P01: Selectins in rheumatoid arthritis: from immunohistochemistry to optical imaging in the mouse Collagen-Induced Arthritis (CIA) model

Charline Alleaume1, Patricia Emmel1, Lucille Vallé1, Philippe Bussat1, Sylvie Henrioud1, David Lazarus1, Samir Cherkaoui1, Thierry Bettinger1, Catherine Botteron1 and Isabelle Tardy1

1Bracco Suisse SA, Switzerland

Rheumatoid arthritis (RA) is a chronic inflammatory disease primarily characterized by a non suppurative and proliferative synovitis. Its diagnosis relies mostly on late clinical, serological and radiological features. Optical imaging (OI) is an emerging imaging modality, adapted to investigate superficial structures such as small joints, particularly affected by the condition. Advanced fluorescent targeted OI agents can improve the sensibility and specificity of detection. The aim of this study was to evaluate the relevance of targeting P&E-selectins, vascular inflammation markers, by optical molecular imaging for the diagnosis and/or therapeutic follow-up of RA.

The collagen-induced arthritis (CIA) model in mouse was chosen as a model of rheumatoid arthritis. After intra-dermal immunization, male DBA/1JRj mice received intra-peritoneal collagen type II to trigger CIA. The clinical evolution of arthritis was scored on the distal appendicular joints (paws) and the animals were followed by optical imaging before being sacrificed. The femoro-tibial and distal appendicular joints were collected and processed for histopathological evaluation (HE stain). Histopathological changes were evaluated qualitatively and the severity of inflammation was graded with a 6-tiered score after visual examination. The expression of P&E-selectins was assessed by immunohistochemistry and the quantity of positive vessels was graded with a 4-tiered score after visual examination. 

Seventy-five mice (75) with collagen-induced arthritis (CIA) versus 11 control mice were analyzed. The lesions as well as distribution of P&E-selectins in the joint were compared with those in 4 joint biopsies from RA patients. In mouse, OI was performed on affected and control joints, after administration of the molecular OI agent, a selectin-ligand conjugated to a liposome-bound fluorophore (DIR). Bound selectin-targeting OI agent was revealed in vivo via fluorescence detection by an infrared camera.

Clinical arthritis was evaluated from 25 up to 50 days after induction and it was bilateral in the CIA model, involving at least one of the distal joint. It consisted mostly in a severe chronic active arthritis with prominent suppuration. Human biopsies revealed a chronic, mostly lymphoplasmacytic synovitis. Unfortunately, it was not possible to obtain joint biopsies with early RA.

Increased vascular P&E-selectins expression in arthritis was confirmed by immunohistochemistry in the CIA mouse model of RA. P&E-selectins were also abundant in human biopsies of chronic RA. 

In mouse, selectin-targeting OI succeeded in enhancing signal in arthritic joint compared to control: the fluorescence in the affected joint was increased with the severity of the inflammation, as well as P&E-selectin expression.

Targeting selectins by optical molecular imaging might be a promising modality for the evaluation of RA. However, to be relevant as an early diagnostic tool, selectin-targeting OI detection should be demonstrated in a different animal model featuring early stages of RA.

P02: Vascular and tissue changes in frequency and magnetic susceptibility in the mouse brain after transient cerebral ischemia

Markus Vaas1, Andres Deistung2, Jürgen R. Reichenbach2, Annika Keller3, Anja Kipar4, Jan Klohs1

1Institute for Biomedical Engineering, ETH & University of Zurich, 8093 Zurich, Switzerland
2Institute of Diagnostic and Interventional Radiology, Friedrich Schiller University Jena, 07743 Jena, Germany
3Division of Neurosurgery, University Hospital Zurich, 8091 Zurich, Switzerland
4Laboratory for Animal Model Pathology, Institute of Veterinary Pathology, University of Zurich, 8057 Zurich Switzerland

Background: Multiple contrasts of magnetic resonance imaging (MRI) are used for diagnosis and treatment stratification of patients suffering from ischaemic stroke. Cerebral microbleeds and haemorrhages are detected by T2*-weighted gradient recalled echo (T2*-GRE), while T2*-GRE data post-processing provide additional contrast information, like phase imaging and quantitative susceptibility mapping (QSM). Here we used these contrasts to assess ischaemic brain injury and validated our findings with histology and immunohistology.

Material and Methods: Experimental focal cerebral ischaemia was induced by 1h of transient middle cerebral artery occlusion using the intraluminal filament model in mice. MRI data were acquired at various time points up to 48h after reperfusion on a 4.7T magnetic resonance scanner. Brains were harvested after 12, 24 and 48h after reperfusion, routinely processed for histology (HE stain) subjected to immunohistology for the demonstration of neutrophils (Ly6G+), microglia (Iba-1+), or vessels (podocalyxin and collagen IV+). 

Results: Phase images and susceptibility maps revealed prominent vessels within the ischaemic hemisphere from 2 to 48h, while vessels appeared more frequently after 12h. Increasing reperfusion time correlated with increased cortical and striatal T2 values and the difference in magnetic susceptibility values, while apparent diffusion coefficient values decreased with time. Histological findings comprised swelling of endothelial cells in micro vessels, capillary closure by neutrophils at around 12h as well as hyperaemia with dilated larger vessels, explaining the contrast of appearing vessels in the MRI data. 

In addition frequency and quantitative susceptibility maps of repetitive measurements showed growing regions with decreased intensity values in the ischaemic territory. Also these regions were at all times smaller than standardly used apparent diffusion coefficient and T2 maps. However, the lesion as it was evident by histology and immunohistology, with necrotic neurons, hyperaemia, oedema and microglial activation in the periphery, did not reflect the observed regions of interest.

Conclusion: Frequency and magnetic susceptibility maps allow visualization of pathological vascular and tissue changes in the ischaemic territory and provides complementary information to evaluate haemodynamic changes, which can be partly underpinned by histopathological findings.

P03: Digital morphometry quantification of dopamine activity in a 6-hydroxydopamine (6-OHDA) Parkinson model in mice

Loeb E.¹, Zuckerman A.1, Margalit R.2, Orgad U.3

¹Patho-Logica, Golda Meir Str 7, Scientific Park, Ness-Ziona, Israel
²Science In Action, Sapir Str. 5, Scientific park, Ness-Ziona, Israel 
³Hebrew University, Koret School for veterinary medicine, Rehovot, Israel

Introduction: Parkinson's disease is a progressive neurodegenerative disorder characterized by degeneration of the dopaminergic neurons of the nigrostriatal pathway. 

Objective: To quantify the amount of damaged dopamine producing cells and fibers by using digital morphometry and to compare the results to those obtained using a traditional semi-quantitative approach.

Materials and Methods: Five mice were injected locally, into the right brain hemisphere, with the toxin (6-OHDA) and the left hemisphere served as control. Intra-striatal injections was done at the following coordinates (in mm relative to bregma, sagittal suture and dural surface): injections of 3 μl at (i) AP=+1.0, L=−2.1, DV=−2.9; Animals were sacrificed 8 weeks later. Brains were dissected to obtain sections from the Substantia Nigra Pars Compacta (SNC) and the striatum (ST). Five mµ thick paraffin sections were cut and stained for tyrosine hydroxylase (TH) activity, a marker for dopamine production using immunofluorescence histochemistry. The intensity of the reaction in SNC and ST was measured by computerized digital morphometry in which the relative percentage of fluorescence was calculated, compared to a semi-quantitative analysis (scale score 0-3). 

Results: The semi-quantitative score method showed a decrease in the activity of TH in the SNC and ST in the right hemisphere compared to the left one hemisphere in four animals. The quantitative digital morphometry score showed also decrease in TH expression of the SNC and the ST. However, here an accurate number for fluorescence intensity and cellular count was also provided. 

Conclusions: TH expression was measured, using a digital morphometric tool, compared to a semi-quantitative method. The digital quantification is highly accurate compared to the traditional semi-quantitative method and therefore is recommended for analysis of histological markers in studies.

P04: Retinal Findings in a Chronic Monkey Toxicity Study monitored by Optical Coherence Tomography (OCT) and Electroretinogram (ERG)

Bjoern Jacobsen1, Nora Denk1, Stefan Kustermann1, Annika Herrmann1, Agnes Poirier1 and Lutz Mueller1

1F. Hoffmann-La Roche Ltd., Roche Innovation Center Basel, Switzerland

Retinal degeneration was observed within a chronic toxicity study (39 weeks) in Cynomolgus monkeys after oral administration of a heterocyclic compound derived from a pyrido-pyrimidinone. During in life phase, multifocal peripheral retina degeneration in the photoreceptor layer and microcystic spaces in the inner retinal layers were detected by optical coherence tomography (OCT) associated with a depressed B-wave in the electroretinogram (ERG). Correlating with OCT, histopathologic findings were mainly confined to the outer nuclear layer (ONL) and photoreceptor layer. In addition, vacuolation of the inner nuclear layer (INL) and retinal pigment epithelium (RPE) hypertrophy were present. Incidence and severity were dose dependent and findings were mostly located in the periphery. In a 22-week recovery phase, retinal degeneration with peripheral photoreceptor loss and RPE hypertrophy remained. In OCT, however, microcystoid spaces decreased significantly. ERG amplitudes noticeably increased by end of the recovery phase showing normal ERGs in 3/4 recovery animals. OCT findings were confirmed histopathologically. Vacuolation of the INL had reversed with the latter being likely the reason for recovery from depressed ERG responses. Follow up investigations in vitro give reasons to assume that the effect might be due to high melanin binding and tissue retention of the compound in the retina and an impaired lysosomal /autophagosomal function in RPE cells. However, no morphological correlate was present in histology and electron microscopy. In conclusion, the excellent correlation between OCT and histopathology allows monitoring of retinal findings during in life phase of toxicity studies. In addition, ERG provides a valuable tool for a functional in vivo monitoring.

P05: Preclinical histopathologic evaluation of ocular medical devices

Elodie Drevon-Gaillot1

1NAMSA, Chasse sur Rhône, France

The complex architecture of the eye is responsible for some of the difficulties in histologic processing of the ocular globe, all the more when eyes are implanted with medical devices (MD). The choice of the histologic technique is outstanding since inadapted fixation or trimming method can preclude histopathologic evaluation of the safety and performance of MD.

This presentation will focus on the histologic process and histopathologic evaluation of the eye in the preclinical testing of MD. After a presentation of the testing guidelines, principles of histologic preparation will be summarized, then illustrated by examples of ocular MD.

A. Guidelines, animal models and study design 

B. Histologic preparation and techniques

- fixation, trimming, embedding, staining

C. Histopathologic evaluation of ocular MD based on examples:

    1. Topical corneal administration: 

"Ocular irritation study in the rabbit following acute or repeated exposure according to ISO 10993 standard"

    2. Intra-ocular injection: 

"Evaluation of an Oil Tamponade Agent in a Rabbit Vitrectomy Model"

    3. Intra-ocular implantation: 

- Intraocular lenses (IOL): "Biocompatibility of an IOL after 6- or 12-month Intraocular Implantation in the Rabbit". Evaluation through paraffin histology on implanted eyes and surface analysis (Scanning Electron Microscopy/Energy-dispersive X-ray spectroscopy) on removed lenses.

