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In MeLiM minipigs, melanomas develop around birth, can metastasize, and have histopathologic characteristics similar to humans. Interestingly, MeLiM melanomas eventually regress. This favorable outcome raises the question of their malignancy, which we investigated. We clinically followed tens of tumors from onset to first signs of regression. Transcriptome analysis revealed an enrichment of all cancer hallmarks in melanomas, although no activating or suppressing somatic mutation were found in common driver genes. Analysis of tumor cell genomes revealed high mutation rates without UV signature. Canonical proliferative, survival and angiogenic pathways were detected in MeLiM tumor cells all along progression stages. Functionally, we show that MeLiM melanoma cells are capable to grow in immunocompromised mice, with serial passages and for a longer time than in MeLiM pigs. Pigs set in place an immune response during progression with dense infiltration by myeloid cells while melanoma cells are deficient in B2M expression. To conclude, our data on MeLiM melanomas reveal several malignancy characteristics. The combination of these features with the successful spontaneous regression of these tumors make it an outstanding model to study an efficient anti-tumor immune response., (© 2024. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 De Wolf, Gouin, Jouneau, Glorion, Premachandra, Pascale, Huriet, Estephan, Leplat, Egidy, Richard, Gelin, Urien, Roux, Le Guen, Schwartz-Cornil and Sage.)

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., (© 2023. The Author(s).)

Background: Normothermic ex vivo lung perfusion (EVLP) increases the pool of donor lungs by requalifying marginal lungs refused for transplantation through the recovery of macroscopic and functional properties. However, the cell response and metabolism occurring during EVLP generate a nonphysiological accumulation of electrolytes, metabolites, cytokines, and other cellular byproducts which may have deleterious effects both at the organ and cell levels, with impact on transplantation outcomes., Methods: We analyzed the physiological, metabolic, and genome-wide response of lungs undergoing a 6-h EVLP procedure in a pig model in 4 experimental conditions: without perfusate modification, with partial replacement of fluid, and with adult or pediatric dialysis filters., Results: Adult and pediatric dialysis stabilized the electrolytic and metabolic profiles while maintaining acid-base and gas exchanges. Pediatric dialysis increased the level of IL-10 and IL-6 in the perfusate. Despite leading to modification of the perfusate composition, the 4 EVLP conditions did not affect the gene expression profiles, which were associated in all cases with increased cell survival, cell proliferation, inflammatory response and cell movement, and with inhibition of bleeding., Conclusions: Management of EVLP perfusate by periodic replacement and continuous dialysis has no significant effect on the lung function nor on the gene expression profiles ex vivo. These results suggest that the accumulation of dialyzable cell products does not significantly alter the lung cell response during EVLP, a finding that may have impact on EVLP management in the clinic., Competing Interests: The authors declare no funding and conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

This study describes the associations between fecal microbiota and vaccine response variability in pigs, using 98 piglets vaccinated against the influenza A virus at 28 days of age (D28) with a booster at D49. Immune response to the vaccine is measured at D49, D56, D63, and D146 by serum levels of IAV-specific IgG and assays of hemagglutination inhibition (HAI). Analysis of the pre-vaccination microbiota characterized by 16S rRNA gene sequencing of fecal DNA reveals a higher vaccine response in piglets with a richer microbiota, and shows that 23 operational taxonomic units (OTUs) are differentially abundant between high and low IAV-specific IgG producers at D63. A stronger immune response is linked with OTUs assigned to the genus Prevotella and family Muribaculaceae, and a weaker response is linked with OTUs assigned to the genera Helicobacter and Escherichia-Shigella. A set of 81 OTUs accurately predicts IAV-specific IgG and HAI titer levels at all time points, highlighting early and late associations between pre-vaccination fecal microbiota composition and immune response to the vaccine., (© 2021. The Author(s).)

Background: The impact of individual genetic and genomic variations on immune responses is an emerging lever investigated in vaccination strategies. In our study, we used genetic and pre-vaccination blood transcriptomic data to study vaccine effectiveness in pigs., Results: A cohort of 182 Large White pigs was vaccinated against Mycoplasma hyopneumoniae (M. hyo) at weaning (28 days of age), with a booster 21 days later. Vaccine response was assessed by measuring seric M. hyo antibodies (Ab) at 0 (vaccination day), 21 (booster day), 28, 35, and 118 days post-vaccination (dpv). Inter-individual variability of M. hyo Ab levels was observed at all time points and the corresponding heritabilities ranged from 0.46 to 0.57. Ab persistence was higher in females than in males. Genome-wide association studies with a 658 K SNP panel revealed two genomic regions associated with variations of M. hyo Ab levels at 21 dpv at positions where immunity-related genes have been mapped, DAB2IP on chromosome 1, and ASAP1, CYRIB and GSDMC on chromosome 4. We studied covariations of Ab responses with the pre-vaccination blood transcriptome obtained by RNA-Seq for a subset of 82 pigs. Weighted gene correlation network and differential expression analyses between pigs that differed in Ab responses highlighted biological functions that were enriched in heme biosynthesis and platelet activation for low response at 21 dpv, innate antiviral immunity and dendritic cells for high response at 28 and 35 dpv, and cell adhesion and extracellular matrix for high response at 118 dpv. Sparse partial least squares discriminant analysis identified 101 genes that efficiently predicted divergent responders at all time points. We found weak negative correlations of M. hyo Ab levels with body weight traits, which revealed a trade-off that needs to be further explored., Conclusions: We confirmed the influence of the host genetics on vaccine effectiveness to M. hyo and provided evidence that the pre-vaccination blood transcriptome co-varies with the Ab response. Our results highlight that both genetic markers and blood biomarkers could be used as potential predictors of vaccine response levels and more studies are required to assess whether they can be exploited in breeding programs.

