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Ex-vivo lung perfusion (EVLP) has extended the number of transplantable lungs by reconditioning marginal organs. However, EVLP is performed at 37°C without homeostatic regulation leading to metabolic wastes' accumulation in the perfusate and, as a corrective measure, the costly perfusate is repeatedly replaced during the standard of care procedure. As an interesting alternative, a hemodialyzer could be placed on the EVLP circuit, which was previously shown to rebalance the perfusate composition and to maintain lung function and viability without appearing to impact the global gene expression in the lung. Here, we assessed the biological effects of a hemodialyzer during EVLP by performing biochemical and refined functional genomic analyses over a 12h procedure in a pig model. We found that dialysis stabilized electrolytic and metabolic parameters of the perfusate but enhanced the gene expression and protein accumulation of several inflammatory cytokines and promoted a genomic profile predicting higher endothelial activation already at 6h and higher immune cytokine signaling at 12h. Therefore, epuration of EVLP with a dialyzer, while correcting features of the perfusate composition and maintaining the respiratory function, promotes inflammatory responses in the tissue. This finding suggests that modifying the metabolite composition of the perfusate by dialysis during EVLP can have detrimental effects on the tissue response and that this strategy should not be transferred as such to the clinic. [ABSTRACT FROM AUTHOR]

The human cutaneous metastatic melanoma is the deadliest skin cancer. Partial, or less frequently complete spontaneous regressions could be observed, mainly mediated by T cells. Nevertheless, the underlying mechanisms are not fully unraveled. We investigated the first events of the immune response related to cancer regression in Melanoma-bearing Libechov Minipigs (MeLiM), a unique swine model of cutaneous melanoma that regresses spontaneously. Using a multiparameter flow cytometry strategy and integrating new clinical and histological criteria of the regression, we show that T cells and B cells are present only in the late stages, arguing against their role in the initial destruction of malignant cells. NK cells infiltrate the tumors before T cells and therefore might be involved in the induction of the regression process. Myeloid cells represent the main immune population within the tumor microenvironment regardless of the regression stage. Among those, MHCII+ CD163− macrophages that differ phenotypically and functionally compared to other tumor-associated macrophages, increase in number together with the first signs of regression suggesting their crucial contribution to initiating the regression process. Our study supports the importance of macrophage reprogramming in humans to improve current immunotherapy for metastatic melanoma. [ABSTRACT FROM AUTHOR]

The effects of CGP-42112, a ligand for angiotensin II subtype II receptors, on human monocyte membranes and whole monocytes were investigated. The saturability of protein concentration influenced the link between the I-CGP-42112 and the membranes. Monocytes incubated with CGP-42112 showed different stages of prostaglandin secretion, tumor necrosis factor levels and the release of other monocyte inflammatory mediators. Results also showed that the monocytes' physiological state also influenced the effects of CGP-42112.

Abstract Background Melanocytic proliferations are common in horses but the diagnosis of malignancy is not always straightforward. To improve diagnosis and prognosis, markers of malignancy are needed. Receptor for activated C kinase 1 (RACK1) protein may be such a marker. RACK1 was originally found to characterize malignant melanocytic lesions in the Melanoblastoma-bearing Libechov minipig (MeLiM) and, later, in human patients. Our purpose was to investigate the value of RACK1 in the classification of cutaneous melanocytic proliferations in horses. Results Using immunofluorescence, we report here that both MITF (Microphthalmia-associated transcription factor) and PAX3 (Paired box 3) allow the identification of melanocytic cells in horse skin samples. Importantly, RACK1 was detected in melanocytic lesions but not in healthy skin melanocytes. Finally, we found that RACK1 labeling can be used in horses to distinguish benign melanocytic tumors from melanomas. Indeed, RACK1 labeling appeared more informative to assess malignancy than individual histomorphological features. Conclusions This study confirms that horses provide an interesting model for melanoma genesis studies. It establishes MITF and PAX3 as markers of horse melanocytic cells. RACK1 emerges as an important marker of malignancy which may contribute to progress in the diagnosis of melanomas in both human and veterinary medicine.

