Antisense Oligonucleotide-Related Macrovesicular Vacuolation of Hippocampal Neurons in Nonhuman Primates.

2'-methoxyethyl (MOE) antisense oligonucleotides (ASOs) tested in multidose intrathecal nonhuman primate (NHP) toxicity studies have consistently revealed the presence of single large vacuoles in pyramidal neurons of the hippocampus in the absence of any cellular response. Termed "macrovesicular," these vacuoles were characterized by immunohistochemistry and transmission electron microscopy which showed that these vacuoles are dilated lysosomes in neurons containing accumulated ASO. Additionally, two NHP studies were conducted to investigate the role of tissue fixation on their histogenesis. In Fixation Study 1, 6 doses of 5 mg 2'-MOE ASO with a full phosphorothioate backbone were administered by lumbar puncture over 5 weeks; in Fixation Study 2, 5 doses of 35 mg 2'-MOE ASO with a mixed phosphorothioate/phosphodiester backbone were administered over 12 weeks. At necropsy in each study, brain slices were either immersion fixed in neutral buffered 10% formalin or Carnoy's fixative; frozen at -80 °C; or perfusion fixed with modified Karnovsky's fixative. Fixed samples were processed to paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and compared with H&E cryosections prepared from the frozen tissue of the same brain. The presence of vacuoles in fixed brain tissue but never in fresh frozen tissue showed that they arose during postmortem tissue fixation, and as such represent a processing artifact that is not relevant to human safety assessment of intrathecally administered 2'-MOE ASOs.

Scientific and Regulatory Policy Committee Points to Consider: Data Visualization for Clinical and Anatomic Pathologists.

Assessment and communication of toxicology data are fundamental components of the work performed by veterinary anatomic and clinical pathologists involved in toxicology research. In recent years, there has been an evolution in the number and variety of software tools designed to facilitate the evaluation and presentation of toxicity study data. A working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee reviewed existing and emerging visualization technologies. This Points to Consider article reviews some of the currently available data visualization options, describes the utility of different types of graphical displays, and explores potential areas of controversy and ambiguity encountered with the use of these tools.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 1 of 2).

Spontaneous rodent progressive cardiomyopathy (PCM) in the Sprague Dawley rat may confound identification and/or interpretation of potential test article (TA)-related cardiotoxicity. Pathologists apply diagnostic term(s) and thresholds for diagnosing and assigning severity grades for PCM and/or PCM-like (PCM/like) lesions consistently within a study, which is necessary to identify and interpret TA-related findings. Due to differences in training and/or experiences, diagnostic terms and thresholds may vary between pathologists. Harmonized terminology and thresholds across studies will generate better historical control data, will likely enhance interpretation of study data, and may further enhance our understanding of the spontaneous change. An assessment of the diagnostic approaches of a group of 37 pathologists identified an approach that is relatively easily applied; and if adopted, it could enhance diagnostic consistency across studies. This approach uses the single "slash" term "necrosis/inflammatory cell infiltrate (NICI)" as the diagnosis for the spectrum of lesions seen in younger rats, uses no threshold for diagnosis (e.g., diagnose all lesions clearly identifiable as PCM/like), and uses aggregate lesion size of approximately ≥45% of the field of view (FOV) using a 10×/22 eyepiece and the 40× objective or approximately ≥100% of the FOV using the 60× objective as the criterion separating minimal from mild severities.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 2 of 2).

To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist's examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions. In exercise #2, there was improved consistency of categorization of small borderline morphologies and mild lesions, but a decrement in consistency of categorizing minimal lesions. Exercises 1 and 2 suggest the described diagnostic approach is representative of that in use by the majority of toxicologic pathologists across companies/agencies and that application by all may improve diagnostic consistency of PCM/like lesions. Additionally, a criterion of approximately 5% heart section involvement is suggested for separating mild from moderate or greater severity. While evidence is not absolute, until further investigation shows otherwise, microscopic changes resembling PCM, but located in the epicardial and subepicardial region of the right ventricle, may be considered as part of the spectrum of PCM.

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

A Mixed-Methods Investigation Into the Acceptability, Usability, and Perceived Effectiveness of Active and Passive Virtual Reality Scenarios in Managing Pain Under Experimental Conditions.

Burn patients often suffer excruciating pain during clinical procedures, even with analgesia. Virtual Reality (VR) as an adjunct to pharmacological therapy has proved promising in the management of burn pain. More evidence is needed regarding specific forms of VR. This mixed-method study examined the impact of active and passive VR scenarios in experimental conditions, gathering data relating to user experience, acceptability, and effectiveness in managing pain. Four scenarios were developed or selected following a consultative workshop with burn survivors and clinicians. Each was trialed using a cold pressor test with 15 University students. Data were gathered regarding pain threshold and tolerance at baseline and during each exposure. Short interviews were conducted afterwards. The two active scenarios were ranked highest and significantly extended participants' pain threshold and tolerance times compared to passive and baseline conditions. Passive scenarios offered little distraction and relief from pain. Active scenarios were perceived to be engaging, challenging, distracting, and immersive. They reduced subjective awareness of pain, though suggestions were made for further improvements. Results suggested that active VR was acceptable and enjoyable as a means of helping to control experimental pain. Following suggested improvements, scenarios should now be tested in the clinical environment.

Feline epitheliotropic intestinal malignant lymphoma: 10 cases (1997-2000).

The clinical, histopathologic, and immunohistochemical features of 10 cats with epitheliotropic intestinal malignant lymphoma (EIL) are described. Intestinal biopsy samples were reviewed by 3 pathologists to confirm the diagnosis of EIL. These samples (n = 10) were compared to the intestinal biopsies of normal cats (n = 11), cats with inflammatory bowel disease (IBD; n = 7), and cats with non-EIL (n = 9) for quantification and immunophenotyping of intraepithelial lymphocytes. Immunophenotypic studies were performed with CD3 and CD79a antibody stains to assess for T- and B-cell immunoreactivity, respectively. EIL biopsies had markedly more intraepithelial lymphocytes than normal intestine (NRL) and samples from cats with IBD. However, no marked difference was observed in the number of intraepithelial lymphocytes in cats with non-EIL compared to cats with EIL. Regardless of the histologic diagnosis, the intraepithelial lymphocytes in all cats were small- to intermediate-sized T cells. Clinical findings and imaging studies in the cats identified minimal or nonspecific findings in affected cats. Most cats fit the typical profile of cats with IBD or alimentary malignant lymphoma. Nine of 10 cats with EIL were treated with prednisone with or without additional chemotherapy. Four cats were refractory to chemotherapy and were euthanized within 3.5 months. The remaining 5 cats had long-term survival times of 11 months or greater. The median survival time was 11 months. Additional studies are warranted to better characterize EIL and its relationship to IBD in cats and non-EIL and to identify optimal treatment strategies for this disease.

Discovery of novel and potent leukotriene B4 receptor antagonists. Part 1.

The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4)) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found to be highly potent inhibitors of LTB(4) binding to BLT1 and BLT2 receptors, with varying levels of selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB(4) and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity experiment provided the rationale for differentiation and selection of one compound (33) for clinical development.