- Glaucoma drainage device: "Evaluation of the local tissue effects of a glaucoma implant following Chronic Implantation in the subscleral space of the Rabbit Eye." Paraffin histology.

- Bioretinal prosthesis: “Evaluation of the local tissue effects following chronic implantation in the pig eye". Plastic embedding of the posterior eyecup implanted with the metallic prosthesis. 

P06: Retinal Degeneration/Atrophy in P301S Mice: Histopathologic and Genetic Characterization

Karen Bodié1, Axel H. Meyer1

1Abbvie Deutschland GmbH & Co.KG

Introduction: P301S mice are frequently used as a model for Alzheimer’s Disease. Retinal degeneration/atrophy can be observed at variable frequency in P301S mice of different age or sex as well as in their wild-type littermates. The breeding background of P301S mice includes the C3H mouse strain which carries the Pde6brd1 mutation (rd1 mutation). Mice homozygous for this mutation have an early onset of severe retinal degeneration.

Material and Methods: The eyes of five, 6 month-old male and female wild-type and P301S mice and six, 4 month-old male and female P301S mice were evaluated in histopathology for retinal degeneration/atrophy. Frozen tissue (liver or kidney or brain) from the same mice was used for evaluation of the presence of the rd1 mutation using polymerase chain reaction. None of the mice received any experimental compound.

Results: All mice with histopathological evidence of retinal degeneration/atrophy were homozygous for the rd1 mutation, while heterozygous mice and mice without the mutation did not show evidence of retinal degeneration. As expected, mice homozygous for the mutation were present in both the wild-type and transgenic groups. 

Conclusion: Retinal degeneration/atrophy in P301S mice and their wild-type littermates due to the rd1 mutation is a background, spontaneous, but potentially confounding  finding to be aware of when using these mice on pharmacological or toxicological studies.  It is therefore recommended to characterize P301S mice before a study by either ophthalmologic examination or genotyping to exclude mice with this spontaneous retinal pathology.  If a compound-related effect needs to be excluded, genotyping is recommended to demonstrate the presence of the rd1 mutation.

P07: Histopathological evaluation of avian gonads from chicks exposed in ovo as an alternative method for endocrine endpoints in birds

Maike Huisinga1, Burkhard Flick1, Nicole Kreling2, Lennart Weltje2, Katharina Ott2, Luzie Jessl3, Jessica Schneider3, Sibylle Gröters1, Bennard van Ravenzwaay1

1BASF SE, Experimental Toxicology and Ecology
2BASF SE, Crop Protection - Ecotoxicology
3Goethe University Frankfurt

In support of the 3Rs concept regarding animal testing at BASF SE, an alternative method investigating endocrine-mediated endpoints during early sexual development in birds was developed: An exposure-dependent shift of steroid hormones before sexual differentiation might lead to sex reversal. The egg injection assay utilizes this effect. 

We adapted the assay, described in literature, to stay within the limits of non-animal testing regulations and developed an evaluation scheme for histopathological staging of sex reversal, using the aromatase inhibitor fadrozole (0.01, 0.03, 0.1, 0.3, 1 mg/egg) as anti-estrogenic positive control.

Fertilized chicken eggs were injected after 1 day and necropsied on day 19 of incubation. The genetic sex was determined by PCR. Gonads were fixed in modified Davidson’s solution and processed routinely. 

Histopathological evaluation was performed on H&E-stained step-sections. Gonads were diagnosed as testes in control males and as left ovary and rudimentary right ovary in control females. The different stages of sex reversal in females were categorized as ovotestis grade 1 to 4, depending on the proportion of testicular transformation of the ovaries in the fadrozole-treated eggs. 

The histopathological staging of ovotestis led to a 100% detection of fadrozole treated females up to 0.03 mg/egg and a 100% coverage with the genetic sex. Ovotestis were not observed in controls.

In conclusion, the histopathological evaluation of chick gonads is a suitable method to evaluate endocrine-mediated endpoints during sexual development of birds and thus a reliable alternative method for animal testing in early screening.

P08: Correlation of Histopathology with Thyroid Hormone Measurement in Rats and Possible Stepwise Approach of Hormone Measurement in OECD 421/422 Studies

Silke Treumann1, Maria Cecilia Rey Moreno1, Volker Strauss1, Steffen Schneider1, Heike-Antje Marxfeld1, Sibylle Gröters1, Bennard van Ravenzwaay1

1BASF SE, Ludwigshafen

With the release of the new OECD TG’s 421/422 the validity of thyroid hormone measurements in juvenile rats with commercially available test kits is re-discussed.

ELISA kits for measurement of T3 and T4 in rats were validated and compared among each other regarding precision and functional sensitivity with established radioimmunoassays (RIA). Blood was sampled from PND4, PND13 and/or PND22 and adult animals in different study types (OECD 421/422). In addition, thyroid glands were preserved in 10% neutral buffered formalin for histopathologic examination and in adult rats the thyroid gland weight was determined.

Measurements of T3 and/or T4 and TSH in PND4, PND 13 pups or adult rats and concurrent thyroid gland histopathology were performed. 

Inter-individual variation of hormone values in control pups were assessed and statistical power was calculated. These parameters were compared with corresponding values observed in adult rats.

No histopathological correlate was observed with T4 decreases in PND13 pups.

Assessment of thyroid hormone measurements should be done in correlation with pathology data (organ weights, histopathology) and in a step-wise approach following a decision tree.

P09: T cell Bispecific Antibodies: Early Assessment of Tumor Targeting Antigens in the Cynomolgus Monkey – Lessons Learned

Anna Maria Giusti1, Barbara Lenz2, Erich Wolz2, Jürgen Bachl2, Matthias Fueth2, Marina Bacac1, Flavio Crameri2

1Roche Innovation Center Zurich
2Roche Innovation Center Basel

Tumor antigen targeted CD3 T cell bispecific (TCB) antibodies represent a highly potent and promising approach for cancer immunotherapy. Availability of tumor specific antigens, however, is very limited and targeting antigens with high tumor prevalence and potential expression in healthy tissues need therefore also to be considered.

Mesothelin (MSLN) is a such candidate antigen, with known serosal expression in normal tissues, already investigated as potential target for anticancer therapies in phase I or phase II clinical trials as tumor vaccine strategy, and antibody-based as well as adoptive CAR T-cell therapy. An early single ascending dose PK/MTD study in the cynomolgus monkey was performed to assess target validity in a CD3 T cell bispecific molecule.

MSLN-TCB treatment did not induce clinical signs. Clinical pathology changes, consistent with an inflammatory response, and microscopic changes of infiltrates of inflammatory cells on serosal surfaces and mesothelial hypertrophy/hyperplasia, as well as haemorrhage and inflammation in the urinary tract were observed in animals sacrificed 3 days after treatment. Chronic serosal adhesions in the lungs were observed in some animals at lower doses sacrificed after 21 days. These findings were attributed to the mechanism of action of the bispecific antigen in organs expressing the targeting antigen.

These results indicate that high selectivity for tumor expression of the targeting antigen is a must for the highly potent TCB molecules to avoid off-tumor on-target effects. A thorough evaluation of expression in normal tissues is of paramount importance in the selection of new targets for TCBs. Furthermore, early in vivo target assessment is recommended for antigens of interest that have unclear or controversial expression data in normal tissues. This allows to address accessibility of the target and investigate the potential for a therapeutic window.

P10: CD40-CD40L pathway activation in tertiary lymphoid organs contributes to disease pathology in primary Sjögren’s syndrome

Grazyna Wieczorek1, Marc Bigaud1, Sabina Pfister1, Sebastian Hoersch1, Katriona McMichael1, Catherine Afatsawo1, Meike Hamburger1, Celine Texier1, Maurane Henry1, Celine Cojean1 and James S. Rush1

1Novartis Institutes for Biomedical Research - Autoimmunity, Transplantation and Inflammation, Basel, Switzerland

Introduction: T cell-dependent activation of B lymphocytes is a key effector arm of the adaptive immune system, resulting in protective antibody responses and long-lived humoral immunity.  Such B cell responses often occur in germinal centers (GCs) within secondary lymphoid organs.  Similar GC-like structures can also be found in various tissues in autoimmune diseases, including the salivary glands of primary Sjögren’s syndrome (pSS) patients. Non-obese diabetic (NOD) mice provide an often used model of autoimmune diseases as they spontaneously develop autoimmune diabetes sharing many similarities to type 1 diabetes (T1D) and sialadenitis resembling pSS in human subjects.

Aim of the study: In the current study, we investigated expression of CD40 and CD40L in minor salivary glands from pSS patients and impact of CD40-CD40L blockade on tertiary lymphoid structures (TLOs) formation, (auto)antibodies production as well as function of the salivary glands in NOD mice.

Material and Methods: Minor salivary gland biopsies from pSS patients were immunostained for CD40 and CD40L expression and transcriptomics data of parotid glands from pSS patients were mined for CD40 gene signature. NOD mice were treated for 10 weeks with MR1 (anti-CD40L) antibody. In parallel, we observed development of TLOs in CD40 knockout NOD mice.  Salivary glands were immunostained for T and B cells, CD138 (plasma cells), Ki-67 (proliferation marker) and aquaporin 5 (a water channel protein). Autoantibody anti-Ro60 were measured by ELISA and frequency of antibody secreting cells was counted using ELISPOT.

Results: Using a published microarray dataset generated using parotid gland biopsies we could demonstrate upregulation of a portion of the B cell CD40 gene signature in biopsies from pSS patients but not from healthy donors or individuals with Sicca symptoms, suggesting that CD40 pathway activation was occurring within disease-relevant tissue in lymphocytes implicated in disease pathology. These results suggested that there might be ongoing T-B cell collaboration in TLOs and we therefore wanted to examine whether there was evidence of CD40 pathway activation in situ.  Immunohistochemistry revealed expression of CD40 and CD40L on B and T cells within the TLOs in minor salivary glands in pSS patients. Wild-type NOD mice developed sialadenitis, displayed evidence of TLOs in salivary glands and had circulating anti-SSA autoantibodies.  In contrast, there was no evidence of TLOs, sialadenitis or autoantibodies in CD40 deficient NOD mice up to one year of age.  Further, anti-CD40L treatment resulted in disaggregation in splenic GCs as well as established TLOs in NOD mice and resulted in decreased levels of IgG secreting cells in salivary glands. 

Conclusion: Our data indicate that CD40 pathway signalling is essential for formation and maintenance of salivary gland TLOs and suggest that CD40 pathway signalling is active in established TLOs from pSS patients, supporting the notion that blockade of CD40-CD40L interactions may provide therapeutic benefit in patients suffering from this autoimmune exocrinopathy.

