Gallbladder mucocele is characterized by the accumulation of immobile mucus in the gallbladder, and it is one of the most common gallbladder diseases in dogs.1–4 Gallbladder mucocele typically is diagnosed during abdominal ultrasonography, which can reveal the immobile and striated or stellate patterns within the gallbladder lumen that are characteristic of GM.1 Studies have found excess secretion of gelforming mucins in dogs with GM5 and described histopathologic features of GM, such as fattened mucosal folds, mucinous metaplasia of surface epithelium, and cystic mucinous hyperplasia.2,5,6
Several factors that predispose dogs to GM have been reported, including age, breed (Shetland Sheepdogs,3,7 Miniature Schnauzers,7 Cocker Spaniels,7 Pomeranians,7 and Chihuahuas7), dyslipidemia (eg, hypercholesterolemia and hypertriglyceridemia7), endocrinopathies (eg, hyperadrenocorticism and hypothyroidism8), and use of imidacloprid drugs in Shetland Sheepdogs.9 Among these predisposing factors, hyperadrenocorticism has a strong association with GM. The odds of GM in dogs with hyperadrenocorticism are 29 times those in dogs without hyperadrenocorticism8; however, the role of glucocorticoids in the pathogenesis of GM has not been clarified.
In another study10 conducted by our research group, we detected a marked decrease in food-induced gallbladder emptying rate in dogs with GM, compared with the motility in healthy dogs. A study11 of mice revealed that a decrease in gallbladder emptying rate is associated with an increase in mucin expression in the gallbladder wall. A decrease in gallbladder emptying rate may cause an increase in mucin expression in the gallbladder wall of dogs and may result in the onset of GM. Several causes for a lower gallbladder emptying rate and gallbladder smooth muscle dysfunction have been reported, including use of CCK antagonists,11 accumulation of cholesterol in the gallbladder wall,12 and changes in bile acid composition.13 However, the effect of glucocorticoids on gallbladder emptying rate has not been reported.
Gallbladder mucin is a main component of gallbladder mucus14,15 that abnormally accumulates in the gallbladders of dogs with GM.3,15 Gallbladder mucin plays an important role in the defense against bile acids,14,16 and mucin secretion by cultured canine gallbladder epithelial cells is accelerated by bile acids such as taurochenodeoxycholic acid and taurodeoxycholic acid.17,18 Prostaglandin E2 also increases mucin secretion by canine gallbladder epithelial cells,19 and mucin expression in the gallbladder of humans increases as the inflammatory score increases.20 Although several factors that increase secretion or expression of mucin have been identified, effects of glucocorticoid administration on gallbladder bile mucin concentration have not been investigated.
Gallbladder emptying rate is significantly lower in dogs with GM, and abnormal mucin accumulation in the gallbladder is one of the main characteristics of GM. Therefore, the purpose of the study reported here was to investigate effects of continuous glucocorticoid administration on gallbladder emptying rate and gallbladder bile composition (concentrations of mucin, cholesterol, and each bile acid). We hypothesized that glucocorticoid administration would decrease the gallbladder emptying rate, increase the gallbladder bile mucin concentration, and ultimately result in the onset of GM in dogs.
Materials and Methods
Animals
Six castrated male Beagles owned by the University of Tokyo were used in the study. Dogs were between 3 and 4 years old, and body weight ranged from 9.0 to 10.2 kg. The dogs were considered healthy on the basis of results of a physical examination, CBC, and plasma biochemical analysis. Experimental and animal care procedures were approved by the Animal Use and Care Committee of the University of Tokyo (P16-216).
Procedures
Although a prednisolone dosage of 4 mg/kg/d was used in other studies,21,22 the dosage in the present study was selected to avoid adverse effects, including vomiting.23 Prednisolonea was administered (2 mg/kg, SC, once daily in the morning for 2 weeks) to each dog. After prednisolone administration at that dosage for 2 weeks, the dosage of prednisolone was gradually decreased over a 2-week period (prednisolone was administered at a dosage of 1 mg/kg, SC, once daily in the morning for 1 week, then at 0.5 mg/kg, SC, once daily in the morning for 1 week). At that time, prednisolone administration ceased. The dogs were examined before and after administration of prednisolone for 2 weeks and 1 week after cessation of prednisolone administration. All examinations, including the measurement of gallbladder emptying rate and sample collections, were performed by 1 veterinarian (TN).
