TPC NEWS Vol.13 No.1, Spring, 1994 (Whole Number 22)

( English Summarry )

Page-3   At Karachi

Yasuhiro Yoshikawa

      If I remember correctly, it was in 1985 that I visited Karachi for  the  first time as a member of Karachi  Encephalitis  Survey Team  of a joint Japan-Pakistan study project by the Ministry  of Education, Science and Culture, the Government of Japan. Since 1988, I have beento Karachi annually seven times in November or  December.

      I  shall never forget the  image that I got on my first arrival at Karachi: crowded streets, shouting and roaring voice,  stuffy hot air, confusion, .....  It was just a culture shocok itself.

      During this decade, Karachi has changed  its all aspects  of  town.  The New  Karachi ir Port was opened, improving the service of  the customs,  taxis and other transportation, but I miss  old  shabby Rikisyas, Donkey cars, and Camel cars.

      I  have  never had rain in Karachi.  Every day it is  fine  and dry.  Life in  Karachi is full of vitality and chaos that I  like very much.

      Karachi  has "Pakistan Time".  Tight schedule has no meaning, and eveything is finally set "Inshallah (  If  Allah  wills.)."

     After I accepted  these cultural difference, I have really come to enjoy staying in Karachi, where there are people with good will, chaos and freedom we once had had in our childhood. I  greatly  appreciate  this project that hasbeen giving me the opportunity to look back myself away from my rutine work.

Page-4   Survey of pathogenic and  nonpathogenic  Entamoeba histlytica  in  nonhuman

primates 

              Ayako Kohno

    We  surveyed  the state of infection with  Entamoeba  histolytica  in  our  cynomolgus monkeys and African green monkeys.

    The direct smear method and the staining technique  were used to isolate Entamoeba

histolytica. The culture test using Robinson's medium, the indirect fluorescent-antibody techniques using monoclonal antibody, and zymodeme analysis were used for identification of Entamoeba

 histolytica.

   The results obtained are shown in Table  1 and 2.

Page-6   Rickets in a cynomolgus monkey

                                                                                                      Ippei Sakakibara

    Only a few cases of rickets in cynomolgus monkeys have been reported. 

Case: A female, three years and eight months old cynomolgus monkey, born in the TPC's  colony.

Pedigree: There have been no rickets case in her 13 brothers and sisters by different mothers and 2 by different fathers .

Clinical symptoms: Her weight little increased, reached the peak at the age of  1 year and  months old, and then decreased.

Biochemical examination: Phosphorus level was extremely low (1.6mg/dl) , and alkaline phosphatase level was high (2832IU / l).   Other test results, including calcium level  (10.5mg / dl), were almost  normal.

X-ray findings: The cortical substance of the thigh bones and ribs were thin, and the linea epiphysialis were irregular.  The shadows of the bones were faint. The epiphysis of the thigh  bones deformed its shape like a cup, broadening the bone diameter.

Autopsy findings:  the animal was dwarfish (29 cm, from the top of the head to the base of the tail), and light in weight.  Rachitic rosary was observed at the junctions of the rib and the rib cartilage.  The thorax was deformed, and the spinal column was scoliotic.  The bone were soft enough to cut with a knife.

Pathological findings:  At the junction of the rib and the rib cartilage, the layer of cartilage columns was thickened, and the columns were arranged irregularly.  Some cartilage columns grew longer like a tongue.  Abnormally large amount of osteoid was yield in the cortical compact bone and spongy bone.  Osteoid was observed around Haversian canals and everywhere in the tubular bone.  The front of the calcified zone was unclear.  Osteoid exceeded the calcified zone in ratio in some pat of the spongy bone.  Fibrosis and osteoclasts were not seen in the bone marrow.  Osteoblasts were inactive and flat in shape.

These findings suggest that the monkey grew normally for about two years after birth until the onset of rickets.     Rickets or osteomalaysia are known to be resulted from vitamin-D, calcium phosphorus deficiency, and some other causes.  We have seen some dwarfish animals in our colony ( one %  of  the total number of the monkeys ), and  now we have to clarify the cause of the rickets in our center, from the stand points of both environmental and genetic effects.