P11: Canine dendritic cells: preliminary results of new immunohistochemical markers on paraffin embedded tissues

Sara Belluco1, Diane Razanajaona-Doll2, Jean-Jacques Pin2, Thierry Marchal1

1ICE UPSP 2016.A104, Axe Cancérologie, VetAgro Sup
2Dendritics, Lyon, France 

In humans two main types of dendritic cells (DC) have been identified: classical (cDC) and plasmacytoid (pDC). Within these 2 groups, several subgroups have been characterized using specific antibodies. Understanding the role of DC in physiological and pathological conditions is crucial, since they are major candidates for the development of immunotherapeutic strategies. Unfortunately, animal models are scarce and murine DC are phenotypically different from humans. In dog, little is known about DC, probably due to the lack of specific antibodies working on formalin-fixed paraffin embedded samples (FFPE). 

In an attempt to identify specific antibodies for the characterization of canine DC subpopulations, we tested antibodies raised against human antigens and cross-reacting with canine cells: CD303 and CD123, known to label pDC, and CD208 and CD209 which should detect cDC, on FFPE canine lymphoid tissues. In hyperplastic lymph nodes, a small amount of scattered cells, positive for CD303 and CD123 and with a plasma cell morphology, were located in the medullary cords and in the paracortex. Interestingly, no labelling was present on normal lymph node and thymus. CD208 antibody strongly labelled cells with a dendritic morphology in lymph nodes and thymus. CD209 gave no specific results. 

In conclusion, we identified three potential canine DC markers working on FFPE tissues: CD303 and CD123 seem to detect cells with a plasma cell morphology, compatible with pDC, and CD208 (DC-LAMP) labels classical antigen presenting DC.Further studies are needed to confirm the dendritic origin by studying the co-expression of the new and known DC’s immune markers and to characterize the distribution of canine DC in physiological and pathological conditions.

P12: Addressing current challenges of emerging therapies: pitfalls in the assessment of irradiated mice in bone marrow transplantation studies

Francesca Sanvito1,2, Ilaria Visigalli1, Nicola Carriglio1, Raisa Jofra Hernandez1, Francesca Cecere1, Michela Vezzoli1, Maura De Simone1, Rossana Norata1, Martina Rocchi1,2, Paola Albertini1, Luigi Naldini1,3, Patrizia Cristofori1,4

2Pathology Unit, San Raffaele Scientific Institute
3“Vita-Salute”San Raffaele University
4GSK David Jack Centre for R&D, United Kingdom

Bone marrow transplantation (BMT) is the treatment of choice for many leukemias, solid tumors, and metabolic diseases. The field of bone marrow research is highly dependent on in vivo experiments, because in vitro techniques do not mimic these complicated in vivo systems. Whole-body irradiation is one of the most common tools for myeloablation of the recipient’s bone marrow.

Ionizing radiation causes breaks in the DNA double-strands thus it mostly affects mitotically active cells. The DNA breaks occur in multiple sites, and damage is so severe that the cellular repair systems are unable to fix the DNA. Consequently, this damage leads to cell death through either necrosis or apoptosis. The cells in the hematopoietic system and gastrointestinal tract are extremely sensitive to irradiation because they are always mitotically active. With any therapy that has the potential to damage cellular DNA and causes immunosuppression comes the inherent risk for developing secondary neoplasias, a variety of infections and a decrease in hematopoietic cell populations. Delayed irradiation-related effects can also occur in kidneys, lungs and other organs. These findings have to be identified and properly considered in the evaluation of results of study. The review of histopathology data, collected from GLP toxicology and tumorigenicity studies, using irradiated and transplanted mice (comprehensive list of tissues examined), has shown some similarities and some significant differences in timing and sensitivity of tissues among strains. Historical data collection is central in recognition of confounding effects and in providing weight of evidence conclusions for the definition of safety profile, in particular when using mouse model of human disease as test system.

P13: Use of molecular pathology and histopathology to characterize different mouse models of colorectal cancer

Marjolein van Heerden1, Karine Smans2, Hillary Millar2, Ad Knaapen1, Lieve Lammens1, Janine Arts2, Sandra De Jonghe1

1Preclinical Development & Safety, Discovery Sciences
2Drug Discovery CRC, Janssen R&D, Beerse, Belgium 

To enable drug development for colorectal cancer prevention and interception there is a need for translational models to assess both pharmacological activity as well as potential safety concerns. In support of our Colorectal Cancer (CRC) initiative, several (mouse) disease models (ApcMin/+, villin-Cre-ER Apcfl/+ KRasG12D/+ and ApcMin/+ /DSS) have been further characterized using extensive immunohistochemistry (e.g. inflammation and proliferation markers) and applying optimized sampling and fixation techniques.

Multiple intestinal neoplasia (ApcMin/+) mice are heterozygous for the Apc gene and considered a classic model for CRC pathogenesis given that Apc mutations are present in most human colorectal cancers (Sears and Garret 2014). However, these mice develop principally small intestinal adenomas (polyps), while humans develop mainly colorectal cancers. The ApcMin/+ /DSS mouse model and a villin-Cre-ER Apcfl/+ KRasG12D/+ (Apc/KRas) mouse model, with colorectal polyp development were also evaluated, and their polyp morphology was compared with the polyps from the ApcMin/+ mice.

The Swiss roll technique was used to allow for assessment of efficacy (eg. polyp count/development) and safety (signs of inflammation and erosions/ulcers, as disruption of the epithelial barrier enables intestinal microbes to access the lamina propria promoting localized inflammation and genetic instability, which can lead to the development of cancer). Polyps were histologically classified (focal hyperplasia/aberrant crypt focus, hyperplastic polyp or adenomatous polyp), annotated, counted and measured on scanned digital slides, and statistical analysis on gross and histological polyp counts was performed to demonstrate efficacy.

The main differences between the polyps of the ApcMin/+ and Apc/KRas model were noted for Paneth cells and neutrophils. For the ApcMin/+ /DSS model the remaining colitis (with high individual variation) was a confounding factor in the evaluation of polyp morphology. Paneth cell hyperplasia in pre-neoplastic lesions and Paneth cell containing polyps were frequently observed in the ApcMin/+ mice model, but not in the Apc/KRas model and the ApcMin/+ /DSS model. In humans colorectal Paneth cell metaplasia was frequently observed in the adjacent mucosa or within colorectal epithelial neoplasias (Wada et al. 2005). The incidence of Paneth cell containing adenomas varies widely ranging from 0.2 to 39% (Pai et al. 2013). Neutrophilic infiltrates were prominently present in polyps of the Apc/KRas model, in contrast to the ApcMin/+ mice.

The characterization of several mouse disease models and comparison of their polyp morphology, allowed the selection of the most appropriate CRC mouse model for efficacy studies.

Pai R. K. et al. Am J Surg Pathol 2013, 37:98–103

Sears C. L. and Garrett W. S. Cell Host Microbe. 2014, 15(3): 317–328

Wada R. et al. Journal of Carcinogenesis 2005, 41477-3163-4-5

P14: Accumulation of naked and N-acetyl galactosamine-conjugated locked nucleic acids in rat liver

Fernando Romero-Palomo1, Matthias Festag1, Barbara Lenz1, Annamaria Braendli-Baiocco1

1Pharmaceutical Research and Early Development (pRED), Pharmaceutical Sciences, Roche Innovation Center Basel, Switzerland

Locked nucleic acids (LNAs) are synthetic RNA nucleotides with a methylene bridge that connects the 2’-oxygen of ribose with the 4’-carbon, increasing their stability and binding affinity to complementarity sequences. These chemically modified nucleic acids have the potential to be used as therapeutic anti-sense oligonucleotides (AON), and currently numerous AONs are being evaluated in clinical trials for a variety of therapeutic indications. Despite their proved therapeutic potential, some LNA have also showed toxic effects mainly associated with their accumulation in liver and kidney. 

We performed a rat toxicity study to further characterize mechanisms of toxicity after single and multiple doses of selected LNAs.  Four rats per group and time point were dosed subcutaneously every 7 days for up to 6 weeks and sacrificed at either 4, 18 or 39 days after first dose, including a vehicle control group. Tool LNAs with three different sequences and safety profiles (safe/medium/high toxicity based on previous studies) were selected. These LNAs were used both as unconjugated (naked) compounds or conjugated with N-acetyl galactosamine (GalNAc), which is a high-affinity ligand for the asialoglycoprotein receptor (ASGPR) expressed on the hepatocyte cell surface. 

We evaluated the liver histopathologically, and we studied the accumulation of LNAs by means of immunohistochemistry (IHC) and in situ hybridization (ISH) in formalin-fixed paraffin-embedded tissues. An antibody targeting phosphorothioate internucleotide modifications present in all LNA compounds was used, and detected with DISCOVERY DAB chromogen. DIG-labeled probes from Exiqon were used for ISH and detected with DISCOVERY Purple chromogen. Ventana Discovery XT® and Discovery Ultra® automated tissue staining systems were used for IHC and ISH, respectively. Signal intensity and cell types were assessed by semiquantitative evaluation. 

The tool LNAs showed different toxicity according to their safety profile, with the “safe” compound showing no evident lesions in the liver despite accumulation. The two GalNac-conjugated LNAs showed higher hepatocellular toxicity than their naked counterparts after both single and multiple doses. Both ISH and IHC confirmed the accumulation of LNAs in the liver and resulted in similar LNA levels, although ISH proved to be more sensitive than IHC for very low amounts of LNAs. However, IHC seemed to be more reliable for quantification when the amount of LNA increases after multiple doses. Presence of naked LNAs was demonstrated in high amounts in Kupffer cells and sinusoid endothelial cells after single and multiple doses, whereas presence in the hepatocytes was very low throughout the study. GalNAc-conjugated LNAs accumulated from the first dose primarily in hepatocytes, and after repeated doses, they were also detected in Kupffer cells and sinusoid endothelial cells. The distribution of naked and GalNAc LNAs in the liver provides a useful starting point for additional investigations on the pathogenesis of LNA-induced toxicity.

P15: Assessment of liver biomarkers following Galactosamine (and LPS) administration in Wistar rats

Maliver Pierre1, Winter Michael1, Bollrath Julia1, Lenz Barbara1, Festag Matthias1, Christen Francois1

1Roche Pharma Research and Early Development, Pharmaceutical Sciences, Pathology, Roche Innovation Center Basel, Switzerland

Introduction:  Galactosamine (galN) is a known hepatotoxicant which renders rodents sensitive for liver toxicity of co-administered Lipopolysaccharide (LPS).  In order to evaluate a panel of liver biomarkers (GLDH, Arginase 1, miR 122), a mechanistic study was conducted using galN and LPS.

Material and Methods: Wistar rats were dosed with galN (intravenous administration, 0-30-80-200 mg/kg/day) up to seven days, with an additional group combining galN at 100 mg/kg/day plus LPS administration (Day 7 only). Clinical pathology parameters were evaluated on Day 2 (acute response) and Day 8 (chronic response) and compared to microscopic results (Day 8 except the 200 mg/kg/day group prematurely sacrificed on Day 4, dose not tolerated).

