between melanoma tissue and healthy skin and between the faecal microbiome of MeLiM piglets and control piglets were observed. Both Principal Coordinate Analysis and Non-metric multidimensional scaling revealed dissimilarities between different bacterial communities. Linear discriminant analysis effect size at the genus level determined different potential biomarkers in multiple bacterial communities. Lactobacillus, Clostridium sensu stricto 1 and Corynebacterium 1 were the most discriminately higher genera in the healthy skin microbiome, while Fusobacterium, Trueperella, Staphylococcus, Streptococcus and Bacteroides were discriminately abundant in melanoma tissue microbiome. Bacteroides, Fusobacterium and Escherichia-Shigella were associated with the faecal microbiota of MeLiM piglets. Potential functional pathways analysis based on the KEGG database indicated significant differences in the predicted profile metabolisms between the healthy skin microbiome and melanoma tissue microbiome. The faecal microbiome of MeLiM piglets was enriched by genes related to membrane transports pathways allowing for the increase of intestinal permeability and alteration of the intestinal mucosal barrier. CONCLUSION: The associations between melanoma progression and dysbiosis in the skin microbiome as well as dysbiosis in the gut microbiome were identified. Results provide promising information for further studies on the local skin and gut microbiome involvement in melanoma progression and may support the development of new therapeutic approaches.

Serum proteomic analysis of melanoma patients with immunohistochemical profiling of primary melanomas and cultured cells: Pilot study.

The steadily increasing incidence of malignant melanoma (MM) and its aggressive behaviour makes this tumour an attractive cancer research topic. The tumour microenvironment is being increasingly recognised as a key factor in cancer biology, with an impact on proliferation, invasion, angiogenesis and metastatic spread, as well as acquired therapy resistance. Multiple bioactive molecules playing cooperative roles promote the chronic inflammatory milieu in tumours, making inflammation a hallmark of cancer. This specific inflammatory setting is evident in the affected tissue. However, certain mediators can leak into the systemic circulation and affect the whole organism. The present study analysed the complex inflammatory response in the sera of patients with MM of various stages. Multiplexed proteomic analysis (Luminex Corporation) of 31 serum proteins was employed. These targets were observed in immunohistochemical profiles of primary tumours from the same patients. Furthermore, these proteins were analysed in MM cell lines and the principal cell population of the melanoma microenvironment, cancer­associated fibroblasts. Growth factors such as hepatocyte growth factor, granulocyte­colony stimulating factor and vascular endothelial growth factor, chemokines RANTES and interleukin (IL)­8, and cytokines IL­6, interferon­α and IL­1 receptor antagonist significantly differed in these patients compared with the healthy controls. Taken together, the results presented here depict the inflammatory landscape that is altered in melanoma patients, and highlight potentially relevant targets for therapy improvement.

Isolation and Characterization of Small Extracellular Vesicles from Porcine Blood Plasma, Cerebrospinal Fluid, and Seminal Plasma.

Extracellular vesicles (EVs) are a highly attractive subject of biomedical research as possible carriers of nucleic acid and protein biomarkers. EVs released to body fluids enable indirect access to inner organs by so-called "liquid biopsies". Obtaining a high-quality EV sample with minimum contaminants is crucial for proteomic analyses using LC-MS/MS or other techniques. However, the EV content in various body fluids largely differs, which may hamper subsequent analyses. Here, we present a comparison of extracellular vesicle yields from blood plasma, cerebrospinal fluid, and seminal plasma using an experimental pig model. Pigs are widely used in biomedical research as large animal models with anatomy and physiology close to those of humans and enable studies (e.g., of the nervous system) that are unfeasible in humans. EVs were isolated from body fluids by differential centrifugation followed by ultracentrifugation. EVs were characterized according to protein yields and to the quality of the isolated vesicles (e.g., size distribution, morphology, positivity for exosome markers). In our experimental setting, substantial differences in EV amounts were identified among body fluids, with the seminal plasma being the richest EV source. The yields of pellet proteins from ultracentrifugation of 1 mL of porcine body fluids may help to estimate body fluid input volumes to obtain sufficient samples for subsequent proteomic analyses.

AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington's Disease Minipig Model.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD (tgHD) minipig model. We detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT. The combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic.

Multiplex immunoassays for quantification of cytokines, growth factors, and other proteins in stem cell communication.

Immunoassays represent valuable and broadly used techniques for detection and quantification of proteins. Thanks to their high sensitivity, such techniques are powerful for analyzing growth factors, trophic factors, angiogenic factors, hormones, cytokines, chemokines, soluble receptors, and other proteins which play key roles in intercellular communication and operate as potent regulators of stem cell survival, proliferation, differentiation, or cell death. Multiplex immunological assays, in contrast to ELISA, offer simultaneous quantification of tens of proteins across multiple samples, and have been developed to save time, costs, and sample volumes. Among them, planar antibody microarrays and xMAP(®) bead-based assays have become particularly popular for characterization of proteins secreted by stem cells, as they are relatively easy, highly accurate, multiplex to a high degree and a broad spectrum of analytes can be measured. Here, we describe protocols for multiplex quantification of secreted proteins using Quantibody(®) microarrays (RayBiotech) and xMAP(®) assays (Luminex and its partners).