Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - Part IV: the ECETOC and CLE Proposal for a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS).

Following the European Commission Endocrine Disruptor Criteria, substances shall be considered as having endocrine disrupting properties if they (a) elicit adverse effects, (b) have endocrine activity, and (c) the two are linked by an endocrine mode-of-action (MoA) unless the MoA is not relevant for humans. A comprehensive, structured approach to assess whether substances meet the Endocrine Disruptor Criteria for the thyroid modality (EDC-T) is currently unavailable. Here, the European Centre for Ecotoxicology and Toxicology of Chemicals Thyroxine Task Force and CropLife Europe propose a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS). In Tier 0, before entering the Thyroid-NDT-TAS, all available in vivo, in vitro and in silico data are submitted to weight-of-evidence (WoE) evaluations to determine whether the substance of interest poses a concern for thyroid disruption. If so, Tier 1 of the Thyroid-NDT-TAS includes an initial MoA and human relevance assessment (structured by the key events of possibly relevant adverse outcome pathways) and the generation of supportive in vitro/in silico data, if relevant. Only if Tier 1 is inconclusive, Tier 2 involves higher-tier testing to generate further thyroid- and/or neurodevelopment-related data. Tier 3 includes the final MoA and human relevance assessment and an overarching WoE evaluation to draw a conclusion on whether, or not, the substance meets the EDC-T. The Thyroid-NDT-TAS is based on the state-of-the-science, and it has been developed to minimise animal testing. To make human safety assessments more accurate, it is recommended to apply the Thyroid-NDT-TAS during future regulatory assessments.

Preclinical toxicity evaluation of erythrocyte-encapsulated thymidine phosphorylase in BALB/c mice and beagle dogs: an enzyme-replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy.

Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is currently under development as an enzyme replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder caused by a deficiency of thymidine phosphorylase. The rationale for the development of EE-TP is based on the pathologically elevated metabolites (thymidine and deoxyuridine) being able to freely diffuse across the erythrocyte membrane where the encapsulated enzyme catalyses their metabolism to the normal products. The systemic toxic potential of EE-TP was assessed when administered intermittently by iv bolus injection to BALB/c mice and Beagle dogs for 4 weeks. The studies consisted of one control group receiving sham-loaded erythrocytes twice weekly and two treated groups, one dosed once every 2 weeks and the other dosed twice per week. The administration of EE-TP to BALB/c mice resulted in thrombi/emboli in the lungs and spleen enlargement. These findings were also seen in the control group, and there was no relationship to the number of doses administered. In the dog, transient clinical signs were associated with EE-TP administration, suggestive of an immune-based reaction. Specific antithymidine phosphorylase antibodies were detected in two dogs and in a greater proportion of mice treated once every 2 weeks. Nonspecific antibodies were detected in all EE-TP-treated animals. In conclusion, these studies do not reveal serious toxicities that would preclude a clinical trial of EE-TP in patients with MNGIE, but caution should be taken for infusion-related reactions that may be related to the production of nonspecific antibodies or a cell-based immune response.

Murid herpesvirus-4 induces chronic inflammation of intrahepatic bile ducts in mice deficient in gamma-interferon signalling.

AIM: Infection of gamma interferon receptor defective mice with murid herpesvirus-4 also known as murine gammaherpesvirus-68 results in multi-organ fibrosis. In this paper we characterise the pathological changes occurring in the liver in this model. METHODS: Standard immunohistochemistry and in situ hybridisation techniques were used to identify the cellular changes and the presence of virus at different times post infection. RESULTS: In liver sections from infected gamma interferon receptor defective mice sampled on day 16 to at least day 120, 79% showed proliferating intrahepatic bile ducts associated with a chronic mononuclear cell inflammation. Only 8% of wild type mice showed similar lesions. Coincident with the inflammatory response bile duct epithelial cells were positive for arginase 1. Around day 50 post infection onwards focal fibrotic lesions appeared in approximately 30% of gamma interferon receptor defective mice resulting in destruction of intrahepatic bile ducts. In contrast to the chronic persisting inflammatory response the presence of virus infected cells were only observed between day 12-20 post-infection. CONCLUSION: Infection of gamma interferon receptor defective mice with a murine gammaherpesvirus initiates a chronic persisting inflammatory response with a pathological profile similar to the human fibrotic liver disorder Primary Sclerosing Cholangitis.