Results: Hepatocyte degeneration/single cell necrosis was noted at 200 mg/kg/day and 100 mg/kg/day (plus LPS) and correlated to liver biomarkers results. Liver inflammation and increased TNF-alpha (mRNA ISH) expression from hepatocytes or macrophages were also observed. On Day 2, miR 122 results correlated well to GLDH or Arginase 1 responses (p<0.0001). All three biomarkers were highly increased at 200 mg/kg/day and presented similar sensitivity. On Day 8 (100 mg/kg/day plus LPS), higher (GLDH) or similar (Arginase 1) serum activities were noted when compared to Day 2 results. GLDH and Arginase 1 presented higher sensitivity than miR 122.

Conclusion: MiR 122, GLDH and Arginase-1 equally detected acute, significant hepatocyte injury. Since Arginase 1 can be secreted during inflammation by macrophages, combination of several liver biomarkers (including mi R122 and GLDH) is considered valuable when evaluating acute liver degeneration associated with inflammation.

P16: Molecular pathological differences between nodular regenerative hepatocellular hyperplasias and hepatocellular adenoma induced by long term exposure of piperonyl butoxid to mice

Shinji Takasu1, Yuh Yokoo1, Yuji Ishii1, Aki Kijima1, Kumiko Ogawa1, Takashi Umemura1,2

1Division of pathology, National Institute of Health Sciences
2Faculty of Animal Science Technology, Yamazaki Gakuen University

Long-term exposure to piperonyl butoxide (PBO) induces hepatocellular tumors in mouse. Previously, we have reported that multiple nodular masses were induced in the liver of mice in addition to development of hepatocellular tumors by treatment of PBO (Tasaki et al., Exp Toxicol Pathol 2014). This lesion was not composed of clonal cells, and the lobular architecture mostly remained intact although severe compression of surrounding liver was presented. In addition to these morphological features, focal necrosis was observed in the nodules. These facts allowed us to speculate that this lesion would be regenerative, but not neoplastic change. On the basis of these histopathological findings, we diagnosed this proliferative lesion as a nodular regenerative hepatocellular hyperplasia (NRH). However, biological characteristics of NRH distinguishable from hepatocellular tumor remain unclear. In this study, molecular characteristics in this lesion were compared with those in hepatocellular adenomas (HCA) by global gene expression. 6-week old male ICR mice were given a diet containing 0 or 6000 ppm PBO for 43 weeks to form NRH and HCA. cDNA microarray was performed using mRNA extracted from frozen sections including NRH, HCA, or non-proliferative area in PBO-treated group or non-proliferative area in basal diet group collected by the laser microdissection. As a result, the expressions of cell cycle-related genes were increased in both lesions compared with non-proliferative area. The expressions of metabolism-related genes including Cyps were decreased only in NRH, those of genes associated with cell differentiation such as Notch pathway being increased only in HCA. These results implied that NRH was different from HCA in term of not only morphological but also molecular characteristics in spite of both lesions having proliferative nature. In addition, the global gene expression analysis might indicate the biological properties of simple proliferative lesions having decreased activities in Cyps and neoplastic lesions possessing activation of Notch pathway.

P17: Comprehensive evaluation of general toxicity, genotoxicity and carcinogenicity of elemicin using gpt delta rats

Yuji Ishii1, Liang Shi1, Shinji Takasu1, Aki Kijima1, Kumiko Ogawa1, Takashi Umemura1,2

1Division of Pathology, National Institute of Health Sciences
2Faculty of Animal Science Technology, Yamazaki University

Introduction: Elemicin is one of natural alkoxybenzene compounds, and is a flavor component of several plant species such as nutmeg and mace. Although some other alkoxybenzene compounds including estragole, safrole and methyleugenol are known to possess genotoxicity and carcinogenicity in the livers of rodents, there are few data concerning elemicin. gpt delta transgenic rat is recognized as an in vivo mutation assay model that is capable of detecting point mutations by 6-thioguanine selection (gpt assay) and deletion mutations by Spi- selection (Spi- assay). Quantitative analyses of glutathione S-transferase placental form (GST-P) positive foci can be performed in gpt delta transgenic rats. Thus, the gpt delta rat animal model is a promising tool for investigating the comprehensive toxicities, including the genotoxicity and carcinogenicity, of chemical agents in target organs. In the present study, to clarify toxicity, genotoxicity, and carcinogenicity of elemicin, comprehensive evaluation was performed using gpt delta transgenic rats.

Materials and Methods: Six-week-old male F344 gpt delta rats (10 rats/group) were given elemicin by gavage at doses of 0, 25, 100 or 400 mg/kg/day for 13 weeks. Blood samples were collected from the abdominal aorta for hematology and serum biochemistry under anesthesia. At necropsy, weights of brain, heart, lungs, liver, kidneys, spleen, thymus, adrenal glands and testes were measured. After weighing the whole livers, left liver lobe was fixed with neutral-buffered formalin for histopathological examination and immunohistopathological analysis of GST-P positive foci. In addition to these organs, the artery, bone/marrow, coagulation gland, esophagus, epididymides, large intestine, lymph node, mammary gland, pancreas, peripheral nerve, prostate gland, pituitary gland, thyroid glands, salivary gland, skeletal muscle, skin, small intestine, spinal cord, stomach, urinary bladder, tongue, and trachea were fixed in 10% neutral buffered formalin. These tissues were routinely embedded in paraffin, sectioned for hematoxylin and eosin staining, and examined under light microscopy. The remaining liver was stored at -80oC for comprehensive DNA adduct analysis by LC-MS/MS (Ishii et al., Anal. Bioanal. Chem., 2014) and reporter gene mutation assays.

Results: Significantly higher liver weights, and serum ALT, γ-GTP and total cholesterol were observed in the 400 mg/kg group, when compared with controls. Histopathologically, diffuse hepatocyte hypertrophy and focus of eosinophilic foci of cellular alteration were found in the 100 mg/kg group and above. Significant increases in the number and areas of GST-P-positive foci were apparent in the 400 mg/kg group. Comprehensive DNA adduct analysis demonstrated the existence of several spots indicating putative DNA adducts at m/z 434, 458, 474 and 496. Subsequent MS spectrum analyses showed four spots to be elemicin-specific deoxyguanosine, deoxyadenosine and deoxycitidine adducts. Evaluation of in vivo mutagenicity by gpt assay and Spi- assay is now in progress.

Discussion: Changes in the serum biochemical parameters and histopathological findings indicate that elemicin is hepatotoxic in rats, and NOAEL in the present study is considered to be 25 mg/kg/day. Quantitative analysis of GST-P positive foci suggests that elemicin is likely to be hepatocarcinogenic in rats. Elemicin-specific DNA adduct formation might imply the involvement of genotoxic mechanisms in its hepatocarcinogenesis.

P18: Genotoxicity Testing of the Natural Food Colorant, Gardenia Blue, and its Derivation Precursor, Genipin

Mihoko Koyanagi1, Cheryl A. Hobbs2, Carol Swartz2, Jeffrey Davis2, Leslie Recio2, Shim-mo Hayashi3, Robert R. Maronpot4

1San-Ei Gen F.F.I., Inc., Osaka, Japan
2ILS, Inc., Research Triangle Park, NC, USA 
3San-Ei Gen F.F.I., Inc., Osaka, Japan 
4Maronpot Consulting LLC, USA

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted in accordance with OECD guidelines. No evidence of mutagenicity of gardenia blue was measured in a bacterial reverse mutation assay in Salmonella strains TA98, TA100, TA97a and TA1535 and in E. coli WP2 uvrA pKM101, with or without metabolic activation; an equivocal response for genipin occurred in Salmonella TA97a without metabolic activation. In micronucleus (MN) and chromosome aberration assays using human TK6 lymphoblast and Chinese hamster ovary cells, respectively, with and without metabolic activation, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. To assess the ability of these compounds to induce DNA damage in a rodent model, combined MN/comet assays were conducted in male and female B6C3F1 mice. Micronucleated reticulocyte (MN-RET) frequencies in blood were determined by flow cytometry and induction of DNA damage in liver, stomach and/or duodenum was assessed using the comet assay. No effects occurred at genipin doses reaching maximal toxicity (74 and 222 mg/kg/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/day) on blood MN-RET frequency or DNA damage in examined tissues of exposed mice.  Thus, although our studies showed some in vitro evidence of genotoxic potential of genipin, there was no evidence of DNA or chromosome structural damage in male and female mice exposed to either genipin or gardenia blue.  Furthermore, use of a modified (“reverse”) comet assay did not provide any evidence of DNA crosslinking induced by either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue or genipin in food products does not pose a significant genotoxic concern for humans.

P19: Hemorrhagic necrosis of the gallbladder smooth muscle layer with an anti-bacterial drug: An uncommon finding in the dog 

K. Broudic1, M. Slaoui2, C. Chalier2, Y. Lopez2, J.M. Guillon2, X. Boulenc4, E. Fontaine5, M. Doubovetzky3

1Sanofi Pasteur Non Clinical Safety, Marcy L’Etoile, France 
2Sanofi R&D Preclinical Safety, Alfortville, France 
3Sanofi R&D Preclinical Safety R&D, Marcy L’Etoile, France 
4Sanofi R&D DMPK, Marcy L’Etoile, France 
5Sanofi R&D Infectious Diseases, Marcy L’Etoile, France 

Tuberculosis (TB) remains a major global health problem that caused an estimated 1.3 million deaths in 2012. A growing percentage of TB infections are multidrug resistant and in this context, Sanofi Infectious Disease is supporting a number of programs to identify and develop new anti TB agents with a novel mechanism of action.

SARXXXXXX is a new anti-tuberculosis antibiotic with a potent activity against Mycobacterium tuberculosis, both in vitro (MIC: 0.05 µg/ml; MBC: 0.5 µg/ml) and in vivo in the mouse model (MED: 50 mg/kg; MBD: <200 mg/kg (oral dose)) and is effective against drug-resistant and evolutionarily distinct lineages of M.tb. Besides, SARXXXXXX was investigated for its binding to a panel of receptors and enzymes (CEREP profiling) as well as its inhibitory effect on a set of key ion channels, with no relevant finding coming out of these assays.

After oral administration in dogs, a good bioavailability (>90%) was observed as well as long t1/2 (69h). SARXXXXXX distributes largely in tissues (liver in particular: tissue/plasma ratio of 10) and is highly bound to plasma proteins (Fup:1.3%). It is primarily eliminated through metabolism, with less than 1% eliminated by urine and bile as unchanged parent drug (rat data). A low clearance was determined in dogs (16% of blood flow) after IV route. In the dog, SARXXXXXX is also a CYP3A4 inhibitor (mechanism-based inhibition with kinact : 0.082 min-1), and a BSEP and PgP transporters inhibitor with IC50s of 7 µM and 30 µM, respectively.

In a mouse oral toxicity study at 100, 300 and 1000 mg/kg over 2 weeks, findings were essentially limited to the liver with dose-related minimal or mild periportal/midzonal or panlobular fatty change and increased glycogen content associated with increases in plasma bilirubin, bile acids, triglycerides and decrease in glucose.