Food-induced gallbladder emptying rate
Gallbladder volume and gallbladder emptying rate were calculated in accordance with methods described elsewhere.10,24 Food was withheld from the dogs for ≥ 18 hours before evaluation. Gallbladder volume was calculated by use of ultrasonography with the ellipsoid method. Dogs were positioned in left lateral recumbency; dogs were not sedated and were manually restrained. Maximum length was measured on the longitudinal plane, and maximum width and depth were measured on the transverse plane. Volume was calculated as follows: volume = length × width × depth × 0.52.
The initial gallbladder volume was measured by use of ultrasonography, and dogs then were fed a test mealb (10 g of diet/kg of body weight). Postprandial gallbladder volumes were measured at 60 and 120 minutes after feeding. Gallbladder emptying rates were calculated by use of the following equation: Et = (V0 – Vt)/V0 × 100, where Et was the emptying rate for time t (ie, 60 or 120 minutes), V0 was the initial gallbladder volume before feeding of the test meal, and Vt was the gallbladder volume measured at time t. The value reported was the percentage change in gallbladder volume from V0.
Sample collection
A venous blood sample (5 mL) was collected before evaluation of food-induced gallbladder emptying rate. Plasma was separated immediately, and activities of alkaline phosphatase, alanine aminotransferase, γ-glutamyltranspeptidase, aspartate aminotransferase, and lipase and concentrations of total cholesterol, triglyceride, glucose, BUN, and creatinine were determined with a dry chemistry analyzer.c
Gallbladder bile was collected on the day after assessment of the gallbladder emptying rate. Food was withheld from the dogs for 18 hours, and dogs were sedated by IV administration of medetomidined (0.02 mg/kg) and midazolame (0.2 mg/kg). Gallbladder bile was aspirated (range, approx 5 to 50 mL) with a 21-gauge needle via ultrasound-guided percutaneous cholecystocentesis as described in another study25 to avoid, as much as possible, bile leakage. After gallbladder bile was collected, atipamezolef (0.1 mg/kg, IV) was administered. All gallbladder bile samples were stored at −20°C until used for analysis.
Bile analysis
Separation and quantification of bile acid fractions were performed with a high-performance liquid chromatography system in a commercial laboratory.g Unconjugated, glycine-conjugated, and taurine-conjugated forms of bile acids (ursodeoxycholic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid) were measured. Cholesterol concentrations in gallbladder bile were determined with absorptiometry by use of the cholesterol dehydrogenase method.
Mucin concentration in gallbladder was determined in a university laboratoryh with a fluorometric assay26,27 for O-glycosylated glycoproteins by use of a fecal mucin assay kit.i Bile was centrifuged at 3,000 × g for 10 minutes to remove gallbladder sludge. Then, 200 μL of bile supernatant was diluted 1:1 (vol/vol) in acetate buffer, and 615 μL of 99.5% ethanol was added; the mixture was stored overnight at −20°C. The next morning, the mixture was centrifuged at 20,000 × g for 10 minutes. The upper phase was discarded, and the pellet was dissolved in 1,000 μL of PBS buffer and used for fluorometric determination of the mucin concentration. An aliquot (24 μL) of alkaline reagent (1:5 [vol/vol] mixture of 0.6% NaOH and 5% 2-cyanoacetamide) was added to 20 μL of the pellet solution, and the mixture was incubated at 100°C for 30 minutes. Then 200 μL of 3.7% borate buffer was added, and fluorescence was measured at 400 nm (excitation, 360 nm).
Statistical analysis
Data were analyzed with statistical software.j Data were compared between each examination point by use of a Wilcoxon signed rank test. The cutoff value of P < 0.05 was modified with a Bonferroni correction; values of P < 0.017 were considered significant.
Results
Blood biochemical analysis
Activities of alkaline phosphatase, alanine aminotransferase, γ-glutamyltranspeptidase, and aspartate aminotransferase and triglyceride concentration increased (P = 0.016) significantly and total cholesterol and creatinine concentrations decreased significantly (P = 0.016) after prednisolone administration for 2 weeks, compared with the values before prednisolone administration (Table 1). One week after cessation of prednisolone administration, activities of alkaline phosphatase, γ-glutamyltranspeptidase, and aspartate aminotransferase were still significantly (P = 0.016) higher than activities before prednisolone administration, and the creatinine concentration was still significantly (P = 0.016) lower than the concentration before administration. No significant changes in lipase activity or concentrations of glucose or BUN were detected among any time points.
