Preclinical Evaluation of a Microwave-Based Accessory Device for Colonoscopy in an In Vivo Porcine Model with Colorectal Polyps.

BACKGROUND AND AIMS: Colonoscopy is currently the most effective way of detecting colorectal cancer and removing polyps, but it has some drawbacks and can miss up to 22% of polyps. Microwave imaging has the potential to provide a 360° view of the colon and addresses some of the limitations of conventional colonoscopy. This study evaluates the feasibility of a microwave-based colonoscopy in an in vivo porcine model. METHODS: A prototype device with microwave antennas attached to a conventional endoscope was tested on four healthy pigs and three gene-targeted pigs with mutations in the adenomatous polyposis coli gene. The first four animals were used to evaluate safety and maneuverability and compatibility with endoscopic tools. The ability to detect polyps was tested in a series of three gene-targeted pigs. RESULTS: the microwave-based device did not affect endoscopic vision or cause any adverse events such as deep mural injuries. The microwave system was stable during the procedures, and the detection algorithm showed a maximum detection signal for adenomas compared with healthy mucosa. CONCLUSIONS: Microwave-based colonoscopy is feasible and safe in a preclinical model, and it has the potential to improve polyp detection. Further investigations are required to assess the device's efficacy in humans.

TNF ΔARE Pigs: A Translational Crohn's Disease Model.

BACKGROUND AND AIMS: Crohn's disease [CD] is a major subtype of inflammatory bowel diseases [IBD] with increasing incidence and prevalence. Results of studies using available small and large animal models are often poorly translatable to patients, and few CD models show small intestinal pathology. Due to its similarities to humans, the pig has emerged as a highly suitable translational disease model, particularly for testing novel nutritional and technological interventions. Our goal was to develop a physiologically relevant porcine CD model to facilitate translation of findings and interventions towards the clinic. METHODS: We generated pigs bearing a 93-bp deletion of the adenosine-uracil-rich element [ARE] and a constitutive-decay element within the 3' untranslated region of the TNF gene. Comparative analysis of physiological, molecular, histological and microbial characteristics was performed between wild-type, TNFΔARE/+ and TNFΔARE/ΔARE animals. Alterations in the microbiome were compared to the TNFΔARE mouse model and IBD patients. RESULTS: TNF ΔARE pigs recapitulate major characteristics of human CD, including ulcerative transmural ileocolitis, increased abundance of proinflammatory cytokines, immune cell infiltration and dysbiotic microbial communities. 16S rRNA gene amplicon sequencing revealed enrichment in members belonging to Megasphaera, Campylobacter, Desulfovibrio, Alistipes and Lachnoclostridum in faecal or mucosa-associated bacteria compared to wild-type littermates. Principal components analysis clustering with a subset of TNFΔARE/+ mice and human IBD patients suggests microbial similarity based on disease severity. CONCLUSIONS: We demonstrate that the TNFΔARE pig resembles a CD-like ileocolitis pathophenotype recapitulating human disease. The ability to conduct long-term studies and test novel surgical procedures and dietary interventions in a physiologically relevant model will benefit future translational IBD research studies.

Elevated circulating Hsp70 levels are correlative for malignancies in different mammalian species.

Circulating Hsp70 levels were determined in feline and porcine cohorts using two different ELISA systems. These comparative animal models of larger organisms often reflect diseases, and especially malignant tumors, better than conventional rodent models. It is therefore essential to investigate the biology and utility of tumor biomarkers in animals such as cats and pigs. In this study, levels of free Hsp70 in the blood of cats with spontaneously occurring tumors were detected using a commercial Hsp70 ELISA (R&D Systems). Sub-analysis of different tumor groups revealed that animals with tumors of epithelial origin presented with significantly elevated circulating Hsp70 concentrations. In addition to free Hsp70 levels measured with the R&D Systems Hsp70 ELISA, levels of exosomal Hsp70 were determined using the compHsp70 ELISA in pigs. Both ELISA systems detected significantly elevated Hsp70 levels (R&D Systems: median 24.9 ng/mL; compHsp70: median 44.2 ng/mL) in the blood of a cohort of APC1311/+ pigs diagnosed with high-grade adenoma polyps, and the R&D Systems Hsp70 ELISA detected also elevated Hsp70 levels in animals with low-grade polyps. In contrast, in flTP53R167H pigs, suffering from malignant osteosarcoma, the compHsp70 ELISA (median 674.32 ng/mL), but not the R&D Systems Hsp70 ELISA (median 4.78 ng/mL), determined significantly elevated Hsp70 concentrations, indicating that in tumor-bearing animals, the dominant form of Hsp70 is of exosomal origin. Our data suggest that both ELISA systems are suitable for detecting free circulating Hsp70 levels in pigs with high-grade adenoma, but only the compHsp70 ELISA can measure elevated, tumor-derived exosomal Hsp70 levels in tumor-bearing animals.

