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Digital toxicologic histopathology has been broadly adopted in preclinical compound development for informal consultation and peer review. There is now increased interest in implementing the technology for good laboratory practice–regulated study evaluations. However, the implementation is not straightforward because systems and work processes require qualification and validation, with consideration also given to security. As a result of the high-throughput, high-volume nature of safety evaluations, computer performance, ergonomics, efficiency, and integration with laboratory information management systems are further key considerations. The European Society of Toxicologic Pathology organized an international expert workshop with participation by toxicologic pathologists, quality assurance/regulatory experts, and information technology experts to discuss qualification and validation of digital histopathology systems in a good laboratory practice environment, and to share the resulting conclusions broadly in the toxicologic pathology community.

Supplementary Table 1 from Alpha- versus Beta-Particle Radiopeptide Therapy in a Human Prostate Cancer Model (213Bi-DOTA-PESIN and 213Bi-AMBA versus177Lu-DOTA-PESIN)

Supplementary Table 3 from Alpha- versus Beta-Particle Radiopeptide Therapy in a Human Prostate Cancer Model (213Bi-DOTA-PESIN and 213Bi-AMBA versus177Lu-DOTA-PESIN)

Supplementary Figures 1-4 from Alpha- versus Beta-Particle Radiopeptide Therapy in a Human Prostate Cancer Model (213Bi-DOTA-PESIN and 213Bi-AMBA versus177Lu-DOTA-PESIN)

Supplementary Table 2 from Alpha- versus Beta-Particle Radiopeptide Therapy in a Human Prostate Cancer Model (213Bi-DOTA-PESIN and 213Bi-AMBA versus177Lu-DOTA-PESIN)

We introduce HistoNet, a deep neural network trained on normal tissue. On 1690 slides with rat tissue samples from 6 preclinical toxicology studies, tissue regions were outlined and annotated by pathologists into 46 different tissue classes. From these annotated regions, we sampled small 224 × 224 pixels images (patches) at 6 different levels of magnification. Using 4 studies as training set and 2 studies as test set, we trained VGG-16, ResNet-50, and Inception-v3 networks separately at each magnification level. Among these model architectures, Inception-v3 and ResNet-50 outperformed VGG-16. Inception-v3 identified the tissue from query images, with an accuracy up to 83.4%. Most misclassifications occurred between histologically similar tissues. Investigation of the features learned by the model (embedding layer) using Uniform Manifold Approximation and Projection revealed not only coherent clusters associated with the individual tissues but also subclusters corresponding to histologically meaningful structures that had not been annotated or trained for. This suggests that the histological representation learned by HistoNet could be useful as the basis of other machine learning algorithms and data mining. Finally, we found that models trained on rat tissues can be used on non-human primate and minipig tissues with minimal retraining.

SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.

Deep learning models have been applied on various tissues in order to recognize malignancies. However, these models focus on relatively narrow tissue context or well-defined pathologies. Here, instead of focusing on pathologies, we introduce models characterizing the diversity of normal tissues. We obtained 1,690 slides with rat tissue samples from the control groups of six preclinical toxicology studies, on which tissue regions were outlined and annotated by pathologists into 46 different tissue classes. From these annotated regions, we sampled small patches of 224 × 224 pixels at six different levels of magnification. Using four studies as training set and two studies as test set, we trained VGG-16, ResNet-50, and Inception-v3 networks separately at each of these magnification levels. Among these models, Inception-v3 consistently outperformed the other networks and attained accuracies up to 83.4% (top-3 accuracy: 96.3%). Further analysis showed that most tissue confusions occurred within clusters of histologically similar tissues. Investigation of the embedding layer using the UMAP method revealed not only pronounced clusters corresponding to the individual tissues, but also subclusters corresponding to histologically meaningful structures that had neither been annotated nor trained for. This suggests that the histological representation learned by the normal histology network could also be used to flag abnormal tissue as outliers in the embedding space without a need to explicitly train for specific types of abnormalities. Finally, we found that models trained on rat tissues can be used on non-human primate and minipig tissues with minimal retraining.Author contributionT.S. and H.H. contributed equally to this work.Significance statementLike many other scientific disciplines, histopathology has been profoundly impacted by recent advances in machine learning with deep neural networks. In this field, most deep learning models reported in the literature are trained on pathologies in specific tissues/contexts. Here, we aim to establish a model of normal tissues as a foundation for future models of histopathology. We build models that are specific to histopathology images and we show that their embeddings are better feature vectors for describing the underlying images than those of off-the shelf CNN models. Therefore, our models could be useful for transfer learning to improve the accuracy of other histopathology models.

Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B virus, and, since the FDA approval of sofosbuvir in 2013, also for hepatitis C virus (HCV). However, many promising nucleotide inhibitors have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of compound 1, a monophosphate prodrug of a 2′-ethynyluridine developed for the treatment of HCV. Compound 1 inhibits multiple HCV genotypes in vitro (50% effective concentration [EC 50 ], 0.05 to 0.1 μM) with a selectivity index of >300 (50% cytotoxic concentration [CC 50 ], 30 μM in MT-4 cells). The active triphosphate metabolite of compound 1, compound 2, does not inhibit human α, β, or γ DNA polymerases but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of compound 1 resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of compound 1 as an anti-HCV compound was terminated.

Peritraumatic dissociation and distress are strong predictors of acute stress disorder (ASD) and posttraumatic stress disorder (PTSD) development. However, there is limited data concerning gender differences in these relations, particularly among victims of violent crimes (VVC). The objective of this study is to examine whether peritraumatic dissociation and distress predict the number of ASD symptoms differently for men and women VVC. In all, 162 adults (97 women, M age = 39.6 years), 63% of whom experienced physical assaults, completed the Acute Stress Disorder Interview, the Peritraumatic Dissociative Experience Questionnaire, and the Peritraumatic Distress Inventory. Analyses included t tests and multiple hierarchical regressions models controlling for known PTSD risk factors. The regression model showed dissociation and distress to be significant predictors of ASD for both men and women (β = .349 and β =.312 respectively; all p < .001). A significant three-way interaction was also observed between peritraumatic distress (PDI), past potentially traumatic experiences, and gender. In simple slopes analyses, the combination of high levels of PDI and of a high number of past potentially traumatic events were associated with greater risk of ASD in men only ( b = 3.78, p < .001). However, women experienced greater PDI, t(157) = 5.844, p = .005, than men, and elevated distress was associated with more ASD symptoms independently of past traumatic events. Gender differences were revealed as a function of past potentially traumatic experiences. There is a cumulative impact of past potential traumas and current distress that predicts ASD in men, while in women, it contributes to ASD via increased distress.

The somatostatin analog pasireotide and the 11β-hydroxylase inhibitor osilodrostat (LCI699) reduce cortisol levels by distinct mechanisms of action. There exists a scientific rationale to investigate the clinical efficacy of these two agents in combination. This manuscript reports the results of a toxicology study in rats, evaluating different doses of osilodrostat and pasireotide alone and in combination. Sixty male and 60 female rats were randomized into single-sex groups to receive daily doses of pasireotide (0.3mg/kg/day, subcutaneously), osilodrostat (20mg/kg/day, orally), osilodrostat/pasireotide in combination (low dose, 1.5/0.03mg/kg/day; mid-dose, 5/0.1mg/kg/day; or high dose, 20/0.3mg/kg/day), or vehicle for 13weeks. Mean body-weight gains from baseline to Week 13 were significantly lower in the pasireotide-alone and combined-treatment groups compared to controls, and were significantly higher in female rats receiving osilodrostat monotherapy. Osilodrostat and pasireotide monotherapies were associated with significant changes in the histology and mean weights of the pituitary and adrenal glands, liver, and ovary/oviduct. Osilodrostat alone was associated with adrenocortical hypertrophy and hepatocellular hypertrophy. In combination, osilodrostat/pasireotide did not exacerbate any target organ changes and ameliorated the liver and adrenal gland changes observed with monotherapy. Cmax and AUC0–24h of osilodrostat and pasireotide increased in an approximately dose-proportional manner.In conclusion, the pasireotide and osilodrostat combination did not exacerbate changes in target organ weight or toxicity compared with either monotherapy, and had an acceptable safety profile; addition of pasireotide to the osilodrostat regimen may attenuate potential adrenal gland hyperactivation and hepatocellular hypertrophy, which are potential side effects of osilodrostat monotherapy.

Contradictory evidence exists regarding the role of peritraumatic distress (PDI) and dissociation (PD) in the development of PTSD. Victims of certain trauma types may be more susceptible to these peritraumatic reactions. Few studies have assessed gender differences in these associations, particularly among victims of violent crimes. This prospective study evaluated the main and interactive effects of peritraumatic reactions with gender on PTSD onset, independently of known risk factors. Methods. 111 adult victims of violent crime (68 women, Mage = 41.23) completed the Structured Clinical Interview, the Modified PTSD symptom Scale, the Peritraumatic Dissociative Experience Questionnaire, and the Peritraumatic Distress Inventory. Hierarchical regressions and chi-square tests were conducted for continuous and categorical measures of PTSD. Results. Extremely elevated scores of PD and PDI were obtained for both sexes. Greater PDI and PD were related to more PTSD symptoms (b =0. 380, p =. 001; b =0. 217, p =. 025, respectively). No significant interaction effects with gender emerged. These results remained when acute stress disorder was controlled for. Analyses on categorical data produced similar results. Conclusion. Victims of violent crimes recall severe peritraumatic reactions. The more severe these are, the more likely they are to develop significant PTSD symptomatology in the months that follow.