Median (range) values for blood biochemical analysis of samples obtained from 6 dogs before and after prednisolone administration for 2 weeks and 1 week after cessation of prednisolone administration.
| Variable | Before | After | 1 week after cessation |
|---|---|---|---|
| ALP (U/L) | 196 (125–229) | 1,378 (1,050–2,413)* | 802 (453–1,578)* |
| ALT (U/L) | 33 (30–48) | 128 (72–352)* | 157 (12–359) |
| GGT (U/L) | 6 (6–10) | 30 (18–130)* | 50 (15–120)* |
| AST (U/L) | 35 (31–38) | 49 (41–89)* | 48 (33–64)* |
| T-Chol (mg/dL) | 205 (137–232) | 162 (89–207)* | 192 (136–261) |
| TG (mg/dL) | 42 (27–76) | 60 (30–85)* | 57 (30–136) |
| Glucose (mg/dL) | 103 (98–110) | 122 (92–143) | 104 (100–111) |
| Lipase (U/L) | 56 (28–127) | 77 (34–191) | 51 (29–65) |
| BUN (mg/dL) | 8.1 (6.7–8.6) | 10.2 (7.4–17.8) | 10.8 (7.9–23.3) |
| Creatinine (mg/dL) | 0.6 (0.4–0.6) | 0.2 (0.1–0.4)* | 0.3 (0.3–0.3)* |
Prednisolone was administered (2 mg/kg, SC, once daily in the morning) for 2 weeks. During the next 2 weeks, the dose of prednisolone was tapered to cessation; gallbladder emptying rate was assessed again 1 week after cessation of prednisolone administration.
Value differs significantly (P < 0.017) from the value before administration.
ALP = Alkaline phosphatase. ALT = Alanine aminotransferase. AST = Aspartate aminotransferase. GGT = γ-Glutamyltranspeptidase. T-Chol = Total cholesterol. TG = Triglyceride.
Food-induced gallbladder emptying rate
Before prednisolone administration, the median initial gallbladder volume (after food was withheld for ≥ 18 hours but before a meal was fed) was 11.0 mL (range, 8.3 to 33.9 mL). After prednisolone was administered for 2 weeks, the median initial gallbladder volume was 17.5 mL (range, 10.0 to 72.6 mL), which was significantly (P = 0.016) higher than that before prednisolone administration. One week after cessation of prednisolone administration, the median initial gallbladder volume was 16.9 mL (range, 11.3 to 45.5 mL), which did not differ significantly from the volume before prednisolone administration.
Gallbladder emptying rate at 60 and 120 minutes after feeding decreased significantly (P = 0.016) after prednisolone administration for 2 weeks, compared with emptying rates before prednisolone administration (Figure 1). The gallbladder emptying rate at 120 minutes after feeding significantly (P = 0.016) decreased 1 week after cessation of prednisolone administration, compared with the corresponding emptying rate after prednisolone administration; however, that gallbladder emptying rate was still significantly (P = 0.016) lower than the corresponding emptying rate before prednisolone administration.
Food-induced gallbladder emptying rate at 60 (A) and 120 (B) minutes after a meal for 6 dogs before and after prednisolone administration (2 mg/kg, SC, once daily in the morning) for 2 weeks; during the next 2 weeks, the dose of prednisolone was tapered to cessation, and gallbladder emptying rate was assessed again 1 week after cessation of prednisolone administration. Each circle represents results for 1 dog. Median values are indicated by horizontal lines. *Value differs significantly (P = 0.016) from the value before prednisolone administration. †Value differs significantly (P = 0.016) from the value after prednisolone administration.
Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1050
Gallbladder bile composition
Gallbladder bile concentrations of mucin and cholesterol decreased significantly (P = 0.016) after prednisolone administration for 2 weeks, compared with concentrations before prednisolone administration (Table 2). The gallbladder bile concentration of cholesterol 1 week after cessation of prednisolone administration was still significantly (P = 0.016) lower than the concentration before prednisolone administration.