Abstract Background Metastatic melanoma is a severe disease. Few experimental animal models of metastatic melanoma exist. MeLiM minipigs exhibit spontaneous melanoma. Cutaneous and metastatic lesions are histologically similar to human's. However, most of them eventually spontaneously regress. Our purpose was to investigate whether the MeLiM model could reveal markers of malignancy in human melanocytic proliferations. Results We compared the serial analysis of gene expression (SAGE) between normal pig skin melanocytes and melanoma cells from an early pulmonary metastasis of MeLiM minipigs. Tag identification revealed 55 regulated genes, including GNB2L1 which was found upregulated in the melanoma library. In situ hybridisation confirmed GNB2L1 overexpression in MeLiM melanocytic lesions. GNB2L1 encodes the adaptor protein RACK1, recently shown to influence melanoma cell lines tumorigenicity. We studied the expression of RACK1 by immunofluorescence and confocal microscopy in tissues specimens of normal skin, in cutaneous and metastatic melanoma developped in MeLiM minipigs and in human patients. In pig and human samples, the results were similar. RACK1 protein was not detected in normal epidermal melanocytes. By contrast, RACK1 signal was highly increased in the cytoplasm of all melanocytic cells of superficial spreading melanoma, recurrent dermal lesions and metastatic melanoma. RACK1 partially colocalised with activated PKCαβ. In pig metastases, additional nuclear RACK1 did not associate to BDNF expression. In human nevi, the RACK1 signal was low. Conclusion RACK1 overexpression detected in situ in human melanoma specimens characterized cutaneous and metastatic melanoma raising the possibility that RACK1 can be a potential marker of malignancy in human melanoma. The MeLiM strain provides a relevant model for exploring mechanisms of melanocytic malignant transformation in humans. This study may contribute to a better understanding of melanoma pathophysiology and to progress in diagnosis.

Increased [AT.sub.1] receptor expression and mRNA in kidney glomeruli of [AT.sub.2] receptor gene-disrupted mice. Am J Physiol Renal Physiol 280: F71-F78, 2001.--The proposed feedback between angiotensin II [AT.sub.2] and [AT.sub.1] receptors prompted us to study [AT.sub.1] receptor expression in kidneys of male [AT.sub.2] receptor-gene disrupted mice (agtr2 -/y). In wild-type (agtr2 +/y) mice, [AT.sub.1] receptor binding and mRNA is abundant in glomeruli, and [AT.sub.1] receptor binding is also high in the inner stripe of the outer medulla. [AT.sub.2] receptors are scarce, primarily associated to cortical vascular structures. In agtr2 -/y mice, [AT.sub.1] receptor binding and mRNA were increased in the kidney glomeruli, and [AT.sub.1] receptor binding was higher in the rest of the cortex and outer stripe of the outer medulla, but not in its inner stripe, indicating different cellular regulation. Although [AT.sub.2] receptor expression is very low in male agtr 2 +/y mice, their gene disruption alters [AT.sub.1] receptor expression. [AT.sub.1] upregulation alone may explain the [AT.sub.2] gene-disrupted mice phenotype such as increased blood pressure, higher sensitivity to angiotensin II, and altered renal function. The indirect [AT.sub.1]/[AT.sub.2] receptor feedback could have clinical significance because [AT.sub.1] antagonists are widely used in medical practice. renin-angiotensin system; angiotensin II receptor types; gene-disrupted models

The endothelin system in normal human colon. Am J Physiol Gastrointest Liver Physiol 279: G211-G222, 2000.--Endothelin (ET)-1 is a potent vasoconstrictor and mitogenic peptide that has a variety of biological effects in noncardiovascular tissues. The precise cellular distribution of the ET-1 system in the wall of the normal human colon was studied to identify the physiological role of ET in the gut. In situ hybridization revealed ET-converting enzyme-1 (ECE-1) mRNA in all vessels, the colon epithelium, and macrophages. Prepro-ET-1 (PPET-1) mRNA had a similar distribution except for a scattered signal in mucosal microvessels. [ET.sub.A] and [ET.sub.B] receptor mRNAs were mainly in the lamina propria, pericryptal myofibroblasts, microvessels, and mononuclear cells, with [ET.sub.A] mRNA more abundant than [ET.sub.B] mRNA. [sup.125]I-ET-1 binding showed [ET.sub.B] along the crypts and in nerve fibers descending from the ganglionic plexus that contained PPET-1, ECE-1, and [ET.sub.B] transcripts, whereas glia contained [ET.sub.A] receptors. The finding of the entire ET system in the normal mucosa suggests its implication in some characteristic functions of the colon and its secretion as both a neuroactive and a vasoactive peptide. in situ hybridization; immunocytochemistry; endothelial cell; smooth muscle cell

The cluster of differentiation 4 (CD4) molecule functions as a co-receptor for MHC class II binding to TCR in T helper cells. A CD4 epitope deficiency was identified in the swine MeLiM (melanoblastoma-bearing Libechov minipig) strain, a model for spontaneous cutaneous melanoma development and regression. Extensive sequencing revealed a high genetic variability of CD4 and the existence of several haplotypes segregating in MeLiM. Forty polymorphisms were identified in the coding sequence, out of which 20 correspond to non-synonymous variants and 10 are located in the 3′UTR of CD4 transcripts. One of the haplotypes segregating in the MeLiM explained the epitope deficiency observed. An association analysis between CD4 genotype and several phenotypes related to tumor regression was performed in 267 animals. An association was evidenced between a MeLiM alternative CD4 haplotype and skin and eye depigmentation, as well as the extent of hair depigmentation. Also, seric IgG concentration was shown to be higher in pigs carrying the alternative haplotype at the homozygous state. In conclusion, the genetic variability of the CD4 gene is associated with immune response-related phenotypes in MeLiM minipigs. [ABSTRACT FROM AUTHOR]

Background Constitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses. Methods Grey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting. Results We found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway. The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases. Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses. Conclusions These findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this pathway during melanomagenesis. These findings are consistent with the universal importance of the ERK pathway in melanomagenesis and may have valuable implications for human melanoma research. [ABSTRACT FROM AUTHOR]

The article presents a study which was aimed to propose a detailed histopathological atlas and classification of melanocytic lesions (ML) encountered in Tyr::NRas Q61K; Cdkn2a-/- or Cdkn2a-/+ transgenic mice along with their main microscopic features, with a particular emphasis on cutaneous ML. The study found that primary cutaneous ML were invariably present in the skin and were observed on gross examination as early as 2 months of age.

