Mapping restricted introgression across the genomes of admixed indigenous African cattle breeds.

BACKGROUND: The genomes of indigenous African cattle are composed of components with Middle Eastern (taurine) and South Asian (indicine) origins, providing a valuable model to study hybridization and to identify genetic barriers to gene flow. In this study, we analysed indigenous African cattle breeds as models of hybrid zones, considering taurine and indicine samples as ancestors. In a genomic cline analysis of whole-genome sequence data, we considered over 8 million variants from 144 animals, which allows for fine-mapping of potential genomic incompatibilities at high resolution across the genome. RESULTS: We identified several thousand variants that had significantly steep clines ('SCV') across the whole genome, indicating restricted introgression. Some of the SCV were clustered into extended regions, with the longest on chromosome 7, spanning 725 kb and including 27 genes. We found that variants with a high phenotypic impact (e.g. indels, intra-genic and missense variants) likely represent greater genetic barriers to gene flow. Furthermore, our findings provide evidence that a large proportion of breed differentiation in African cattle could be linked to genomic incompatibilities and reproductive isolation. Functional evaluation of genes with SCV suggest that mitonuclear incompatibilities and genes associated with fitness (e.g. resistance to paratuberculosis) could account for restricted gene flow in indigenous African cattle. CONCLUSIONS: To our knowledge, this is the first time genomic cline analysis has been applied to identify restricted introgression in the genomes of indigenous African cattle and the results provide extended insights into mechanisms (e.g. genomic incompatibilities) contributing to hybrid differentiation. These results have important implications for our understanding of genetic incompatibilities and reproductive isolation and provide important insights into the impact of cross-breeding cattle with the aim of producing offspring that are both hardy and productive.

IL-9 Producing Tumor-Infiltrating Lymphocytes and Treg Subsets Drive Immune Escape of Tumor Cells in Non-Small Cell Lung Cancer.

Although lung cancer is the leading cause of cancer deaths worldwide, the mechanisms how lung cancer cells evade the immune system remain incompletely understood. Here, we discovered IL-9-dependent signaling mechanisms that drive immune evasion in non-small cell lung cancer (NSCLC). We found increased IL-9 and IL-21 production by T cells in the tumoral region of the lung of patients with NSCLC, suggesting the presence of Th9 cells in the lung tumor microenvironment. Moreover, we noted IL-9 producing Tregs in NSCLC. IL-9 target cells in NSCLC consisted of IL-9R+ tumor cells and tumor-infiltrating lymphocytes. In two murine experimental models of NSCLC, and in vitro, IL-9 prevented cell death and controlled growth of lung adenocarcinoma cells. Targeted deletion of IL-9 resulted in successful lung tumor rejection in vivo associated with an induction of IL-21 and reduction of Treg cells. Finally, anti-IL-9 antibody immunotherapy resulted in suppression of tumor development even in established experimental NSCLC and was associated with reduced IL-10 production in the lung. In conclusion, our findings indicate that IL-9 drives immune escape of lung tumor cells via effects on tumor cell survival and tumor infiltrating T cells. Thus, strategies blocking IL-9 emerge as a new approach for clinical therapy of lung cancer.

[Cough and Exertional Dyspnoea after Oily Mentholine Inhalation]. / Husten und Belastungsdyspnoe nach ölhaltiger Mentholinhalation.

A 48-year-old-male presented with a five-week history of non-productive cough and exertional dyspnoea. A pulmonary function test showed a mild diffusion disorder. A CT scan revealed an atypical pneumonia with bipulmonary consolidations, which were accentuated in the right upper lobe. The transbronchial biopsy showed lipid-loaded macrophages. These findings confirmed the diagnosis of a lipoid pneumonia, which developed in the context of inhalation of substances containing menthol. After discontinuation of the causative agent and high-dose steroid administration the symptoms were reversible within a few weeks.

Co-targeting MCL-1 and ERK1/2 kinase induces mitochondrial apoptosis in rhabdomyosarcoma cells.

