TPC NEWS Vol.15, No.1 Spring 1996 (Whole Number 25)

(English Summary)

Page-3  Message from the chief director of the Corporation for Production and                                        Research of Laboratory Primates (CPRLP)

                   Dr. Matsuura, chief director of the CPRLP, describes here the present state of the CPRLP and his visions of new business, looking back the last year's big disaster and accidents, including the Hanshin great earthquake, the sarin murder in the subway and ebola outbreaks.  Among which ebola outbreaks in Zaire have resulted in an increase of concern about safety in using nonhuman primates.  He is demonstrating that since 1993 the CPRLP has entrusted with quarantine work and virus tests of imported monkeys for laboratory use, and his resolution to go on making effort toward new era of the CPRLP and TPC.

Page-4  New Mission : Joint use facility for research with monkeys

                   The TPC's Director, Dr. Yoshikawa announces that a new joint use facility for research with monkeys, which has been in the TPC’s plan for a log time, will be realized by the revised budget in fiscal 1995.  The facility will be constructed in the TPC's site.  Dr. Yoshikawa outlines the role and object of the new facility.

                   The Tsukuba Primate Center for Medical Science (TPC)  is the unique breeding facility of large scale, featuring the research with monkeys for medical science in Japan.  However, there are large differences between the activities of the TPC and those of the NIH Primate Research Centers in the USA.   The reasons why the USA has made marvelous progress in medical science with nonhuman primates, despite that it is not natural habitat of monkeys, are: a) the USA is well aware of the importance of development and maintenance of research resources for medical science, and has heavily been investing in that area, b) each primate research center is affiliated with universities to maintain research activities, educating young researchers, and c) the end users such as pharmaceutical enterprises, universities and research laboratories are fully aware of the importance of the development of research resources, either, and as well as being supporting the devlopment.  On the other hand, in Japan at present, above three conditions are not fulfilled yet.

                   Japan is faced with comming aging society.  Accordingly, the research on aging is critical to prevent and control senile diseases such as diabetes, hyperlipemia, osteoporosis, dementia, Parkinson disease, and optical and auditory disorders with age.  Establishment of an animal model is an essential prerequisite of the research on human diseases, and development new techniques in medical science, either.  Nonhuman primates are man's closest relatives, therefore, they are most suitable animals in the study to understand and control these senile diseases, for which mice and rats have been used so far as animal models are not fully applicable.

                   TPC has bred nonhuman primates under the large-scale indoor breeding system since its establishment.  Every animal has been controlled physiologically, genetically, and environmentally.  This breeding system has resulted in a database on individuals since their birth, including clinical history.  TPC’s animals have a great advantage over others’, and we are planing to produce aged monkeys from now on.

                   These aged disease model animals should be used effectively as common research resources for health science.  Breeding nonhuman primates needs various research supporting requirements such as buildings, experts of husbandry, veterinary care and laboratory technology.  Therefore, establishing the joint use facility for research with monkeys in the TPC is more economical and reasonable than each research institution houses a small number of monkeys. Effective and comprehensive research projects will come to be possible only after the system like this joint use facility is established.

Page-6  Research Project at the TPC:  Nonhuman primate models　for senile                            disease

III  Development of nonhuman primate models for lipid research                                       

Teruhiko MATSUSHIMA

                   Lipoprotein(a) (Lp(a)) has been known to be one of the potent and independent risk factors for atherosclerotic diseases.  Although the metabolism of Lp(a) has been attracting keen attention, Lp(a) is found only in human and old world monkeys so that the basic research for Lp(a) is behind.  We have tried to develop cynomolgus monkeys as the model for Lp(a) research and found they have high serum level of Lp(a) and rich distribution of S2 apo(a) polymorphism.  In primary culture of hepatocytes it was found that Lp(a) secretion from the cells was enhanced by supplement of IL-6, as well as in the whole body study by the injection of IL-6, which suggest that the high level of serum Lp(a) observed in inflammatory diseases is mediated by IL-6.

Page-9  IV  Diabetes mellitus in cynomolgus monkeys

                                                    Mitsuya YASUDA

                   Diabetic signs of spontaneous diabetes mellitus in cynomolgus monkeys were

evaluated; then, monkeys were classified into non-diabetic, impaired glucose tolerant and overt diabetic groups and changes in glucose metabolism were analyzed.  Longitudinal studies in the course of development of diabetes from pre-diabetic phase to overt diabetic phase were also conducted.  Characteristics of diabetes in cynomolgus monkeys were as follows;

1) Clinical signs of diabetes in cynomolgus monkeys are similar in certain aspects to human non-insulin dependent diabetes mellitus (NIDDM) .  2) Intravenous glucose tolerance test is adequate method for detecting pre-diabetic phase.  3) Although monkeys in impaired glucose tolerant group show defects in both early phase and secondary phase of insulin release in response to direct glucose stimuli, they has normal insulin response to increased glucose levels after having meal or nonglucose stimulant, arginine.  Diabetic monkeys has minimal insulin response to these stimuli.  4) Abnormalities of glucose metabolism might be referred to attenuated insulin-mediated peripheral glucose utilization as well as insufficient insulin release in diabetic and impaired glucose tolerant groups .  5) Since longitudinal studies revealed development of diabetes in three descendants of diabetic monkeys and deterioration of glucose tolerance in several of them, genetic factors seem to be involved in the onset of diabetes.