A humanized minipig model for the toxicological testing of therapeutic recombinant antibodies.

The safety of most human recombinant proteins can be evaluated in transgenic mice tolerant to specific human proteins. However, owing to insufficient genetic diversity and to fundamental differences in immune mechanisms, small-animal models of human diseases are often unsuitable for immunogenicity testing and for predicting adverse outcomes in human patients. Most human therapeutic antibodies trigger xenogeneic responses in wild-type animals and thus rapid clearance of the drugs, which makes in vivo toxicological testing of human antibodies challenging. Here we report the generation of Göttingen minipigs carrying a mini-repertoire of human genes for the immunoglobulin heavy chains γ1 and γ4 and the immunoglobulin light chain κ. In line with observations in human patients, the genetically modified minipigs tolerated the clinically non-immunogenic IgG1κ-isotype monoclonal antibodies daratumumab and bevacizumab, and elicited antibodies against the checkpoint inhibitor atezolizumab and the engineered interleukin cergutuzumab amunaleukin. The humanized minipigs can facilitate the safety and efficacy testing of therapeutic antibodies.

360º optoacoustic capsule endoscopy at 50 Hz for esophageal imaging.

Gastrointestinal (GI) endoscopy is a common medical diagnostic procedure used for esophageal cancer detection. Current emerging capsule optoacoustic endoscopes, however, suffer from low pulse repetition rates and slow scanning units limit attainable imaging frame rates. Consequently, motion artifacts result in inaccurate spatial mapping and misinterpretation of data. To overcome these limitations, we report a 360º, 50 Hz frame rate, distal scanning capsule optoacoustic endoscope. The translational capability of the instrument for human GI tract imaging was characterized with an Archimedean spiral phantom consisting of twelve 100 µm sutures, a stainless steel mesh with a pitch of 3 mm and an ex vivo pig esophagus sample. We estimated an imaging penetration depth of ~0.84 mm in vivo by immersing the mesh phantom in intralipid solution to simulate light scattering in human esophageal tissue and validated our findings ex vivo using pig esophagus. This proof-of-concept study demonstrates the translational potential of the proposed video-rate endoscope for human GI tract imaging.

Tumor Targeting with Bacterial Shiga Toxin B Subunit in Genetic Porcine Models for Colorectal Cancer and Osteosarcoma.

The B subunit of bacterial Shiga toxin (STxB) is nontoxic and has low immunogenicity. Its receptor, the glycosphingolipid Gb3/CD77, is overexpressed on the cell surface of human colorectal cancer. We tested whether genetic porcine models, closely resembling human anatomy and pathophysiology, can be used to exploit the tumor-targeting potential of STxB. In accordance with findings on human colorectal cancer, the pig model APC1311 bound STxB in colorectal tumors, but not in normal colon or jejunum, except for putative enteroendocrine cells. In primary tumor cells from endoscopic biopsies, STxB was rapidly taken up along the retrograde intracellular route to the Golgi, whereas normal colon organoids did not bind or internalize STxB. Next, we tested a porcine model (TP53LSL-R167H) for osteosarcoma, a tumor entity with a dismal prognosis and insufficient treatment options, hitherto not known to express Gb3. Pig osteosarcoma strongly bound StxB and expressed the Gb3 synthase 1,4-galactosyltransferase (A4GALT). Primary osteosarcoma cells, but not normal osteoblasts, rapidly internalized fluorescently labeled STxB along the retrograde route to the Golgi. Importantly, six of eight human osteosarcoma cell lines expressed A4GALT mRNA and showed prominent intracellular uptake of STxB. The physiologic role of A4GALT was tested by CRISPR/Cas9 mutagenesis in porcine LLC-PK1 kidney epithelial cells and RNAi in MG-63 human osteosarcoma cells. A4GALT deficiency or knockdown abolished STxB uptake and led to significantly reduced cell migration and proliferation, hinting toward a putative tumor-promoting role of Gb3. Thus, pig models are suitable tools for STxB-based tumor targeting and may allow "reverse-translational" predictions on human tumor biology.