The RAS/MEK/ERK genetic axis is commonly altered in rhabdomyosarcoma (RMS), indicating high activity of downstream effector ERK1/2 kinase. Previously, we have demonstrated that inhibition of the RAS/MEK/ERK signaling pathway in RMS is insufficient to induce cell death due to residual pro-survival MCL-1 activity. Here, we show that the combination of ERK1/2 inhibitor Ulixertinib and MCL-1 inhibitor S63845 is highly synergistic and induces apoptotic cell death in RMS in vitro and in vivo. Importantly, Ulixertinib/S63845 co-treatment suppresses long-term survival of RMS cells, induces rapid caspase activation and caspase-dependent apoptosis. Mechanistically, Ulixertinib-mediated upregulation of BIM and BMF in combination with MCL-1 inhibition by S63845 shifts the balance of BCL-2 proteins towards a pro-apoptotic state resulting in apoptosis induction. A genetic silencing approach reveals that BIM, BMF, BAK and BAX are all required for Ulixertinib/S63845-induced apoptosis. Overexpression of BCL-2 rescues cell death triggered by Ulixertinib/S63845 co-treatment, confirming that combined inhibition of ERK1/2 and MCL-1 effectively induces cell death of RMS cells via the intrinsic mitochondrial apoptotic pathway. Thus, this study is the first to demonstrate the cytotoxic potency of co-inhibition of ERK1/2 and MCL-1 for RMS treatment.

[Anxiety about Health of Patients Suffering from Epilepsy]. / Krankheitsängste bei Epilepsiepatient*innen.

BACKGROUND: The presence of somatic diseases makes diagnosis of health-related anxiety difficult. Studies have shown that a significant proportion of patients suffering from somatic diseases also suffer from illness anxiety. OBJECTIVE: Patients suffering from epilepsy were examined, for whom no data regarding their illness anxiety had previously been obtained. METHOD: 76 epileptics were analyzed regarding their psychological distress (Mini-SCL), fear of recurrence (PA-F) and hypochondriacal traits (MIHT). RESULTS: In general, epileptics experienced the same level of distress and fear in comparison to a norm sample. At the same time, 28% (overall distress) to 45% (anxiety) of the study participants were observed to have abnormal values. The fear of recurrence was low in comparison to other somatic diagnosis groups. Hypochondriac traits were seen in 7% (perceptive) to 13% (affective/behavioral) of the respondents. CONCLUSION: An increased awareness of illness anxiety in patients can help improve healthcare and quality of life of epileptics.

Genome-wide association studies for canine hip dysplasia in single and multiple populations - implications and potential novel risk loci.

BACKGROUND: Association mapping studies of quantitative trait loci (QTL) for canine hip dysplasia (CHD) can contribute to the understanding of the genetic background of this common and debilitating disease and might contribute to its genetic improvement. The power of association studies for CHD is limited by relatively small sample numbers for CHD records within countries, suggesting potential benefits of joining data across countries. However, this is complicated due to the use of different scoring systems across countries. In this study, we incorporated routinely assessed CHD records and genotype data of German Shepherd dogs from two countries (UK and Sweden) to perform genome-wide association studies (GWAS) within populations using different variations of CHD phenotypes. As phenotypes, dogs were either classified into cases and controls based on the Fédération Cynologique Internationale (FCI) five-level grading of the worst hip or the FCI grade was treated as an ordinal trait. In a subsequent meta-analysis, we added publicly available data from a Finnish population and performed the GWAS across all populations. Genetic associations for the CHD phenotypes were evaluated in a linear mixed model using 62,089 SNPs. RESULTS: Multiple SNPs with genome-wide significant and suggestive associations were detected in single-population GWAS and the meta-analysis. Few of these SNPs overlapped between populations or between single-population GWAS and the meta-analysis, suggesting that many CHD-related QTL are population-specific. More significant or suggestive SNPs were identified when FCI grades were used as phenotypes in comparison to the case-control approach. MED13 (Chr 9) and PLEKHA7 (Chr 21) emerged as novel positional candidate genes associated with hip dysplasia. CONCLUSIONS: Our findings confirm the complex genetic nature of hip dysplasia in dogs, with multiple loci associated with the trait, most of which are population-specific. Routinely assessed CHD information collected across countries provide an opportunity to increase sample sizes and statistical power for association studies. While the lack of standardisation of CHD assessment schemes across countries poses a challenge, we showed that conversion of traits can be utilised to overcome this obstacle.

