Translational safety biomarkers of colonic barrier integrity in the rat.

The intestinal barrier controls intestinal permeability, and its disruption has been associated with multiple diseases. Therefore, preclinical safety biomarkers monitoring barrier integrity are essential during the development of drugs targeting the intestines, particularly if starting treatment early after onset of disease. Classical toxicology endpoints are not sensitive enough and therefore our objective was to identify non-invasive markers enabling early in vivo detection of colonic barrier perturbation. Male Sprague-Dawley rats were dosed intracolonically via the rectum, using sodium caprate or ibuprofen as tool compounds to alter barrier integrity. Several potentially translational biomarkers and probe molecules related to permeability, inflammation or tissue damage were evaluated, using various analytical platforms, including immunoassays, targeted metabolomics and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry. Several markers were identified that allow early in vivo detection of colonic barrier integrity changes, before histopathological evidence of tissue damage. The most promising permeability markers identified were plasma fluorescein isothiocyanate-dextran 4000 and a lactulose/mannitol/sucralose mixture in urine. These markers showed maximum increases over 100-fold or approximately 10-50-fold, respectively. Intracolonic administration of the above probe molecules outperformed oral administration and inflammatory or other biomarkers, such as α2 -macroglobulin, calprotectin, cytokines, prostaglandins and a panel of metabolic molecules to identify early and subtle changes in barrier integrity. However, optimal timing of probe administration and sample collection is important for all markers evaluated. Inclusion of these probe molecules in preclinical toxicity studies might aid in risk assessment and the design of a clinical biomarker plan, as several of these markers have translational potential.

Modeling the Time Course of the Tissue Responses to Intramuscular Long-acting Paliperidone Palmitate Nano-/Microcrystals and Polystyrene Microspheres in the Rat.

Long-acting injectable (LAI) drug suspensions consist of drug nano-/microcrystals suspended in an aqueous vehicle and enable prolonged therapeutic drug exposure up to several months. The examination of injection site reactions (ISRs) to the intramuscular (IM) injection of LAI suspensions is relevant not only from a safety perspective but also for the understanding of the pharmacokinetics. The aim of this study was to perform a multilevel temporal characterization of the local and lymphatic histopathological/immunological alterations triggered by the IM injection of an LAI paliperidone palmitate suspension and an analog polystyrene suspension in rats and identify critical time points and parameters with regard to the host response. The ISRs showed a moderate to marked chronic granulomatous inflammation, which was mediated by multiple cyto-/chemokines, including interleukin-1ß, monocyte Chemoattractant Protein-1, and vascular endothelial growth factor. Lymphatic uptake and lymph node retention of nano-/microparticles were observed, but the contribution to the drug absorption was negligible. A simple image analysis procedure and empirical model were proposed for the accurate evaluation of the depot geometry, cell infiltration, and vascularization. This study was designed as a reference for the evaluation and comparison of future LAIs and to support the mechanistic modeling of the formulation-physiology interplay regulating the drug absorption from LAIs.

Expression of inflammation-related genes is associated with adipose tissue location in horses.

BACKGROUND: In humans, adipose tissue (AT) originating from different depots shows varying gene expression profiles. In horses, the risk of certain metabolic disorders may also be influenced by the impact of specific AT depots. Macrophage infiltration in human and rat AT is considered to be a source of inflammatory changes. In horses, this relationship has not been extensively studied yet. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), a useful method to evaluate differences in mRNA expression across different tissues, can be used to evaluate differences between equine AT depots. For a correct interpretation of the RT-qPCR results, expression data have to be normalized by the use of validated reference genes. The main objectives of this study were to compare mRNA expression of inflammation-related genes, as well as adipocyte morphology and number between different equine AT depots; and in addition, to investigate the presence of antigen presenting cells in equine AT and any potential relationship with adipokine mRNA expression. RESULTS: In this study, the mRNA expression of inflammation-related genes (leptin, chemokine ligand 5, interleukin 1ß, interleukin 6, interleukin 10, adiponectin, matrix metalloproteinase 2, and superoxide dismutase 2) and candidate reference gene stability was investigated in 8 different AT depots collected from the nuchal, abdominal (mesenteric, retroperitoneal, and peri-renal) and subcutaneous (tail head and loin) AT region. By using GeNorm analysis, HPRT1, RPL32, and GAPDH were found to be the most stable genes in equine AT. The mRNA expression of leptin, chemokine ligand 5, interleukin 10, interleukin 1ß, adiponectin, and matrix metalloproteinase 2 significantly differed across AT depots (P < 0.05). No significant AT depot effect was found for interleukin 6 and superoxide dismutase 2 (P > 0.05). Adipocyte area and number of antigen presenting cells per adipocyte significantly differed between AT depots (P < 0.05). CONCLUSIONS: Adipose tissue location was associated with differences in mRNA expression of inflammation-related genes. This depot-specific difference in mRNA expression suggests that the overall inflammatory status of horses could be partially determined by the relative proportion of the different AT depots.