The gut microbiota comprises a large and diverse community of bacteria that play a significant role in swine health. Indeed, there is a tight association between the enteric immune system and the overall composition and richness of the microbiota, which is key in the induction, training and function of the host immunity, and may therefore, influence the immune response to vaccination. Using vaccination against Mycoplasma hyopneumoniae ( M. hyo ) as a model, we investigated the potential of early-life gut microbiota in predicting vaccine response and explored the post-vaccination dynamics of fecal microbiota at later time points. At 28 days of age (0 days post-vaccination; dpv), healthy piglets were vaccinated, and a booster vaccine was administered at 21 dpv. Blood samples were collected at 0, 21, 28, 35, and 118 dpv to measure M. hyo -specific IgG levels. Fecal samples for 16S rRNA gene amplicon sequencing were collected at 0, 21, 35, and 118 dpv. The results showed variability in antibody response among individual pigs, whilst pre-vaccination operational taxonomic units (OTUs) primarily belonging to Prevotella , [ Prevotella ], Anaerovibrio , and Sutterella appeared to best-predict vaccine response. Microbiota composition did not differ between the vaccinated and non-vaccinated pigs at post-vaccination time points, but the time effect was significant irrespective of the animals' vaccination status. Our study provides insight into the role of pre-vaccination gut microbiota composition in vaccine response and emphasizes the importance of studies on full metagenomes and microbial metabolites aimed at deciphering the role of specific bacteria and bacterial genes in the modulation of vaccine response.

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.)

DNA vaccination is an attractive technology, based on its well-established manufacturing process, safety profile, adaptability to rapidly combat pandemic pathogens, and stability at ambient temperature; however an optimal delivery method of DNA remains to be determined. As pigs are a relevant model for humans, we comparatively evaluated the efficiency of vaccine DNA delivery in vivo to pigs using dissolvable microneedle patches, intradermal inoculation with needle (ID), surface electroporation (EP), with DNA associated or not to cationic poly-lactic-co-glycolic acid nanoparticles (NPs). We used a luciferase encoding plasmid (pLuc) as a reporter and vaccine plasmids encoding antigens from the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a clinically-significant swine arterivirus. Patches were successful at inducing luciferase expression in skin although at lower level than EP. EP induced the cutaneaous recruitment of granulocytes, of MHC2 pos CD172A pos myeloid cells and type 1 conventional dendritic cells, in association with local production of IL-1β, IL-8 and IL-17; these local responses were more limited with ID and undetectable with patches. The addition of NP to EP especially promoted the recruitment of the MHC2 pos CD172A pos CD163 int and CD163 neg myeloid subsets. Notably we obtained the strongest and broadest IFNγ T-cell response against a panel of PRRSV antigens with DNA + NPs delivered by EP, whereas patches and ID were ineffective. The anti-PRRSV IgG responses were the highest with EP administration independently of NPs, mild with ID, and undetectable with patches. These results contrast with the immunogenicity and efficacy previously induced in mice with patches. This study concludes that successful DNA vaccine administration in skin can be achieved in pigs with electroporation and patches, but only the former induces local inflammation, humoral and cellular immunity, with the highest potency when NPs were used. This finding shows the importance of evaluating the delivery and immunogenicity of DNA vaccines beyond the mouse model in a preclinical model relevant to human such as pig and reveals that EP with DNA combined to NP induces strong immunogenicity., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The porcine reproductive and respiratory syndrome virus (PRRSV), an RNA virus inducing abortion in sows and respiratory disease in young pigs, is a leading infectious cause of economic losses in the swine industry. Modified live vaccines (MLVs) help in controlling the disease, but their efficacy is often compromised by the high genetic diversity of circulating viruses, leading to vaccine escape variants in the field. In this study, we hypothesized that a DNA prime with naked plasmids encoding PRRSV antigens containing conserved T-cell epitopes may improve the protection of MLV against a heterologous challenge. Plasmids were delivered with surface electroporation or needle-free jet injection and European strain-derived PRRSV antigens were targeted or not to the dendritic cell receptor XCR1. Compared to MLV-alone, the DNA-MLV prime- boost regimen slightly improved the IFNγ T-cell response, and substantially increased the antibody response against envelope motives and the nucleoprotein N. The XCR1-targeting of N significantly improved the anti-N specific antibody response. Despite this immuno-potentiation, the DNA-MLV regimen did not further decrease the serum viral load or the nasal viral shedding of the challenge strain over MLV-alone. Finally, the heterologous protection, achieved in absence of detectable effective neutralizing antibodies, was not correlated to the measured antibody or to the IFNγ T-cell response. Therefore, immune correlates of protection remain to be identified and represent an important gap of knowledge in PRRSV vaccinology. This study importantly shows that a naked DNA prime immuno-potentiates an MLV, more on the B than on the IFNγ T-cell response side, and has to be further improved to reach cross-protection.