Chitosan (CS)-based thermosensitive solutions that turn into semi-solid hydrogels upon injection at body temperature have increasingly drawn attention over the last decades as an attractive new type of in situ forming depot (ISFD) drug delivery system. Despite the great potential of the standard CS/β-glycerophosphate (β-GP) thermogelling solutions, their lack of stability over time at room temperature as well as at refrigerated conditions renders them unsuitable as ready-to-use drug product. In the present study, we investigated Glucose-1-Phosphate (G1-P) as an alternative gelling agent for improving the stability of CS-based ISFD solutions. The in vitro release performance of CS/G1-P formulations was assessed using several model compounds. Furthermore, the local tolerance of subcutaneously implanted CS/G1-P hydrogels was investigated by histological examination over three weeks. The thermogelling potential of CS/G1-P solutions, determined by rheology, is dependent on the polymer molecular weight (Mw) and concentration as well as on the G1-P concentration. Differential scanning calorimetry (DSC) measurements confirmed that sol/gel transition takes place at around body temperature and is not fully thermo-reversible. The long term storage stability was evaluated through the appearance, pH, viscosity and gelation time at 37°C of the solution. The results emphasized an enhanced stability of the CS/G1-P system compared to the standard CS/β-GP. CS solution with 0.40 mmol/g G1-P is stable for at least 9 months at 2-8°C, versus less than 1 month when using β-GP as gelling agent. Furthermore, the solution is easy to inject, as evidenced from injectability evaluation using 23-30 G needles. In vitro release experiments showed a sustained release over days to weeks for hydrophilic model compounds, demonstrating thereby that CS/G1-P may be suitable for the prolonged delivery of drugs. The inflammatory reaction observed in the tissue surrounding the hydrogel in rats was a typical foreign body reaction, similar to the one observed for CS/β-GP hydrogels. These features confirm the potential of CS/G1-P solutions as an injectable ready-to-use in situ forming hydrogel.

Hostility may be associated with greater systemic inflammation. However, contradictory evidence exists. Certain individuals or dimensions of hostility may be more susceptible to these effects. Main and interactive effects of hostility with sex and/or age were evaluated on markers of inflammation, independently of traditional risk factors for coronary artery disease.199 healthy men (81) and women (118), aged 20-64 years (M=41 ± 11 years) were recruited. Hostility was assessed using the Cook-Medley Hostility Inventory (CMHo) and ecological momentary assessments (EMA) of quarrelsome behavior and angry affect in daily living. Blood samples were drawn to measure inflammatory activity (Il-6, TNF-α, hsCRP, Il-8, Il-10, Il-18, MCP-1) and lipid oxidation (Myeloperoxidase; MPO). Correlations and hierarchical regression analyses were performed controlling for pertinent behavioral, psychological, medical, and socio-demographic factors.Significant univariate associations emerged between CMHo and Il-6, TNF-α, MCP-1 (p.05). Hierarchical regressions showed interactions of hostility with sex (Il-6, TNF-α; p.05) and age (hsCRP, Il-6, TNF-α; p.05). For example, in simple slope analyses, hostility was positively related to TNF-α in women (b=0.009, p=0.006) but not men. Greater hostility was also related to greater Il-6 levels among younger women (b=. 027, p=0.000).Hostility, particularly cynical hostility, may be detrimental to (younger) women. The TNF-α, Il-6, CRP triad appears vulnerable to psychological and behavioral factors, and may be one mechanism by which cynical hostility (CMHo) contributes to increased cardiovascular risk in women. Prospective research is needed to verify this.

