(1) Background: Huntington's disease (HD) is rare incurable hereditary neurodegenerative disorder caused by CAG repeat expansion in the gene coding for the protein huntingtin (HTT). Mutated huntingtin (mHTT) undergoes fragmentation and accumulation, affecting cellular functions and leading to neuronal cell death. Porcine models of HD are used in preclinical testing of currently emerging disease modifying therapies. Such therapies are aimed at reducing mHTT expression, postpone the disease onset, slow down the progression, and point out the need of biomarkers to monitor disease development and therapy efficacy. Recently, extracellular vesicles (EVs), particularly exosomes, gained attention as possible carriers of disease biomarkers. We aimed to characterize HTT and mHTT forms/fragments in blood plasma derived EVs in transgenic (TgHD) and knock-in (KI-HD) porcine models, as well as in HD patients' plasma. (2) Methods: Small EVs were isolated by ultracentrifugation and HTT forms were visualized by western blotting. (3) Results: The full length 360 kDa HTT co-isolated with EVs from both the pig model and HD patient plasma. In addition, a ~70 kDa mutant HTT fragment was specific for TgHD pigs. Elevated total huntingtin levels in EVs from plasma of HD groups compared to controls were observed in both pig models and HD patients, however only in TgHD were they significant (p = 0.02). (4) Conclusions: Our study represents a valuable initial step towards the characterization of EV content in the search for HD biomarkers.

• Keywords
• Huntington´s disease, KI-HD, TgHD, biomarker, exosome, extracellular vesicle, fragment, huntingtin, neurodegenerative disease, pig model,
• MeSH
• Biomarkers MeSH
• Extracellular Vesicles * metabolism   MeSH
• Huntington Disease * metabolism   MeSH
• Plasma metabolism   MeSH
• Humans MeSH
• Swine MeSH
• Nerve Tissue Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers MeSH
• Nerve Tissue Proteins MeSH

National cancer databases document that melanoma is the most aggressive and deadly cutaneous malignancy with worldwide increasing incidence in the Caucasian population. Around 10% of melanomas occur in families. Several germline mutations were identified that might help to indicate individuals at risk for preventive interventions and early disease detection. More than 50% of sporadic melanomas carry mutations in Ras/Raf/mitogen-activated protein kinase (MAPK/MEK) pathway, which may represent aims of novel targeted therapies. Despite advances in targeted therapies and immunotherapies, the outcomes in metastatic tumor are still unsatisfactory. Here, we review animal models that help our understanding of melanoma development and treatment, including non-vertebrate, mouse, swine, and other mammal models, with an emphasis on those with spontaneously developing melanoma. Special attention is paid to the melanoma-bearing Libechov minipig (MeLiM). This original swine model of hereditary metastatic melanoma enables studying biological processes underlying melanoma progression, as well as spontaneous regression. Current histological, immunohistochemical, biochemical, genetic, hematological, immunological, and skin microbiome findings in the MeLiM model are summarized, together with development of new therapeutic approaches based on tumor devitalization. The ongoing study of molecular and immunological base of spontaneous regression in MeLiM model has potential to bring new knowledge of clinical importance.

• Keywords
• MeLiM, animal model, devitalization, genetics, melanoma, mutation, progression, spontaneous regression, swine,
• MeSH
• Melanoma, Cutaneous Malignant MeSH
• Melanoma genetics   MeSH
• Swine, Miniature genetics   MeSH
• Disease Models, Animal MeSH
• Skin Neoplasms genetics   MeSH
• Swine genetics   MeSH
• Disease Progression MeSH
• Neoplasms, Second Primary genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

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.

• Keywords
• body fluid, cerebrospinal fluid, exosome, extracellular vesicle, pig model, plasma, proteomics, seminal plasma,
• Publication type
• Journal Article MeSH

BACKGROUND: Treatment of advanced cutaneous melanoma remains challenging, and new data on melanoma biology are required. The most widely accepted criteria for the prognostic evaluation of melanoma are histopathological and clinical parameters, and the identification of additional tumor markers is thus of paramount importance. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI), an important tool in cancer research, is useful for unraveling the molecular profile of melanoma. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we used the melanoma-bearing Libechov minipig (MeLiM), a unique animal model that allows observation of the complete spontaneous regression of invasive cutaneous melanoma, to investigate i) the differences between melanoma and healthy skin protein profiles and ii) the proteins potentially involved in spontaneous regression. The MeLiM tissues were cryosected, histologically characterized, analyzed by MALDI MSI, and immunohistologically stained. Multivariate statistical analyses of the MALDI MSI data revealed ten relevant m/z ions, of which the expression levels varied significantly among the studied MeLiM tissues. These ion peaks were used to create mass ion images/maps and visualize the differences between tumor and healthy skin specimens, as well as among histologically characterized tissue regions. CONCLUSIONS/SIGNIFICANCE: Protein profiles comprising ten statistically significant mass ion peaks useful for differentiating cutaneous melanoma and healthy skin tissues were determined. Peaks at m/z 3044, 6011, 6140 and 10180 were overexpressed in melanoma compared with healthy skin tissue. More specifically, m/z 6140 was expressed at significantly (p < 0.05) higher levels in normally growing melanoma regions than in regions with early and late spontaneous regression. This study demonstrates the clinical utility of MALDI MSI for the analysis of tissue cryosections at a molecular level.