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an arterivirus responsible for highly contagious infection and huge economic losses in pig industry. Two species, PRRSV-1 and PRRSV-2 are distinguished, PRRSV-1 being more prevalent in Europe. PRRSV-1 can further be divided in subtypes. PRRSV-1.3 such as Lena are more pathogenic than PRRSV-1.1 such as Lelystad or Flanders13. PRRSV-1.3 viruses trigger a higher Th1 response than PRRSV-1.1, although the role of the cellular immune response in PRRSV clearance remains ill defined. The pathogenicity as well as the T cell response inductions may be differentially impacted according to the capacity of the virus strain to infect and/or activate DCs. However, the interactions of PRRSV with in vivo -differentiated-DC subtypes such as conventional DC1 (cDC1), cDC2, and monocyte-derived DCs (moDC) have not been thoroughly investigated. Here, DC subpopulations from Lena in vivo infected pigs were analyzed for viral genome detection. This experiment demonstrates that cDC1, cDC2, and moDC are not infected in vivo by Lena. Analysis of DC cytokines production revealed that cDC1 are clearly activated in vivo by Lena. In vitro comparison of 3 Europeans strains revealed no infection of the cDC1 and cDC2 and no or little infection of moDC with Lena, whereas the two PRRSV-1.1 strains infect none of the 3 DC subtypes. In vitro investigation of T helper polarization and cytokines production demonstrate that Lena induces a higher Th1 polarization and IFNγ secretion than FL13 and LV. Altogether, this work suggests an activation of cDC1 by Lena associated with a Th1 immune response polarization.

Lung inflammation is frequently involved in respiratory conditions and it is strongly controlled by mononuclear phagocytes (MNP). We previously studied porcine lung MNP and described a new population of cells presenting all the features of alveolar macrophages (AM) except for their parenchymal location, that we named AM-like cells. Herein we showed that AM-like cells are macrophages phagocytosing blood-borne particles, in agreement with a pulmonary intravascular macrophages (PIM) identity. PIM have been described microscopically long time ago in species from the Laurasiatheria superorder such as bovine, swine, cats or cetaceans. We observed that PIM were more inflammatory than AM upon infection with the porcine reproductive and respiratory syndrome virus (PRRSV), a major swine pathogen. Moreover, whereas PRRSV was thought to mainly target AM, we observed that PIM were a major producer of virus. The PIM infection was more correlated with viremia in vivo than AM infection. Finally like AM, PIM-expressed genes were characteristic of an embryonic monocyte-derived macrophage population, whose turnover is independent of bone marrow-derived hematopoietic precursors. This last observation raised the interesting possibility that AM and PIM originate from the same lung precursor.

The epithelium of the intestinal mucosa and the gut-associated lymphoid tissues (GALT) constitute an essential physical and immunological barrier against pathogens. In order to study the specificities of the GALT transcriptome in pigs, we compared the transcriptome profiles of jejunal and ileal Peyer's patches (PPs), mesenteric lymph nodes (MLNs) and peripheral blood (PB) of four male piglets by RNA-Seq. We identified 1,103 differentially expressed (DE) genes between ileal PPs (IPPs) and jejunal PPs (JPPs), and six times more DE genes between PPs and MLNs. The master regulator genes FOXP3, GATA3, STAT4, TBX21 and RORC were less expressed in IPPs compared to JPPs, whereas the transcription factor BCL6 was found more expressed in IPPs. In comparison between IPPs and JPPs, our analyses revealed predominant differential expression related to the differentiation of T cells into Th1, Th2, Th17 and iTreg in JPPs. Our results were consistent with previous reports regarding a higher T/B cells ratio in JPPs compared to IPPs. We found antisense transcription for respectively 24%, 22% and 14% of the transcripts detected in MLNs, PPs and PB, and significant positive correlations between PB and GALT transcriptomes. Allele-specific expression analyses revealed both shared and tissue-specific cis-genetic control of gene expression.

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.

