Genomic evidence for the suitability of Göttingen Minipigs with a rare seizure phenotype as a model for human epilepsy.

Epilepsy is a complex genetic disorder that affects about 2% of the global population. Although the frequency and severity of epileptic seizures can be reduced by a range of pharmacological interventions, there are no disease-modifying treatments for epilepsy. The development of new and more effective drugs is hindered by a lack of suitable animal models. Available rodent models may not recapitulate all key aspects of the disease. Spontaneous epileptic convulsions were observed in few Göttingen Minipigs (GMPs), which may provide a valuable alternative animal model for the characterisation of epilepsy-type diseases and for testing new treatments. We have characterised affected GMPs at the genome level and have taken advantage of primary fibroblast cultures to validate the functional impact of fixed genetic variants on the transcriptome level. We found numerous genes connected to calcium metabolism that have not been associated with epilepsy before, such as ADORA2B, CAMK1D, ITPKB, MCOLN2, MYLK, NFATC3, PDGFD, and PHKB. Our results have identified two transcription factor genes, EGR3 and HOXB6, as potential key regulators of CACNA1H, which was previously linked to epilepsy-type disorders in humans. Our findings provide the first set of conclusive results to support the use of affected subsets of GMPs as an alternative and more reliable model system to study human epilepsy. Further neurological and pharmacological validation of the suitability of GMPs as an epilepsy model is therefore warranted.

Choline-deficient high-fat diet-induced MASH in Göttingen Minipigs: Characterisation and effects of a chow reversal period.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) is increasing, and translational animal models are needed to develop novel treatments for this disease. The physiology and metabolism of pigs have a relatively high resemblance to humans, and the present study aimed to characterise choline-deficient, and high-fat diet (CDAHFD) fed Göttingen Minipigs as a novel animal model of MASLD/MASH. Göttingen Minipigs were fed CDAHFD for up to 5 months, and the phenotype was investigated by analysis of plasma parameters and repeated collection of liver biopsies. Furthermore, changes in hepatic gene expression during the experiment were explored by RNA sequencing. For a subset of the minipigs, the diet was changed from CDAHFD back to chow to investigate if the liver pathology was reversible. Göttingen Minipigs on CDAHFD gained bodyweight, and plasma levels of cholesterol, AST, ALT, ALP and GGT were increased. CDAHFD-fed minipigs developed hepatic steatosis, inflammation, and fibrosis, which in 5 of 16 animals progressed to cirrhosis. During an 11-week chow reversal period, steatosis regressed while fibrosis persisted. Regarding inflammation, the findings were less clear, depending on the type of readout. MASH Human Proximity Scoring (combined evaluation of transcriptional, phenotypic and histopathological parameters) showed that CDAHFD-fed Göttingen Minipigs resemble human MASLD/MASH better than most rodent models. In conclusion, CDAHFD-fed minipigs develop a MASH-like phenotype which in several aspects resemble the changes observed in human patients with MASLD/MASH. Furthermore, repeated collection of liver biopsies allow detailed characterisation of histopathological changes over time in individual animals.

Skin immune cell characterization in juvenile and adult Göttingen Minipigs.

The skin hosts a sophisticated immune system involving responses from both innate and adaptive immune cell populations. Swine skin is close to human skin by its structure, composition and function. In addition, the minipig is considered the model of choice in toxicology studies for drugs applied by the dermal route and developed for both the adult and paediatric indications. However, knowledge on the skin immune system in minipigs, particularly in Göttingen Minipigs, is still limited. The objective of our work was first to characterize the main skin immune populations (Langerhans cells, dermal dendritic cells, macrophages and T lymphocytes) in Göttingen Minipigs. In parallel, we compared the skin immune populations from healthy and immunocompromised piglets following oral treatment with cyclosporin A (CsA) at 10 mg/kg/day. We also explored other pathological conditions using a contact dermatitis model in minipigs challenged with a sensitizer, 2,4-dinitrochlorobenzene (DNCB). Langerhans cells and dermal MHCIIlowCD163+ cells were increased one month after oral treatment with CsA at 10 mg/kg/day. The contact dermatitis model in Göttingen Minipigs challenged by DNCB suggested migration of Langerhans cells and dermal dendritic cells as well as T cell recruitment into the skin. These data bring new information in skin immunotoxicology in Göttingen Minipigs and could contribute to a better understanding of the effects of new therapeutic drugs on the developing immune system.