Whole-Genome Sequence Data Suggest Environmental Adaptation of Ethiopian Sheep Populations.

Great progress has been made over recent years in the identification of selection signatures in the genomes of livestock species. This work has primarily been carried out in commercial breeds for which the dominant selection pressures are associated with artificial selection. As agriculture and food security are likely to be strongly affected by climate change, a better understanding of environment-imposed selection on agricultural species is warranted. Ethiopia is an ideal setting to investigate environmental adaptation in livestock due to its wide variation in geo-climatic characteristics and the extensive genetic and phenotypic variation of its livestock. Here, we identified over three million single nucleotide variants across 12 Ethiopian sheep populations and applied landscape genomics approaches to investigate the association between these variants and environmental variables. Our results suggest that environmental adaptation for precipitation-related variables is stronger than that related to altitude or temperature, consistent with large-scale meta-analyses of selection pressure across species. The set of genes showing association with environmental variables was enriched for genes highly expressed in human blood and nerve tissues. There was also evidence of enrichment for genes associated with high-altitude adaptation although no strong association was identified with hypoxia-inducible-factor (HIF) genes. One of the strongest altitude-related signals was for a collagen gene, consistent with previous studies of high-altitude adaptation. Several altitude-associated genes also showed evidence of adaptation with temperature, suggesting a relationship between responses to these environmental factors. These results provide a foundation to investigate further the effects of climatic variables on small ruminant populations.

Methods to Improve Joint Genetic Evaluation of Canine Hip Dysplasia Across BVA/KC and FCI Screening Schemes.

The BVA/KC (British Veterinary Association/Kennel Club) and FCI (Fédération Cynologique Internationale) are the main screening schemes used to evaluate the status of canine hip dysplasia (HD) in Europe. Jointly utilizing HD records from both BVA/KC and FCI schemes could improve the reliability of genetic evaluation within and across countries. In this study, HD scores for German shepherd dogs (GSDs) in the UK (using the BVA/KC scheme) and Sweden (using the FCI scheme) were used to investigate how to better operate joint genetic evaluations across the two schemes. It was shown that under a bivariate model, which regarded BVA/KC and FCI scores as different traits, the estimated genetic correlations between the UK and Swedish GSD populations were the same when using BVA/KC total or worse hip scores and for single-country or joint analysis of both the UK and Swedish populations. Under a univariate model that converted BVA/KC scores into FCI scores, the predictability of estimated breeding values was slightly improved by performing a joint analysis.

Unravelling selection signatures in a single dog breed suggests recent selection for morphological and behavioral traits.

Strong selection has resulted in substantial morphological and behavioral diversity across modern dog breeds, which makes dogs interesting model animals to study the underlying genetic architecture of these traits. However, results from between-breed analyses may confound selection signatures for behavior and morphological features that were coselected during breed development. In this study, we assess population genetic differences in a unique resource of dogs of the same breed but with systematic behavioral selection in only one population. We exploit these different breeding backgrounds to identify signatures of recent selection. Selection signatures within populations were found on chromosomes 4 and 19, with the strongest signals in behavior-related genes. Regions showing strong signals of divergent selection were located on chromosomes 1, 24, and 32, and include candidate genes for both physical features and behavior. Some of the selection signatures appear to be driven by loci associated with coat color (Chr 24; ASIP) and length (Chr 32; FGF5), while others showed evidence of association with behavior. Our findings suggest that signatures of selection within dog breeds have been driven by selection for morphology and behavior. Furthermore, we demonstrate that combining selection scans with association analyses is effective for dissecting the traits under selection.

Genetic dissection of complex behaviour traits in German Shepherd dogs.

