animals : the research was approved and overseen by the animal experiments 
 committee of riken ( wako , japan ) and was conducted in accordance with the institutional 
 guidelines for experiments using animals .
common marmosets were reared at the riken brain science institute ( wako , japan ) , maintained 
 at 27c and 50% humidity on a 12/12-hr light / dark cycle . all marmosets in this study were 
 chosen from animals between 2 and 6 years old .
marmosets were allowed ad 
 libitum access to water and food pellets ( cms-1 m , clea japan inc . , tokyo , japan ) 
 with added vitamin c , d , calcium and acidophilus .
hot water and comb honey were also added 
 to soften the pellets and to improve the animals preference for the food .
animals were 
 given a piece of calorie mate ( otsuka pharmaceutical co. , ltd .
, tokyo , japan ) or castella 
 ( castella , yamazaki baking co. , ltd . ,
briefly , individuals with body weights less than 325 g with 0.05% body weight 
 loss per day were defined as the wms group ( n=7 ) .
individuals with body 
 weight higher than 375 g ( a median value of 350400 g was reported as the average weight of 
 adult marmosets ) , with 0.036% weight gain per 
 day ( average weight gain ratio of normal marmosets reported in ) , were defined as the control group ( n=7 ) .
body weight , the highest body weight , 
 current body weight / the highest body weight ratio and gain / loss ratio per day are listed in 
 table 1table 1.sex , age , body weight , the highest body weight , current body weight / the highest 
 body weight ratio and body weight gain / loss ratio per day in individual animals are 
 representedsexage(years)bw ( g)the highestbw ( g)bw / the highestbw ratio ( % ) bw gain or lossratio per day ( % ) wms 1female5274.3437.062.80.63wms 2female3220.4351.162.80.10wms 3female4207.8348.159.71.23wms 4female4201.6290.069.50.35wms 5female4220.3375.258.70.44wms 6female4250.0320.078.10.40wms 7male4255.0360.070.80.30control 1female4455.0464.598.00.04control 2male3397.1476.483.40.04control 3female4387.9391.599.10.04control 4female2439.7470.893.40.07control 5female4461.2461.21000.18control 6female4434.3434.31000.14control 7male2432.8432.81000.29 .
appearance check : first , all animals underwent an appearance check , 
 including fur and posture ( with or without unkempt fur , pale face , undervitalized 
 appearance , alopecia , curved back , pigeon - toed and stiff movement ) .
blood collection : blood samples were drawn from an individual s femoral 
 vein using 26-gauge needles . for the duration of blood collection , animals were under manual 
 retention .
the rest of the blood was 
 centrifuged ( 1,800g , 20 min and 4c ) after standing for 1 hr at room 
 temperature .
the purified serum was stored at 80c until used for serum chemistry and serum 
 mmp9 concentration tests .
cbc and serum chemistry tests : a cbc analysis , including white blood 
 cells , red blood cells , hematocrit , hemoglobin , platelets , mean corpuscular volume ( mcv ) , 
 mean corpuscular hemoglobin ( mch ) and mean corpuscular hemoglobin concentration ( mchc ) , was 
 performed using celltac  ( mek-6450 , nihon kohden co. , ltd . ,
tokyo , japan ) . a serum 
 chemistry panel ( albumin and calcium ) was performed using a drychem 4,000 system ( fujifilm 
 co. , ltd . ,
serum mmp9 concentration test : we performed serum tests of mmp9 
 concentrations using commercial elisa kits ( quantikine elisa human mmp-9 , smp900 ; r&d 
 systems , inc . ,
anatomical study : marmoset colons were dissected after perfusion with 
 saline followed by 4% paraformaldehyde under deep anesthesia . the dissected colons were 
 fixed with tissue fixative ( genostaff , co. , ltd . ,
tokyo , japan ) , then embedded in paraffin 
 wax and sectioned at 6 m for hematoxylin and eosin ( he ) staining .
statistical analysis : two - tailed mann - whitney u - tests were used to compare 
 the wms and control groups , and spearman s rank coefficient ( two tailed ) was used to measure 
 correlations between body weight and each data point ( graphpad prism , ver . 6 for windows ; 
 graphpad software inc . , la jolla , ca , u.s.a . ) .
data are presented as means  standard error 
 of the mean ( sem ) .
appearance check : pigeon - toe , which was considered to be caused by stiff 
 movement in the hind limb ( fig . 1a and 1bfig .