                   From above, it is suggested that the family of diabetic monkeys may be useful as an animal model for studies on human NIDDM.

Page-13 Breeding Topics:  Spontaneous malformations in non-human primates

                                        Fumiaki CHO and Fumiko ONO

                   Early reports indicated that the spontaneous malformation rates in nonhuman primate, mostly in macaques (Macaca spp.) and baboons, were 0.44 - 0.52%, but more recent reviews have reported somewhat higher incidence ( 1.02 - 1.62% for Old World species and 6.0% in squirrel monkeys) *.  These rises in malformation rates seem to be derived from the　substantial increases in number of the nonhuman primates which are subjected to  research and observations in captive conditions.

                   Tsukuba Primate Center for Medical Science (TPC), whose indoor breeding facilities are being managed by a unique system in the world, has produced more than 4.000 pregnancies since its establishment.  Newborn babies are subjected to detailed external examinations at birth, and their records are computerized.  Malformations so far observed in the newborn cynomolgus babies (Macaca fascicularis) in the Center are: each single case of funnel chest and scoliosis, two cases of hydrocephalus, three cases of cataract, one case of urethra/vaginal atresia and two cases of cleft palate.  A triple-handicapped cynomolgus baby (Macaca fascicularis) was also born in the Center.  He neither showed the righting reflex action nor had auditory ability.  His eyeballs rarely moved, indicating that the eyes were never focused on objects.  Vocalization was very rare and short.  He also showed dysphagia and had trouble in evacuation.  This baby eventually died at 7 months of age.

                   One of the two cleft palate cases was a cynomolgus F2 baby (Macaca fascicularis) born October 15, 1995 (Fig. 1).  She was the seventh baby of her mother, who had normally had six babies before.  The F2 baby with cleft palate did not exhibit any other malformations like lip palate.  The palate of the baby was bilaterally cleft along the median line, expanding from the hard palate to the soft palate.  Milk was given orally to her with a gastric tube.  As seen in Fig.2, her body weight gain was much slower than that of the normal newborns.  Showing neither nasal-infectious symptoms nor nasal-inflammations at this moment, the baby is growing up normally in our Center.   (*From:Nonhuman Primates I,  T.C. Jones U. Mohr R.D.Hunt(Eds.) Springer-Verlag)

Page-14  Laboratory Tests: Alkaline phosphatase isozyme in cynomolgus                    monkeys

                                                      Keiko OHTO

                   We measured total alkaline phosphatase (ALP) and alkaline phosphatase isozyme activities in the sera from 54 healthy female cynomolgus monkeys to know the change with age.  Total ALP activity of the female cynomolgus monkeys decreased with age until the age of seven, and after that the ALP activity kept low level of about 500IU/l (Fig.1).

                   In the cynomolgus monkeys, only liver (ALP2 ) and bone (ALP 3) isozymes fraction were detected according to the patterns of human ALP.  The ALP2 activity was low in each age, whereas the APL 3 activity showed the same change with age as that of the total ALP activity (Fig. 2 and 3).

                   As for the five cynomolgus monkeys with growth disorder aged 7 to 11 years, three of them showed higher total ALP and ALP 3 isozyme activities than those of the normally grown animals.　 However, each animal differed in both total ALP and ALP isozyme activities ( Fig.4).  Therefore, we found that the cause of growth disorder is not single but complicated.

Page-15  Techniques of Care and Management of monkeys: Water supply system                    in Biosafety Level 3 laboratories

                                                     Katsuhiko KOMATSUZAKI

                   We developed a new water supply system for the animals housed in Biosafety Level 3 laboratories.

                    This system is composed of a conbination of a vinyl water bag , a three-way cock and a back flow stopper, and it should be set every cage (Fig.1).  As shown in Fig.1, the back flow stopper, three-way cock and vinyl water bag were equipped to Teflon water tube ( 6mm inside- and 12mm outside-diameter) connecting water supply pipe and the nozzle.  The amount of the water in the bag was controlled 500-1000 ml/day.  Water flow from the pipe to the bag (intake) and that from the bag to the nozzle (supply) were switched by the three-way cock with L-shape revolving part, and the cock was usually set on supply (Fig.2).