New concept for colonoscopy including side optics and artificial intelligence.

BACKGROUND AND AIMS: Adenoma detection rate is the crucial parameter for colorectal cancer screening. Increasing the field of view with additional side optics has been reported to detect flat adenomas hidden behind folds. Furthermore, artificial intelligence (AI) has also recently been introduced to detect more adenomas. We therefore aimed to combine both technologies in a new prototypic colonoscopy concept. METHODS: A 3-dimensional-printed cap including 2 microcameras was attached to a conventional endoscope. The prototype was applied in 8 gene-targeted pigs with mutations in the adenomatous polyposis coli gene. The first 4 animals were used to train an AI system based on the images generated by microcameras. Thereafter, the conceptual prototype for detecting adenomas was tested in a further series of 4 pigs. RESULTS: Using our prototype, we detected, with side optics, adenomas that might have been missed conventionally. Furthermore, the newly developed AI could detect, mark, and present adenomas visualized with side optics outside of the conventional field of view. CONCLUSIONS: Combining AI with side optics might help detect adenomas that otherwise might have been missed.

The Missing Link: Cre Pigs for Cancer Research.

The Cre/loxP system is a powerful tool for the generation of animal models with precise spatial and temporal gene expression. It has proven indispensable in the generation of cancer models with tissue specific expression of oncogenes or the inactivation of tumor suppressor genes. Consequently, Cre-transgenic mice have become an essential prerequisite in basic cancer research. While it is unlikely that pigs will ever replace mice in basic research they are already providing powerful complementary resources for translational studies. But, although conditionally targeted onco-pigs have been generated, no Cre-driver lines exist for any of the major human cancers. To model human pancreatic cancer in pigs, Cre-driver lines were generated by CRISPR/Cas9-mediated insertion of codon-improved Cre (iCre) into the porcine PTF1A gene, thus guaranteeing tissue and cell type specific function which was proven using dual fluorescent reporter pigs. The method used can easily be adapted for the generation of other porcine Cre-driver lines, providing a missing tool for modeling human cancers in large animals.

Role of Methylation in Period2 (PER2) Transcription in the Context of the Presence or Absence of Light Signals: Natural and Chemical-Studies on the Pig Model.

It has been proposed that carbon monoxide (CO) is a chemical light carrier that is transferred by the humoral pathway from the retina to the brain. Here, we aimed to study how deeply CO is involved in regulating the expression of Period2 gene (PER2), one of the genes maintaining the intrinsic biological clock. In our in vivo experiment, we studied whether CO may be a chemical signal and is also equivalent to natural light in three groups of pigs: Normal: housed in natural conditions without any procedures, Control: adapted and kept in constant darkness, infused with blank plasma, and CO treated: adapted and kept in constant darkness infused with CO-enriched plasma. After the experiment, the animals were slaughtered at two times of day: 12 p.m. and 12 a.m. Next, hypothalamus samples were collected. Quantitative PCR, the DNA methylation of the promoter sequence containing enhancers (E-box) and a functional analysis of the PER2 promoter was performed. qPCR showed a differential pattern of PER2 mRNA expression at daytime oscillation in the examined groups. Pyrosequencing revealed daytime changes in the methylation level of regulatory sites of the examined sequence. Luciferase reporter assay confirmed that E-boxes (CANNTG) drive the expression of the porcine PER2 in vitro. In conclusion, changes in methylation over 24 h may regulate the oscillatory manner of PER2 expression.

Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma.

Osteosarcoma (OS) is a primary bone malignancy that mainly occurs during adolescent growth, suggesting that bone growth plays an important role in the aetiology of the disease. Genetic factors, such as heritable mutations of Rb1 and TP53, are associated with an increased risk of OS. Identifying driver mutations for OS has been challenging due to the complexity of bone growth-related pathways and the extensive intra-tumoral heterogeneity of this cancer. We previously generated pigs carrying a mutated TP53 gene, which develop OS at high frequency. RNA sequencing and allele expression imbalance (AEI) analysis of OS and matched healthy control samples revealed a highly significant AEI (p = 2.14 × 10-39) for SNPs in the BIRC3-YAP1 locus on pig chromosome 9. Analysis of copy number variation showed that YAP1 amplification is associated with the AEI and the progression of OS. Accordingly, the inactivation of YAP1 inhibits proliferation, migration, and invasion, and leads to the silencing of TP63 and reconstruction of p16 expression in p53-deficient porcine OS cells. Increased p16 mRNA expression correlated with lower methylation of its promoter. Altogether, our study provides molecular evidence for the role of YAP1 amplification in the progression of p53-dependent OS.