In the dog, SARXXXXXX toxicity was investigated in an exploratory protocol over 3 weeks, at a daily dose of 50 mg/kg (2/sex treated dogs vs 2/sex controls). There was no clinical signs reported, but changes in hematology (i.e. decreased RBC mass parameters and reticulocytes), and in clinical chemistry (i.e. increased AST, ALT, GLDH, bile acids, total bilirubin, ALP, phosphorus and urea nitrogen and of decreased potassium and chloride). Hepatocellular single cell necrosis and eosinophilic cytoplasmic inclusions and cytoplasmic glycogen accumulation were observed in the liver.

One treated male and in one treated female exhibited a multifocal mild or moderate hemorrhagic necrosis of the muscular wall of the gall bladder. This uncommon gall bladder change displayed acute features in the male, or was associated with subchronic inflammation in the female. 

Potential root causes for gall bladder changes are discussed as well as experimental and derisking plan – with particular reference to gallbladder contractility.

P20: Histopathological characterization of lung lesions in preclinical Mycobacterium tuberculosis murine models: from mice to humans 

Isabelle Blanc1, Sylvain Ribeyro1, Carla Thevenet2, Laurence Somody1, Caroline Michel1, Pierre Cortez1, Michel Doubovetzky3, Jean-François Gallas4, Laurent Fraisse1

1SANOFI R&D TA Infectious Diseases 
2trainee TA ID
3SANOFI Clinical safety  RNCS/preclinical safety
4SANOFI Preclinical safety 

One-third of the world population is latently infected with Mycobacterium tuberculosis Mtb and active Tuberculosis (TB) is estimated to cause 1.5 million deaths per year. When inhaled, Mtb reaches the lungs and is internalized by lung macrophages. The latter triggers the accumulation of macrophages and lymphocytes at the infectious site producing granuloma, which is a major histo-pathological feature of TB. Caseous granulomas are the hallmark of human tuberculosis providing a unique bacterial microenvironment which is important to reproduce in preclinical models used for new anti-TB drug development.

Preclinical mice models allow the investigation of different mechanisms of the disease with different growth rates ranging from exponential growth to stabilization of bacterial numbers up to a non-replicative persistence state. 

We validated and routinely use three different murine Mtb models with different inoculation rates: (i) the highly acute model (10E6 CFU/mouse), (ii) the acute model  (10E4) and (iii) the chronic model (10E2) that differ on the replicative state of Mtb.

Using formalin-fixed, paraffin-embedded lung tissues from infected mice, different specific stainings allowed us to characterize the lung lesions in our various different models. First, using Haematoxylin and Eosin-staining, we compared the lung organization and observed a massive infiltration of immune cells into the lung in the three Mtb models. Next, the Ziehl-Neelsen staining enabled the Mtb identification within lung cells and its colocalization with the infiltrated macrophages. Then, red oil coloration allowed staining lipids that accumulated into foamy macrophages (FM). These FM, a specific feature of human disease, were found within granulomatous structures in the three animal models. Furthermore, because macrophages are important contributors to fibrosis in lesions caused by TB, we assessed the presence of collagen through red sirius staining in our different Mtb murine models and identified extensive fibrosis forming a sort of matrix in the chronic model. Finally, we showed the presence of pulmonary granulomatous lesions in murine model but didn’t observe any necrotic granuloma as it is reported in the C3HeB/FeJ mice model (Kramnik model) and in human tuberculosis lesions.

In summary, as previously reported, we confirmed that pulmonary granulomatous lesions observed in the chronic Mtb murine model did not display any necrosis as it is observed in the C3HeB/FeJ mice model  and in human tuberculosis lesions.  However, the histopathological characterization together with in vivo performance permit to conclude the suitability of the three models, acute, highly acute and chronic for anti-TB drug development.

P21: Mast cells assessment in a murine Atopic Dermatitis model with a new semi automated counting method

AS. Dugaret1, B. Gauthier1, F. Weltzer1, B. Bertino1, M. Motte1, A. Lamit1, P. Rossio1, D. Froude1, F. Hacini-Rachinel1, F. Bihl1

1Galderma R&D

Mast cells are important immune cells found predominantly in tissues at the host-environment interface such as gastrointestinal and genitourinary tracts, airways, and skin. Mast cells express at their cell surface the high-affinity IgE receptor (FcεRI). Upon IgE-mediated allergic reaction, this receptor binds IgE released by plasma cells that leads to mast cells activation and immediate release of preformed mediators, such as histamine, as well as TH2-polarizing cytokines, such as IL-4 and IL-13. Therefore, mast cells are important orchestrating effectors of IgE-mediated allergic responses in various pathologies such as atopic dermatitis.

Atopic dermatitis (AD) is the most common chronic pruritic inflammatory skin disease that usually starts in early infancy but also affects substantial number of adults [1]. AD is recognized as a multifactorial heterogeneous disease characterized by different clinical phenotypes based on complex interactions between susceptibility genes, host’s environment, and defects in skin barrier function. Histopathology remains the reference for the lesion characterization and microscopic examination of AD lesions reveals an epidermal hyperplasia associated with spongiosis and a mixed cell infiltrate consisting of mast cells, eosinophils and T lymphocytes. An animal model based on epicutaneous sensitization to house dust mite allergens extract was developed in female BALB/c ByJ Rj mice to evaluate new drug candidates [2]. Amongst all study readouts, mast cells manual counting is a time limiting factor.

We therefore developed a new semi-automated counting of mast cells using Definiens® Developer (Definiens AG, Münich) on toluidine blue (TB)-stained histological skin slides of AD-induced lesions. This method relies on the metachromatic properties of the TB dye. Mast cells granulations appear red-purple on a pale blue background. Two separated algorithms were set up: the first one for automated detection of the different tissue sections on the TB-stained virtual slides and the second one for masts cells segmentation using color deconvolution [3]. The full process includes a visual review step. We validated this new approach by two complementary methods, one requiring a direct photo review process and the other based on retrospective results of four previous studies.

Overall, this new semi -automated method was considered adequate and valid for mast cells counting. It is now currently in place and henceforth replaces the manual counting for further AD model studies and closely-related other models. 


1. Weidinger S, Novak N. Atopic dermatitis. Lancet. 2016, 0387 (10023): 1109-1122

2. JM Spergel, E Mizoguchi, JP Brewer, TR Martin, AK Bhan, RS Geha. Epicutaneous sensitization with protein antigen induces localized allergic dermatitis and hyperresponsiveness to methacholine after single exposure to aerosolized antigen in mice. J Clin Invest, 101 (1998), pp. 1614–1622

3. Ruifrok AC, Johnston DA. Quantification of histological staining by color deconvolution. Anal Quant Cytol Histol 23: 291- 299, 2001.

P22: In vivo measurement of reporter protein expression in a fibrotic mouse model using whole-body optical imaging and probe-based confocal laser endomicroscopy

Philippe Pujuguet1, Corinne Saccomani1, Emile Berrocal1, Aymeric Blanc2, Angelina Cauvin1, Philippe Clément-Lacroix1, Sonia Dupont1, Ziad Ben El Kahdi2, Catherine Jagerschmidt1, Elodie Ly2, Gilles Cestelli2

1Galapagos SASU, Romainville, France
2Mauna Kea Technologies, Preclinical and Translational Medicine Business Unit, France

Enhanced Episomal Vectors (EEV) are non-viral, non-integrating plasmid-based expression vector systems that enable sustained transgene expression for several months in both in vitro and in vivo applications. In vivo efficient gene transfer can be achieved by hydrodynamic injection (rapid injection of a large volume of DNA solution) via the tail vein (1). It is generally accepted that transforming growth factor-β (TGFβ) is a key factor of fibrosis induction, regulating the expression of important fibrosis genes such as collagen 1a1 (Col1a1).

Here, we evaluate EEV with Luciferase (Luc) and Green Fluorescent Protein (GFP) expression vector in which both GFP and Luc genes are under the control of TGFβ-inducible Col1a1 promoter. The level of gene reporter must be overexpressed with the presence of fibrosis-induced treatment (CCL4 or TGFβ). After EEV hydrodynamic injection, GFP or Luc expression level was measured in liver and kidney. The goal is to establish a mouse fibrosis model in which the evaluation of the extent of fibrosis is made possible over time and can be quantified non-invasively. Two imaging modalities, whole-body optical imaging and probe-based confocal laser endomicroscopy (pCLE), were used to monitor in vivo the expression of this reporter protein involved in fibrosis. With the latter technology, fluorescent dyes were used to highlight the cellular structure or the vasculature of the organs of interest (acriflavin and Evans blue, respectively). Additionally, we assessed the ability of a fluorescent collagen binding probe (col-F) to measure in vivo and non-invasively kidney fibrosis.

Different doses of pCol1a1-GFP-Luc EEV (3 and 5 µg) were injected in BALB/c N mice via the tail vein. After fibrosis activation, the expression of the reporter protein was monitored in the liver and kidney of the same animal both non-invasively by whole-body luminescence imaging and in a minimally invasive manner by pCLE. At necropsy, ex vivo imaging analysis was also performed. Using pCLE, the fluorescence of the reporter protein was detected in vivo, in the liver (right lobe: high expression level, caudate lobe: lower expression level) as well as in the kidney, at the cellular level. While whole-body optical luminescence imaging could easily measure the expression of the reporter protein in the liver of fibrosis mouse model, the reporter protein was not detected in other organs. In addition, for the first time to our knowledge, we report the ability of col-F to measure non-invasively kidney fibrosis.

These findings show that pCLE is complementary to whole-body optical imaging as in addition to in vivo quantitative information of the expression level it allows the precise localization of a reporter molecule at the cellular level.

Such information is important for the precise in vivo follow-up of fibrosis extent in mouse fibrosis models.


(1) Lui et al. Gene Ther 1999, 6(7): 1258-66

P23: Imaging-guided Laser Based Tissue Preparation for Advanced Histology and Pathology

Heiko Richter1, Isabel Bleeker1, Diego Ramirez1, Fabian Will1, Birgitta Stolze1

1LLS ROWIAK LaserLabSolutions GmbH, Hannover, Germany 

Introduction: Preclinical studies in the field of regenerative medicine become more and more important to compete in the market. Histological analysis often is a mandatory, yet laborious part of preclinical study design. Especially the preparation of hard tissue samples or samples containing implants is challenging. Ground section technologies are restricted in section thickness and suffer from high material loss. Alternatively, decalcification of hard tissue for microtomy has to be applied, resulting in loss of biological information. Beyond histology, it becomes more important not only to analyze morphology, but rather to match it with biochemical analysis. Femtosecond laser based cutting of tissue provides novel options of preparing histology sections and 3D-site specific tissue sectioning of further biochemical analysis in one tool. In this contribution we investigate the influence of the laser cutting on the integrity of the tissue. 

Materials and Methods: For laser microtomy different types of tissue were plastic embedded, hard tissue without prior decalcification. Sections were prepared by laser cutting (Amplitude T-Pulse 500) at 10 µm thickness. Histological, immune- and histochemical stainings were applied as indicated in the results. For site-specific 3D-sectioning of native tissue for further molecular analysis, samples from bone-titanium interfaces of a fresh rat tibia were cut at volumes of ~300x300x300 µm. Regions of interest were determined by Optical Coherence Tomography (OCT). Harvested samples were subjected to RNA-extraction and TNF-alpha gene expression analysis.