Median (range) values for gallbladder bile composition of samples obtained from 6 dogs before and after prednisolone administration for 2 weeks and 1 week after cessation of prednisolone administration.
| Variable | Before | After | 1 week after cessation |
|---|---|---|---|
| Mucin (mg/dL) | 13.1 (10.7–21.7) | 8.8 (6.2–11.3)* | 14.3 (9.6–26.7) |
| T-Chol (mg/dL) | 97.0 (61.0–114.0) | 46.0 (35.0–70.0)* | 72.0 (53.0–83.0)* |
| TUDCA (mmol/L) | 0.5 (0.2–1.1) | 0.3 (0.2–0.4) | 0.4 (0.2–0.6) |
| UDCA (mmol/L) | 0.2 (0.1–0.2) | 0.1 (0.0–0.4) | 0.1 (0.1–0.2) |
| GCA (mmol/L) | 1.7 (1.0–2.0) | 1.3 (0.0–1.7) | 1.4 (0.0–1.6) |
| TCA (mmol/L) | 145.2 (127.9–164.2) | 101.9 (78.1–187.2) | 150.8 (141.0–183.8) |
| CA (mmol/L) | 0.3 (0.0–16.5) | 10.5 (0.0–13.7) | 1.0 (0.3–14.7) |
| GCDCA (mmol/L) | 0.4 (0.1–0.5) | 0 (0–0.1)* | 0.1 (0–0.2) |
| TCDCA (mmol/L) | 27.2 (22.0–31.9) | 8.1 (6.8–15.2)* | 26.4 (15.1–31.5)† |
| DCA (mmol/L) | 0.0 (0.0–0.9) | 0.2 (0.0–0.3) | 0.0 (0.0–0.4) |
| TDCA (mmol/L) | 45.1 (42.5–65.4) | 32.3 (22.9–78.4) | 37.4 (20.9–50.0) |
| CDCA (mmol/L) | 0.0 (0.0–0.4) | 0.3 (0.0–1.1) | 0.1 (0.0–0.2) |
| TLCA (mmol/L) | 0.4 (0.2–0.9) | 0.2 (0–1.0) | 0.4 (0.1–0.5) |
| TBA (mmol/L) | 234.2 (214.4–241.0) | 179.2 (147.4–231.6)* | 221.4 (207.6–267.1) |
Value differs significantly (P < 0.017) from the value after administration.
CA = Cholic acid. CDCA = Chenodeoxycholic acid. DCA = Deoxycholic acid. GCA = Glycocholic acid. GCDCA = Glycochenodeoxycholic acid. TBA = Total bile acids. TCA = Taurocholic acid. TCDCA = Taurochenodeoxycholic acid. TDCA = Taurodeoxycholic acid. TLCA = Taurolithocholic acid. TUDCA = Tauroursodeoxycholic acid. UDCA = Ursodeoxycholic acid.
See Table 1 for remainder of key.
Glycoursodeoxycholic acid was detected in only 2 of 18 bile samples (both with a concentration of 0.1 mmol/L), glycodeoxycholic acid was detected in only 1 of 18 bile samples (concentration, 0.5 mmol/L), glycolithocholic acid was detected in only 3 of 18 bile samples (all with a concentration of 0.1 mmol/L), and lithocholic acid was not detected in any bile samples. Concentrations of glycochenodeoxycholic acid, taurochenodeoxycholic acid, and total bile acids decreased significantly (P = 0.016) after prednisolone administration for 2 weeks, compared with concentrations before prednisolone administration (Table 2). The concentration of taurochenodeoxycholic acid then increased significantly (P = 0.016) 1 week after cessation of prednisolone administration, compared with the concentration after prednisolone administration for 2 weeks. No significant changes in the concentrations of tauroursodeoxycholic acid, ursodeoxycholic acid, glycocholic acid, taurocholic acid, cholic acid, chenodeoxycholic acid, taurodeoxycholic acid, deoxycholic acid, or taurolithocholic acid were detected among any time points.
Discussion
Food-induced gallbladder emptying rate decreased significantly after prednisolone was administered to healthy Beagles for 2 weeks. Impairment of gallbladder emptying may have pathological effects on the gallbladder through prolonged exposure to concentrated bile. Cytotoxic effects of bile acids on gallbladder epithelial cells have been investigated.28–30 Although the concentration of each bile acid did not increase in the present study, prolonged exposure may be harmful to canine gallbladder epithelium. One week after cessation of prednisolone administration, food-induced gallbladder emptying rate was significantly increased; for this reason, the decrease in gallbladder emptying rate caused by prednisolone administration was thought to be reversible. However, the gallbladder emptying rate 1 week after cessation of prednisolone administration was still significantly lower than that before prednisolone administration.