A favourable genetic structure and diversity of behavioural features highlights the potential of dogs for studying the genetic architecture of behaviour traits. However, behaviours are complex traits, which have been shown to be influenced by numerous genetic and non-genetic factors, complicating their analysis. In this study, the genetic contribution to behaviour variation in German Shepherd dogs (GSDs) was analysed using genomic approaches. GSDs were phenotyped for behaviour traits using the established Canine Behavioural Assessment and Research Questionnaire (C-BARQ). Genome-wide association study (GWAS) and regional heritability mapping (RHM) approaches were employed to identify associations between behaviour traits and genetic variants, while accounting for relevant non-genetic factors. By combining these complementary methods we endeavoured to increase the power to detect loci with small effects. Several behavioural traits exhibited moderate heritabilities, with the highest identified for Human-directed playfulness, a trait characterised by positive interactions with humans. We identified several genomic regions associated with one or more of the analysed behaviour traits. Some candidate genes located in these regions were previously linked to behavioural disorders in humans, suggesting a new context for their influence on behaviour characteristics. Overall, the results support dogs as a valuable resource to dissect the genetic architecture of behaviour traits and also highlight the value of focusing on a single breed in order to control for background genetic effects and thus avoid limitations of between-breed analyses.

Increased expression of the immunosuppressive interleukin-35 in patients with non-small cell lung cancer.

BACKGROUND: The immunosuppressive role of the cytokine IL-35 in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, we analysed the localisation and regulation of IL-35 in the lung of patients with non-small cell lung cancer (NSCLC) to further elucidate the immune-escape of cancer cells in perioperative course of disease. METHODS: Interleukin 35 (IL-35) was measured by ELISA in postoperative serum from 7 patients with NSCLC as well as 8 samples from healthy controls. Immunohistochemistry, FACS analysis, real-time PCR, as well as western blot from samples of the control (CTR), peri-tumoural (PT) and the tumoural (TU) region of the lung derived from patients with NSCLC and 10 controls were performed. RESULTS: Here we found elevated levels of IL-35 in the TU region as well as postoperative serum from patients with lung adenocarcinoma. Consistently, we found an increased expression of IL-35+Foxp-3+ cells, which associated with ARG1 mRNA expression and decreased TNFA in the TU region of the lung of patients with NSCLC as compared to their CTR region. Furthermore, in the CTR region of the lung of patients with NSCLC, CD68+ macrophages were induced and correlated with IL-35+ cells. Finally, IL-35 positively correlated with TTF-1+PD-L1+ cells in the TU region of NSCLC patients. CONCLUSIONS: Induced IL-35+Foxp3+ cell numbers in the TU region of the lung of patients with NSCLC associated with ARG1 mRNA expression and with TTF-1+PD-L1+ cells. In the tumour-free CTR area, IL-35 correlated with CD68+ macrophages. Thus inhibitors to IL-35 would probably succeed in combination with antibodies against immune checkpoints like PD-L1 and PD-1 currently used against NSCLC because they would inhibit immunosuppressive macrophages and T regulatory cells while promoting T cell-mediated anti-tumoural immune responses in the microenvironment as well as the TU region of NSCLC patients.

NFATc1 Promotes Antitumoral Effector Functions and Memory CD8+ T-cell Differentiation during Non-Small Cell Lung Cancer Development.

Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor activated by T-cell receptor (TCR) and Ca2+ signaling that affects T-cell activation and effector function. Upon tumor antigen challenge, TCR and calcium-release-activated channels are induced, promoting NFAT dephosphorylation and translocation into the nucleus. In this study, we report a progressive decrease of NFATc1 in lung tumor tissue and in tumor-infiltrating lymphocytes (TIL) of patients suffering from advanced-stage non-small cell lung cancer (NSCLC). Mice harboring conditionally inactivated NFATc1 in T cells (NFATc1ΔCD4) showed increased lung tumor growth associated with impaired T-cell activation and function. Furthermore, in the absence of NFATc1, reduced IL2 influenced the development of memory CD8+ T cells. We found a reduction of effector memory and CD103+ tissue-resident memory (TRM) T cells in the lung of tumor-bearing NFATc1ΔCD4 mice, underlining an impaired cytotoxic T-cell response and a reduced TRM tissue-homing capacity. In CD4+ICOS+ T cells, programmed cell death 1 (PD-1) was induced in the draining lymph nodes of these mice and associated with lung tumor cell growth. Targeting PD-1 resulted in NFATc1 induction in CD4+ and CD8+ T cells in tumor-bearing mice and was associated with increased antitumor cytotoxic functions. This study reveals a role of NFATc1 in the activation and cytotoxic functions of T cells, in the development of memory CD8+ T-cell subsets, and in the regulation of T-cell exhaustion. These data underline the indispensability of NFATc1 for successful antitumor immune responses in patients with NSCLC.Significance: The multifaceted role of NFATc1 in the activation and function of T cells during lung cancer development makes it a critical participant in antitumor immune responses in patients with NSCLC. Cancer Res; 78(13); 3619-33. ©2018 AACR.