Intravenous Administration of Ad26.COV2.S Does Not Induce Thrombocytopenia or Thrombotic Events or Affect SARS-CoV-2 Spike Protein Bioavailability in Blood Compared with Intramuscular Vaccination in Rabbits.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a very rare but serious adverse reaction that can occur after Ad26.COV2.S vaccination in humans, leading to thrombosis at unusual anatomic sites. One hypothesis is that accidental intravenous (IV) administration of Ad26.COV2.S or drainage of the vaccine from the muscle into the circulatory system may result in interaction of the vaccine with blood factors associated with platelet activation, leading to VITT. Here, we demonstrate that, similar to intramuscular (IM) administration of Ad26.COV2.S in rabbits, IV dosing was well tolerated, with no significant differences between dosing routes for the assessed hematologic, coagulation time, innate immune, or clinical chemistry parameters and no histopathologic indication of thrombotic events. For both routes, all other non-adverse findings observed were consistent with a normal vaccine response and comparable to those observed for unrelated or other Ad26-based control vaccines. However, Ad26.COV2.S induced significantly higher levels of C-reactive protein on day 1 after IM vaccination compared with an Ad26-based control vaccine encoding a different transgene, suggesting an inflammatory effect of the vaccine-encoded spike protein. Although based on a limited number of animals, these data indicate that an accidental IV injection of Ad26.COV2.S may not represent an increased risk for VITT.

Molecular cloning and characterization of the porcine prostaglandin transporter (SLCO2A1): evaluation of its role in F4 mediated neonatal diarrhoea.

BACKGROUND: Because prostaglandins are involved in many (patho)physiological processes, SLCO2A1 was already characterized in several species in an attempt to unravel specific processes/deficiencies. Here, we describe the molecular cloning and characterization of the porcine ortholog in order to evaluate its possible involvement in F4 enterotoxigenic E. coli mediated neonatal diarrhoea, based on a positional candidate gene approach study. RESULTS: Porcine SLCO2A1 is organized in 14 exons, containing an open reading frame of 1935 bp, encoding a 12-transmembrane organic anion cell surface transporter of 644 aa. The -388 to -5 upstream region comprises a (CpG)48 island containing a number of conserved promoter elements, including a TATA box. A potential alternative promoter region was found in the conserved -973 to -700 upstream region. No consensus polyadenylation signal was discovered in the 3' UTR. Repeat sequences were found in 15% of all the non coding sequences.As expected for a multifunctional protein, a wide tissue distribution was observed. mRNA expression was found in the adrenal gland, bladder, caecum, colon (centripetal coil/centrifugal coil), diaphragm, duodenum, gallbladder, heart, ileum, jejunum, kidney, liver, longissimus dorsi muscle, lung, lymph node, mesenterium, rectum, spleen, stomach, tongue and ureter, but not in the aorta, oesophagus and pancreas.The promoter region and the exons (including the splice sites) of SLCO2A1 were resequenced in 5 F4ab/ac receptor positive and 5 F4ab/ac receptor negative pigs. Two silent and 2 missense (both S --> L at position 360 and 633) mutations were found, but none was associated with the F4ab/ac receptor phenotype. In addition, no phenotype associated differential mRNA expression or alternative/abberant splicing/polyadenylation was found in the jejunum. CONCLUSION: The molecular cloning and characterization of porcine SLCO2A1 not only contributes to the already existing knowledge about the transporter in general, but enables studies on porcine prostaglandin related processes/deficiencies as patient and/or model. Here we examined its possible involvement as receptor in F4 enterotoxigenic E. coli mediated neonatal diarrhoea. Because no phenotype associated differences could be found in the gene sequence nor in its jejunal transcription profile of F4ab/ac receptor positive/negative pigs, SLCO2A1 can most likely be excluded as receptor for F4 bacteria.