( b ) magnification of the white square in a. hind limbs 
 are pigeon - toed .
red arrow indicates an 
 area of alopecia . the black square indicates tail - base alopecia .
( d ) magnification of 
 the black square in c. red arrow indicates an area of alopecia , and the black arrow 
 indicates ulceration . ) , and alopecia ( fig .
1d ) , were observed in all 
 seven animals in the wms group . neither abnormality was observed in any of the seven animals 
 in the control group .
( b ) magnification of the white square in a. hind limbs 
 are pigeon - toed .
( d ) magnification of 
 the black square in c. red arrow indicates an area of alopecia , and the black arrow 
 indicates ulceration .
cbc : the hematocrit and hemoglobin values differed significantly between 
 the animals in the two groups ( p<0.01 , fig .
hematocrit 
 values and ( b ) hemoglobin values of marmosets in the wms and control groups .
correlations between ( c ) body 
 weight and hematocrit value and ( d ) body weight and hemoglobin value in individual 
 animals .
the dotted 
 lines represent best - fit lines of the data points . , and p<0.01 , fig . 2b , 
 respectively )
the mean hematocrit values of the wms group and the control group were 24.8  
 1.92% and 40.7  1.48% , respectively .
the mean hemoglobin values of the wms group and the 
 control group were 8.26  0.808 g / dl and 12.97  0.451 
 g / dl , respectively .
significant correlations between body weight and 
 hematocrit and hemoglobin values were observed ( p<0.01 , 
 rs=0.837 , fig .
2c , and 
 p<0.01 , rs=0.851 , fig . 
 2d , respectively ) .
hematocrit 
 values and ( b ) hemoglobin values of marmosets in the wms and control groups .
correlations between ( c ) body 
 weight and hematocrit value and ( d ) body weight and hemoglobin value in individual 
 animals .
the platelet value was also significantly different between the two groups 
 ( p<0.01 , fig .
the mean 
 platelet values of the wms group and control group were 118.3  11.61  
 10/l and 45.9  4.36  10/l , 
 respectively .
platelet values showed a significant negative correlation with body weight 
 ( p<0.01 , rs=0.695 , fig . 

although there was also a significant difference between the two groups in red blood cell 
 values ( mean 335.4  80.75  10/l in the mws group and 618.9  
 66.04  10/l in the control group , 
 p<0.01 ) , there was no significant difference 
 ( p>0.05 ) between the groups in mcv ( mean 67.8  0.82 fl 
 in the wms group and 65.8  1.74 fl in the control group ) , mch ( mean 22.4  
 0.87 pg in the wms group and 21.0  0.34 pg in the control 
 group ) , mchc ( mean 33.2  1.42 g / dl in the wms group and 31.9  0.16 
 g / dl in the control group ) or white blood cell values ( mean 71.4  28.23 
  10/l in the mws group and 57.9  7.30  
 10/l in the control group ) .
serum chemistry test : the levels of serum albumin differed significantly 
 between animals in the two groups ( p<0.01 , fig . 3afig .
( a ) 
 serum albumin values and ( b ) serum calcium values of marmosets in the wms and control 
 groups .
. correlations 
 between ( c ) body weight and serum albumin value and ( d ) body weight and serum calcium 
 value in individual animals .
the dotted lines represent best - fit lines of the data 
 points . ) .
the mean albumin levels in the wms and control groups were 3.49  0.221 
 g / dl and 5.53  0.300 g / dl , respectively .
a significant 
 correlation between body weight and serum albumin level was observed 
 ( p<0.01 , rs=0.879 , fig . 