                   Using this cock has resulted in preventing big water leaking accidents by animals' mischief, and in easy switching of water intake to the bag and supply to the animals.  The reason why we used L-shaped cock not T-shaped is: the T-shaped makes through passage from water intake side to supply side, even though for a moment.  Therefore, the possibility of back-flow from the vinyl water bag to the upper side of the cock in case of the T-shaped is higher than that of the L-shaped (Fig. 3).  Addition to that, we equipped the back flow stopper at the top of the Teflon tube.  This double blocking prevents mutual contamination through water supply equipment.  This system, therefore, is considered to be suitable as a water supply system in Biosafety Level-3 laboratories.

Page-17  Case Report: Dermoid cyst in a cynomolgus monkey

Ippei SAKAKIBARA

                   Only a few cases have been reported on dermoid cyst and on teratoma in the primate.  Here I report the first case of dermoid cyst in our center.

Case:  Female cynomolgus monkey born in our center ( 13 years old).

Clinical findings:  She has given two normal births, after that seven times mating with three different male breeders did not result in any pregnancy.  Tumor in abdominal cavity was palpated by a veterinarian at periodical physical examination, and it was removed.  Menstrual cycle around the operation time was approximately normal but showing slightly prolonging tendency.

Macroscopic findings: The cyst was 4.8x4.4cm in size, and its surface was smooth.  It was policystic, containing grayish brown turbid fluid, in which hair and cottage cheese-like white materials were floating.  Two dermoid plugs of 1.5cm diameter having hair on the top were seen.

X-ray findings: Lime-like deposition or tooth-like processes were seen.

Pathological findings: Histologic structures of ectodermal, mesodermal and endodermal were observed, that is, the cyst wall had mostly normal skin tissue structure.  From the outside, the layers of keratin and keratinized materials, squamous epithelium, melanocytes, sebum glands, nerves, hair follicles and hair were seen.   Although dermoid plugs had almost the same tissue structures as those of the cyst wall, its central area mostly was comprised of lipocytes.  Intercellular bridges were cleanly observed in the squamous cell layers.  In the dermis, many number of  blood capillaries, sebaceous glands, sweat glands, fibrocytes, lymphocytes, plasma cells, eosinophils and deposit of lime-like materials were seen.

It is said that dermoid cyst is a benign tumor of which tissue originates in germ cells.  The incidence of this tumor at our center was 1/980 (except the number of aborted and dead cases). 

Page-18  Clinology: Squamous cell carcinoma in the oral cavity of a cynomolgus                            monkey

Fumiko ONO

                   Neoplastic disease of the oral cavity in juvenile nonhuman primates has rarely been reported.   The reported cases such as squamous cell carcinoma in squirrel, capuchin and cynomolgus monkeys, and odontoameloblastoma in Japanese monkeys were all over 12 years old.  Here, I report  squamouse cell carcinoma in a juvenile macaque.

Case:

                    One year old colony-bred F2 cynomolgus monkey (male).  Swelling on the left side of the buccal part was observed.   The physical examination was performed.    The buccapharyngeal became indurated, the hemorrhagic gingivae got into lockjaw, and the mandibuloar lymph nodes enlarged ( Fig. 1). 

                   Since the blood examination showed the increase of CRP and WBC level,  antibiotic therapy was done.  Although the monkey was in good physical and nutritional conditions, the swelling went on enlarging.  Ten days later, biopsy of the gingival area was performed, and it was diagnosed to be squamous cell carcinoma.

                   We accessed to Tokyo Medial and Dental University, School of Medicine, and surgical operation was done by Dr. Kamata and other staff members of the Second Department of Oral Maxillofacial Surgery.

                   Hemimandibulectomy and extirpation of the affected area of zygomatic region including the tumor mass and the enlarged mandibular lymph nodes were performed.　 The monkey recovered, and returned to the TPC.  He could eat well with hemimandibula and his general health condition looked good.  After one month, however, swelling with oncolysis and continuous hemorrhage was noticed again in the region of the lateral orbitalis at the surgical site.   Since reoperation was impossible for him, the monkey was sacrificed at 80 days after the operation.

Page-20 Report on the visit to Microbiological Associates, Inc.

Koji FUJIMOTO 

                   Dr. Fujimoto, Chief Researcher of the Corporation For Production and Research of Laboratory Primates (CPRLP), reports his visit to Microbiological Associates, Inc. in the U.S.A., from which the CPRLP are carrying a project in partnership

Page-22  Literature Guide:

                   Dr. Fujimoto introduces and reviews "Guidelines for the Prevention and Treatment of B-virus infection in exposed Persons"  Gray p. Holmes et al., Clinical Infectious Diseases 20:421-439, 1995.