The gallbladder contracts in response to stimulation by CCK released from the small intestine after meals. There is no significant difference in postprandial serum CCK concentration between healthy dogs and dogs with pituitary-dependent hyperadrenocorticism.31 On the basis of results of the study reported here, we do not believe that postprandial CCK content was deficient in dogs receiving prednisolone, although serum CCK concentration was not evaluated in the present study. An association between abnormalities in lipid metabolism and a decrease in gallbladder emptying rate has been reported.32,33 In cholesterol-supersaturated bile, cholesterol is absorbed by biliary epithelium cells and accumulates in smooth muscle in the gallbladder; cholesterol accumulation in smooth muscle results in dysfunction of gallbladder smooth muscle.12 However, the bile cholesterol concentration decreased significantly in the present study, and similar results have been reported in another study.21 Further evaluation of changes in gallbladder smooth muscle function caused by prednisolone is necessary to determine the cause of the lower gallbladder emptying rate.
The mucin concentration in the gallbladder bile decreased significantly after prednisolone administration for 2 weeks in the present study. This result did not support our hypothesis that the gallbladder bile concentration of mucin would increase owing to glucocorticoid administration. Although it has been reported34 that bile is diluted by hydrocortisone in dogs, hydrocortisone treatment for 84 days increased concentrations of unconjugated bile acids such as cholic acid, chenodeoxycholic acid, and deoxycholic acid.22 We thought that the gallbladder bile concentration of mucin could increase if mucin secretion was greatly increased by prednisolone administration. Explanations for the decrease in mucin concentration in gallbladder bile potentially include a combination of impaired secretion and dilution. Taurochenodeoxycholic acid promotes mucin secretion from cultured canine gallbladder epithelial cells17,18; therefore, the decrease in taurochenodeoxycholic acid concentration in the present study might have caused a decrease in mucin secretion. Moreover, mucin secretion is decreased by aspirin in cultured canine gallbladder epithelial cells, and this effect is caused by inhibition of cyclooxygenase-2.35,36 Glucocorticoids also inhibit cyclooxygenase-2 through suppression of the nuclear factor κB pathway.37 Therefore, prednisolone may similarly decrease mucin secretion by canine gallbladder epithelial cells. In addition, hydrocortisone has weak choleretic and dilutional effects in dogs, although the mechanisms of choleretic effects caused by hydrocortisone are not fully understood.34 It is possible that decreased absorption by, or increased secretion of water from, the gallbladder epithelium contributed to the decrease in mucin concentration; however, to our knowledge, there have been no reports on water transportation into the gallbladder of healthy dogs or dogs with GM.
Results of the present study cannot be used to explain the relationship between high glucocorticoid concentrations and abnormal mucin accumulation in dogs with GM. Although we believe that glucocorticoids may play a primary role in the pathogenesis of GM in dogs, other factors must also contribute to the abnormal accumulation of mucin. The type of mucin secreted in dogs with GM differs from that of healthy dogs, with disproportionally significant increases in Muc5ac relative to Muc5b, defective mucin unpackaging, and increases in mucin-interacting innate defense protein.5 Although we did not investigate the type of mucins secreted and concentrations of mucin-interacting innate defense protein in the study reported here, it might be possible that high amounts of glucocorticoids would alter the type of mucins secreted and lead to the onset of GM.
The decrease in total cholesterol concentration in gallbladder bile in the present study was consistent with results of a study21 of dogs administered hydrocortisone. The decrease in the gallbladder bile concentration of cholesterol would contribute to the choleretic effects of glucocorticoids,34 thus increasing gallbladder filling and distention, which were observed in the present study. In addition, the decrease in plasma cholesterol concentration after glucocorticoid administration in the study reported here might also have contributed to a decrease in the cholesterol concentration in gallbladder bile because cholesterol is secreted into bile in the liver. However, the mechanism for the decrease in the plasma cholesterol concentration was not investigated in the present study.