Assessing breed integrity of Göttingen Minipigs.

BACKGROUND: Göttingen Minipigs (GMP) is the smallest commercially available minipig breed under a controlled breeding scheme and is globally bred in five isolated colonies. The genetic isolation harbors the risk of stratification which might compromise the identity of the breed and its usability as an animal model for biomedical and human disease. We conducted whole genome re-sequencing of two DNA-pools per colony to assess genomic differentiation within and between colonies. We added publicly available samples from 13 various pig breeds and discovered overall about 32 M loci, ~ 16 M. thereof variable in GMPs. Individual samples were virtually pooled breed-wise. FST between virtual and DNA pools, a phylogenetic tree, principal component analysis (PCA) and evaluation of functional SNP classes were conducted. An F-test was performed to reveal significantly differentiated allele frequencies between colonies. Variation within a colony was quantified as expected heterozygosity. RESULTS: Phylogeny and PCA showed that the GMP is easily discriminable from all other breads, but that there is also differentiation between the GMP colonies. Dependent on the contrast between GMP colonies, 4 to 8% of all loci had significantly different allele frequencies. Functional annotation revealed that functionally non-neutral loci are less prone to differentiation. Annotation of highly differentiated loci revealed a couple of deleterious mutations in genes with putative effects in the GMPs . CONCLUSION: Differentiation and annotation results suggest that the underlying mechanisms are rather drift events than directed selection and limited to neutral genome regions. Animal exchange seems not yet necessary. The Relliehausen colony appears to be the genetically most unique GMP sub-population and could be a valuable resource if animal exchange is required to maintain uniformity of the GMP.

Neonatal and Juvenile Ocular Development in Göttingen Minipigs and Domestic Pigs: A Histomorphological and Immunohistochemical Study.

Pigs are considered one of the relevant animal models for ocular research as they share several histological and anatomical similarities with the human eye. With the increasing interest in juvenile animal models, this study aimed to describe the postnatal development of ocular structures in 16 Göttingen minipigs and 25 F2 domestic pigs, between birth and 6 months of age, using histopathology and immunohistochemistry against Ki-67, caspase-3, calbindin, glial fibrillary acidic protein, rhodopsin, and synaptophysin. All ocular structures in both pig breeds were incompletely developed at birth and for variable periods postnatally. Noteworthy histological features of immaturity included vascularization in the corneal stroma in neonatal Göttingen minipigs, increased cellularity in different substructures, remnants of the hyaloid vasculature, short and poorly ramified ciliary body processes, and a poorly developed cone inner segment. Increased cellular proliferation, highlighted by abundant Ki-67 immunolabeling, was observed in almost all developing structures of the pig eye for variable periods postnatally. Apoptosis, highlighted with caspase-3 immunolabeling, was observed in the retinal inner nuclear layer at birth and in the regressing hyaloid vasculature remnants. Immunohistochemistry against rhodopsin, synaptophysin, and calbindin demonstrated the short size of the developing photoreceptors and the immature cone inner segment morphology. Calbindin labeling revealed significant differences in the amount of positively labeled cone nuclei between the retinal area centralis and the non-area centralis regions. The elongation of Müller cell processes in the developing retina was shown with glial fibrillary acidic protein. In both pig breeds, the eyes reached histomorphological and immunohistochemical maturity at 6 months of age.

Hyperglycemia-induced transcriptional regulation of ROCK1 and TGM2 expression is involved in small artery remodeling in obese diabetic Göttingen Minipigs.