Peripartal feeding strategy with different n-6:n-3 ratios in sows: effect on gene expression in backfat white adipose tissue postpartum.

The aim of this study was to describe the effects of two diets differing in n-6:n-3 ratio and prepartal feeding regime on gene expression of PPARgamma1a/1b, PPARgamma1c/1d, PPARgamma2, PPARgamma coactivator 1A (PPARGC1A), GLUT4, TNFalpha, adiponectin, leptin, leptin receptor (LEPR), fatty acid binding protein 4 (FABP4), lipoprotein lipase (LPL) in sows' white adipose tissue on the first day of lactation. The relationship between mRNA expression of these genes and circulating insulin, leptin and thyroid hormones was also considered. Diets contained a low (supplemented with fish oil; f group) or a high (supplemented with sunflower oil; s group) n-6:n-3 ratio and were provided from 8 (f8, s8) or 3d (f3, s3) before parturition (onset day 8 or 3). A low n-6:n-3 ratio reduced the 1d postpartum expression of PPARgamma2 and PPARGC1A but only when applied from 3 d before parturition. Circulating leptin was negatively correlated with mRNA expression of adiponectin, LEPR and LPL, whereas thyroxine was positively correlated with levels of PPARGC1A. In conclusion, the effect of dietary treatments, e.g. altering the n-6:n-3 ratio, around parturition on the expression of crucial genes in nutrient metabolism can be modulated by the duration of application before parturition.

Elimination of amplification artifacts in real-time reverse transcription PCR using laser capture microdissected samples.

Gene expression analysis on laser capture microdissected samples can be hampered because of the small sample size. The interference of PCR inhibitors increases with smaller sample size. Real-time reverse transcription PCR (RT-PCR) is usually performed directly after the reverse transcription step, enabling PCR inhibitors to remain in the complementary DNA (cDNA). A protocol was optimized for real-time RT-PCR with SYBR Green I. The introduction of an additional cDNA purification step after reverse transcription removed PCR inhibitors, making the reaction more efficient.

Analytical performance of a commercial multiplex Luminex-based cytokine panel in the rat.

INTRODUCTION: Multiplex immunoassays are an important tool in biomarker research during preclinical drug development. However, information regarding analytical performance of commercial multiplex assays for animal species is often limited. To be able to correctly interpret study results, a fit-for-purpose validation approach is recommended. The objective of our study was to provide a realistic example of what level of validation can be expected from this type of assay, using a rat cytokine panel. METHODS: The analytical performance of a commercial Luminex-based multiplex assay comprising IFN-γ, IL-6, IL-10, IL-12p70, IP-10 and TNF-α was evaluated in Sprague-Dawley rat plasma and serum. Calibration curve, working range, precision, accuracy, selectivity, parallelism, dilutional linearity, prozone effect and sample stability were assessed. RESULTS: Analytical performance in plasma and serum was comparable. Precision and accuracy results for all analytes were acceptable with coefficient of variation (CV) and relative error (RE) often below 15%, except for serum IL-6. Selectivity results varied per analyte with several cytokines showing CV>30% and no single minimum required dilution (MRD) could be identified. In addition, some striking differences between recombinant and endogenous protein results were observed. A pronounced prozone effect was detected for IP-10. Analytes in samples stored at -70°C were stable (RE<30%) from 1 up to 6months depending on the analyte. DISCUSSION: The results illustrate the challenges encountered during validation of commercial animal Luminex-based multiplex assays, revealing analytical limitations such as matrix and prozone effects. The Milliplex rat cytokine panel under investigation was deemed suitable for relative quantification of exploratory type biomarkers.

