Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

BACKGROUND: Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. METHODS: Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. RESULTS: Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. CONCLUSION: We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

Association of circulating PLA2G7 levels with cancer cachexia and assessment of darapladib as a therapy.

BACKGROUND: Cancer cachexia (CCx) is a multifactorial wasting disorder characterized by involuntary loss of body weight that affects many cancer patients and implies a poor prognosis, reducing both tolerance to and efficiency of anticancer therapies. Actual challenges in management of CCx remain in the identification of tumour-derived and host-derived mediators involved in systemic inflammation and tissue wasting and in the discovery of biomarkers that would allow for an earlier and personalized care of cancer patients. The aim of this study was to identify new markers of CCx across different species and tumour entities. METHODS: Quantitative secretome analysis was performed to identify specific factors characteristic of cachexia-inducing cancer cell lines. To establish the subsequently identified phospholipase PLA2G7 as a marker of CCx, plasma PLA2G7 activity and/or protein levels were measured in well-established mouse models of CCx and in different cohorts of weight-stable and weight-losing cancer patients with different tumour entities. Genetic PLA2G7 knock-down in tumours and pharmacological treatment using the well-studied PLA2G7 inhibitor darapladib were performed to assess its implication in the pathogenesis of CCx in C26 tumour-bearing mice. RESULTS: High expression and secretion of PLA2G7 were hallmarks of cachexia-inducing cancer cell lines. Circulating PLA2G7 activity was increased in different mouse models of CCx with various tumour entities and was associated with the severity of body wasting. Circulating PLA2G7 levels gradually rose during cachexia development. Genetic PLA2G7 knock-down in C26 tumours only partially reduced plasma PLA2G7 levels, suggesting that the host is also an important contributor. Chronic treatment with darapladib was not sufficient to counteract inflammation and tissue wasting despite a strong inhibition of the circulating PLA2G7 activity. Importantly, PLA2G7 levels were also increased in colorectal and pancreatic cancer patients with CCx. CONCLUSIONS: Overall, our data show that despite no immediate pathogenic role, at least when targeted as a single entity, PLA2G7 is a consistent marker of CCx in both mice and humans. The early increase in circulating PLA2G7 levels in pre-cachectic mice supports future prospective studies to assess its potential as biomarker for cancer patients.

High levels of modified ceramides are a defining feature of murine and human cancer cachexia.

BACKGROUND: Cancer cachexia (CCx) is a multifactorial energy-wasting syndrome reducing the efficiency of anti-cancer therapies, quality of life, and survival of cancer patients. In the past years, most studies focused on the identification of tumour and host-derived proteins contributing to CCx. However, there is still a lack of studies addressing the changes in bioactive lipids. The aim of this study was to identify specific lipid species as a hallmark of CCx by performing a broad range lipid analysis of plasma from well-established CCx mouse models as well as cachectic and weight stable cancer patients. METHODS: Plasma from non-cachectic (PBS-injected mice, NC26 tumour-bearing mice), pre-cachectic and cachectic mice (C26 and LLC tumour-bearing mice, ApcMin/+ mutant mice), and plasma from weight stable and cachectic patients with gastrointestinal cancer, were analysed using the Lipidyzer™ platform. In total, 13 lipid classes and more than 1100 lipid species, including sphingolipids, neutral and polar glycerolipids, were covered by the analysis. Correlation analysis between specific lipid species and readouts of CCx were performed. Lipidomics data were confirmed by gene expression analysis of metabolic organs to analyse enzymes involved in sphingolipid synthesis and degradation. RESULTS: A decrease in several lysophosphatidylcholine (LPC) species and an increase in numerous sphingolipids including sphingomyelins (SMs), ceramides (CERs), hexosyl-ceramides (HCERs) and lactosyl-ceramides (LCERs), were mutual features of CCx in both mice and cancer patients. Notably, sphingolipid levels gradually increased during cachexia development. Key enzymes involved in ceramide synthesis were elevated in liver but not in adipose, muscle, or tumour tissues, suggesting that ceramide turnover in the liver is a major contributor to elevated sphingolipid levels in CCx. LPC(16:1), LPC(20:3), SM(16:0), SM(24:1), CER(16:0), CER(24:1), HCER(16:0), and HCER(24:1) were the most consistently affected lipid species between mice and humans and correlated negatively (LPCs) or positively (SMs, CERs and HCERs) with the severity of body weight loss. CONCLUSIONS: High levels of sphingolipids, specifically ceramides and modified ceramides, are a defining feature of murine and human CCx and may contribute to tissue wasting and skeletal muscle atrophy through the inhibition of anabolic signals. The progressive increase in sphingolipids during cachexia development supports their potential as early biomarkers for CCx.

On the language specificity of basic number processing: transcoding in a language with inversion and its relation to working memory capacity.

Transcoding Arabic numbers from and into verbal number words is one of the most basic number processing tasks commonly used to index the verbal representation of numbers. The inversion property, which is an important feature of some number word systems (e.g., German einundzwanzig [one and twenty]), might represent a major difficulty in transcoding and a challenge to current transcoding models. The mastery of inversion, and of transcoding in general, might be related to nonnumerical factors such as working memory resources given that different elements and their sequence need to be memorized and manipulated. In this study, transcoding skills and different working memory components in Austrian (German-speaking) 7-year-olds were assessed. We observed that inversion poses a major problem in transcoding for German-speaking children. In addition, different components of working memory skills were differentially correlated with particular transcoding error types. We discuss how current transcoding models could account for these results and how they might need to be adapted to accommodate inversion properties and their relation to different working memory components.

Intransparent German number words complicate transcoding - a translingual comparison with Japanese.

Superior early numerical competencies of children in several Asian countries have (amongst others) been attributed to the higher transparency of their number word systems. Here, we directly investigated this claim by evaluating whether Japanese children's transcoding performance when writing numbers to dictation (e.g., "twenty five" → 25) was less error prone than that of German-speaking children - both in general as well as when considering language-specific attributes of the German number word system such as the inversion property, in particular. In line with this hypothesis we observed that German-speaking children committed more transcoding errors in general than their Japanese peers. Moreover, their error pattern reflected the specific inversion intransparency of the German number-word system. Inversion errors in transcoding represented the most prominent error category in German-speaking children, but were almost absent in Japanese-speaking children. We conclude that the less transparent German number-word system complicates the acquisition of the correspondence between symbolic Arabic numbers and their respective verbal number words.