Results: The results of the histology and histochemistry of tissue sections prepared show that established routine stainings can be successfully applied to thin sections performed by laser microtomy as shown by Hematoxilin and Eosin, Leval Laczko, McNeal or Masson Goldner staining. The sections show tissue architecture and cellular details clearly. Special detection of enzyme activity inside bone samples was demonstrated by Tartrate-Resistant Acid Phosphatase (TRAP) staining after laser microtomy, implicating that the laser cutting did not destroy the enzymes. Immunohistochemistry was demonstrated for antigens as Col-I, CD31 or SMA. The results are very encouraging and indicate that thin sections prepared by laser microtomy can be applied to investigative histopathology applying immunohistochemistry and that antigens are not impaired by laser sectioning. RNA-analysis of 3D-cell clusters cut out of native tissue along an implant interface for biochemical analysis showed that laser cutting does not destroy the biochemical information of the tissue. The opposite is true. Overall RNA-expression was significantly higher (>1000-fold) than from tissue samples prepared by mechanical methods due to the gentle and fast preparation. Clear site-specific TNF-alpha expression at the bone-implant interface could be demonstrated.

Conclusions: Femtosecond laser based tissue sectioning does not interfere with enzyme activity, antigen integrity or gene expression of the tissue. It opens a new range of possibilities for histological analysis of hard and implanted tissues and beyond, for site specific biochemical tissue analysis.

P24: High-fructose diet feeding accelerates diabetic nephropathy in Spontaneously Diabetic Torii (SDT) rats.

Kaoru Toyoda1, Yusuke Suzuki1, Kyotaka Muta1, Taku Masuyama1, Kochi Kakimoto1, Toshiyuki Shoda1 and Shoichiro Sugai1

1Toxicology Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc.

Background: Diabetic nephropathy (DN) is one of the complications of diabetes and is now the most common cause of end-stage renal disease (ESRD). Fructose has been reported to have the potential to exacerbate diabetes and DN in humans and experimental animals even though fructose itself does not increase postprandial plasma glucose levels. In this study, we investigated the effects of a high fructose diet on the nephropathy in Spontaneously Diabetic Torii (SDT) rats, which are known as a useful model for non-obese type 2 diabetes in that they spontaneously develop hyperglycemia and glucose intolerance resulting from impaired insulin secretion due to β cell degeneration in the pancreas. 

Materials & Methods: Male Sprague-Dawley (SD) and SDT rats were divided into two groups and allowed free access to either high fructose (65% fructose diet) or standard diet. Three animals were assigned to each group, a standard diet fed-SD rats (C-SD) group, a high fructose diet fed-SD rats (HF-SD) group, a standard diet fed-SDT rats (C-SDT) group and high fructose diet fed-SD rats (HF-SDT) group. Animals in each group were fed their assigned diets for 4 weeks. Measurement of the body weights and food consumption, clinical chemistry, urinalysis, necropsy, measurement of kidney weights and histopathological evaluation were conducted.

Results: The urinary protein, KIM-1, Clusterin and TIMP-1 levels were higher in the HF-SDT group than that in the C-SDT group at post-examination. In the histopathological examination, the severity of some lesions including hyaline casts, epithelial regeneration in tubules, tubular dilatation, mineralization and fibrosis were higher in the HF-SDT group than those in the C-SDT group. Immunohistochemistry revealed that the number of the vimentin, Kim-1 and ED-1 positive cells was increased in the HF-SDT group when compared with that in the C-SDT group.

Conclusion: Four-week feeding of a high fructose diet induced increased urinary excretion of kidney injury makers and accelerated the development of microscopic tubular and interstitial lesions in SDT rats. This study reveals apparent adverse effects of fructose on renal lesions in SDT rats. It suggests that the SDT rat is a useful model to investigate human DN and excessive fructose intake can be a risk factor of the development and progression of DN in diabetic patients.

P25: Toxicity and toxicokinetics of alpha-glycosyl isoquercitrin: a 90-day study in Sprague-Dawley rats

Shim-mo Hayashi1, Abraham Nyska2, Jeffrey P. Davis3, Micheal P. Jokinen3, Yuval Ramot4, Mihoko Koyanagi1, Robert R. Maronpot5

1Global Scientific and Regulatory Affairs, San-Ei Gen, F.F.I., Inc., Osaka, Japan
2Sackler School of Medicine, Tel Aviv University, and Consultant in Toxicologic Pathology, Timrat, Israel
3Integrated Laboratory Systems, Research Triangle Park, NC, USA
4Hadassah - Hebrew University Medical Center, Jerusalem, Israel
5Maronpot Consulting LLC, Raleigh, NC, USA

Alpha-Glycosyl isoquercitrin (AGIQ) is a highly absorbable mixture of isoquercitrins with antioxidative properties. It has been confirmed as generally recognized as safe (GRAS) compound by the FDA, and approved by the Japanese Ministry of Health and Welfare for use as a food additive. Nevertheless, safety and toxicity information for AGIQ is still sparse and outdated. The aim of this study was to comprehensively evaluate the toxicity of AGIQ in Sprague-Dawley rats exposed orally for 90 days and to explore the toxicokinetics (TK) of AGIQ. 60 male and 60 female Sprague-Dawley rats were daily administered with AGIQ at dietary doses up to 5%, and followed for 90 days. TK of AGIQ was tested in twenty male Sprague Dawley rats up to 24 hours following a single gavage dose. All animals survived to the scheduled sacrifice with no animals showing signs of morbidity, and there was no evidence for systemic toxicity. AGIQ was rapidly absorbed with metabolism to quercetin and quercetin glucuronide at all dose levels. Dose dependent yellow discoloration of bones was observed, but no changes were found microscopically, and this observation was concluded as toxicologically insignificant. The overall lack of adverse clinical signs, changes in body weight, feed consumption, clinical pathology parameters, and histopathological endpoints in animals administered AGIQ supports no observable adverse effect levels (NOAEL) of 5.0% in diet for both male and female rats. Based on this study, AGIQ can be safely used as a food additive.

P26: Action for FDA SEND: what pathologists need to know

T. Anzai1, H. Hatakeyama1, S. Horikawa2, J. Sakurai2, D. Potenta1, M. Wasko1, R. Aerni, H. Iwata3

1PDS Lifesciences
2INA Research
3LunaPath LLC

The Study Data Tabulation Model (SDTM), established by the Clinical Data Interchange Standards Consortium (CDISC) for US Food and Drug Administration (FDA) pharmaceutical submissions serves as the model for clinical study data. SEND is the implementation of SDTM for nonclinical studies and specifies the rules for standardizing nonclinical study data. SDTM is already used in over 60% of current clinical study applications. For studies starting on or after December 18, 2016, SDTM and SEND are required for studies starting after that date that support NDAS, BLAS, and ANDDAS. Studies supporting INDs will be required to conform to these standards by December 18, 2017.

Both SEND and SDTM require the mapping of controlled terminology (CT), and dealing with pathological terms is a challenge.

This poster emphasizes on the precautions and the roles of pathologists in SEND conversion of pathological terms.


Rittinghausen Susanne1, Baker Julia2, Bradley Alys3, Goodman Dawn G4, Harada Takanori5, Hayashi Shim-mo6, Herbert Ronald7, Kellner Rupert1, Mahler Beth7, Meseck Emily8, Nolte Thomas9, Ruehl-Fehlert Christine10, Vahle John11, Yoshizawa Katsuhiko12, Keenan Charlotte M13 

1Fraunhofer ITEM, Hannover, Germany
2Charles River, Frederick, MD
3Charles River, Tranent, Scotland, UK
4Independent Consultant, Potomac, MD
5The Institute of Environmental Toxicology, Joso-shi, Ibaraki, Japan
6San-Ei Gen F.F.I., Inc., Toyonaka, Osaka, Japan
7NIEHS, Research Triangle Park, NC
8Novartis Institute for Biomedical Research, East Hanover, NJ
9Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
10Bayer AG, Wuppertal, D-42096 Wuppertal, Germany
11Eli Lilly & Company, Indianapolis, IN
12Mukogawa Women's University, Nishinomiya, Hyogo, Japan
13CM Keenan ToxPath Consulting, Doylestown, PA

The INHAND Proposal (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) has been operational since 2005.  A Global Editorial Steering Committee (GESC) helps coordinate overall objectives of the project. Development of harmonized terminology for each rodent organ system or non-rodent species is the responsibility of the Organ Working Groups (OWG) or Non-rodent Working Groups (NRWG) respectively, drawing upon experts from North America, Europe and Japan.

Great progress has been made with 12 rodent organ systems published to date – Respiratory, Hepatobiliary, Urinary, Central/Peripheral Nervous Systems, Male Reproductive and Mammary, Zymbals, Clitoral and Preputial Glands in Toxicologic Pathology and the Integument and Soft Tissue, Female Reproductive System, Digestive System, Cardiovascular System and Skeletal System in the Journal of Toxicologic Pathology as supplements and on a web site – www.goReni.org.  Recommendations of the Apoptosis/Necrosis Working Group have been published. INHAND nomenclature guides offer terminology, diagnostic criteria, differential diagnoses and guidelines for recording lesions observed in toxicity and carcinogenicity studies.  The guides provide representative photo-micrographs of morphologic changes, information regarding pathogenesis, and key references. 

INHAND GESC representatives attended meetings with representatives of the FDA Center for Drug Evaluation and Research (CDER), Clinical Data Interchange Standards Consortium (CDISC), and the National Cancer Institute (NCI) Enterprise Vocabulary Services (EVS) to assist with incorporating INHAND terminology as preferred terminology for SEND (Standard for Exchange of Nonclinical Data) submissions to the FDA.  Interest in utilizing INHAND nomenclature, based on input from industry and government toxicologists as well as information technology specialists, is encouraging wide acceptance of this nomenclature.