The ecological interactions within the gut microbial communities are complex and far from being fully understood. Here we report the first study that aims at defining the interaction network of the gut microbiota in pigs and comparing it with the enterotype-like clustering analysis. Fecal microbiota of 518 healthy piglets was characterized by 16S ribosomal RNA gene sequencing. Two networks were constructed at the genus and operational taxonomic unit levels. Within-network interactions mirrored the human gut microbiota relationships, with a strong co-exclusion between Prevotella and Ruminococcus genera, and were consistent with the two enterotype-like clusters identified in the pig microbiota. Remarkably, the cluster classification of the individuals was significantly associated with the body weight at 60 days of age (P=0.005) and average daily gain (P=0.027). To the best of our knowledge, this is the first study to provide an integrated overview of the porcine gut microbiota that suggests a conservation of the ecological community interactions and functional architecture between humans and pig. Moreover, we show that the microbial ecosystems and porcine growth traits are linked, which allows us to foresee that the enterotype concept may have an important role in the animal production industry.

Human and mouse respiratory tracts show anatomical and physiological differences, which will benefit from alternative experimental models for studying many respiratory diseases. Pig has been recognized as a valuable biomedical model, in particular for lung transplantation or pathologies such as cystic fibrosis and influenza infection. However, there is a lack of knowledge about the porcine respiratory immune system. Here we segregated and studied six populations of pig lung dendritic cells (DCs)/macrophages (Mθs) as follows: conventional DCs (cDC) 1 and cDC2, inflammatory monocyte-derived DCs (moDCs), monocyte-derived Mθs, and interstitial and alveolar Mθs. The three DC subsets present migratory and naive T-cell stimulation capacities. As observed in human and mice, porcine cDC1 and cDC2 were able to induce T-helper (Th)1 and Th2 responses, respectively. Interestingly, porcine moDCs increased in the lung upon influenza infection, as observed in the mouse model. Pig cDC2 shared some characteristics observed in human but not in mice, such as the expression of FCɛRIα and Langerin, and an intra-epithelial localization. This work, by unraveling the extended similarities of the porcine and human lung DC/Mθ networks, highlights the relevance of pig, both as an exploratory model of DC/Mθ functions and as a model for human inflammatory lung pathologies.

Early bacterial colonization and succession within the gastrointestinal tract has been suggested to be crucial in the establishment of specific microbiota composition and the shaping of host phenotype. Here, the composition and dynamics of faecal microbiomes were studied for 31 healthy piglets across five age strata (days 14, 36, 48, 60 and 70 after birth) together with their mothers. Faecal microbiome composition was assessed by 16S rRNA gene 454-pyrosequencing. Bacteroidetes and Firmicutes were the predominant phyla present at each age. For all piglets, luminal secretory IgA concentration was measured at day 70, and body weight was recorded until day 70. The microbiota of suckling piglets was mainly represented by Bacteroides, Oscillibacter, Escherichia/Shigella, Lactobacillus and unclassified Ruminococcaceae genera. This pattern contrasted with that of Acetivibrio, Dialister, Oribacterium, Succinivibrio and Prevotella genera, which appeared increased after weaning. Lactobacillus fermentum might be vertically transferred via breast milk or faeces. The microbiota composition coevolved with their hosts towards two different clusters after weaning, primarily distinguished by unclassified Ruminococcaceae and Prevotella abundances. Prevotella was positively correlated with luminal secretory IgA concentrations, and body weight. Our study opens up new possibilities for health and feed efficiency manipulation via genetic selection and nutrition in the agricultural domain., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Increasing robustness via improvement of resistance to pathogens is a major selection objective in livestock breeding. As resistance traits are difficult or impossible to measure directly, potential indirect criteria are measures of immune traits (ITs). Our underlying hypothesis is that levels of ITs with no focus on specific pathogens define an individual's immunocompetence and thus predict response to pathogens in general. Since variation in ITs depends on genetic, environmental and probably epigenetic factors, our aim was to estimate the relative importance of genetics. In this report, we present a large genetic survey of innate and adaptive ITs in pig families bred in the same environment., Methodology/principal Findings: Fifty four ITs were studied on 443 Large White pigs vaccinated against Mycoplasma hyopneumoniae and analyzed by combining a principal component analysis (PCA) and genetic parameter estimation. ITs include specific and non specific antibodies, seric inflammatory proteins, cell subsets by hemogram and flow cytometry, ex vivo production of cytokines (IFNα, TNFα, IL6, IL8, IL12, IFNγ, IL2, IL4, IL10), phagocytosis and lymphocyte proliferation. While six ITs had heritabilities that were weak or not significantly different from zero, 18 and 30 ITs had moderate (0.10.4) heritability values, respectively. Phenotypic and genetic correlations between ITs were weak except for a few traits that mostly include cell subsets. PCA revealed no cluster of innate or adaptive ITs., Conclusions/significance: Our results demonstrate that variation in many innate and adaptive ITs is genetically controlled in swine, as already reported for a smaller number of traits by other laboratories. A limited redundancy of the traits was also observed confirming the high degree of complementarity between innate and adaptive ITs. Our data provide a genetic framework for choosing ITs to be included as selection criteria in multitrait selection programmes that aim to improve both production and health traits.