Obesity and diabetes in humans are associated with hypertrophic remodeling and increased media:lumen ratio of small resistance arteries, which is an independent predictor of cardiovascular events. In order to minimize increases in media:lumen ratio, hypertrophic remodeling should be accompanied by outward remodeling. We aimed to investigate the mechanisms of structural remodeling in small pial arteries (PAs) and terminal mesenteric arteries (TMAs) from obese Göttingen Minipigs with or without diabetes. Göttingen Minipigs received either control diet (lean control (LC)), high fat/high fructose/high cholesterol diet (FFC), or FFC diet with streptozotocin (STZ)-induced diabetes (FFC/STZ) for 13 months. At the end of the study (20 months), we assessed body weight, fasting plasma biochemistry, passive vessel dimensions, mRNA expression (matrix metallopeptidases 2/9 (MMP2, MMP9), tissue inhibitor of metallopeptidase 1 (TIMP1), transglutaminase 2 (TGM2), Rho-kinase 1 (ROCK1), TGFß-receptor 2 (TGFBR2), and IGF1-receptor (IGFR1) genes), and immunofluorescence in PAs and TMAs. We performed multiple linear correlation analyses using plasma values, structural data, and gene expression data. We detected outward hypertrophic remodeling in TMAs and hypertrophic remodeling in PAs from FFC/STZ animals. ROCK1 and TGM2 genes were up-regulated in PAs and TMAs from the FFC/STZ group. Passive lumen diameter (PLD) of TMAs was correlated with plasma values of glucose (GLU), fructosamine (FRA), total cholesterol (TC), and triglycerides (TGs). ROCK1 and TGM2 expressions in TMAs were correlated with PLD, plasma GLU, fructosamine, and TC. ROCK1 and TGM2 proteins were immunolocalized in the media of PAs and TMAs, and their fluorescence levels were increased in the FFC/STZ group. Hyperglycemia/hyperlipidemia is involved in regulation of ROCK1 and TGM2 expression leading to outward remodeling of small resistance arteries in obese diabetic Göttingen Minipigs.

A comparative analysis of gut microbiota disturbances in the Gottingen minipig and rhesus macaque models of acute radiation syndrome following bioequivalent radiation exposures.

In rodent studies, the gut microbiota has been implicated in facilitating both radioresistance, by protecting the epithelium from apoptotic responses and radiosensitivity, inducing endothelial apoptotic responses. Despite the observation that large animal models, such as the Chinese Rhesus macaque and the Gottingen Minipig, demonstrate similarity to human physiologic responses to radiation, little is known about radiation-induced changes of the gut microbiome in these models. To compare the two models, we used bioequivalent radiation doses which resulted in an LD50 for Gottingen Minipigs and Chinese Rhesus macaques, 1.9 Gy and 6.8 Gy, respectively. Fecal samples taken prior and 3 days post-radiation were used for 16S rRNA gene sequence amplicon high throughput sequencing (Illumina MiSeq). Baseline gut microbiota profiles were dissimilar between minipigs and rhesus macaques. Irradiation profoundly impacted gut microbiota profiles in both animals. Significant increases of intracellular symbionts were common to both models and to reported changes in rodents suggesting universality of these findings post-radiation. Remarkably, opposite dynamics were observed for the main phyla, with increase of Firmicutes and decrease of Bacteroidetes and Proteobacteria in minipigs but with enrichment of Bacteroidetes in rhesus macaques. Minipig changes in magnitude and in variety of species affected were more extensive than those observed in rhesus macaques. This pilot study provides an important first step in comparing the radiosensitive pig model to the comparatively more radioresistant macaque model, for the identification of microbial elements which may influence radiosensitivity.

Spontaneous background pathology in Göttingen minipigs.

