Th1/17 polarization and potential treatment by an anti-interferon-γ DNA aptamer in Hunner-type interstitial cystitis.

Hunner-type interstitial cystitis (HIC) is a rare, enigmatic inflammatory disease of the urinary bladder with no curative treatments. In this study, we aimed to characterize the unique cellular and immunological factors specifically involved in HIC by comparing with cystitis induced by Mycobacterium bovis bacillus Calmette-Guérin, which presents similar clinicopathological features to HIC. Here, we show that T helper 1/17 +polarized immune responses accompanied by prominent overexpression of interferon (IFN)-γ, enhanced cGAS-STING cytosolic DNA sensing pathway, and increased plasma cell infiltration are the characteristic inflammatory features in HIC bladder. Further, we developed a mouse anti-IFN-γ DNA aptamer and observed that the intravesical instillation of the aptamer significantly ameliorated bladder inflammation, pelvic pain and voiding dysfunction in a recently developed murine HIC model with little migration into the blood. Our study provides the plausible basis for the clinical translation of the anti-IFN-γ DNA aptamer in the treatment of human HIC.

Motility Profile of Captive-Bred Marmosets Revealed by a Long-Term In-Cage Monitoring System.

A quantitative evaluation of motility is crucial for studies employing experimental animals. Here, we describe the development of an in-cage motility monitoring method for new world monkeys using off-the-shelf components, and demonstrate its capability for long-term operation (e.g., a year). Based on this novel system, we characterized the motility of the common marmoset over different time scales (seconds, hours, days, and weeks). Monitoring of seven young animals belonging to two different age groups (sub-adult and young-adult) over a 231-day period revealed: (1) strictly diurnal activity (97.3% of movement during daytime), (2) short-cycle (∼20 s) transition in activity, and (3) bimodal diurnal activity including a "siesta" break. Additionally, while the mean duration of short-cycle activity, net daily activity, and diurnal activity changed over the course of development, 24-h periodicity remained constant. Finally, the method allowed for detection of progressive motility deterioration in a transgenic marmoset. Motility measurement offers a convenient way to characterize developmental and pathological changes in animals, as well as an economical and labor-free means for long-term evaluation in a wide range of basic and translational studies.

Generation of transgenic marmosets using a tetracyclin-inducible transgene expression system as a neurodegenerative disease model.

Controllable transgene expression systems are indispensable tools for the production of animal models of disease to investigate protein functions at defined periods. However, in nonhuman primates that share genetic, physiological, and morphological similarities with humans, genetic modification techniques have not been well established; therefore, the establishment of novel transgenic models with controllable transgene expression systems will be valuable tools to understand pathological mechanism of human disease. In the present study, we successfully generated transgenic marmosets using a tetracyclin-inducible transgene expression (tet-on) system as a neurodegenerative disease model. The mutant human ataxin 3 gene controlled by the tet-on system was introduced into marmoset embryos via lentiviral transduction, and 34 transgene-introduced embryos were transferred into the uteri of surrogate mothers. Seven live offspring (TET1-7) were obtained, of which four were transgenic. Fibroblasts from TET1 and 3 revealed that inducible transgene expression had occurred after treatment with 10 µg/mL of doxycycline, while treatment with doxycycline via drinking water resulted in 1.7- to 1.8-fold inducible transgene expression compared with before treatment. One transgenic second-generation offspring (TET3-3) was obtained from TET3, and doxycycline-inducible transgene expression in its fibroblasts showed that TET3-3 maintained a high transgene expression level that matched its parent. In conclusion, we established a novel transgenic marmoset line carrying the mutant human ataxin 3 gene controlled by the tet-on system. The development of nonhuman primate models with controllable transgene expression systems will be useful for the identification of disease biomarkers and evaluation of the efficacy and metabolic profiles of therapeutic candidates.

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms.

Age-associated neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3-4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.

Gentamicin inhibits HSP70-assisted protein folding by interfering with substrate recognition.

We previously reported that gentamicin (GM) specifically binds to heat-shock protein with subunit molecular masses of 70 kDa (HSP70). In the present study, we have investigated the effects of GM binding on HSP70-assisted protein folding in vitro. The C-terminal, and not the N-terminal of HSP70 was found to bind to GM. GM significantly suppressed refolding of firefly luciferase in the presence of HSP70 and HSP40, although the ATPase activity of HSP70 was unaffected by GM. A surface plasmon resonance analysis revealed that GM specifically interferes with the binding of HSP70 to a model peptide that mimics the exposed hydrophobic surface of the folding intermediates. These results indicated that GM inhibits the chaperone activity of HSP70 and may suppress protein folding via inhibition of HSP70 in vivo.