Incomplete spontaneous regression of melanoma is common. However, complete melanoma regression is still a very rare phenomenon. Because melanoma is the most immunogenic human malignancy, the mechanisms leading to regression, based on accumulative evidence, are the host's immune responses. Unfortunately, therapies aiming to enhance the patient's natural immunity against melanoma have yet to meet their expectations. Reasons for failure include various immune escape mechanisms, induced by the tumor, that subsequently lead to tolerance. Here, we performed time-dependent gene expression profiling to unravel molecular changes involved in the transition of progressive melanoma to complete tumor regression using a porcine model. The melanoblastomabearing Libechov minipigs are highly suitable for this study because these animals exhibit naturally occurring and regressing melanomas. We were able to identify a molecular signature of the melanoma regression process. Genes regulated in this signature were associated with 1) cell cycle, 2) immune response, and 3) melanocyte differentiation. These genes may shed light on molecular mechanisms involved in complete melanoma regression and indicate what improvements are needed for successful antimelanoma therapy.

Background: Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. We studied the reconstitution of myeloid and lymphoid compartments after the transplantation of autologous CD34+ bone marrow cells following gamma irradiation in cynomolgus macaques., Results: The bone marrow cells were first transduced ex vivo with a lentiviral vector encoding eGFP, with a mean efficiency of 72% +/- 4%. The vector used was derived from the simian immunodeficiency lentivirus SIVmac251, VSV-g pseudotyped and encoded eGFP under the control of the phosphoglycerate kinase promoter. After myeloid differentiation, GFP was detected in colony-forming cells (37% +/- 10%). A previous study showed that transduction rates did not differ significantly between colony-forming cells and immature cells capable of initiating long-term cultures, indicating that progenitor cells and highly immature hematopoietic cells were transduced with similar efficiency. Blood cells producingeGFP were detected as early as three days after transplantation, and eGFP-producing granulocyte and mononuclear cells persisted for more than one year in the periphery., Conclusion: The transplantation of CD34+ bone marrow cells had beneficial effects for the ex vivo proliferation and differentiation of hematopoietic progenitors, favoring reconstitution of the T- and B-lymphocyte, thrombocyte and red blood cell compartments.

Partial and some few cases of complete spontaneous regression have been observed in cutaneous melanoma patients but little is known about the molecular mechanisms involved. The Melanoblastoma-bearing Libechov Minipig (MeLiM) is a suitable animal model to study the phenomenon of spontaneous regression because MeLiM pigs exhibit naturally occurring melanomas which regress completely 6 months after birth. In this study, we used suppression subtractive hybridization (SSH) to identify molecular determinants of melanoma regression within swine melanoma tissues and melanoma cell cultures. Several markers involved in cell-adhesion, -communication, -motility, signal transduction, negative regulation of cell proliferation, transport and immune response were identified that correlated with melanoma regression whereas the main genes involved in melanin synthesis showed a strong downregulation. For the most differentially expressed genes, we validated the results obtained by SSH with qRT-PCR and with immunohistochemistry for some of them (CD9, MITF, RARRES1). Most notable, for the first time in melanoma, we identified the retinoic acid responder 1 gene (RARRES1) as a main actor of the regression process in melanoma. This first gene expression study in swine melanoma regression, may contribute to the finding of new therapeutic targets for human melanoma treatment.

Human cutaneous melanoma is a complex trait inherited in about 10% of cases. Although 2 high-risk genes, CDKN2A and CDK4, and 1 low risk gene, MC1R, have been identified, susceptibility genes remain to be discovered. Here, we attempted to determine new genomic regions linked to melanoma using the pig MeLiM strain, which develops hereditary cutaneous melanomas. We applied quantitative trait loci (QTL) mapping method to a significant genome-wide scan performed on 331 backcross pigs derived from this strain. QTLs were detected at chromosome-wide level for a melanoma synthetic trait corresponding to the development of melanoma. The peak positions on Sus scrofa chromosomes (SSC) were at 49.4 and 88.0 cM (SSC1), 56.0 cM (SSC13), 86.5 cM (SSC15) and 39.8 cM (SSC17), and, on SSC2, at 16.9 cM, in families derived from F1 males only (p < 0.05, except for SSC13, p < 0.01). Analysis of 7 precise specific traits revealed highly significant QTLs on SSC10 (ulceration), on SSC12 (presence of melanoma at birth), on SSC13 (lesion type), and on SSC16 and SSC17 (number of aggressive melanomas) at the respective positions 42.0, 95.6, 81.0, 45.3 and 44.8 cM (p < 0.001 and p < 0.05 respectively at the chromosome- and genome-wide levels). We also showed that MeLiM MC1R*2 allele, which determines black coat colour in pigs, predisposes significantly to melanoma. Interactions were observed between MC1R and markers located on SSC1 (p < 0.05). Taken together, these results indicate that MeLiM swine is a model for human multigenic diseases. Comparative mapping revealed human regions of interest to search for new melanoma susceptibility candidates., ((c) 2006 Wiley-Liss, Inc.)