( a ) 
 serum albumin values and ( b ) serum calcium values of marmosets in the wms and control 
 groups .
correlations 
 between ( c ) body weight and serum albumin value and ( d ) body weight and serum calcium 
 value in individual animals .
similarly , the levels of serum calcium differed significantly between animals in the two 
 groups ( p<0.01 , fig .
the 
 mean calcium levels in the wms and control groups were 8.8  0.40 mg / dl and 
 11.3  0.56 mg / dl , respectively . a significant correlation between body 
 weight and serum calcium level
serum mmp9 concentration : there was a significant difference in serum mmp9 
 concentration between the two groups ( p<0.01 , fig .
( a ) blood mmp9 values of 
 marmosets in the wms and control groups .
( b ) correlations between body weight , and blood mmp9 values in 
 individual animals .
the mean mmp9 concentrations in the wms and control groups were 91.7  21.79 
 ng / ml and 17.4  2.73 
 ng / ml , respectively .
a significant negative correlation 
 between body weight and serum mmp9 concentration was observed ( p<0.05 , 
 rs=0.660 , fig .
( a ) blood mmp9 values of 
 marmosets in the wms and control groups .
( b ) correlations between body weight , and blood mmp9 values in 
 individual animals .
bowel section of animals in the wms group : there were no polyps or cancer 
 in the colons in either the wms or control group ( data not shown ) .
epithelial damage and 
 disruption of crypt architecture were observed in the transverse colon samples ( fig . 5afig .
( c ) magnification of the black square in a. scale bar : 50 
 m .
( d ) magnification of the black square in b. scale bar : 50 
 m .
red arrows in c and d indicate goblet cells . and 5b ) and ascending colon samples ( fig . 

the colonic tissues revealed inflammatory cell 
 infiltration in the lamina propria of the mucosa , atrophy of intestinal villus and decreased 
 numbers of goblet cells ( fig . 5b and 5d ) .
there 
 were no abnormalities in the colons in the control group ( data not shown ) .
( c ) magnification of the black square in a. scale bar : 50 
 m .
( d ) magnification of the black square in b. scale bar : 50 
 m .
common marmosets are often used for long - term research experiments , such as behavioral 
 analysis , in the field of brain science .
a long 
 duration is needed for the production of transgenic marmosets , at least 5 months for the 
 duration of pregnancy and a litter size of 24 infants , so each animal is very valuable .
wms can occur in any animal used for ongoing research . due to this morbidity and mortality
it was reported that some 
 treatments , such as long - term administration of steroids with relatively few side effects , 
 such as budesonide , can cause temporary remission . however , because steroid treatment is relatively ineffective in animals in the 
 terminal stage and is not persistent , steroid treatment can not be said to be adequate .
thus , 
 early identification of diseased animals , removal from experiments and prompt treatment of 
 these animals are important .
as other reports have mentioned , alopecia [ 18 , 25 ] , pigeon - toe [ 18 , 25 ] , anemia , thrombocytosis , 
 hypoalbuminemia [ 2 , 18 ] and hypocalcemia   were also observed 
 in the wms group in this study .
these results support the hypothesis that hematocrit , 
 hemoglobin , platelets , serum albumin and serum calcium can be used for the diagnosis of 
 wms .
it was suggested that the anemia and hypoalbuminemia seen in the animals reflect poor 
 nutritional condition , caused by an uptake disorder or protein and/or iron absorption 
 defect . however , because no significant difference or even trend was seen in mcv , mch or 
 mchc between the wms and control groups , we suggest that the anemia and hypoalbuminemia 
 observed in this study were due to other factor(s ) depending on the individuals .
anemia is a 
 major symptom of ibd in humans [ 13 , 29 ] , and iron deficiency is the largest contributing 
 factor .
however , it has been suggested that erythropoietin depression , vitamin insufficiency 
 and autoimmune hemolytic disease in ibd patients 
 could also cause anemia .
given that several factors other than iron deficiency could have 
 caused anemia in wms , similar to the situation in human ibd , further investigation is 
 needed .
it is known that mmps function to process several inflammatory cytokines , including tumor 
 necrosis factor alpha ( tnf- ) .
mmp9 is thought 
 to be the main metalloproteinase implicated in the development of ibd , and the increase in mmp9 in mouse and rat ibd has been reported 
 [ 4 , 22 ] . 