Page-7 Clinography: Diabetes mellitus in colony-bred cynomolgus monkeys

Fumiko Ono                  

      Producing  the  animal model of diabetes mellitus (DM) is  important  for  the basic study on human DM.  On the other hand, controlling diabetes mellitus is one of the most important subjects in our colony to improve  the quality of life of the  monkeys. 

     Recently,  it  has been considered that some genetic factors  cause  both insulin-dependent (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) in human.  IDDM is considered to be occurred by an immune-disorder, and  the patients  have  some  common characteristics: 1) young, 2) thin,  and  3)  diabetic ketoacidotic,  while  NIDDM  is  considered  to  associate  with    abnormal methabolism, and its patients are characterized by 1) adult, 2) obesity, and  3) non-ketoacidosis.   NIDDM is the most common type clinically  recognized  in our cynomolgus monkeys. 

    In the past two years, 26 % of the monkeys who died at the age of over 10 years old  had  the medical history of diabetes mellitus or  such a disease as rapid weight loss  after obesity, fatty liver, and hyperglycemia.   Fig. 1 showes the blood glucose value of the colony-bred cynomolgus monkeys at each age. The average value was 52.0 mg/dl (S.D. 22.7) in female, and 58.3  mg/dl (S.D.  24.2) in male.   High blood glucose values indicating  diabetes  were found  in  the monkeys which were more than 10 years old.   In  the  glucose tolerance test (IV-GTT), five animals of the monkeys with high blood glucose values  showed low K value (0.8 + 0.56), being diagnosed  diabetes  mellitus. 

(K value in 13 controls : 3.48 + 1.35).

      Fig.  2  shows the clinical course of a monkey treated with  insulin.  The timing  of initiation of the treatment is usually judged from the  clinikal findings  such as weight loss, hypertriglycemia and hyperglycemia.  Now  the  monkey is under successful control.  It  is inevitable for controlling diabetes mellitus that we diagnose  the disease  as  early  as possible.  One of  the  first markers  indicating diabetes  mellitus  is  blood glucose value, although  its  specificity  and sensitivity  are  not  enough.   Now, we  are  investigating  another  more reliable indicator, such as blood 1.5-anhydroglycitol.

Page-8  Opthalmoscopic observations in the ocular fundi of the  cynomolgus  monkeys

Michiro Suzuki

        This  note  describes two topics as follows:  (1)the abnormal findings and their incidence in the ocular  fundi and  (2) hereditary macula degeneration  (Fig. 1) we  have  recently found in our colony-bred and wild-originated cynomolgus monkeys.  

     We  have observed more than 17 abnormal findings in the ocular  fundi  of  our  monkeys.  The abnormal findings are as follows:  micropapilla, optic disc  ectasia, myeli-nation   of   retinal  nerve  fibers,  arterial   tortuosity,   venous tortuosity, arterial & venous tortousity, artery-vein crossing,  copper-wire artery, inosculation of the artery, vascularization of the vein,  persistent hyaloid artery, embolia artery, retinal degeneration, macular degeneration, peripheral degeneration, retinochoroidal coloboma, and  retinal hemorrhage (Table 1).

     Hereditary  macula degeneration  was first found in the monkeys  having  a certain pedigree (Fig. 2).  As the findings of this disease,  many  fine spots, which were whitish gray or whitish yellow in color, were found in the macula.

     Experimental  cross breeding  between  F1 animals with this  degeneration and  those without degeneration was carried out.  Abnormal  ocular  findings were  observed  in F2 animals derived the F1 with degeneration, but  not  in  F2 animals derived from the F1 without degeneration.

Page 11  Animal Model: Immune Functions in aged-cynomolgus monkeys

Keiji Terao

      Age-associated  decline in immune functions leads to the  disfunction  of natural defense mechanism, resulting in an increase of sensitivity of  a  host to  infectious diseases.  To prevent aged  humans  from  tragic  diseases, nonhuman primate models must be established to study the age-related changes in  immune functions.  The characteristics of age-associate disfunction  are multiple  and  widely vary in individuals.