P28: Goals of INHAND Non-Rodent Fish Working Group (NRFWG)

Heike Schmidt-Posthaus1, James Baily2, Wes Baumgartner3, Thomas Braunbeck4, Stephen Feist5, Satoshi Furukawa6, Yuki Katou7, Christine Rühl-Fehlert8, Stephen Schmidt9, Helmut Segner1, Jan Spitsbergen10, Jeff Wolf11

1Centre for Fish and Wildlife Health, University of Bern, Bern, Switzerland
2Charles River Laboratories, Edinburgh, UK
3Mississippi State University, United States
4University of Heidelberg, Heidelberg, Germany
5Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UK
6Nissan Chemical Industries, Ltd., Japan
7Shionogi & Co., Ltd., Osaka, Japan
8Pathology Wuppertal, Bayer AG, Wuppertal, Germany
9Iowa State University, Wisconsin, USA
10Oregon State University, Corvallis, USA
11Experimental Pathology Laboratories, Inc. Sterling, VA, USA

INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted system of preferred diagnostic criteria and nomenclature for non-proliferative and proliferative microscopic lesions in laboratory animals. The NRFWG (Non-rodent Fish Working Group) has been tasked with adapting standardized terminology that has been assembled for rodent studies to fish.  Fish are widely used laboratory models for studies that involve environmental contamination, mechanisms of human disease, and the safety and efficacy of pharmaceuticals employed in aquaculture or mariculture.  Advantages of commonly employed biomedical research species such as zebrafish (Danio rerio), medaka (Oryzias latipes), fathead minnow (Pimephales promelas), rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio) for bioassays with histopathology endpoints include the ability to dose animals via multiple exposure routes simultaneously (e.g. dermal, respiratory, and digestive), responsiveness to chemical manipulation, the ability to view multiple organ systems in a few whole body sections, the investigation of human toxicologic or carcinogenic responses not displayed by laboratory rodents, and in embryo models, the ability to perform high throughput screening.  The alignment of rodent and fish terminology is not without inherent challenges, however.  Foremost are fundamental anatomic and physiologic differences between fish and mammals, and likewise among various fish species. Consequently, although many diagnoses may be shared, others are unique to rodents or fish. The ultimate goals of the NRFWG are to produce a fish-relevant diagnostic lexicon, followed by a peer-reviewed publication that describes and illustrates diagnostic criteria to be used by toxicologic pathologists for the evaluation of studies intended for submission to regulatory agencies.  Examples will involve both chemically-induced and spontaneous lesions, the latter of which will include background infections.  Sources of material for this publication will include histopathology databases from government, academia, and industrial laboratories throughout the world, in addition to the scientific literature.  A widely-accepted and internationally generated system of harmonized nomenclature will serve to improve the exchange of information among toxicologists and pathologists, and will promote compatibility and comparability among vertebrate studies.

P29: Current activities of the RITA CEPA group – results of an interlaboratory study

Susanne Rittinghausen1 and Rupert Kellner1

1Fraunhofer Institute for Toxicology and Experimental Medicine ITEM

Immunohistochemical investigations are an essential prerequisite for the detection of specific antigens by biomarkers to determine cell types, cell cycle phase, and apoptotic events in preclinical studies performed for the development of new pharmaceuticals and chemicals. Mechanistic information and cell proliferation studies, especially if addressed to a specific cell type, are used as predictive tools for prospective tumor formation and such data are requested increasingly by the regulatory authorities. However, such studies are performed devoid of any regulatory guidelines and might be misinterpreted as a result of little or no experience in terms of immunohistochemical staining and analysis. The aim of the RITA-associated group CEPA (CEll Proliferation and Apoptosis) is to exchange and increase the knowledge in the field of cell proliferation assays, apoptosis research, immunohistochemistry techniques, and image analysis, and thus to enhance their quality.

In order to advance the quality and reproducibility of preclinical study results, the group has undertaken substantial activities for an interlaboratory comparison of immunohistochemistry and quality assessment with optional participation of the member companies. The consolidated results were kept coded for the different laboratories, but were presented to scientists and technicians and discussed intensively. All details of staining protocols and evaluation results, except company names, were made accessible to all CEPA members via the group’s website. For the interlaboratory comparison study, paraffin slides from predefined tissue blocks of formalin-fixed rodent organs were distributed to the volunteering laboratories by Fraunhofer ITEM. The technicians were requested to perform immunostaining of rat and mouse tissues with the cell proliferation marker Ki-67 and the macrophage marker F4/80, respectively. The organizing team offered proposals for the selection of antibodies, but these were not binding on the laboratories. Eleven laboratories sent back stained slides, which were then scanned by a slide scanner (Zeiss Mirax). For the evaluation and scoring of the immunostainings, a protocol was developed including criteria for proper demonstration of the stained antigen, false-positive results, nuclear counterstain, background and artifacts. The evaluation was performed via telepathology by a team of five pathologists from participating CEPA member companies. For Ki-67 immunohistochemical evaluation, liver, kidney, spleen and duodenum were scored. For F4/80, liver and spleen were evaluated. The poster presents and discusses the present CEPA activities and results of the interlaboratory study.

P30: Adversity of Lysosomal Accumulation in Toxicity Studies: The Pathologists' Point of View – Results from the 5th ESTP International Expert Workshop in Barcelona, Sept. 23 – 24, 2016

Popp, Andreas, AbbVie Deutschland GmbH & Co. KG, Germany; Braendli-Baiocco, Annamaria, Roche Innovation Center, Switzerland; Engelhardt, Jeff, Ionis, USA; Fant, Pierlugi, Charles River, France; Fischer, Holger, Roche Innovation Center, Switzerland; Francke, Sabine, US-FDA, USA; Fukuda, Ryo, Takeda, Japan; Gröters, Sibylle, BASF SE, Germany; Harada, Takanori, Institute of Environmental Toxicology, Japan; Harleman, Johannes, Fresenius Kabi, Germany; Kaufmann, Wolfgang, Merck KGaA, Germany; Kustermann, Stefan, Roche Innovation Center, Switzerland; Lenz, Barbara, Roche Innovation Center, Switzerland; Nolte, Thomas, Boehringer Ingelheim Pharma GmbH & Co. KG, Germany; Palazzi, Xavier, Pfizer, USA; Pohlmeyer-Esch, Gabriele, Boehringer Ingelheim Pharma GmbH & Co. KG, Germany; Romeike, Annette, Covance Laboratories Inc., France; Schulte, Agnes, Federal Institute for Risk Assessment, Germany; Silva Lima, Beatriz, Faculdade de Farmácia, Universidade de Lisboa, Portugal; Tomlinson, Lindsay, Pfizer, USA; Willard, James, US FDA, USA; Wood, Charles, US-EPA, USA; Yoshida, Midori, Food Safety Commission of Japan, Japan

The European Society of Toxicologic Pathology (ESTP) organized a series of international workshops due to the recognized need of the toxicological community to better align on the determination of nonclinical adversity. An introductory workshop in 2015 defined and characterized adversity and developed general recommendations for a practical approach to evaluate the adversity in toxicology studies (Palazzi et al., 2016). Building on this, a lesion-specific workshop entitled “Adversity of Lysosomal Accumulation” was held in Barcelona, Spain, on September 23–24, 2016. 

Lysosomal accumulation is a common issue in nonclinical studies, as a result of the specific role of lysosomes in cellular uptake and metabolism of endogenous and exogenous substances, and it can be influenced by the physicochemical properties of drugs and chemical agents.

Twenty-three international expert pathologists, toxicologists and research scientists representing pharmaceutical and chemical industries, contract research organizations and regulatory authorities from Europe, the United States, and Japan met in Barcelona after a 7-month preparatory phase of teleconferences with several expert contributions. The approach of this workshop was holistic, taking into account analytical and research methods available for assessing lysosomal accumulation, in addition to the routine microscopic evaluation of H&E-stained tissues.

The expert working group agreed that the diversity of lysosomal accumulations requires a case-by-case weight-of-evidence approach and outlined several factors to consider in the adversity assessment, including location and type of cell affected, lysosomal contents, the severity of the accumulation, and related pathological effects, including evidence of cellular or organ dysfunction. Lysosomal accumulations associated with cytotoxicity, inflammation, or fibrosis would generally be considered adverse, while those found in isolation may or may not have functional consequences and could represent a diagnostic challenge. Workshop examples highlighted the importance of thoroughly characterizing the biological context of lysosomal effects, including mechanistic data and functional in-vitro read-outs if available. This information should support greater consistency and transparency in the interpretation of lysosomal effects.

This poster reflects the views of the authors and should not be construed to represent FDA’s or other authorities’ views or policies.

P31: Hypertrophic osteopathy in two cynomolgus monkeys secondary to opportunistic infections during a preclinical study with an immunomodulator

Sobry Cécile1 , Flandre Thierry2, Forster Roy1, and Palate Bernard1 

1 CiToxLAB France
2Novartis Pharma AG

Hypertrophic osteopathy (HO) is characterized by pathologic periosteal new bone formation in the long bones. This syndrome is reported in humans and many animal species, and develops secondary to various severe, mostly intra-thoracic, neoplastic or non-neoplastic conditions. The exact pathogenesis of secondary HO is still unknown. One popular theory involves the presence in the systemic circulation of angiogenic and bone-remodeling growth factors that are normally inactivated in the lungs. There are only two reports of HO in laboratory species used in preclinical studies, in rats, and three reports in monkeys, in zoo animals. We describe HO in monkeys associated with severe cytomegalovirus (CMV) systemic infection and pneumonia following treatment by an immunomodulatory drug. Bone lesions characteristic for HO were identified in 2/28 treated monkeys, while pharmacological immune depression and secondary opportunistic CMV infections were present in all animals at mid and high dose-levels. These two individuals showed the most severe clinical signs and pulmonary changes. To our knowledge, this is the first report of HO in cynomolgus macaques (Macaca fascicularis) occurring in a preclinical toxicological study.

P32: Intranodal angiomyomatous hamartoma in a cynomolgus monkey (Macaca fascicularis)

Catherine Thirion-Delalande1, André Almeida Schenka1, Frédéric Gervais1, Roy Forster1 and Bernard Palate1

1CiToxLAB France, Evreux

An angiomyomatous hamartoma is described in the right axillary lymph node of a three-year old male Mauritius monkey (Macaca fascicularis), used as a control subject in a short-term toxicity study. This is a very rare lesion which has been reported almost exclusively in inguinal lymph nodes, and to date only in human beings. Light microscopy revealed partial replacement of the lymph node parenchyma by a disorganized, irregular vascular network, sparsely distributed smooth muscle cells and a fibro-adipocytic stroma confirmed by the immunohistochemical analysis. It was considered to be a congenital abnormality, given the age of the animal, with no special clinical or toxicological significance. To the best of our knowledge, this is the first report of an intranodal angiomyomatous hamartoma in an animal species.

P33: Dual Infection with Canine Circovirus and Canine Parvovirus in a Beagle Dog at a Nonclinical Research Laboratory in Michigan, USA

Laura Zwick1, Dale Cooper1, Marci Harter1, and Daniel Patrick1

1MPI Research, Mattawan, Michigan, USA

6-month-old male beagle dog with a current vaccination history in the high dose group of a 91-day oral toxicity study of a pharmaceutical compound at a nonclinical contract research laboratory in Michigan, USA died on study day 9. No premonitory clinical signs were present and there were no abnormalities in pretest clinical pathology data. Gross findings included red discoloration of the lungs and a small gallbladder. Microscopic findings included meningoencephalitis with necrotizing vasculitis, enterocolitis with rare epithelial amphophilic intranuclear inclusion bodies, and multicentric lymphoid necrosis and hemorrhage. At the scheduled study termination, none of the remaining dogs exhibited similar microscopic lesions, and there were no significant compound-related findings in any animal. Polymerase chain reaction (PCR) testing of paraffin-embedded formalin-fixed sections of intestine from the affected dog tested positive for canine circovirus and canine parvovirus. The lesions in this dog were consistent with reported findings associated with these viruses. Canine circovirus infection has recently been identified in dogs and has been associated with fibronecrotizing vasculitis, hemorrhage, and lymphoid necrosis, although the pathogenesis is unknown and no current vaccine is available. Dual infection with canine circovirus and canine parvovirus associated with enteritis and lymphoid necrosis has been documented in dogs despite up-to-date vaccination against canine parvovirus, and may indicate a synergistic relationship of these pathogens. Lesions associated with infection with these viruses could confound drug safety evaluation, and additional investigation is needed to elucidate the pathogenesis and potential preventative measures. No additional cases have been identified at this laboratory.

