Prolonged dialysis during ex vivo lung perfusion promotes inflammatory responses.

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.

Malignant features of minipig melanomas prior to spontaneous regression.

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.

Deciphering the immune reaction leading to spontaneous melanoma regression: initial role of MHCII+ CD163- macrophages.

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.

Challenging the Ex Vivo Lung Perfusion Procedure With Continuous Dialysis in a Pig Model.

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.

Links between fecal microbiota and the response to vaccination against influenza A virus in pigs.

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.

Influence of genetics and the pre-vaccination blood transcriptome on the variability of antibody levels after vaccination against Mycoplasma hyopneumoniae in pigs.

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.

Discovery of Predictors of Mycoplasma hyopneumoniae Vaccine Response Efficiency in Pigs: 16S rRNA Gene Fecal Microbiota Analysis.

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.

The Composition of Circulating Leukocytes Varies With Age and Melanoma Onset in the MeLiM Pig Biomedical Model.

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- CD14low 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.

Electroporation of a nanoparticle-associated DNA vaccine induces higher inflammation and immunity compared to its delivery with microneedle patches in pigs.

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 MHC2posCD172Apos myeloid cells and type 1 conventional dendritic cells, in association with local production of IL-1ß, 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 MHC2posCD172Apos CD163int and CD163neg 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.

A DNA Prime Immuno-Potentiates a Modified Live Vaccine against the Porcine Reproductive and Respiratory Syndrome Virus but Does Not Improve Heterologous Protection.

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 Type 1.3 Lena Triggers Conventional Dendritic Cells 1 Activation and T Helper 1 Immune Response Without Infecting Dendritic Cells.

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.

Porcine Alveolar Macrophage-like cells are pro-inflammatory Pulmonary Intravascular Macrophages that produce large titers of Porcine Reproductive and Respiratory Syndrome Virus.

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.

Impact of a CD4 gene haplotype on the immune response in minipigs.

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.

Phylogenetic network analysis applied to pig gut microbiota identifies an ecosystem structure linked with growth traits.

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.

Early-life establishment of the swine gut microbiome and impact on host phenotypes.

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.

Immunity traits in pigs: substantial genetic variation and limited covariation.

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.

Reconstitution of the myeloid and lymphoid compartments after the transplantation of autologous and genetically modified CD34+ bone marrow cells, following gamma irradiation in cynomolgus macaques.

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.

Detection of novel quantitative trait loci for cutaneous melanoma by genome-wide scan in the MeLiM swine model.

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.

Low-dose exposure to gamma rays induces specific gene regulations in normal human keratinocytes.

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.

Activation of an energy providing response in human keratinocytes after gamma irradiation.

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