Hostility has been associated with higher basal levels of inflammation. The present study evaluated the association of hostility with acute stress-induced changes in inflammatory activity. One hundred and ninety-nine healthy men and women, aged 19-64 years, were exposed to a stress protocol involving four interpersonal stressors. Participants completed the Cook-Medley Hostility questionnaire and provided two blood samples for the measurement of inflammatory biomarkers (CRP, Il-6, MPO, TNF-α, MCP-1, Il-8, Il-10, and Il-18), prior to and following exposure to a standardized stress protocol. In univariate analyses, hostility was associated with significantly higher TNF-α, but lower Il-8 and Il-18 values post-stress, though only Il-8 remained significant after controlling for baseline differences. In multivariate analyses, a significant Age by Hostility interaction emerged for Il-6, while sex moderated the relation between hostility and Il-10 reactivity. Following stress, hostility was associated with greater pro-inflammatory Il-6 activity among younger individuals and to decreased anti-inflammatory Il-10 activity in women. Future research is needed to replicate these findings and to evaluate their implication for disease.

Recurrent prostate cancer presents a challenge to conventional treatment, particularly so to address micrometastatic and small-volume disease. Use of α-radionuclide therapy is considered as a highly effective treatment in such applications due to the shorter range and exquisite cytotoxicity of α-particles as compared with β-particles. 213Bi is considered an α-emitter with high clinical potential, due to its short half-life (45.6 minutes) being well matched for use in peptide-receptor radionuclide α-therapy; however, there is limited knowledge available within this context of use. In this study, two novel 213Bi-labeled peptides, DOTA-PEG4-bombesin (DOTA-PESIN) and DO3A-CH2CO-8-aminooctanoyl-Q-W-A-V-G-H-L-M-NH2 (AMBA), were compared with 177Lu (β-emitter)-labeled DOTA-PESIN in a human androgen-independent prostate carcinoma xenograft model (PC-3 tumor). Animals were injected with 177Lu-DOTA-PESIN, 213Bi-DOTA-PESIN, or 213Bi-AMBA to determine the maximum tolerated dose (MTD), biodistribution, and dosimetry of each agent; controls were left untreated or were given nonradioactive 175Lu-DOTA-PESIN. The MTD of 213Bi-DOTA-PESIN and 213Bi-AMBA was 25 MBq (0.68 mCi) whereas 177Lu-DOTA-PESIN showed an MTD of 112 MBq (3 mCi). At these dose levels, 213Bi-DOTA-PESIN and 213Bi-AMBA were significantly more effective than 177Lu-DOTA-PESIN. At the same time, 177Lu-DOTA-PESIN showed minimal, 213Bi-DOTA-PESIN slight, and 213Bi-AMBA marked kidney damage 20 to 30 weeks posttreatment. These preclinical data indicate that α-therapy with 213Bi-DOTA-PESIN or 213Bi-AMBA is more efficacious than β-therapy. Furthermore, 213Bi-DOTA-PESIN has a better safety profile than 213Bi-AMBA, and represents a possible new approach for use in peptide-receptor radionuclide α-therapy treating recurrent prostate cancer. Cancer Res; 71(3); 1009–18. ©2011 AACR.

Poly(ethyleneglycol) 500 dimethylether (PEG500DME) was tested as a novel solvent for the manufacture of an injectable in situ forming depot (ISFD) containing poly(D,L-lactide-co-glycolide) (PLGA). The sustained release of pasireotide from the ISFD was evaluated in vitro and in vivo. Furthermore, the local tolerability of the delivery system using PEG500DME was investigated in subcutaneous (s.c.) tissue over 48 days. A flow-through cell was used to determine the in vitro drug release from the ISFD in comparison to a peptide suspension without polymer. The biocompatibility as well as the pharmacokinetic profile of the ISFD was investigated in rabbits. A prolonged peptide release over at least 48 days with an initial burst lower than 1% was observed in vitro for the ISFD compared to the suspension without polymer. A similar tissue response as it was observed for other common PLGA delivery systems was found upon histopathological examination of tissue from the administration site in rabbits. A sustained release of at least 48 days in vivo confirmed the in vitro observation including the low initial plasma concentration levels. Two ISFDs with different peptide loads were used to correlate the in vitro and in vivo data (IVIVC). Overall, the functionality of the ISFD containing PEG500DME as a novel solvent was demonstrated in vitro and in vivo. In addition, the local tolerability of the system confirmed the biocompatibility of PEG500DME in parenteral depots.

