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Determination of peripheral lymphocyte subset levels becomes important method for analyzing immune function in nonhuman primates. However, It has been well-known that the separation of peripheral lymphocytes by density gradient centrifuge is relatively difficult in nonhuman primates, because the gravity of red blood cells (RBC) varies among individuals. The purpose of this paper is to compare the major lymphocyte subset levels, the recovery rate of lymphocyte and the contamination of RBC in lymphocyte fraction among four different methods of lymphocyte separation, Method-1: conventional density gradient centrifuge using Ficoll, Method-2: density gradient centrifuge after sedimentation of RBC using 5% dextran, Method-3: whole blood method A; hemolysis before staining lymphocyte, and Method-4: whole blood method B; hemolysis after staining lymphocytes. The lymphocyte recovery rate as well as RBC contamination were also compared between two different hemolysis methods, 1) hemolysis with distilled water and 2) hemolysis with NH4Cl solution. The results obtained are as follows.I. There was no difference in lymphocyte subset levels between Method-1 and Method-3, indicating that loss of specific lymphocyte subset(s) did not occur during density gradient centrifuge in method-1. The density gradient centrifugal method using Ficoll (d=1.077) was able to seem to adapt for rhesus monkeys.II. CD16-positive lymphocytes could not be detected by method-4. It might be necessary to wash cells to remove IgG in sample before staining with anti-CD16, Leu-11a monoclonal antibody in the case of applying Method-4 to analyzing CD16-positive NK cells.III. Method-2 was effective to remove RBC from lymphocyte fraction, but the lymphocyte recovery rate was significantly lower as compared to Method-1.VI. The lymphocyte recovery rate was significantly higher when contaminated RBC were removed by NH4Cl solution as compared when RBC were removed by distilled water.

The mitogen (PHA and ConA)-induced lymphocyte blastogenesis was determined with 15 obese cynomolgus monkeys who were classified into spontaneously occurred obese with hyperlipamic and/or diabetes. There was no difference in the blastogenesis induced by PHA and ConA between obese and normal monkeys when their lymphocytes were cultured in medium supplemented with fetal calf serum. The blastogenesis of obese monkey lymphocytes, however, were significantly lower than that of normal monkey in the case of culturing them in the medium supplemented with autoserum. The wide variation was observed in blastogenesis of normal monkey lymphocytes when they were cultured with obese monkey serum, and some of obese monkey sera significantly inhibited blastogenic response of normal monkey lymphocytes. These results indicate that the responsiveness to mitogensin obese monkey lymphocytes might be normal but their serum might inhibit blastogenesis. Then, the relationship was determined between inhibitory effect of serum and contents of several biochemical markers associated with obese. The significant negative correlation was observed between inhibitory effect and total cholesterol as well as low-density cholesterol (VLDLC+LDLC). This result indicates that the level of serum LDLC+VLDLC possibly correlates with the inhibitory effect, and the increase of serum LDLC+VLDLC with obese might depress the lymphocyte function, resulting in the decrease of immune function in obese patients.

A cytogenetic study on the Bolivian squirrel monkeys (Saimiri boliviensis boliviensis), maintained in Tsukuba Primate Center for Medical Science of the National Institute of Health, has been carried out. The chromosome number is 44. The autosomes consist of 15 non-acrocentric and 6 acrocentric chromosome pairs. The X and the Y are the metacentric and the smallest acrocentric chromosomes, respectively. The Nos. 4 and 6 chromosomes show interstitial C-band variations. The variations of chromosome 4 are confirmed as a Mendelian genetic character by a genetic analysis of the offspring among them. The C-band size differences of the chromosome 6, however, are not clear whether a genetical or a artificially induced difference at present.

In vitro responses of lymphocytes to PHA, ConA and PWM as well as the mixed lymphocyte reactivity (MLR) were determined with 83 pregnant cynomolgus monkeys (Macaca fascicularis) from 39 to 157 days of gestation age. The mitogenesis to PHA in pregnant monkeys was significantly lower during 5 to 8 weeks and more than 20 weeks after conception than in non-pregnant animals. The responses to ConA and PWM did not change during the first 18 weeks of gestation, but decreased remarkably after 20th week.The mixed lymphocyte reactivity was also significantly decreased after 20 weeks of gestation age. This highly significant reduction was observed regardless of the origin of stimulator cells; mated male lymphocytes or third party male lymphocytes. This finding suggests that the depression of MLR in the present study is not specific to fetal histocompatibility antigens but nonspecific. In addition, MLR was apparently suppressed when maternal lymphocytes were incubated with autologous serum (pregnant serum) as compared with that when incubated with pooled young male serum. From these results, it can be postulated that the mixed lymphocyte reactivity of maternal lymphocytes is suppressed by some soluble factors that may be contained in pregnant serum 20 or more weeks after conception.