although there are other biomarkers used for diagnosis of ibd , such as c - reactive proteins 
 ( crp ) or erythrocyte sedimentation rate ( esr ) , it was reported that mmp9 is a more useful 
 biomarker for diagnosis of human ibd than other biochemical markers in terms of that mmp9 
 can reflect disease state because mmp9 has much higher specificity in distinguishing between 
 active ibd and inactive ibd than other parameters . in our study , the wms group showed significantly increased serum mmp9 
 concentrations , suggesting that mmp9 is connected with the pathological condition of wms . 

our results suggest that serum mmp9 might have an important role in the mechanism of wms and 
 could be useful for the diagnosis of wms .
a mmp9-targeted inhibitor was developed in rats 
 and is hoped to be a specific medicine for ibd . 

to our knowledge , this is the first 
 report of increased mmp9 levels in marmosets with wms .
we previously reported that fecal 
 calprotectin can be a marker for detecting colonic inflammation , and it was reported that fecal mmp9 can be used for detecting human 
 ibd as well as fecal calprotectin .
so , further 
 investigations of fecal mmp9 as a marker for detecting wms should be performed .
we propose 
 that the wms marmoset could be an animal model of human ibd with respect to mmp9 .
further 
 investigations of other molecules involved in human ibd need to be performed in marmosets to 
 explore this possibility . tnf- and interleukin-6 ( il-6 ) are major cytokines in 
 inflammation , and there is a report regarding the involvement of tnf- and il-6 in human 
 autoimmune diseases , including ibd .
although 
 tnf- and il-6 might be involved in wms , little has been reported on the behavior of this 
 molecule . because it is known that mmp9 is regulated by il-6   and affects tnf- , future 
 experiments aimed at investigating the relationship between mmp9 and tnf- , or il-6 in wms 
 are needed .
 use of the common marmoset ( callithrix jacchus ) as a non - human primate 
 experimental animal has increased in recent years . although wasting marmoset syndrome 
 ( wms ) is one of the biggest problems in captive marmoset colonies , the molecular 
 mechanisms , biochemical markers for accurate diagnosis and a reliable treatment remain 
 unknown . in this study , as a first step to finding biochemical marker(s ) for the accurate 
 diagnosis of wms , we conducted blood cell counts , including hematocrit , hemoglobin and 
 platelets , and examined serum chemistry values , including albumin , calcium and levels of 
 serum matrix metalloproteinase 9 ( mmp9 ) , using a colony of marmosets with and without 
 weight loss . 
 mmp9 is thought to be an enzyme responsible for the degradation of 
 extracellular matrix components and participates in the pathogenesis of inflammatory 
 conditions , such as human and murine inflammatory bowel disease , which , like wms , are 
 characterized histologically by inflammatory cell infiltrations in the intestines . 
 the 
 values of hematocrit and hemoglobin and levels of serum albumin and calcium in the wms 
 group were significantly decreased versus the control group . 
 the platelet values and serum 
 mmp9 concentrations were increased significantly in the wms group compared with the 
 control group . 
 mmp9 could be a new and useful marker for the diagnosis of wms in addition 
 to hematocrit , hemoglobin , serum albumin and calcium . 
 our results also indicate that mmp9 
 could be a useful molecular candidate for treatment . 
MATERIALS AND METHODS
RESULTS
DISCUSSION