In dogs, taurine-conjugated bile acid constitutes 99.04%, glycine-conjugated bile acid constitutes 0.15%, and unconjugated bile acid constitutes 0.81% of the bile acids.22 In the study reported here, the bile acid profile (taurine-conjugated bile acid constituted 98.80%, glycine-conjugated bile acid constituted 0.89%, and unconjugated bile acid constituted 0.20% of the bile acids) supported the results of that study.22 However, changes in the bile acid composition detected in the present study differed from those of previous experiments at several points. First, the concentrations of glycochenodeoxycholic acid and taurochenodeoxycholic acid significantly decreased after once-daily prednisolone administration for 2 weeks, although there were no significant differences in these bile acid concentrations in a study22 of dogs administered hydrocortisone twice daily for 84 days. The cause of this discrepancy is presumed to be the different drugs used in each study, duration of administration, and frequency of administration. Second, the concentrations of cholic acid, chenodeoxycholic acid, and deoxycholic acid did not change significantly in the present study, although the concentrations of these bile acids significantly increased in dogs administrated hydrocortisone.22 Because significant changes in concentrations of these bile acids were detected for the first time at day 56 in that previous study,22 the duration of prednisolone administration in the present study would appear to have been insufficient to cause significant changes in the cholic acid, chenodeoxycholic acid, and deoxycholic acid concentrations.
The effect of the duration of prednisolone administration could be considerable. We chose administration for 2 weeks on the basis of a previous study38 in which a high-fat diet was fed for 2 weeks and significantly changed the gallbladder emptying rate and gallbladder bile composition of dogs and another previous study39 in which dexamethasone treatment for 2 weeks significantly increased gallbladder volume in mice. In the present study, gallbladder concentrations of mucin, cholesterol, and bile acids significantly decreased after prednisolone administration for 2 weeks. It is possible that gallbladder bile was diluted by administration of glucocorticoid, as was reported in another study.34 Hydrocortisone treatment for 3 months significantly increased concentrations of some unconjugated bile acids22; therefore, the effect of prednisolone administration on gallbladder bile composition would not be expected to plateau within 2 weeks. A longer duration of prednisolone administration might yield different results.
The present study had several limitations. Gallbladder tissues were not collected, so gene and protein expression (including mucin expression) were not evaluated, nor was a histologic examination of the gallbladder wall conducted. Moreover, the duration of prednisolone administration was insufficient to investigate the effect of hyperadrenocorticism, which is typically an insidious, chronic condition resulting from long-term use of glucocorticoids that are continuously administered for several months or years. A small sample size and a homogenous population were also potential limitations of the present study.
In the present study, food-induced gallbladder emptying rate significantly decreased in healthy dogs after prednisolone administration for 2 weeks. A lower gallbladder emptying rate may play a role in the pathogenesis of GM in dogs with hyperadrenocorticism. However, the mucin concentration in gallbladder bile significantly decreased after prednisolone administration for 2 weeks. It is apparent that other factors, such as dyslipidemia, which is also reportedly a predisposing factor for development of GM,7 must also contribute to abnormal accumulation of mucin. Further studies will be required to determine the cause of abnormal accumulation of mucin in dogs.
Acknowledgments
Supported in part by Scholarship Program 2016 of the Japanese Society of Veterinary Clinical Nutrition.
None of the authors had a financial or personal relationship with other individuals or organizations that could have inappropriately influenced or biased the content of this report.
The authors thank Hajime Asada, Takahiro Mizukami, Kotoko Ogawa, and Yukiko Nakazono for technical assistance.
ABBREVIATIONS
| CCK | Cholecystokinin |
| GM | Gallbladder mucocele |
Footnotes
Prednisolone injection solution KS, Kyoritsu Seiyaku Co, Tokyo, Japan.
Hill's Prescription Diet a/d, Hill's Pet Nutrition Inc, Topeka, Kan.
Fuji Dri-Chem 5000V, Fujifilm Corp, Tokyo, Japan.
Domitor, Nippon Zenyaku Kogyo Co, Fukushima, Japan.
Sandoz Inc, Yamagata, Japan.
Antisedan, Orion Corp, Espoo, Finland.
SRL Inc, Tokyo, Japan.
Laboratory of Molecular Immunology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.
Cosmo Bio Ltd, Tokyo, Japan.
JMP, version 11.2, SAS Institute Inc, Cary, NC.
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