Tsuneo Ito

An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. One of the most common models used in NASH research is a nutritional model where NASH is induced by feeding a diet deficient in both methionine and choline. However, the dietary methionine‐/choline‐deficient model in mice can cause severe weight loss and liver atrophy, which are not characteristics of NASH seen in human patients. Exclusive, long‐term feeding with a high‐fat diet (HFD) produced fatty liver and obesity in mice, but the HFD for several months did not affect fibrosis. We aimed to establish a mouse model of NASH with fibrosis by optimizing the methionine content in the HFD. Male mice were fed a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) consisting of 60 kcal% fat and 0.1% methionine by weight. After 1–14 weeks of being fed CDAHFD, the mice were killed. C57BL/6J mice maintained or gained weight when fed CDAHFD, while A/J mice showed a steady decline in body weight (of up to 20% of initial weight). In both strains of mice, plasma levels of alanine aminotransferase increased from week 1, when hepatic steatosis was also observed. By week 6, C57BL/6J mice had developed enlarged fatty liver with fibrosis as assessed by Massons trichrome staining and by hydroxyproline assay. Therefore, this improved CDAHFD model may be a mouse model of rapidly progressive liver fibrosis and be potentially useful for better understanding human NASH disease and in the development of efficient therapies for this condition.

C-reactive protein (CRP) measurement in canine serum following experimentally-induced acute gastric mucosal injury

To establish the diagnostic significance of canine C-reactive protein (CRP) in gastrointestinal disorders, the serum canine CRP concentration was measured in dogs with experimentally-induced acute gastric mucosal injury. Gastric injury was induced in one male and one female beagle by a single dose oral administration of acetylsalicylic acid (200 mg/kg body weight) or indomethacin (60 mg/kg body weight), or sodium chloride (1000 mg/kg body weight). CRP was measured prior to dose, and 1, 3, 7, and 14 days after the administration of the drugs, together with the total leucocyte counts and serum iron. Changes in the serum CRP in dogs with gastric injury were similar for the three test compounds, and reflected by the endoscopic findings. CRP values increased from 87 to 390 mg/l within 1 to 3 days after the compound administration but returned nearly to the predose levels within 14 days. Endoscopy revealed haemorrhagic erosion of the gastric mucosa in all dogs one day after dosing, with no evidence of the erosions observed after 7 days in many of the dogs. Changes of the total leucocyte and serum iron also occurred following gastric injury, but these changes were not as marked as those observed for CRP. The results of this study suggest that serum CRP level may be a useful indicator of a gastrointestinal mucosal injury in dogs.

Collagenofibrotic Glomerulonephropathy in a Cynomolgus Macaque (Macaca fascicularis)

Collagenofibrotic glomerulonephropathy (CFGN) is characterized by the deposition of type III collagen within the mesangial matrix and the absence of mesangial cell proliferation. A case of CFGN in a 2.7-year-old female cynomolgus macaque was investigated in the present study. Clinically, the animal was shown to have severe systemic edema along with hypoproteinemia. At necropsy, the kidneys were swollen and pale. The glomerular lesions were characterized by massive diffuse and global accumulation of fibrous materials in the mesangial areas. Neither mesangial cell proliferation nor changes in other organs were found. The fibrous materials were confirmed by the results of immunohistochemical and electron microscopic findings to consist mainly of randomly arranged, curve-shaped, twisted, and entwined type III collagen. This is the first case report of CFGN in nonhuman primates to date.

Spontaneous Glomerular and Tubulointerstitial Lesions in Common Marmosets (Callithrix jacchus)