Signal changes within the bone marrow adjacent to osteoarthritic joints are commonly seen on magnetic resonance (MR) images in humans and in dogs. The histological nature of these lesions is poorly known. In this study, we describe the MR imaging of bone marrow lesions adjacent to the stifle joints of dogs with experimental osteoarthritis over 13 months. Histology of the proximal tibia at the end of the study was compared with the last MR imaging findings. In five adult dogs, the left cranial cruciate ligament was transected. Post-operatively, MR imaging was performed at 1, 2, 3, 4, 6, 8, and 13 months. Dogs were euthanised after 13 months and histological specimen of the proximal tibia were evaluated. Bone marrow edema like MR imaging signal changes were seen in every MR examination of all dogs in one or more locations of the proximal tibia and the distal femur. Lesions varied in size and location throughout the whole study with the exception of constantly seen lesions in the epiphyseal and metaphyseal region at the level of the tibial eminence. On histology, hematopoiesis and myxomatous transformation of the bone marrow and/or intertrabecular fibrosis without signs of bone marrow edema were consistent findings in the areas corresponding to the MR imaging signal changes. We conclude that within the bone marrow, zones of increased signal intensity on fat suppressed MR images do not necessarily represent edema but can be due to cellular infiltration. Contrary to humans, hematopoiesis is seen in bone marrow edema-like lesions in this canine model of osteoarthritis.

Little is known about the magnetic resonance imaging (MRI) appearance of canine meniscal lesions. The aim of this study is to describe the MR appearance of meniscal lesions in dogs with experimentally induced cranial cruciate ligament (CCL) deficiency. The pilot study revealed dogs weighing approximately 10 kg to be too small for meniscal evaluation on low-field MRI. In the main study, dogs weighing approximately 35 kg were used. The left CCL was transected and low-field MRI was performed regularly until 13 months post-surgery. Normal menisci were defined as grade 0. Intrameniscal lesions not reaching any surface corresponded to grade 1 if focal and to grade 2 if linear or diffuse. Grade 3 lesions consisted in linear tears penetrating a meniscal surface. Grade 4 lesions included complex signal changes or meniscal distortion. Between 2 and 13 months post-surgery, all dogs developed grade 4 lesions in the medial meniscus. Most of them corresponded to longitudinal or bucket handle tears on arthroscopy and necropsy. Two dogs showed grade 3 lesions reaching the tibial surface of the lateral meniscus on MRI but not in arthroscopy. Such tears are difficult to evaluate arthroscopically; MRI provides more accurate information about the tibial meniscal surface. Grades 1 and 2 lesions could not be differentiated from presumably normal menisci with our imaging technique. An MRI grading system better adapted to canine lesions has yet to be developed. MRI is a helpful tool for the diagnosis of complete tears in the canine meniscus, especially in larger dogs.

Conventional approaches to produce transgenic mice recurrently yield unpredictable patterns and levels of transgene expression, a situation calling for the development of new techniques to overcome these drawbacks in the context of overexpression studies. Here we present an efficient method for rapid and reproducible transgenesis using the recombinase mediated cassette exchange (RMCE) (Bouhassira et al.: Blood 90:3332-3344, 1997) procedure. A lox511-EGFP-TK/neo-loxP cassette was placed under the control of the endogenous mouse beta-actin promoter. Heterozygous mice revealed strong and ubiquitous EGFP expression throughout embryogenesis and adulthood. Reproducibly, the same expression pattern was obtained with RMCE when it was used to replace the EGFP-harboring cassette by ECFP or placental alkaline phosphatase (PLAP) reporter genes (DePrimo et al.: Transgenic Res 5:459-466, 1996). Furthermore, the RMCE procedure proved efficient as well in embryonic stem (ES) cells as directly in zygotes. Our results demonstrate ubiquitous expression of floxed transgenes in the endogenous beta-actin locus and they support the general use of the beta-actin locus for targeted transgenesis.

We determined the prevalence of lung and mammary gland lesions associated with maedi-visna (MV) infection, the prevalence of paratuberculosis (PTB), and the prevalence and lesions distribution of caseous lymphadenitis (CL) in culled sheep. Total of 451 ewes and 34 rams were selected randomly from two slaughterhouses in Quebec, Canada. MV serostatus was determined by recombinant ELISA test. PTB diagnosis was based on characteristic histological lesions in the terminal ileum, ileocecal lymph node and/or ileocecal valve and CL by gross detection of abscesses and isolation of Corynebacterium pseudotuberculosis. Seroprevalence of MV was 44% (95% CI: 40, 48). Seropositivity increased with age and was higher in ewes than in rams. The percentages of lung and mammary gland lesions in seropositive sheep were 14 and 40%, respectively, but mammary gland lesions lack specificity. The prevalence of PTB was 3% (95% CI: 2, 5). PTB increased with age and was lower among sheep with abscesses. The prevalence of CL was >/=21% (95% CI: 17, 24). The most-prevalent site of caseous lymphadenitis lesions was the thoracic cavity. The risk of carcass condemnation was significantly associated with region, body score and abscesses. Only the presence of abscesses was associated with an increase in trimming of carcasses.