Effect of time and temperature on anticoagulant-dependent pseudothrombocytopenia in Göttingen minipigs.

BACKGROUND: A marked decrease in thrombocyte count was observed between subsequent measurements of the same EDTA blood sample in several minipigs, but little information was available explaining this finding. OBJECTIVES: The objective was to evaluate the impact of several preanalytic variables on thrombocyte counts in minipigs, in order to improve understanding of the in vitro thrombocyte decrease observed. MATERIALS AND METHODS: Hematology blood samples from male and female Göttingen minipigs were collected using EDTA or citrate as an anticoagulant. Samples were stored under different conditions (room temperature, 4°C, or 37-38°C) and were analyzed approximately 0.5 to one h, 3.5-4 h, 7-7.5 h, and 28-29 h after collection. RESULTS: In EDTA blood samples from male minipigs stored at room temperature, there was a progressive thrombocyte decrease over time up to -71% after 29 h, caused by in vitro aggregation. In females, no consistent change was seen up to 7.5 h, but there was a decrease up to -47% after 29 h. Thrombocyte count was most stable during storage at 4°C. No consistent marked decrease in thrombocyte counts was seen for citrated blood at room temperature, although such a decrease was present in a few individual animals. CONCLUSIONS: Study results provide evidence for an anticoagulant-dependent pseudothrombocytopenia in minipigs progressing over time and depending on the storage temperature of the blood sample. It is therefore recommended to perform thrombocyte counts as soon as possible after blood collection, and in case of low counts, investigate for the presence of artifactual platelet clumping.

Young to Middle-Aged Dogs with High Amyloid-ß Levels in Cerebrospinal Fluid are Impaired on Learning in Standard Cognition tests.

Understanding differences in Alzheimer's disease biomarkers before the pathology becomes evident can contribute to an improved understanding of disease pathogenesis and treatment. A decrease in amyloid-ß (Aß)42 in cerebrospinal fluid (CSF) is suggested to be a biomarker for Aß deposition in brain. However, the relevance of CSF Aß levels prior to deposition is not entirely known. Dogs are similar to man with respect to amyloid-ß protein precursor (AßPP)-processing, age-related amyloid plaque deposition, and cognitive dysfunction. In the current study, we evaluated the relation between CSF Aß42 levels and cognitive performance in young to middle-aged dogs (1.5-7 years old). Additionally, CSF sAßPPα and sAßPPß were measured to evaluate AßPP processing, and CSF cytokines were measured to determine the immune status of the brain. We identified two groups of dogs showing consistently low or high CSF Aß42 levels. Based on prior studies, it was assumed that at this age no cerebral amyloid plaques were likely to be present. The cognitive performance was evaluated in standard cognition tests. Low or high Aß concentrations coincided with low or high sAßPPα, sAßPPß, and CXCL-1 levels, respectively. Dogs with high Aß concentrations showed significant learning impairments on delayed non-match to position (DNMP), object discrimination, and reversal learning compared to dogs with low Aß concentrations. Our data support the hypothesis that high levels of CSF Aß in dogs coincide with lower cognitive performance prior to amyloid deposition. Further experiments are needed to investigate this link, as well as the relevance with respect to Alzheimer's disease pathology progression.

Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A.