• MeSH
• Melanoma metabolism   pathology   MeSH
• Neoplasm Proteins metabolism   MeSH
• Swine MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Neoplasm Proteins MeSH

We describe a new procedure for the parallel mapping of selected metals in histologically characterized tissue samples. Mapping is achieved via image registration of digital data obtained from two neighbouring cryosections by scanning the first as a histological sample and subjecting the second to laser ablation inductively coupled plasma mass spectrometry. This computer supported procedure enables determination of the distribution and content of metals of interest directly in the chosen histological zones and represents a substantial improvement over the standard approach, which determines these values in tissue homogenates or whole tissue sections. The potential of the described procedure was demonstrated in a pilot study that analysed Zn and Cu levels in successive development stages of pig melanoma tissue using MeLiM (Melanoma-bearing-Libechov-Minipig) model. We anticipate that the procedure could be useful for a complex understanding of the role that the spatial distribution of metals plays within tissues affected by pathological states including cancer.

• MeSH
• Histological Techniques methods   MeSH
• Mass Spectrometry methods   MeSH
• Copper analysis   MeSH
• Melanoma pathology   MeSH
• Disease Models, Animal MeSH
• Pilot Projects MeSH
• Image Processing, Computer-Assisted methods   MeSH
• Swine MeSH
• Zinc analysis   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Copper MeSH
• Zinc MeSH

Spontaneous regression (SR) of human melanoma is a rare, well-documented phenomenon that is not still fully understood. Its detailed study cannot be performed in patients due to ethical reasons. Using the Melanoma-bearing Libechov Minipig (MeLiM) animals of various ages (from 3 weeks to 8 months) we implemented a long-term monitoring of melanoma growth and SR. We focused on immunohistochemical detection of two important extracellular matrix proteins, collagen IV and laminin, which are associated with cancer. We showed that SR of melanoma is a highly dynamic process. The expression of collagen IV and laminin correlated with changes in population of melanoma cells. Tumours of 3-week-old animals consisted primarily of melanoma cells with a granular expression of collagen IV and laminin around them. Thereafter, melanoma cells were gradually destroyed and tumour tissue was rebuilt into the connective tissue. Collagen IV expression slightly increased in tumours of 10-week-old pigs showing extracellular fibrous appearance. In tumours of older animals, areas lacking melanoma cells demonstrated a low expression and areas still containing melanoma cells a high expression of both proteins. We considered the age of 10 weeks as a turning point in the transition between tumour growth and SR of the MeLiM melanoma.

• Keywords
• MeLiM, collagen IV, laminin, porcine melanoma, spontaneous regression,
• Publication type
• Journal Article MeSH

Adenosines, endogenous purine nucleosides, appear in the extracellular space under metabolically stressful conditions associated with ischemia, inflammation, and cell damage. Their activity on innate immunity is prevalently inhibitory and can develop both in infectious and neoplastic diseases. During cancer development, tumor cells that release high concentrations of adenosines can impair the function of tumor-infiltrating lymphocytes and assist tumor growth by neo-angiogenesis. We evaluated the influence of A(2) adenosine receptor (A(2)AR) agonist on cytotoxic-cell response comparing human with other mammalian species (rodents, pigs, goats), both in healthy and in cancer conditions. The A(2)AR agonist developed dose-dependent inhibition of the cytotoxic activity of immune effector cells in all studied species. However, variability of the response was observed in relation to the species and the target cells that were used. Altogether, our data indicate that the A(2)AR plays a central role in adenosine-mediated inhibition of immune response to tumors.

• MeSH
• Adenosine analogs & derivatives   immunology   pharmacology   MeSH
• Adenosine A2 Receptor Agonists * MeSH
• Killer Cells, Natural drug effects   immunology   MeSH
• Cytotoxicity, Immunologic drug effects   MeSH
• Rodentia MeSH
• Goats MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Neoplasms immunology   MeSH
• Rats, Wistar MeSH
• Swine MeSH
• Receptors, Adenosine A2 immunology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine MeSH
• Adenosine A2 Receptor Agonists * MeSH
• N-cyclopropyl adenosine-5'-carboxamide MeSH Browser
• Receptors, Adenosine A2 MeSH

In the paper we utilized the adsorptive transfer stripping differential pulse voltammetry Brdicka reaction for the determination of metallothioneins (MT) in melanoma cells, animal melanoma tissues (MeLiM miniature pig) and blood serum of patients with malignant melanoma. Primarily we attempted to investigate the influence of dilution of real sample on MT electrochemical response. Dilution of samples of 1 000 times was chosen the most suitable for determination of MT level in biological samples. Then we quantified the MT level in the melanoma cells, the animal melanoma tissues and the blood serum samples. The MT content in the cells varied within the range from 4.2 to 11.2 μM. At animal melanoma tissues (melanomas localized on abdomen, back limb and dorsum) the highest content of MT was determined in the tumour sampled on the back of the animal and was nearly 500 μg of MTs per gram of a tissue. We also quantified content of MT in metastases, which was found in liver, spleen and lymph nodes. Moreover the average MT level in the blood serum samples from patients with melanoma was 3.0 ± 0.8 μM. MT levels determined at melanoma samples were significantly (p < 0.05) higher compared to control ones at cells, tissues and blood serum.

• Keywords
• Adsorptive Transfer Stripping Technique, Animal Tissue, Brdicka Reaction, Cell, Differential Pulse Voltammetry, Human blood serum, Metallothionein, Protein, Tumour Marker,
• Publication type
• Journal Article MeSH