Immortalization of cells derived from domestic dogs through expressing mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase.

Dog is the first animal that was established as a close partner of human beings. Based on the vast genetic diversity and breeding, dogs exhibit unique genetic evolution and diversity from Chihuahua to St. Bernard. The safety tests of the pharmacological products also included domestic dogs as the test subjects. Although the safety confirmation test of chemicals for human use is important, the welfare of experimental animals requires special consideration. In this study, we cultured domestic dog-derived primary fibroblasts isolated from their muscle tissues. Furthermore, we successfully immortalized them through lentivirus-mediated gene transfer of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). We further demonstrated that the established immortalized domestic dog-derived fibroblasts retained the characteristics of the original parental cells. These cells might act a suitable in vivo model system to replace the implication of animals for evaluating the potential toxicity of pharmacological chemicals. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00504-0.

[Usefulness of an anti-mouse cerebellar tissue-derived antigen antibody test in predicting immunotherapy efficacy in patients with idiopathic cerebellar ataxia].

BACKGROUND: Autoimmune cerebellar ataxia (AICA) is a general term for diseases in which the cerebellum is damaged by an autoimmune mechanism. For the diagnosis of the AICA, anti-thyroid antibodies (anti-thyroid peroxidase antibody and anti-thyroglobulin antibody), anti-glutamic acid decarboxylase (GAD) antibodies, and anti-gliadin antibodies are measured. Immunotherapy is known to be effective for AICA, but some patients with effective immunotherapy lack autoantibodies associated with cerebellar ataxia. The purpose of this study was to clarify whether the effectiveness of immunotherapy in patients with suspected AICA could be predicted by anti-mouse cerebellar tissue-derived antigen antibody tests. METHODS: This study was conducted on 25 patients with idiopathic cerebellar ataxia (excluding multiple system atrophy, hereditary spinocerebellar degeneration, cancer-bearing patients, and patients taking phenytoin) who received immunotherapy from 2005 to 2016 at Tokyo Medical University Hachioji Medical Center. The patients were suspected of having AICA because they were positive for cerebellar ataxia-related autoantibodies (anti-thyroid antibody, anti-GAD antibody, anti-gliadin antibody, or anti-transglutaminase 6 antibody) or other autoantibodies. Antibodies that bind to mouse cerebellar tissue-derived antigens were defined as "anti-mouse cerebellar tissue-derived antigen antibodies" in this study, and their IgG-class antibodies were comprehensively measured using a slot blot. RESULTS: Anti-mouse cerebellar tissue-derived antigen antibody test results were correlated with immunotherapy efficacy. Furthermore, the combination of anti-mouse cerebellar tissue-derived antigen and anti-GAD antibody tests could predict the effectiveness of immunotherapy with 83% sensitivity and 100% specificity, while the combination of the anti-mouse cerebellar tissue-derived antigen, anti-GAD, and anti-gliadin (IgA class) antibody tests could predict the effectiveness of immunotherapy with 94% sensitivity and 86% specificity. CONCLUSION: Anti-mouse cerebellar tissue-derived antigen antibody tests could help to provide useful information for immunotherapy administration to patients with idiopathic cerebellar ataxia suspected to be AICA.

Calpain-1 C2L domain peptide protects mouse hippocampus-derived neuronal HT22 cells against glutamate-induced oxytosis.

Calpains are Ca2+-dependent cysteine proteases; their aberrant activation is associated with several neurodegenerative diseases. The µ-calpain catalytic subunit, calpain-1, is located in the cytoplasm as well as in the mitochondria. Mitochondrial calpain-1 cleaves apoptosis-inducing factor (AIF), leading to apoptotic cell death. We have previously reported that short peptides of calpain-1 C2-like domain conjugated with cell penetrating peptide HIV-Tat (Tat-µCL) selectively inhibit mitochondrial calpain-1 and effectively prevent neurodegenerative diseases of the eye. In this study, we determined whether mitochondrial calpain-1 mediates oxytosis (oxidative glutamate toxicity) in hippocampal HT22 cells using Tat-µCL and newly generated polyhistidine-conjugated µCL peptide and compared their efficacies in preventing oxytosis. TUNEL assay and single strand DNA staining revealed that both µCL peptides inhibited glutamate-induced oxytosis. Additionally, both the peptides suppressed the mitochondrial AIF translocation into the nucleus. All polyhistidine-µCL peptides (containing 4-16 histidine residues) showed higher cell permeability than Tat-µCL. Notably, tetrahistidine (H4)-µCL exerted the highest cytoprotective activity. Thus, H4-µCL may be a potential peptide drug for calpain-1-mediated neurodegenerative diseases such as Alzheimer's disease.