The Göttingen minipig is gaining increasing popularity as a nonrodent species in nonclinical testing. The Göttingen minipig is easy to handle; has many anatomical and physiological similarities to man; and causes fewer ethical concerns than usage of the traditional nonrodent species, nonhuman primates, and dogs. The increasing usage of the Göttingen minipig has raised the need of appropriate background data. This article summarizes the background pathology of 835 untreated control Göttingen minipigs of both sexes used at CiToxLAB Scantox A/S during the period of 1995 to 2007.

Quantifying energy expenditure in Göttingen Minipigs with the 13C-bicarbonate method under basal and drug-treated conditions.

Effective treatments of obesity focusing on energy expenditure (EE) are needed. To evaluate future EE-modulating drug candidates, appropriate animal models and methods to assess EE are needed. This study aimed to evaluate the stable isotope 13C-bicarbonate method (13C-BM) for estimating EE in Göttingen minipigs under basal and drug-treated conditions. Four experiments (Expt.1-4) were conducted to assess: 1) the 13C-BM reproducibility using breath sample collection (n = 8), on two consecutive days, 2) the effect of two dose levels (5 and 10 mg/kg body weight (BW)) of the mitochondrial uncoupler dinitrophenol (DNP) in a crossover design (n = 8), 3) sampling method agreement; blood vs. exhaled air (n = 6) and 4) 13C-BM using constant isotope infusion compared with indirect calorimetry (IC) (n = 3). Results correlated significantly (p < 0.001) between days (Expt.1), with an average coefficient of variance of 5.4 ± 2.3%. Administration of 10 mg DNP/kg BW increased (p < 0.01) EE by 33.2 ± 6.4% (Expt.2). Results based on different sampling methods correlated significantly (p < 0.001) and EE increased after 10 mg DNP/kg BW (p < 0.05) in Expt.3. However, results based on blood sampling were significantly higher (p < 0.01) than those of exhaled air. No effect of DNP and significantly different EE results (p < 0.05) was observed in a limited number of animals, when constant isotope infusion and blood sampling was compared with IC (Expt.4). In conclusion, the 13C-BM is useful for investigating treatment effects on EE in minipigs. However, further validation under standardized conditions is needed to provide accurate estimates of the 13C recovery factor and respiratory quotient, both of decisive importance when using the 13C-BM.

Characterization of the immune system of Ellegaard Göttingen Minipigs - An important large animal model in experimental medicine.

Interest in Ellegaard Göttingen Minipigs (EGMs) as a model in experimental medicine is continuously growing. The aim of this project is to increase the knowledge of the immune system of EGMs as information is still scarce. Therefore, we studied the postnatal maturation of their immune system from birth until 126 weeks of age. For the first 26 weeks of the study, animals were kept under pathogen-reduced conditions (SPF) and afterwards under conventional housing conditions. The development of the immune system was analyzed by monitoring changes in total numbers of leukocytes and lymphocytes of ten individuals and the composition of leukocyte populations by multi-color flow cytometry (FCM). We followed the presence of monocytes using monoclonal antibodies (mAbs) against CD172a+ and CD163+ and B cells based on the expression of CD79a. NK cells were distinguished as CD3-CD16+CD8α+/dim cells and further subdivided using NKp46 (CD335) expression into NKp46-, NKp46+, and NKp46high NK cells. T-cell receptor (TCR) γδ T cells were defined by the expression of TCR-γδ and different subsets were determined by their CD2 and perforin expression. TCR-αß T cells were classified by their CD8ß+ or CD4 expression. For monitoring their differentiation, expression of CD27 and perforin was investigated for CD8ß++ T cells and CD8α together with CD27 for CD4+ T cells. We clearly detected a postnatal development of immune cell composition and identified phenotypes indicative of differentiation within the respective leukocyte subsets. Examination of the development of the antigen-specific immune system after transfer to different distinct housing conditions and after vaccination against common porcine pathogens such as porcine circovirus 2 (PCV2) revealed a markedly increased presence of more differentiated CD8+ and CD4+ T cells with central and effector memory T-cell phenotypes. To complement the findings, a PCV2 vaccine-specific antigen was used for in vitro restimulation experiments. We demonstrated antigen-specific proliferation of CD4+CD8α+CD27+ central and CD4+CD8α+CD27- effector memory T cells as well as antigen-specific production of TNF-α and IFN-γ. This study of postnatal immune development defines basic cellular immune parameters of EGMs and represents an important milestone for the use of EGMs for immunological questions in experimental medicine.