     In our  survey, the  cynomolgus monkeys  aged  over  20 years also showed the multiple  decline  in various immune functions  including  T  cell  functions  (   e.g. blastogenesis, interleukin-2  (IL-2)  production and IL-2 receptor expression ), hemolytic complement activity and NK activity.

     The most significant changes in the immune system resulted from aging  is the impairment of antibody response to most of foreign antigens,  although  the concentration of serum immunoglobulins (Igs) and the Ig forming cells do not decline with  age.  For example, as shown in Figure 1, anti-A  blood  group antibody levels declined in the aged cynomolgus monkeys despite their  serum IgG and IgM levels were the same as those in the young adult.  This  finding suggests that the antibody response to foreign antigens decreases but that to others including "auto antigen(s)"  might be increased in aged  monkeys.

     To demonstrate this hypnosis, the levels of two auto-antibodies including anti-single stranded  DNA  antibody  (α  ssDNA  Ab)  and  anti-phospholipid (cardiolipin) antibody  (α  CL Ab) were compared between  aged-  and  young adult- monkeys (Fig.2).

     As  we  had  suspected, both auto-antibody level  and  the  frequency  of antibody-positive  monkeys  were significantly high in the aged  monkeys  as compared  with the young adults in  α ssDNA as well as α CL  antibodies.   On the other hand, the differences in mean  α  CL Ab levels between the aged- and the young adult monkeys was not remarkable as compared with that in  α  ssDNA Ab  levels, because there were several monkeys with relatively high α CL  Ab levels in the young adult monkeys.

     It  is  well known that the patients with  systemic  lupus  erythematosis (SLE), which is one of the typical autoimune diseases in human, produce  high levels  of auto-antibodies including  α  ssDNA and   α  CL antibodies which  have IgG  with  acidic isoelectric point. 

     We determined the isoelectric point (PI) of immunoglobulin with sera from aged- and young  adult monkeys to clarify the differences in physical  characteristics  of  auto-antibodies.     Fig.3 showed the distribution of total IgG,  α  ssDNA Ab and  α CL Ab among fractions with different PI.  As a result, there were at least two different auto antibodies in the aged monkeys regarding to isoelectric point ( peak  at p1 5.5 6 and that at pI over 7).  On the other hand, single peak at pI  over 7 was observed in the young-adult monkeys.

     These   results  indicate  that  the  aged  monkeys  must   produce  the autoantibody with abnormal isoelectric point, which never appears in  normal young-adult monkey sera.  These  abnormal autoantibodies are known  to  appear  in  SLE patients and may bind to a phosphodiester-containing epitope that is present in diverse biological molecules which cause severe autoimmune diseases.

    If the mechanism of autoantibody synthesis in aged monkeys is the same as that  in SLE patients, it becomes important that clarifying the mechanism  of autoantibody  producing  pathway as well as the action  of  autoantibody  on endothelial cells in aged monkeys.

Page-15  Control mechanism for growth and differentiation of mouse mammary epithelial cells in serum-free collagen gel culture

                                                        Takuya Kanazawa

        It is during pregnancy that drastic changes are induced in mammary glands in  order  to  initiate  milk secretion  after  parturition.   It  has  been documented  that  growth  and  differentiation of  the  cells  in  vivo  are controlled by multiple hormones secreted from endocrine organs.  To  clarify whether  hormones exert their controls directly to the cells or not and  how hormones  interact  with growth factors, the serum-free culture  of  primary mouse  mammary epithelial cells was devised using collagen gel as a  culture substratum.   Growth promoting effects were demonstrated with  hormones  and growth  factors,  excepting estradiol.  Synthesis of casein,  a  major  milk protein,  was  stimulated  by  a set of  hormones,  prolactin,  insulin  and hydrocortisone.   Other  hormones and growth factors were also  involved  but were  not  essential.  These and previous reported results account  for  the phenomenon that mammary epithelial cells continue to grow without  secreting milk during pregnancy.