We performed a microarray study on human differentiated HaCaT keratinocytes exposed to ionizing radiation (2 or 10 Gy). At 3 h after exposure, more than 150 known and unknown genes were found regulated in irradiated HaCaT keratinocytes. Among the genes regulated at 3 h, those involved in cell energy metabolism appeared to be the most abundant and the most responsive. Two mitochondrial ATP-synthases and several other genes involved in energy producing pathways, such as glucose metabolism, were induced, whereas many genes from energy requiring pathways were shut down. These changes in energy metabolism were confirmed both in normal primary keratinocytes and in HaCaT keratinocytes by RT-PCR and proteins studies. Moreover, measures of intracellular ATP revealed a 50% increase in keratinocytes immediately after irradiation, supporting an energy procurement response. The overall results indicate that irradiation induces an immediate burst of ATP that seems to be a general response of human differentiated keratinocytes to the radiation stress. This article contains Supplementary Material available at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/v95.html, ((c) 2005 Wiley-Liss, Inc.)

Skin is the organ most exposed to various environmental aggressors, including ionizing radiation. Low-dose and low-dose-rate exposures to gamma rays account for most occupational, medical or environmental irradiations. To examine whether this type of exposure triggers specific molecular responses, cultured primary keratinocytes isolated from adult normal skin were irradiated with single acute doses of 1 cGy or 2 Gy. DNA microarrays containing 10,500 probes were used to assess transcriptional changes over a time course between 3 and 72 h postirradiation. Keratinocytes were studied at a differentiated stage to mimic the response of cells from the suprabasal layers of the epidermis. A major finding of this study was the identification of an important number of low-dose-specific genes (140), most of which were modulated at 48 h. Clustering analysis also revealed low-dose-specific profiles. One of these clusters (17 known genes) was further analyzed using Gibbs sampling algorithm, which led to the identification of 7 putative promoter sequences. These results show for the first time that low-dose ionizing radiation is able to induce specific transcriptional responses in human keratinocytes. Our findings support the potential usefulness of microarrays in biological dosimetry studies after low-dose exposures.

In human familial melanoma, 3 risk susceptibility genes are already known, CDKN2A, CDK4 and MC1R. However, various observations suggest that other melanoma susceptibility genes have not yet been identified. To search for new susceptibility loci, we used the MeLiM swine as an animal model of hereditary melanoma to perform a genome scan for linkage to melanoma. Founders of the affected MeLiM stock were crossed with each other and with healthy Duroc pigs, generating MeLiM, F1 and backcross families. As we had previously excluded the MeLiM CDKN2A gene, we paid special attention to CDK4 and MC1R, as well as to other candidates such as BRAF and the SLA complex, mapping them on the swine radiation hybrid map and/or isolating close microsatellite markers to introduce them into the genome scan. The results revealed, first, that swine melanoma was inherited as an autosomal dominant trait with incomplete penetrance, preferably in black animals. Second, 4 chromosomal regions potentially involved in melanoma susceptibility were identified on Sus Scrofa chromosomes (SSC) 1, 2, 7 and 8, respectively, in intervals 44-103, 1.9-18, 59-73 and 47-62 cM. A fifth region close to MC1R was revealed on SSC 6 by analyzing an individual marker located at position 7.5 cM. Lastly, CDK4 and BRAF were unlikely to be melanoma susceptibility genes in the MeLiM swine model. The 3 regions on SSC 1, 6 and 7, respectively, have counterparts on human chromosomes (HSA) 9p, 16q and 6p, harboring melanoma candidate loci. The 2 others, on SSC 2 and 8, have counterparts on HSA 11 and 4, which might therefore be of interest for human studies., (Copyright 2004 Wiley-Liss, Inc.)

Spontaneous animal tumors appear to be highly suitable models to study human oncology and cancer therapy. The aim of this study was to characterize the clinical and histological features of hereditary melanocytic lesions found in the French herd of melanoblastoma-bearing Libechov minipigs (MeLiM) and their Duroc crossbreeds. Clinically, we discriminated between three types of melanocytic skin lesions, which offer a lesion continuum from lentigo to metastatic melanomas. More than 70% of these lesions appear on piglets before they are 3 months old and preferentially on homogeneous black coat piglets. The incidence of melanoma reaches 50% in MeLiM. Most of the highly invasive melanomas regressed spontaneously in the first year of the piglet's life and the regression was followed by hair, skin and iris depigmentation. A histopathological study was conducted according to the human melanoma classification. Except for lentigo maligna, we observed the three main types of human melanoma in swine [superficial spreading melanoma (SSM), nodular or unclassified melanoma] with an excess of SSM (59-67%). The histological events leading to total spontaneous regression are chronologically described. The genetic predisposition, the high incidence of melanoma, the clinical and histopathological features similar to the human disease and the high rate of spontaneous regression offer an opportunity to use this model for studying genetic events controlling melanoma development and regression and the biological mechanisms involved in oncogenesis and anti-cancerous self-defense.