A tissue- and gender-specific regulation of the SARS-CoV-2 receptor ACE2 by p53 in pigs.

The current COVID-19 pandemic is caused by infections with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A sex-bias has been observed, with increased susceptibility and mortality in male compared to female patients. The gene for the SARS-CoV-2 receptor ACE2 is located on the X chromosome. We previously generated TP53 mutant pigs that exhibit a sex-specific patho-phenotype due to altered regulation of numerous X chromosome genes. In this study, we explored the effect of p53 deficiency on ACE2 expression in pigs. First, we identified the p53 binding site in the ACE2 promoter and could show its regulatory effect on ACE2 expression by luciferase assay in porcine primary kidney fibroblast cells. Later, quantitative PCR and western blot showed tissue- and gender-specific expression changes of ACE2 and its truncated isoform in p53-deficient pigs. We believe these findings will broaden the knowledge on ACE2 regulation and COVID-19 susceptibility.

Cas9-expressing chickens and pigs as resources for genome editing in livestock.

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Porcine model elucidates function of p53 isoform in carcinogenesis and reveals novel circTP53 RNA.

Recent years have seen an increasing number of genetically engineered pig models of human diseases including cancer. We previously generated pigs with a modified TP53 allele that carries a Cre-removable transcriptional stop signal in intron 1, and an oncogenic mutation TP53R167H (orthologous to human TP53R175H) in exon 5. Pigs with the unrecombined mutant allele (flTP53R167H) develop mainly osteosarcoma but also nephroblastomas and lymphomas. This observation suggested that TP53 gene dysfunction is itself the key initiator of bone tumorigenesis, but raises the question which aspects of the TP53 regulation lead to the development of such a narrow tumour spectrum. Molecular analysis of p53 revealed the presence of two internal TP53 promoters (Pint and P2) equivalent to those found in human. Consequently, both pig and human express TP53 isoforms. Data presented here strongly suggest that P2-driven expression of the mutant R167H-Δ152p53 isoform (equivalent to the human R175H-Δ160p53 isoform) and its circular counterpart circTP53 determine the tumour spectrum and play a critical role in the malignant transformation in flTP53R167H pigs. The detection of Δ152p53 isoform mRNA in serum is indicative of tumorigenesis. Furthermore, we showed a tissue-specific p53-dependent deregulation of the p63 and p73 isoforms in these tumours. This study highlights important species-specific differences in the transcriptional regulation of TP53. Considering the similarities of TP53 regulation between pig and human, these observations provide useful pointers for further investigation into isoform function including the novel circTP53 in both the pig model and human patients.

A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions.

Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 µm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.

A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity.

Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called 'Pig intestinal bacterial collection' (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota.

Placental distribution of endogenous and exogenous substances: A pilot study utilizing cryo-sampled specimen off delivery room.

INTRODUCTION: Reliability in the use of placentome (including placenta, umbilical cord, and cord blood) biomarkers requires an understanding of their distributions. Here we aim to develop a simple and proper placenta sampling scheme, and to evaluate the placental distributions of biomarkers. METHODS: We developed a continuous cooling chain protocol off delivery room and cryo-subsampling method for placenta sampling. The levels of thyroid hormones (THs), elements, persistent organic pollutants (POPs), monoamines, and vitamin E were measured using UPLC-Q-TOF-MS, HPLC-ICP-MS, HPLC-EcD, and HRGC-HRMS, respectively. The distributions of biomarkers were assessed. RESULTS: In human placentome, l-thyroxine (T4), Cd, Se, Zn, Cu, Fe, Ca, K, Mg, α-tocopherol, ß-tocopherol, and ß-tocotrienol levels were higher in placenta than in umbilical cord, while Pb and Mn were concentrated in human cord. In porcine placentome, T4, 3,3',5'-triiodo-l-thyronine (rT3), 3,3'-diiodo-l-thyronine, Cd, Pb, Zn, K, and Al levels were higher in the cord. The intraclass correlation coefficient (ICC) was <0.4 for 3,3',5-triiodo-l-thyronine, rT3, α-tocopherol, and 7 elements in human basal plate, indicating low reliability. rT3, Cd, Zn, Mn, and Cu were significantly concentrated in the central region in human placenta, while higher levels of As, Cd, Cr, and Al were found in the periphery region in porcine placenta. Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) showed moderate reliability (ICC: 0.40-0.98) except PCB-81, -126, and BDE-208, while polychlorinated dibenzo-p-doixins/furans (PCDD/Fs) showed poor reliability (ICC: 0.07-0.31). DISCUSSION: These results highlight the complexity of placenta sampling. This study provides a novel and simple sampling approach in investigating placental exposomics.