STAT1 deficiency supports PD-1/PD-L1 signaling resulting in dysfunctional TNFα mediated immune responses in a model of NSCLC.

In this study we described that Signal Transducer and Activator of Transcription 1 (STAT1) is a key point regulator of PD-1 in tumour infiltrating lymphocytes and PD-L1 in Tumour associated macrophages (TAM) in NSCLC. In our murine model of adenocarcinoma targeted deletion of Stat1 was found associated with enhanced tumour growth, impaired differentiation into M1-like macrophages from the bone marrow, the accumulation of tumor associated macrophages overexpressing PD-L1 and impaired T cell responses in the tumor microenvironment by affecting TNFα responses. In our human NSCLC patient cohort we found that loss of isoforms STAT1 α and STAT1ß mRNA in the tumoural region of the lung correlates with increased tumor size in NSCLC patients. Therefore, STAT1 isoform regulation could be considered for future therapeutical strategies associated to current immune-checkpoint blockade therapy in NSCLC.

Interleukin-10-regulated tumour tolerance in non-small cell lung cancer.

BACKGROUND: Lung cancer is the most life-threatening cancer type worldwide. Treatment options include surgery, radio- and chemotherapy, as well as the use of immunomodulatory antibodies. Interleukin (IL)-10 is an immunosuppressive cytokine involved in tumour immune escape. METHODS: Immunohistochemistry (IHC) on human lung surgery tissue as well as human tumour cell line cultures, FACS analysis, real-time PCR and experimental lung cancer. RESULTS: Here we discovered a positive correlation between IL-10 and IL-10 receptor (IL-10R) expression in the lung with tumour diameter in patients with lung cancer (non-small cell lung cancer), the most life-threatening cancer type worldwide. IL-10 and IL-10R were found induced in cells surrounding the lung tumour cells, and IL-10R was mainly expressed on the surface of Foxp-3+ T-regulatory lymphocytes infiltrating the tumour of these patients where its expression inversely correlated with programmed cell death 1. These findings were confirmed in translational studies. In a human lung adenocarcinoma cell line, IL-10R was found induced under metabolic restrictions present during tumour growth, whereby IL-10 inhibited PDL1 and tumour cell apoptosis. CONCLUSIONS: These new findings suggest that IL-10 counteracts IFN-γ effects on PD1/PDL1 pathway, resulting in possible resistance of the tumour to anti-PD1/PDL1 immunotherapy.

Adrenocortical Expression Profiling of Cattle with Distinct Juvenile Temperament Types.

Temperament affects ease of handling, animal welfare, and economically important production traits in cattle. The use of gene expression profiles as molecular traits provides a novel means of gaining insight into behavioural genetics. In this study, differences in adrenocortical expression profiles between 60 F2 cows (Charolais × German Holstein) of distinct temperament types were analysed. The cows were assessed in a novel-human test at an age of 90 days. Most of the adrenal cortex transcripts which were differentially expressed (FDR <0.05) were found between temperament types of 'fearful/neophobic-alert' and all other temperament types. These transcripts belong to several biological functions like NRF2-mediated oxidative stress response, Glucocorticoid Receptor Signalling and Complement System. Overall, the present study provides new insight into transcriptional differences in the adrenal cortex between cows of distinct temperament types. Genetic regulations of such molecular traits facilitate uncovering positional and functional gene candidates for temperament type in cattle.

Role of the receptor Mas in macrophage-mediated inflammation in vivo.