Page-18  Audio  test  by auditory brainstem  response  (ABR)  in  cynomolgus  monkeys

    Hayato Narita

     Audio  test  by auditory brainstem response was done  in  our  cynomolgus monkeys to know the age related change in the audio function.

     Nineteen  colony-bred and age-known animals were  divided into three  age groups:  the young (8 animals aged 5 ), the adult(5 animals aged 9  to  10), and  the aged (6 animals aged 22 to 27).   Data were obtained from  13,  10, and 11 ears in each group, respectively (Table 1).

    The  animals were anesthetized by intramuscular injection of  0.3ml/kg  of  mixed  solution  of  ketamine-HCl and xylazine ( 2:1 ) before the  test.   A total  of sixty minutes was needed to test one ear.  The animals  were  laid in the position of face  downward  in  a  sealed box (sized 1x1x1 m) placed  in  a  quiet  small  room.   Electrodes  were  set  to the vertex, the earlobe and under the  nose  as  a  ground.  Headphones were set to both of  the ears.

     The intensity of clicks was varied from 90 to 0 by every 10 dB SPL.  Each  ear  was  examined in response to each intensity level with  the  ABR  waves  from I to V.  The amplifier was set to the sensitivity of 10 V/DIV, and to a filter bands of 50 to 3000 Hz.  Click stimuli were given at a  repetition rate of 10Hz, 10 msec and 2000 times.

     The  result obtained were shown in Table 2.  In the groups of  young  and adult, all ears responded to the levels until to 40 dB, in the aged group , however, 6 of 11 ears responded to 40 dB level.

    Seven  of  fifteen animals which were examined with both right  and  left ears,  have  no difference in the response between each ear.  One of  the  eight animals  which showed difference in the response, had the  difference of 20 dB.   The animal was 22 years old.

   The  aged  group showed significantly longer latency of  peaks  of  ABR waves  I  to  V at the level of 70 dB comparing with  the  young  and  adult groups.

   These  results  show that the aged group have auditory dysfunction.

Page-19   The Corporation for Production and Research of Laboratory Primates           (CPRLP) starts virus tests

     The Corporation for Production and Research of Laboratory Primates (CPRLP)  started virus tests as a  business, after making contract with Microbiological Associates Inc., Maryland,  USA.

     The following six viruses can be tested: B virus, simian filovirus, simian immunodeficiency virus (SVI), simian retrovirus, measles virus, and simian varicella-like virus.

     In addition to the virus tests, the CPRLP can offer users consultation and guidance on the test results, and the CPRLP collects the information on the laboratory primates from all over the world, so that the information will be useful for the surveillance of infectious diseases.

     The following services such as , housing and quarantining laboratory animals on consignment, and dispatching animal technicians are also available.  For further information , please contact the CPRLP.

Page-21   Report on "The 11th Annual Symposium on Nonhuman Primate Models for AIDS"    

     Dr. Yoshikawa, Director of the TPC, attended at  "The 11th Annual Symposium on Nonhuman Primate Models for AIDS", held in The University of Wisconsin, Madison, USA, from 20 to 22 in September, 1993.  He reports on the outline of the symposium in a diary format.

Page-25    From the Primate-Talk

     The Primate-Talk is an open forum of the Wisconsin Regional Primate Research Center (WRPRC) for the discussion of primatology and related subjects.  Here some topics summarized from the Primate-Talk are listed.

Page-25   From the TPC's  "Primate Forum"

     Before one and a half  years ago,  the  TPC began "Primate Forum", a  closed PC-net forum for the people who have interest in primatology.   The number of the members is now about 50.   Steering committee was held on January 18th to discuss the present condition and problems of the Forum.

     Mr. Ida, who has joined the committee from Eizai Co., a pharmacy that has a laboratory in Tsukuba, describes his thought on the forum.