Polymorphisms of CSF1R and WISP1 genes are associated with severity of familial adenomatous polyposis in APC1311 pigs.

Hereditary familial adenomatous polyposis (FAP) in humans significantly increases the risk of development of colorectal cancer (CRC). Germline mutations in the APC (adenomatous polyposis coli) gene are responsible for FAP. Despite having the same causative mutation, the severity of the disease differs from patient to patient. The porcine FAP model carrying a truncating APC1311 mutation, orthologous to the dominant human mutation that leads to severe form of the disease (APC1309), mirrors the severity of polyposis. Earlier RNAseq studies have revealed the differential expression of WISP1 and CSF1R in samples derived from low-grade (LG-IEN) and more advanced high-grade (HG-IEN) colon polyps of APC1311/+ pigs. The grade of dysplasia was correlated with the severity of polyposis in APC1311/+ pigs characterized by a low (LP) and high (HP) numbers of polyps. The goal of this work was to find DNA variants that regulate the expression of CSF1R and WISP1 in LP and HP pigs. In total, 32 and 36 polymorphisms in CSF1R and WISP1 were found, respectively. Of these, the genotype frequency of four silent SNPs in the coding region of WISP1 differed significantly between LP and HP lines. In silico analysis revealed an elevated minimum free energy (MFE) for three of these SNPs, suggesting their role in mRNA structure stability. Furthermore, four polymorphisms in the promoter region of CSF1R, cosegregating as a common haplotype, were associated with polyp number in APC1311/+ pigs. A secreted alkaline phosphatase (SEAP) assay showed, however, that these variants have no direct effect on the activity of the CSF1R promoter. Concluding, our study identified polymorphisms in CSF1R and WISP1 that are potentially associated with the severity of polyposis in APC1311/+ pigs.

Activation of cryptic splicing in bovine WDR19 is associated with reduced semen quality and male fertility.

Cattle are ideally suited to investigate the genetics of male reproduction, because semen quality and fertility are recorded for all ejaculates of artificial insemination bulls. We analysed 26,090 ejaculates of 794 Brown Swiss bulls to assess ejaculate volume, sperm concentration, sperm motility, sperm head and tail anomalies and insemination success. The heritability of the six semen traits was between 0 and 0.26. Genome-wide association testing on 607,511 SNPs revealed a QTL on bovine chromosome 6 that was associated with sperm motility (P = 2.5 x 10-27), head (P = 2.0 x 10-44) and tail anomalies (P = 7.2 x 10-49) and insemination success (P = 9.9 x 10-13). The QTL harbors a recessive allele that compromises semen quality and male fertility. We replicated the effect of the QTL on fertility (P = 7.1 x 10-32) in an independent cohort of 2481 Brown Swiss bulls. The analysis of whole-genome sequencing data revealed that a synonymous variant (BTA6:58373887C>T, rs474302732) in WDR19 encoding WD repeat-containing protein 19 was in linkage disequilibrium with the fertility-associated haplotype. WD repeat-containing protein 19 is a constituent of the intraflagellar transport complex that is essential for the physiological function of motile cilia and flagella. Bioinformatic and transcription analyses revealed that the BTA6:58373887 T-allele activates a cryptic exonic splice site that eliminates three evolutionarily conserved amino acids from WDR19. Western blot analysis demonstrated that the BTA6:58373887 T-allele decreases protein expression. We make the remarkable observation that, in spite of negative effects on semen quality and bull fertility, the BTA6:58373887 T-allele has a frequency of 24% in the Brown Swiss population. Our findings are the first to uncover a variant that is associated with quantitative variation in semen quality and male fertility in cattle.