Objectives: Endothelin-1 (ET-1), plays an important role in the pathophysiology of CHF and the pulmonary endothelium is an early hemodynamic target in diastolic left ventricular dysfunction. Therefore we hypothesized that the lung is a main source of humoral endothelin in CHF and that its secretion is proportional to the degree of heart failure. Methods and results: We used rats with coronary artery ligation as an experimental model of either compensated or decompensated heart failure, depending on infarct size. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that in the lung, the expression of preproET-1 mRNA was higher in decompensated HF than in control and compensated HF rats (P<0.001). Run-on assay demonstrated that ET-1 overexpression is regulated at a transcriptional level (P<0.01). In contrast, there was no change in ET-1 mRNA expression in aortae, left ventricular myocardium and skeletal muscle. The expression of endothelin-converting enzyme (ECE)-1 mRNA was not modified and the expression of ETB receptor mRNA in the congestive lung was significantly lower than in control and compensated HF rats (P<0.0001), while the expression of ETA receptor mRNA did not differ between groups. The lung and plasma ET-1 peptide levels were respectively 4.2 and 9 fold higher in the rats with decompensated HF than in control rats (P<0.05; P<0.0001). Organoculture experiments showed that the lung ET-1 peptide secretion level in rats with decompensated HF was higher than that in control rats (P<0.01). In contrast, there was no change in ET-1 peptide secretion by the left ventricular myocardium and skeletal muscle. In plasma of rats with decompensated HF, the rate of bigET-1 conversion to ET-1 was 22%. ET-1 peptide was also present in the pleural effusion of decompensated heart failure. Plasma ET-1 concentration was significantly correlated with upstream markers of left ventricular diastolic dysfunction, with the expression of preproET-1 mRNA in the lung, with lung and pleural ET-1 concentration and with the expression ratio of ET-1/ETB receptor mRNA. Conclusion: Taken together, these data suggest that overexpression of ET-1 and down-regulation of ETB receptors in the lung are determinants of circulating endothelin in CHF. As a corollary, increased plasma endothelin may provide evidence of pulmonary endothelial dysfunction in CHF. [ABSTRACT FROM PUBLISHER]

Immunological research in pigs benefits from many improvements with a direct impact on the veterinary control of pig husbandry and on biomedical models. We compiled the available knowledge to develop gating strategies to monitor simultaneously all blood immune cell types by multicolor flow cytometry in Melanoblastoma-bearing Libechov Minipigs (MeLiM). The MeLiM pig spontaneously develops cutaneous melanomas that regress few months later. We monitored lymphoid and myeloid cell subsets in 3 to 21 weeks old pigs. Interestingly, neutrophils, type III monocytes (CD163 + CD14 + MHC II - ) and CD4 - CD8α - T cells are less abundant in oldest animals in contrast to eosinophils, type II monocytes (CD163 - CD14 low MHC II + ), B cells, γδ T cells, CD4 + CD8α + and CD4 - CD8α + T cells. Melanoma occurrence led to changes in the blood cell composition. Higher proportions of NK cells, CD4 + and CD4 + CD8α + T cells, and CD21 - B cells among B cells are found in young melanoma-bearing piglets, consistent with the immune-mediated spontaneous regression in the MeLiM model., (Copyright © 2020 Blanc, Prévost-Blondel, Piton, Bouguyon, Leplat, Andréoletti, Egidy, Bourneuf, Bertho and Vincent-Naulleau.)

The transcriptome sequencing of melanoma cells from two mouse models differing in the expression level of the scaffold protein Receptor for activated C kinase (RACK1) are presented. Primary melanoma cells were harvested from Tyr:NRas Q61K ; Pax3 GFP/+ mice, with or without the Tyr:Rack1-HA transgene. Cells were cultured and infected with scramble shRNA or Rack1 -targeting shRNA , on technical triplicates of viral infection. Libraries were prepared by selecting polyadenylated mRNAs and RNA Sequencing (RNASeq) was performed. Samples are described in the SRA portal (SRP096162) and FASTQ files have been deposited in Sequence Read Archive (accession numbers: SRR5150106 to SRR5150117). The interpretation of these data is presented in the following research article: "RACK1 cooperates with NRAS Q61K to promote melanoma in vivo " (Campagne et al., 2017, doi: 10.1016/j.cellsig.2017.03.015) [1].