The aim of this study was to evaluate the Melanoblastoma-bearing Libechov Minipigs (MeLiM) as an animal model of melanoma for in vivo imaging. Serial whole-body 2-deoxy-2-[(18)F]fluoro- d-glucose positron emission tomography (FDG PET) scans were conducted on five MeLiM. In order to explore different clinical stages of the tumoural lesions, each animal was scanned two to four times, at intervals of 30-155 days. PET images were analysed by a semiquantitative method based on the tumour to muscle metabolic ratio. Histology was performed on biopsies taken between or after the scans and the histological grading of the tumours was compared with the FDG uptake. The overall sensitivity of FDG PET for the detection of cutaneous melanoma was 75%; 62.5% of involved lymph nodes were positive. Sensitivity was better for tumours with vertical growth than for flat lesions. FDG PET did not detect tumours with epidermal involvement only, nor did it detect small metastatic foci. The metabolic ratio was correlated with the evolution of the melanoma. FDG PET is effective in the staging of cutaneous melanoma and the follow-up of tumoural extension and regression in Melanoblastoma-bearing Libechov Minipigs. The results obtained in this animal model correlate well with those described in human melanoma. Accordingly, this model may be useful in testing new tracers specific for melanoma and in helping to detect molecules expressed early during tumoural regression.

Some herds of miniature swine are genetically predisposed to cutaneous melanoma. To test if swine melanoma susceptibility could be linked to the CDKN2A gene, which is involved in a proportion of 9p21-linked human familial melanoma, we performed a genetic analysis of miniature pigs of the MeLiM strain. F(1) and backcross animals were generated by crossing 1 MeLiM boar with healthy Duroc sows. We isolated the swine CDKN2A gene and characterized a linked informative microsatellite marker, the S0644 marker. Using this marker and 2 flanking markers, we analyzed the segregation of the CDKN2A gene in a 3-generation pedigree. Allelic association, linkage analysis and haplotype analysis of these data led to exclusion of the CDKN2A gene as a candidate for melanoma susceptibility. Nonetheless, this analysis suggests an association with the swine 1q25 chromosomal region, which is homologous to the human 9p21 region., (Copyright 2002 Wiley-Liss, Inc.)

We present in this paper two imaging techniques using contrast agents assessed with in vivo experiments. Both methods are based on the same physical principle, and were implemented at the European Synchrotron Radiation Facility medical beamline. The first one is intravenous coronary angiography using synchrotron radiation X-rays. This imaging technique has been planned for human studies in the near future. We describe the first experiments that were carried out with pigs at the ESRF. The second imaging mode is computed tomography using synchrotron radiation on rats bearing brain tumors. Owing to synchrotron radiation physical properties, these new imaging methods provide additional information compared to conventional techniques. After infusion of the contrast agent, it is possible to derive from the images the concentration of the contrast agent in the tumor area for the computed tomography and in any visible vessel for the angiography method.

Purpose: To establish a successful treatment of subcutaneous fibrosis developing after high doses of gamma rays, suitable for use in clinical practice., Methods and Materials: We used an animal model of acute localized gamma irradiation simulating accidental overexposure in humans. Three groups of 5 Large White pigs were irradiated using a collimated 192Ir source to deliver a single dose of 160 Gy onto the skin surface (100%) of the outer side of the thigh. A well-defined block of necrosis developed within a few weeks which had healed after 26 weeks to leave a block of subcutaneous fibrosis involving skin and skeletal muscle. One experimental group of 5 pigs was dosed orally for 26 weeks starting 26 weeks after irradiation with 1600 mg/120 kg body weight of pentoxifylline (PTX) included in the reconstituted food during its fabrication, and another group of 5 was dosed orally for the same period with a daily dose of 1600 mg/120 kg body weight of PTX combined with 2000 IU/120 kg body weight of alpha-tocopherol. Five irradiated control pigs were given normal food only. Animals were assessed for changes in the density of the palpated fibrotic block and in the dimensions of the projected cutaneous surface. Depth of scar tissue was determined by ultrasound. Physical and sonographic findings were confirmed by autopsy 26 weeks after treatment started. The density, length, width, and depth of the block of fibrotic scar tissue, and the areas and volume of its projected cutaneous surface, were compared before treatment, 6 and 13 weeks thereafter, and at 26 weeks., Results: The experimental animals exhibited no change in behavior and no abnormal clinical or anatomic signs. No modifications were observed in the block of fibrotic scar tissue of pigs dosed with PTX alone. However, significant softening and shrinking of this block were noted in the pigs dosed with PTX + alpha-tocopherol 13 weeks after treatment started and at autopsy, when mean regression was approximately 30% for length, approximately 50% for width and depth, and approximately 70% for area and volume. Histologic examination showed completely normal muscle and subcutaneous tissue surrounding the residual scar tissue. The 50% decrease in the linear dimensions of the scar tissue, were comparable to the results obtained in our previous clinical studies, and were highly significant compared to the clinical and autopsy results for the controls. Histologic examination of the residual scar tissue revealed tissue which was more homogenous and less cellular and inflammatory than in control and PTX-dosed pigs. The tissular and cellular immunolocalization of tumor necrosis factor alpha (TNFalpha) was similar in the residual fibrotic tissues of all three groups of pigs, whereas the immunostaining of transforming growth factor beta-1(TGFbeta-1) diminished much more in the residual fibrotic scar tissue of the PTX + alpha-tocopherol-dosed pigs than in the two other groups., Conclusions: The present results showed a striking regression of the subcutaneous fibrotic scar tissue that develops as a consequence of high doses of gamma rays.