Patient-Specific Bioimplants and Reconstruction Plates for Mandibular Defects: Production Workflow and In Vivo Large Animal Model Study.

A major challenge with extensive craniomaxillofacial bone reconstruction is the limited donor-site availability to reconstruct defects predictably and accurately according to the anatomical shape of the patient. Here, patient-specific composite bioimplants, consisting of cross-linked poly(trimethylene carbonate) (PTMC) networks and ß-tricalcium phosphate (ß-TCP), are tested in vivo in twelve Göttingen minipigs in a large mandibular continuity defect model. The 25 mm defects are supported by patient-specific titanium reconstruction plates and receive either osteoconductive composite bioimplants (PTMC+TCP), neat polymer network bioimplants (PTMC), autologous bone segments (positive control), or are left empty (negative control). Postoperatively, defects treated with bioimplants show evident ossification at 24 weeks. Histopathologic evaluation reveals that neat PTMC bioimplant surfaces are largely covered with fibrous tissue, while in the PTMC+TCP bioimplants, bone attached directly to the implant surface shows good osteoconduction and histological signs of osteoinductivity. However, PTMC+TCP bioimplants are associated with high incidence of necrosis and infection, possibly due to rapid resorption and/or particle size of the used ß-TCP. The study highlights the importance of testing bone regeneration implants in a clinically relevant large animal model and at the in situ reconstruction site, since results on small animal models and studies in nonloadbearing areas do not translate directly.

Electrocardiography and heart rate variability in Göttingen Minipigs: Impact of diurnal variation, lead placement, repeatability and streptozotocin-induced diabetes.

BACKGROUND: The Göttingen Minipig is widely used in preclinical research and safety pharmacology, but standardisation of porcine electrocardiography (ECG) is lacking. The aim of this study was to investigate diurnal effects, change over time and choice of lead on ECG morphology and heart rate variability (HRV) in healthy and streptozotocin (STZ) induced diabetic Göttingen Minipigs. METHODS: Diabetes was experimentally induced using STZ in 11 Göttingen Minipigs (DIA). Seven controls (CON) were included. 24-h ECG was recorded at baseline and four months. Morphological parameters (QRS and T wave duration, P- and T-wave amplitude, PR and QT (Bazett's (QTcb) or Fridericia (QTcf) correction) intervals and ST segment), presence of cardiac arrhythmias, heart rate (HR) and HRV (time and frequency domain) were analysed. RESULTS: Four months after induction, DIA had decreased P-wave amplitude (P < 0.0001) and T-wave duration (P = 0.017), compared to CON. QTcb was lower in DIA, but not in CON. Both groups had decreased HR (P < 0.0001) and QRS duration (lead II, P = 0.04) and length of PR-segment increased (lead I and II, P < 0.01) while selected HRV parameters also increased (all P < 0.01). Time of day influenced HR, QRS duration, PR segment, ST segment, T- and P-wave amplitude and some parameters of HRV. Inter- and intra-observer variability of morphological measurements was low (<6%). CONCLUSION: ECG parameters were influenced by time setting, diurnal variation and lead. Some ECG and HRV changes were found in diabetic minipigs four months after STZ induction. The findings underline the need for standardisation of ECG and HRV in Göttingen Minipigs.

The CRISPR/Cas9 Minipig-A Transgenic Minipig to Produce Specific Mutations in Designated Tissues.