Recently, an alternative renin-angiotensin system pathway has been described, which involves binding of angiotensin-(1-7) to its receptor Mas. The Mas axis may counterbalance angiotensin-II-mediated proinflammatory effects, likely by affecting macrophage function. Here we investigate the role of Mas in murine models of autoimmune neuroinflammation and atherosclerosis, which both involve macrophage-driven pathomechanisms. Mas signaling affected macrophage polarization, migration, and macrophage-mediated T-cell activation. Mas deficiency exacerbated the course of experimental autoimmune encephalomyelitis and increased macrophage infiltration as well as proinflammatory gene expression in the spleen and spinal cord. Furthermore, Mas deficiency promoted atherosclerosis by affecting macrophage infiltration and migration and led to increased oxidative stress as well as impaired endothelial function in ApoE-deficient mice. In summary, we identified the Mas axis as an important factor in macrophage function during inflammation of the central nervous and vascular system in vivo. Modulating the Mas axis may constitute an interesting therapeutic target in multiple sclerosis and/or atherosclerosis.

Adrenal cortex expression quantitative trait loci in a German Holstein × Charolais cross.

BACKGROUND: The importance of the adrenal gland in regard to lactation and reproduction in cattle has been recognized early. Caused by interest in animal welfare and the impact of stress on economically important traits in farm animals the adrenal gland and its function within the stress response is of increasing interest. However, the molecular mechanisms and pathways involved in stress-related effects on economically important traits in farm animals are not fully understood. Gene expression is an important mechanism underlying complex traits, and genetic variants affecting the transcript abundance are thought to influence the manifestation of an expressed phenotype. We therefore investigated the genetic background of adrenocortical gene expression by applying an adaptive linear rank test to identify genome-wide expression quantitative trait loci (eQTL) for adrenal cortex transcripts in cattle. RESULTS: A total of 10,986 adrenal cortex transcripts and 37,204 single nucleotide polymorphisms (SNPs) were analysed in 145 F2 cows of a Charolais × German Holstein cross. We identified 505 SNPs that were associated with the abundance of 129 transcripts, comprising 482 cis effects and 17 trans effects. These SNPs were located on all chromosomes but X, 16, 24 and 28. Associated genes are mainly involved in molecular and cellular functions comprising free radical scavenging, cellular compromise, cell morphology and lipid metabolism, including genes such as CYP27A1 and LHCGR that have been shown to affect economically important traits in cattle. CONCLUSIONS: In this study we showed that adrenocortical eQTL affect the expression of genes known to contribute to the phenotypic manifestation in cattle. Furthermore, some of the identified genes and related molecular pathways were previously shown to contribute to the phenotypic variation of behaviour, temperament and growth at the onset of puberty in the same population investigated here. We conclude that eQTL analysis appears to be a useful approach providing insight into the molecular and genetic background of complex traits in cattle and will help to understand molecular networks involved.

Detection of genetic variants affecting cattle behaviour and their impact on milk production: a genome-wide association study.

Behaviour traits of cattle have been reported to affect important production traits, such as meat quality and milk performance as well as reproduction and health. Genetic predisposition is, together with environmental stimuli, undoubtedly involved in the development of behaviour phenotypes. Underlying molecular mechanisms affecting behaviour in general and behaviour and productions traits in particular still have to be studied in detail. Therefore, we performed a genome-wide association study in an F2 Charolais × German Holstein cross-breed population to identify genetic variants that affect behaviour-related traits assessed in an open-field and novel-object test and analysed their putative impact on milk performance. Of 37,201 tested single nucleotide polymorphism (SNPs), four showed a genome-wide and 37 a chromosome-wide significant association with behaviour traits assessed in both tests. Nine of the SNPs that were associated with behaviour traits likewise showed a nominal significant association with milk performance traits. On chromosomes 14 and 29, six SNPs were identified to be associated with exploratory behaviour and inactivity during the novel-object test as well as with milk yield traits. Least squares means for behaviour and milk performance traits for these SNPs revealed that genotypes associated with higher inactivity and less exploratory behaviour promote higher milk yields. Whether these results are due to molecular mechanisms simultaneously affecting behaviour and milk performance or due to a behaviour predisposition, which causes indirect effects on milk performance by influencing individual reactivity, needs further investigation.