Purpose: To establish how far liposomal copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD), respectively, reduce radiation-induced fibrosis (RIF), using a well-characterized pig model of RIF permitting the design of a controlled laboratory experiment., Methods and Materials: In this model of acute localized gamma irradiation simulating accidental overexposure in humans, three groups of five large white pigs were irradiated using a collimated 192Ir source to deliver a single dose of 160 Gy onto the skin surface (100%) of the outer side of the thigh. A well-defined block of subcutaneous fibrosis involving skin and skeletal muscle developed 6 months after irradiation. One experimental group of five pigs was then injected i.m. with 10 mg/10 kg b.wt. of Cu/Zn-SOD, twice a week for 3 weeks, and another experimental group of five was injected with 10 mg/10 kg b.wt. of Mn-SOD, three times a week for 3 weeks. Five irradiated control pigs were injected with physiological serum. Animals were assessed for changes in the density of the palpated fibrotic block and in the dimensions of the projected cutaneous surface. Block depth was determined by ultrasound. Physical and sonographic findings were confirmed by autopsy 12-14 weeks after completing SOD injections. The density, length, width, and depth of the fibrotic block, and the areas and volume of its projected cutaneous surface were compared before treatment, 1, 3, and 6 weeks thereafter, and at autopsy, 12-14 weeks after treatment ended., Results: The experimental animals exhibited no change in behavior and no abnormal clinical or anatomic signs. Whether they were given Cu/Zn- or Mn-SOD, significant and roughly equivalent softening and shrinking of the fibrotic block were noted in all treated animals between the first week after treatment ended and autopsy, when mean regression was 45% for length and width, 30% for depth, and 70% for area and volume. Histologic examination showed completely normal muscle and subcutaneous tissue surrounding the residual scar. This replacement of scar tissue by normal tissue in experimental animals and the 50% decrease in the linear dimensions of the scar were comparable to the results obtained in previous clinical studies and highly significant compared to the clinical and autopsy results for the control animals., Conclusions: Our results are striking and comparable to the results obtained in our previous clinical study after liposomal Cu/Zn-SOD treatment. To our knowledge, this is the first time that two agents have been shown to reverse the radiation-induced fibrotic process in experimental animals and to permit the regeneration of normal tissue in a zone of well-established postirradiation fibrosis.

Sub-cutaneous and muscular fibrosis are common and irreversible late effect of radiation on normal tissues. An experiment was designed to test the effectiveness of superoxide dismutase in reducing late radiation injury. This study was performed in an experimental porcine model of acute localized gamma irradiation simulating human accidental overexposure: 12 Large White pigs were irradiated on the thigh with a collimated gamma 192Ir source, so that the dose was 160 Gy/skin (100%) and 40 Gy/2 cm depth (25%). In this model, fibrosis appears in 4 to 5 months. The heterogeneous sclerotic tissue is composed of stable fibrotic areas poorly cellularized and active areas with a high density of myofibroblasts and inflammatory perifibrotic part. Lipsod administration modalities were six intramuscular injections during 3 weeks (twice weekly) either 10 mg/inj (five pigs) or of 100 mg/inj (five pigs). A methodic evaluation by two examiners consisted of measurements being taken before and after treatment: sum of the two largest perpendicular measurable dimensions, cutaneous projected surface of palpated fibrotic block, ultrasound fibrosis deepness and extrapolated volume. We conclude that Lipsod is the first drug ever described that reduces radiation-induced fibrosis. Its efficacy in this model was highly significant, with a regression higher than 40% in size and 70% in surface and volume, 12 weeks after the end of treatment. This response was rapid, reproducible without dose-effect or toxicity in the limits studied. This work confirms previously published results in humans.