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

Göttingen Minipigs as a Model to Evaluate Longevity, Functionality, and Memory of Immune Response Induced by Pertussis Vaccines.

Evaluation of the short-term and long-term immunological responses in a preclinical model that simulates the targeted age population with a relevant vaccination schedule is essential for human vaccine development. A Göttingen minipig model was assessed, using pertussis vaccines, to demonstrate that vaccine antigen-specific humoral and cellular responses, including IgG titers, functional antibodies, Th polarization and memory B cells can be assessed in a longitudinal study. A vaccination schedule of priming with a whole cell (DTwP) or an acellular (DTaP) pertussis vaccine was applied in neonatal and infant minipigs followed by boosting with a Tdap acellular vaccine. Single cell RNAsequencing was used to explore the long-term maintenance of immune memory cells and their functionality for the first time in this animal model. DTaP but not DTwP vaccination induced pertussis toxin (PT) neutralizing antibodies. The cellular immune response was also characterized by a distinct Th polarization, with a Th-2-biased response for DTaP and a Th-1/Th-17-biased response for DTwP. No difference in the maintenance of pertussis-specific memory B cells was observed in DTaP- or DTwP-primed animals 6 months post Tdap boost. However, an increase in pertussis-specific T cells was still observed in DTaP primed minipigs, together with up-regulation of genes involved in antigen presentation and interferon pathways. Overall, the minipig model reproduced the humoral and cellular immune responses induced in humans by DTwP vs. DTaP priming, followed by Tdap boosting. Our data suggest that the Göttingen minipig is an attractive preclinical model to predict the long-term immunogenicity of human vaccines against Bordetella pertussis and potentially also vaccines against other pathogens.

Autologous Cartilage Chip Transplantation Improves Repair Tissue Composition Compared With Marrow Stimulation.

BACKGROUND: Repair of chondral injuries by use of cartilage chips has recently demonstrated clinical feasibility. PURPOSE: To investigate in vivo cartilage repair outcome of autologous cartilage chips compared with marrow stimulation in full-thickness cartilage defects in a minipig model. STUDY DESIGN: Controlled laboratory study. METHODS: Six Göttingen minipigs received two 6-mm chondral defects in the medial and lateral trochlea of each knee. The two treatment groups were (1) autologous cartilage chips embedded in fibrin glue (ACC) (n = 12) and (2) marrow stimulation (MST) (n = 12). The animals were euthanized after 6 months, and the composition of repair tissue was quantitatively determined using histomorphometry. Semiquantitative evaluation was performed by means of the International Cartilage Repair Society (ICRS) II score. Collagen type II staining was used to further evaluate the repair tissue composition. RESULTS: Significantly more hyaline cartilage was found in the ACC (17.1%) compared with MST (2.9%) group ( P < .01). Furthermore, the ACC group had significantly less fibrous tissue (23.8%) compared with the MST group (41.1%) ( P < .01). No significant difference in fibrocartilage content was found (54.7% for ACC vs 50.8% for MST). The ACC group had significantly higher ICRS II scores for tissue morphological characteristics, matrix staining, cell morphological characteristics, surface assessment, mid/deep assessment, and overall assessment ( P < .05). The ACC-treated defects had significantly more collagen type II staining (54.5%) compared with the MST-treated defects (28.1%) ( P < .05). CONCLUSION: ACC transplant resulted in improved quality of cartilage repair tissue compared with MST at 6 months postoperatively. CLINICAL RELEVANCE: Further studies are needed to investigate ACC as a possible alternative first-line treatment for focal cartilage injuries in the knee.

Development of a minipig model for lung injury induced by a single high-dose radiation exposure and evaluation with thoracic computed tomography.