Murine gammaherpesvirus-induced fibrosis is associated with the development of alternatively activated macrophages.

Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen of rodents closely related to the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and EBV. Following intranasal infection, the virus replicates in the lung epithelium prior to establishing latent infection in lymphoid tissue. Infection of mice deficient in IFN-gammaR signaling (IFN-gammaR-/-) results in a multiple organ fibrosis, in which the spleen is severely affected. We show here that by Day 12 postinfection, prior to development of fibrosis in the spleens of IFN-gammaR-/- mice, different subsets of splenic macrophages (Mvarphis) are morphologically activated and enter latently infected germinal centers (GCs). Mvarphis coexpressing arginase I (ARG1), a marker of alternative activation of Mvarphis, and murine Mvarphi markers F4/80, ER-TR9, and MOMA-1 are found in GCs of IFN-gammaR-/- mice but not of wild-type mice. Quantitative RT-PCR of spleen RNA confirms induction of ARG1 and in addition, shows up-regulation of found in inflammatory zone 1/resistin-like molecule-alpha, tissue inhibitor of metalloproteinase-1, matrix metalloproteinase-12, fibronectin, and factor XIIIA in IFN-gammaR-/- mice. In contrast, inducible NO synthase, associated with classical Mvarphi activation, is up-regulated following infection of wild-type mice but not IFN-gammaR(-/-) mice. Concomitant with the aaMvarphis, transcription of the Th2 cytokines IL-13, IL-21, and IL-5 is up-regulated. Thus, in the absence of IFN-gammaR signaling, MHV-68 initiates a Th2 immune response, leading to alternative activation of macrophages and induction of fibrosis. This system provides an important model for studying the pathogenesis of fibrosis initiated by a latent herpesvirus infection.

The M4 gene of murine gammaherpesvirus 68 modulates latent infection.

Murine gammaherpesvirus 68 (MHV-68) encodes a set of unique genes, M1, M2, M3 and M4, and eight non-translated tRNA-like molecules that are thought to be important in virus-host interactions and latent infection. The M4 gene is predicted to encode a novel secreted protein. To investigate the role of M4 in viral pathogenesis, a mutant MHV-68 that did not express M4 was constructed and its replication was characterized in vitro and in vivo. Virus replication was identical to the wild type in vitro and no difference could be detected in virus replication in the lung following intranasal infection. However, in the spleen, virus deficient in M4 expression was severely attenuated in the establishment of latency. These results indicate a critical role for M4 in MHV-68 pathogenesis.

Prevalence of antibodies to Toxoplasmagondii and Neosporacaninum in red foxes (Vulpesvulpes) from around the UK.

The purpose of this study was to establish the prevalence of antibodies to Toxoplasma gondii and Neospora caninum in a random sample of red foxes from around the UK. Lung fluid from over 500 foxes was examined using an indirect fluorescent antibody test. Reciprocal titres of specific antibodies to T. gondii or N. caninum ranged from < 1:16 to 1:1024. A titre of 1:128 or greater was deemed indicative of exposure to the parasite. One hundred and eleven (20%) of the 549 foxes tested were seropositive to T. gondii, and only five (0.9%) were seropositive to N. caninum. No correlation could be made between positive samples and geographical distribution, as sample numbers varied greatly between regions. The results of this study indicate that red foxes of the UK have more exposure to T. gondii than to N. caninum in their environment.

Identification of a region of the virus genome involved in murine gammaherpesvirus 68-induced splenic pathology.

Infection with the murine gammaherpesvirus MHV-68 has profound effects on splenic and mediastinal lymph node pathology in mice which lack the interferon-gamma receptor (IFN-gamma R(-/-)). In these mice MHV-68 infection causes fibrosis and loss of lymphocytes in the spleen and the mediastinal lymph node as well as interstitial pulmonary fibrosis and fibrotic changes in the liver. The changes are associated with transient elevated latent virus loads in the spleen. Four independent virus mutants with insertions and/or deletions in the left end of the genome fail to induce the pathological changes and establish latency at normal levels in the spleen. The data indicate that the pathology does not correlate with any of the known genes encoded within this region of the genome, genes M1-M4 and the eight vtRNAs. Northern analysis of mRNAs transcribed by wild-type and mutant viruses shows that at least two uncharacterized transcripts are encoded within this region. These transcripts are absent in the mutant viruses and are candidates for the virus genes responsible for the aberrant pathology in IFN-gamma R(-/-) mice.