BACKGROUND: An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition, peroxisome proliferative activated receptor gamma coactivator 1alpha (PPARGC1A) is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types. RESULTS: The mRNA expression stability of 10 reference genes was determined. The expression of RPL13A and SDHA appeared to be highly unstable. After normalization to the geometric mean of the three most stably expressed reference genes (ACTB, TBP and TOP2B), the results not only showed that the mRNA expression of PPARGC1A was significantly higher in each of the longissimus dorsi muscle samples than in backfat (P < 0.05), but also that the expression was significantly higher in the most cranial of the three muscle samples (P < 0.05). CONCLUSION: This study provides a new set of reference genes (ACTB, TBP and TOP2B) suitable for normalization of real-time RT-PCR data of backfat and longissimus dorsi muscle in the pig. The obtained PPARGC1A expression results, after application of this set of reference genes, are a first step in unravelling the PPARGC1A expression pattern in the pig and provide a basis for possible selection towards improved meat quality while maintaining a lean carcass.

Expression and localization of angiogenic growth factors in developing porcine mesonephric glomeruli.

The development and growth of renal glomeruli is regulated by specific angiogenic growth factors, including vascular endothelial growth factor (VEGF) and the angiopoietins (ANGPT1 and ANGPT2). The expression of these factors has already been studied during metanephric glomerulogenesis, but it remains to be elucidated during the development of the embryonic mesonephros, which can function as an interesting model for glomerular development and senescence. In this study, the presence of the angiogenic growth factors was studied in developing porcine mesonephroi, using IHC and real-time RT-qPCR on laser capture microdissected glomeruli. In addition, mesonephric glomerular growth was measured by using stereological methods. ANGPT2 remained upregulated during maturation of glomeruli, which may be explained by the continuous growth of the glomeruli, as observed by stereological examination. The mRNA for VEGFA was expressed in early developing and in maturing glomeruli. The VEGF receptor VEGFR1 was stably expressed during the whole lifespan of mesonephric glomeruli, whereas VEGFR2 mRNA was only upregulated in early glomerulogenesis, suggesting that VEGFR2 is important for the vascular growth but that VEGFR1 is important for the maintenance of endothelial fenestrations.

Positive correlation between relative mRNA expression of PRNP and SPRN in cerebral and cerebellar cortex of sheep.

SPRN is an interesting new member of the PRNP family because of its sequence homology with the hydrophobic region of PRNP, its expression in brain tissue and its PrP-like properties in functional experiments on Prnp(0/0) mice. In this study, we investigated by quantitative real-time PCR the relative mRNA expression levels of SPRN and PRNP in sheep cerebrum and cerebellum and the mutual relationship between these expression levels. Analysis of PRNP and SPRN mRNA expression levels in 45 cerebral cortex and 47 cerebellar cortex samples showed that the PRNP expression level was significantly higher (p<0.05) in cerebellum than in cerebrum, while no significant difference was detected for SPRN between these tissues. The expression level varied clearly more in cerebrum than in cerebellum for both genes tested, and the variation was larger for SPRN than for PRNP in both types of brain tissue. Remarkably, the mRNA expression levels of PRNP and SPRN showed a highly significant positive correlation in both cerebrum (p<0.0001) and cerebellum (p<0.001). This positive correlation might indicate co-regulation between these genes. Further investigation on the causal nature of this correlation may provide new insights into prion pathogenesis.

Two new splice variants in porcine PPARGC1A.

BACKGROUND: Peroxisome proliferator-activated receptor gamma coactivator 1alpha (PPARGC1A) is a coactivator with a vital and central role in fat and energy metabolism. It is considered to be a candidate gene for meat quality in pigs and is involved in the development of obesity and diabetes in humans. How its many functions are regulated, is however still largely unclear. Therefore a transcription profile of PPARGC1A in 32 tissues and 4 embryonic developmental stages in the pig was constructed by screening its cDNA for possible splice variants with exon-spanning primers. FINDINGS: This led to the discovery of 2 new splice variants in the pig, which were subsequently also detected in human tissues. In these variants, exon 8 was either completely or partly (the last 66 bp were conserved) spliced out, potentially coding for a much shorter protein of respectively 337 and 359 amino acids (aa), of which the first 291 aa would be the same compared to the complete protein (796 aa). CONCLUSION: Considering the functional domains of the PPARGC1A protein, it is very likely these splice variants considerably affect the function of the protein and alternative splicing could be one of the mechanisms by which the diverse functions of PPARGC1A are regulated.