Spontaneous progressive nephropathy dominated by glomerular lesions in common marmosets has been reported. However, the histopathologic characteristics, including the relationship between glomerular and tubulointerstitial lesions, have not been described in detail. In the present study, the authors examined the histopathologic characteristics of the background renal lesions in common marmosets (3 males and 9 females, 3 to 8 years old). The severity of glomerular lesions was graded into 3 classes: grade I, no alteration; grade II, hilar/focal increase of mesangial matrix; grade III, global/diffuse increase of mesangial matrix. Tubulointerstitial lesions (tubular regeneration and hyperplasia and interstitial inflammation and fibrosis) were scored according to the area of each lesion. The renal lesions were characterized by enlargement of glomeruli, expanded mesangial area with increase of periodic acid–Schiff reaction-positive matrix, tubular regeneration and hyperplasia, and interstitial inflammation and fibrosis. Glomerular lesions progressed with increasing mesangial matrix and aging. Additionally, the tubulointerstitial lesions became exacerbated with progressing glomerular lesions. Tubular hyperplasia was divided into 4 types according to the structure of the cell layer (simple or stratified-like), the area of increased lining cells (partial or entire), cytoplasmic staining (eosinophilic or basophilic), brush border and thickness of basement membrane, and the activity of cell proliferation. In conclusion, the background renal lesions in common marmosets were characterized by glomerular lesions with increase of mesangial matrix, which progressed with aging, and secondary tubulointerstitial lesions, including tubular hyperplasia. Those lesions were thus diagnosed as progressive glomerulonephropathy in common marmosets.

Expression of Membrane Complement Regulatory Proteins Crry and CD55 in Normal Rats

Some anticancer therapeutic antibodies are designed to act through complement-dependent cytotoxicity (CDC). It has been reported that there are many membrane complement regulatory proteins (mCRPs) that inhibit CDC. In the present study, we examined the expression of two mCRPs, the complement receptor 1-related gene/protein Y (Crry) and the decay-accelerating factor CD55, in three normal rats by immunohistochemistry. Crry and CD55 were detected widely in rat organs and tissues. Crry was found mainly in the urinary, digestive, respiratory, immunohematopoietic, circulatory and neuroendocrine systems. CD55 was found in the urinary, digestive and neuroendocrine systems. However, the two molecules were expressed in separate cells within the same organ. These results suggest that the distribution of mCRPs is related to the strict regulation of CDC activation in these organs and tissues and that the two molecules have a nonoverlapping expression pattern, a fact indicating specific roles in CDC regulation.

Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3–mediated therapeutics

T cell–mediated immunotherapy is an attractive strategy for treatment in various disease areas. In this therapeutic approach, the CD3 complex is one of the key molecules to modulate T cell functions; however, in many cases, we cannot evaluate the drug candidates in animal experiments because the therapeutics, usually monoclonal antibodies specific to human CD3, cannot react to mouse endogenous Cd3. Although immunodeficient mice transfused with human hematopoietic stem or precursor cells, known as humanized mice, are available for these studies, mice humanized in this manner are not completely immune competent. In this study we have succeeded in establishing a novel mouse strain in which all the three components of the Cd3 complex — Cd3ε, Cd3δ, and Cd3γ — are replaced by their human counterparts, CD3E, CD3D, and CD3G. Basic immunological assessments have confirmed that this strain of human CD3 EDG–replaced mice are entirely immune competent, and we have also demonstrated that a bispecific antibody that simultaneously binds to human CD3 and a tumor-associated antigen (e.g. ERBB2 or GPC3) can be evaluated in human CD3 EDG–replaced mice engrafted with tumors. Our mouse model provides a novel means to evaluate the in vivo efficacy of human CD3–mediated therapy.

Stimulation of adrenal chromaffin cell proliferation by hypercalcemia induced by intravenous infusion of calcium gluconate in rats.

Increased incidence of adrenal pheochromocytoma is frequently encountered in rat carcinogenicity studies. In some of the studies, the finding is judged to be due to a rat-specific mechanism of carcinogenesis caused by a disturbance of calcium homeostasis. However, direct evidence that the proliferation of chromaffin cells in the adrenal medulla is induced solely by hypercalcemia is not available. In this study, calcium gluconate was intravenously infused for 7 days to rat chromaffin cells by a tail cuff method, and cumulative labeling with bromodeoxyuridine (BrdU) was carried out to evaluate the proliferative activity. The serum calcium concentration was dose-dependently increased, and a high calcium concentration was stably sustained from day 2 to 7. In the adrenal medulla, BrdU-positive chromaffin cells increased in the calcium gluconate-treated animals, and the BrdU-labeling index increased in a dose-dependent manner. In addition, an increased BrdU-labeling index of chromaffin cells was shown to correlate with the serum calcium concentration. Our results demonstrate that hypercalcemia directly enhances the proliferative activity of chromaffin cells and that the proliferative activity is correlated with the serum calcium concentration.

Corrigendum: Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3–mediated therapeutics

This corrects the article DOI: 10.1038/srep45839.