P34: Spontaneous Periductal Cholangiofibrosis in the Liver and Pancreas of Male and Female Harlan Sprague Dawley Rats

Torrie A. Crabbs1, Margarita M. Gruebbel1, Jerry F. Hardisty1, Cynthia C. Shackelford1, David E. Malarkey2, Mark Cesta2

1Experimental Pathology Laboratories, Inc. (EPL)
2National Institute of Environmental Health Services/National Toxicology Program

Hepatic cholangiofibrosis in rats is a chronic, focal to diffuse process characterized by abnormal bile ducts that exhibit dilation, hyperplasia, and/or intestinal metaplasia, and are surrounded by dense collagenous connective tissue. In rats, hepatic cholangiofibrosis has only been recognized as an induced change following administration of certain compounds, such as furan. It has also been associated with a genetically related hepatotoxicity in the Long-Evans Cinnamon (LEC) rat, a rodent model of Wilson's disease. A recent review of an on-going carcinogenicity/chronic toxicity study included the histopathologic examination of the liver and pancreas from 630 male and 630 female Harlan Sprague Dawley (HSD) rats. Focal cholangiofibrosis was noted in the liver or pancreas of eight male and twenty-one female HSD rats; two affected animals (one male and one female) were unexposed controls. Incidences in exposed groups were low, sporadic, and unrelated to dose, and thus, considered incidental and unrelated to exposure. In males, 100% of cases (8/8) were located in or closely associated with the liver; were almost always near, if not external to, the liver capsule; and were almost always in close proximity to the main hepatic bile duct. In contrast, 76% of cases in females (16/21) were associated with the pancreas, specifically in a region adjacent and/or immediately proximal to the entrance of the common bile duct into the proximal duodenum. Given the focal distribution of the lesions in the HSD rat and their frequent close proximity to a large bile duct, these findings were recorded as “periductal cholangiofibrosis” (in the liver or pancreas) to differentiate this apparent spontaneous finding from the chemically induced or genetically related hepatic cholangiofibrosis that has previously been reported in rats. To the authors’ knowledge, this is the first report of cholangiofibrosis as a spontaneous change in rats and also the first report of cholangiofibrosis in an extrahepatic location (pancreas).

P35: Malignant mast cell tumor of the thymus in Royal College of Surgeons (RCS) rat

Kiyokazu Ozaki1, Yui Terayama1 and Tetsuro Matsuura1 

1Laboratory of Pathology, Faculty of Pharmaceutical Sciences, Setsunan University

Introduction: Mast cell tumors are extremely rare in rats: among case reports and National Toxicology Program (NTP) pathology database entries, there have only been two and twelve cases of rat mast cell tumors reported, respectively. This report describes the histological characteristics of a spontaneous malignant mast cell tumor with thymic epithelial hyperplasia in a male Royal College of Surgeons (RCS) rat.

Case description: A 152-week-old RCS male rat was kept as a non-treated animal in a long-term study and fed a standard diet and chlorinated water ad libitum. Apart from coarse hair, no clinical signs were apparent until the scheduled sacrifice at 152 weeks of age. 

Grossly, a mass of 40 × 30 × 15 mm was observed in the anterior mediastinum.  Small masses that were adherent to the lung and the esophagus were identified around the main mass. The mass was soft and the cut surface was milky-white to yellow and gray-white in color.

Histopathologically, the mass mainly consisted of round to short spindle-shaped tumor cells that had infiltrated through the hyperplastic thymic tissue. The tumor cells were arranged in loose to dense sheets. Nuclei were moderate in size and round to spindle-shaped, with small nucleoli. Almost all tumor cells exhibited abundant eosinophilic cytoplasm, including eosinophilic granules of a range of sizes. The granules of tumor cells exhibited metachromasia with toluidine blue stain and were positive for c-kit- and mast cell protease II.

Discussion: These findings indicate that the tumor described here represents a rare case of spontaneous malignant mast cell tumor with thymic epithelial hyperplasia.

P36: Phenotyping analysis of p53-mutant mice produced by TALEN and comparison with conventional p53-mutant mice

Ukjin Kim1, C-Yoon Kim1, Ji Min Lee1, Seo-Na Chang1, Hanseul Oh1, Bokyeong Ryu1, Jin Kim1 and Jae-Hak Park1

1Department of Laboratory Animal Medicine, BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National University

Introduction: Transgenic (Tg) Animal models developed by homologous recombination (HR)-mediated gene knockout became useful tools to elucidate functions of the gene. However, HR had low knockout efficiency and required much time, labor and expenses. Based on application of nucleases combined with sequence-specific DNA-binding domain, 'Genome editing' received attention for its efficient gene editing. Transcription activator-like effector nucleases (TALENs) contain FokI nuclease fused to DNA binding domain. This DNA binding domain known as transcription activator-like effectors (TALEs) contain 33-35 amino acid repeats domains that recognize a single base pair of the DNA. The TALE repeats use four repeat-variable di-residues (RVDs) domains NN, NI, HD and NG which recognize guanine, adenine, cytosine and thymidine respectively to determine the TALE specificity. As producing Tg animals became easier with genome editing, re-producing and utilizing Tg animals which were already produced by HR is expected to be realized.

Objective: However, phenotyping analysis and comparison between same gene-targeted animal models produced by two techniques must be prioritized. 

Material & Methods: We made p53-mutant mice with TALEN and analyzed phenotype by comparing with HR-mediated p53-mutant mice. 

Results: Tumors occurred in 36 (74%) of 49 homozygous mutant mice and the mean age of occurrence was 23 weeks. Lymphoma (64%) and sarcoma (23%) were the most common. Heterozygotes developed tumors in 12 mice and the mean age of occurrence was 40 weeks. Sarcoma (54%) and lymphoma (46%) were observed in high proportion. Homozygotes had a mean life span of 157 ± 52 days and developmental abnormalities (P <0.05 and P <0.001) were found in the female compared to male. 

Conclusion: We have analyzed the basic phenotype of p53-mutant mice and found that these mice were not significantly different from conventional HR-mediated p53-mutant mice.

P37: A survey on the working conditions for toxicologic pathologists in Europe

Lars Mecklenburg1, Erio Barale2

1Foell, Mecklenburg & Partner GmBH, Münster, Germany
2Jannsen Research & Development, Beerse, Belgium

Throughout the last decade, the (bio)pharmaceutical and chemical industry has undergone major structural changes, which also affected toxicologic pathologists. To gain a better understanding of the current and future working conditions for toxicologic pathologists in Europe, the European Society of Toxicologic Pathologists (ESTP) and the French Society of Toxicologic Pathology (SFPT) conducted a survey in 2015. Data from this survey shows that there is an almost equal distribution of male versus female toxicologic pathologists in Europe. The majority of them work for big pharmaceutical companies located in Western Europe and reads slides from GLP or non-GLP studies.

About 60% of the toxicologic pathologists in Europe will retire within the next 15 years, and there appears to be an insufficient number of young people to fill the open positions that are forecasted for the next decade. Availability of qualified toxicologic pathologists appears to be an obstacle when filling open positions, although remuneration is rather competitive. The limited geographical flexibility of candidates may be a factor that could be overcome by developing new remote working models.

The information in this survey is not easily available and is expected to assist both pathologists in training, who intend to apply for a toxicologic pathology position in Europe, and  established toxicologic pathologist and their employers, who need to adapt to an ever changing working environment.

P38: Pathological study of pulmonary toxicity induced by intratracheally instilled Asian sand dust (Kosa): effects of lowered serum zinc level on the toxicity

Akinori Shimada, Kotaro Miyake, Yuri Kenmotsu, Kikumi Ogihara, Yuko Naya

Department of Pathology, School of Life and Environmental Science, Azabu University


Exposure to Asian sand dust (ASD) is associated with enhanced pulmonary morbidity and mortality, and the reporting of such cases has rapidly increased in East Asia since 2000. We reported ASD induced acute and chronic pulmonary inflammatory changes in mice (Naota et al., 2010, 2013, Shimada et al., 2015). Zinc (Zn) is reported to have an ability to influence inflammation by the production and signaling of numerous inflammatory cytokines including TNF-α and IL-1β in a variety of cell types. The purpose of the study was to assess the effects of lowered level of serum Zn on the lung toxicity induced by ASD.

Material and methods

A total of 40 ICR mice fed a CE-2 diet (containing normal level of Zn, CLEA, Japan) and 40 ICR mice fed an A12551 diet (containing low level of Zn, CLEA) were randomly divided into 5 control (n=3) and 5 exposure (n=5) groups. Suspensions of 3.0 mg of ASD particles (CJ-2, General Science Corporation, Tokyo, Japan) in saline were intratracheally instilled into mice showing normal level (average 93.46ug/dl) of serum Zn and mice showing low level of serum Zn (less than 40 ug/dl), followed by sacrifice at 24 hours, 2 weeks, and 1, 2 and 3 months after instillation. Paraffin sections of lung tissues were stained by hematoxylin and eosin and by immunohistochemistry to detect cytokines (TNF-α, IL1-β), an autophagy marker (LC-3) and a lysosome marker (Lamp1). Selected lung tissues were examined by electron microscopy.


A lung histological examination revealed similar patterns in the lesions of normal and low level of serum Zn groups treated with ASD. Interstitial inflammation characterized by neutrophil infiltration and macrophage accumulation was observed throughout the time course observed; the inflammatory changes were more prominent and persistent in low level of serum Zn groups. Neutrophil infiltration dominated at 24 hours after the treatment. Thereafter, an increase in the number of macrophage at the alveolar septa was observed over time. Cytoplasmic vacuolar change of enlarged macrophages containing ASD fine particles was observed in the lesion; the changes dominated in low level of serum Zn groups. The cytoplasm of macrophages in the inflammatory lesions showed positive immunolabeling for cytokines including TNF-α and IL1-β throughout the time period examined and semi-quantative analysis showed larger number of TNF-α and IL1-β positive macrophages in normal and low level of serum Zn groups, respectively. In addition, positive immunolabeling for LC-3 (an autophagy marker) and Lamp1 (a lysosome marker) were also shown in the cytoplasm of macrophages containing ASD fine particles; LC-3 positivity was less prominent in low level of serum Zn groups. Electron microscopy demonstrated swollen mitochondrion and dilated lysosomes containing ASD fine particles in the cytoplasm of low level of serum Zn groups.


These findings suggest that lowered serum Zn level may induce modulation of cytokine expression and lysosomal malfunction including phagocytosis and/or autophagy pathway and resultant persistence of interstitial pyogranulomatous inflammation in the lungs of mice treated with ASD.