Radiation-induced lung injury (RILI) due to nuclear or radiological exposure remains difficult to treat because of insufficient clinical data. The goal of this study was to establish an appropriate and efficient minipig model and introduce a thoracic computed tomography (CT)-based method to measure the progression of RILI. Göttingen minipigs were allocated to control and irradiation groups. The most obvious changes in the CT images after irradiation were peribronchial opacification, interlobular septal thickening, and lung volume loss. Hounsfield units (HU) in the irradiation group reached a maximum level at 6 weeks and decreased thereafter, but remained higher than those of the control group. Both lung area and cardiac right lateral shift showed significant changes at 22 weeks post irradiation. The white blood cell (WBC) count, a marker of pneumonitis, increased and reached a maximum at 6 weeks in both peripheral blood and bronchial alveolar lavage fluid. Microscopic findings at 22 weeks post irradiation were characterized by widening of the interlobular septum, with dense fibrosis and an increase in the radiation dose-dependent fibrotic score. Our results also showed that WBC counts and microscopic findings were positively correlated with the three CT parameters. In conclusion, the minipig model can provide useful clinical data regarding RILI caused by the adverse effects of high-dose radiotherapy. Peribronchial opacification, interlobular septal thickening, and lung volume loss are three quantifiable CT parameters that can be used as a simple method for monitoring the progression of RILI.

Temperature dependent embryonic development of Trichuris suis eggs in a medicinal raw material.

The therapeutic potential of infective pig whipworm eggs, Trichuris suis ova (TSO), is currently tested in several clinical trials on immune-mediated diseases. This paper studied the embryonic development of TSO in a medicinal raw product, where the parasite eggs were suspended in sulphuric acid (pH1). Unembryonated T. suis egg batches were stored at 5, 10, 15, 20, 25, 30, and 40°C (±1°C) and examined at 2, 4, 8, and 14 weeks. Subsequently, sub-batches from each temperature were allowed to embryonate for additional 14 weeks at 25°C, and selected samples were tested for infectivity in Göttingen minipigs. Both male and female pigs were used to evaluate eventual gender specific infectivity. Storage at 30°C up to 14 weeks and subsequent embryonation for 14 weeks at 25°C did not significantly reduce the overall larval establishment in minipigs, as compared to storage at 5°C and subsequent embryonation at 25°C. As marked impairment of egg development was observed during storage at 40°C, a second set of unembryonated egg batches were incubated at 30, 32, 34, 36, 38, and 40°C (±1°C) for 1-8 weeks. The development of the eggs was repeatedly examined by manual light microscopy, multispectral analysis (OvaSpec), and an egg hatching assay prior to the final testing in minipigs (Trial 1). These methods showed that the development started earlier at higher temperatures, but the long-term storage at higher temperature affected the egg development. The present study further documents tolerance of the TSO to storage at temperature 5-15°C, at which temperature development of larvae is not initiated.

Dose-dependent establishment of Trichuris suis larvae in Göttingen minipigs.

Embryonated eggs of the pig whipworm Trichuris suis (TSOee) constitute the active pharmaceutical ingredient (API) in a medicinal product explored in human clinical trials against several immune-mediated diseases. The measurement of TSO biological potency (hatchability and infectivity) is a requirement for the assessment of TSO's pharmacological potency in human clinical trials. The present study aims to validate the dose-dependent establishment of T. suis larvae in Göttingen minipigs and eventual clinical implication of a dose range (1000-10,000 TSO). Four groups of 5 minipigs were inoculated with doses of 1000, 2500, 7500, and 10,000 TSOee, respectively, to evaluate a range of concentrations of TSOee in a minipig infectivity model. Unembryonated eggs (TSOue) were added to keep the total egg number in the inoculum constant at 10,000 eggs. Two groups received 2500 and 7500 TSOee per pig without the addition of TSOue as controls. The intestinal larval establishment at 21 days post inoculation (dpi) demonstrated a clear positive linear dose-response relationship between numbers of inoculated TSOee and recovered larvae. There was a low level of variation in larval counts in all study groups. Thus, the infectivity model in minipigs within the tested dose range offers a reliable, sensitive and accurate assay for testing biological potency of TSO.