Attempts were made to elaborate a technique of the mixed leukocyte culture (MLC) and to determine whether this technique can be used as a quantitative in vitro histocompatibility test on human beings.
Responding cells were suspended in TC 199 containing 20% autologous serum at the concentration of 1×10⁶ cells/1.3ml. Stimulating cells prepared by treatment with mitomycin C were suspended in the same manner. MLC were set up by mixing each 0.65ml of responding cells from 2 persons (two way) or each 0.65ml of responding cells from one person and stimulating cells from the other (one way). After 168 hours of incubation, 1.3μc of tritiated thymidine was added to each culture. Cultures were harvested in 2 hours and radioactivity of cultured cells was measured by liquid scintillation counter after discarding unincorporated tritiated thymidine.
Following results were obtained.
1) The highest radioactivity of MLC reaction is obtained on the 7th day after the initiation of MLC.
2) The leukocytes should be subjected to the initiation of culture within 23 hours after collection of peripheral blood.
3) Homologous serum and erythrocytes present in MLC as admixture have no stimulating effect on cultured leukocyte, and do not exert any appreciable effect on the result of MLC.
4) Abnormally low cpm values are obtained in the cases of polymorphonuclear leukocytosis. After purification of lymphocytes using tetron fiber column, cpm values are normalized.
5) The combinations in which no stimulation is obtained in mutual one way and two way reactions are certified to be MLC identical.
To confirm the reliability of MLC as a histocompatibility test, MLC was performed in various pairs (monozygotic twins, dizygotic twins, siblings, parent-child and unrelated subjects).
1) Seven monozygotic twin pairs tested were all MLC identical.
2) One out of 5 dizygotic twin pairs tested was MLC identical.
3) Seventeen pairs out of 67 sibling pairs (25%) were MLC identical.
4) No MLC identical pair was found among parent-child pairs or unrelated pairs.
These results lead to the conclusion that, at present time, MLC may probably be the most reliable test, in the selection of a living donor among siblings of prospecive recipient.

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For the purpose of early serological diagnosis predicting renal allograft rejection, detektion of changes in serum α₂-globulin, serum γ-globulin and BUN, detection of cytotoxic antibodies and histological and immunopathological studies (direct immunofluorescent technique using anti-canine-γ-globulin-rat-globulin-F. I. T. C.) were performed in four groups of canine renal allografts.
Group 1. Bilateral nephrectomy and renal allotransplantation.
Group 2. Group 1 with immunosuppression.
Group 3. Unilateral nephrectomy and renal allotransplantation, followed by graftectomy 7 days after transplantation.
Group 4. Group 3 with immunosuppression.
Immunosuppression was carried out with prednisolone 5mg/kg/day and azathioprine 2mg/kg/day. Aminobenzyl penicillin 250mg/kg/day and methylchlorophenyl isoxazolil penicillin 250mg/day were given for five days after the operation to prevent infection.
1. α₂-globulin increased after renal allotransplantation and the graftectomy in almost all cases, However because of possible overlapping with α₂-Acute-Phase-globulin, it seems that immediate postoperative rise of α₂-globulin level does not indicate directly acute or superacute rejection of the grafts.
2. Serum γ-globulin fraction increased with the increase of BUN or after the destruction of the grafts was completed. Significant increase of γ-globulin was observed after graftectomy in some cases. Rise of γ-globulin fraction in groups 2 and 4 was not so marked with contrast to groups 1 and 3.
3. Appearance of cytotoxic antibodies in the serum following renal allotransplantation indicated graft rejection. On the other hand, there were some cytotoxin-negative cases although histological and immunopathological studies revealed graft destruction and azotemia (cytotoxin false negative). Cytotoxin false negative cases might be due to (a) no common histocompatibility antigens between a kidney donor and lymphocyte donor dogs because lymphocyte donor dogs were relatively small in number. (b) absorption of the cytotoxic antibodies by the grafts which contain very large amount of antigen. Serum cytotoxic antibodies were detected after graftectomy, or low titers of cytotoxin frequently increased more significantly after graftectomy. (c) The cytotoxin could be so-called C-N-A-P antibodies.
4. The appearance of cytotoxic antibodies and the increase of serum γ-globulin level were not always simultaneous.
5. It seems to be possible to predict graft rejection by cytotoxic titration in near future. So storage method of the lymphocytes and collection of pure lymphocyte suspension should be popularized for higher and earlier cytotoxic antibodies detection, if test lymphocytes are obtained from the same graft donor.
6. Kidney sections of four groups of renal allotransplants were examined by immunofluorescence technique to detect deposits of γ-globulin. They were detected from fifth days after transplantation, and they appeared in the glomerular tufts, peritubular capillaries and venules. As the rejection progressed γ-globulin deposits appeared in the relatively large vessels such as interlobular arteries and arcuate arteries, especially in the endothelium and internal elastic layer. In completely rejected allografts, there were γ-globulin deposits in the interstitial tissue of the cortex and medulla. But no specific fluolescence was demonstrated in the tubular cells. γ-globulin was shown in a significant number of rejected kidneys in comparison with the non-rejecting kidneys. There were two types of γ-globulin deposition in the glomeruli, linear and granular. The demonstration of these deposits suggests that humoral antibodies play an important role in the rejection of canine renal allografts.

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1. Differential renal function test was performed by ureteral catheterization on 33 cases with unilateral disturbance of renal function and the excretion of various chemotherapeutic agents was studied. The results summerized as follows:
a) Creatinin clearance (Ccr) of affected kidney/total Ccr was proved to correlate to PAH clearance (C_PAH) of affected kidney/total C_PAH. Therefore Ccr was selected as an indicator representin t excretory function.
b) Unaffected kidney compensated the affected kidney by drug excretion test.
c) Though it has been generally accepted that C_drug (drug clearance)/Ccr is constant, it was suggested that C_drug/Ccr of affected kidney was lower than that of unaffected one, according to the degree of damage.
2. No remarkable difference was obtained in C_drug/C_PAH between unaffected and affected kidney by the experimental unilateral stricture of renal artery.
3. Analysis of urinary concentration and recovery of drug was performed from the pharmacodynamic standpoint.

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Isolated ileal segments used for ileocystoplasty were studied histologically.
Specimens were obtained from 7 cases (3 Scheele's method, 1 Cat-tail method and 3 Goodwin's method) at the time of reoperation or autopsy.
Histologic appearance of ileal segments from the cases operated by Scheele's method showed slight changes on the structure of the intestinal mucosa except interstitial edema and evident infiltration with cells associated with inflammation.
On the other hand, on the specimens from the cases of cat-tail method and Goodwin's method remarkable mucosal changes, showing partly metaplasia-like appearance, are seen with marked cell infiltration.

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Statistical studies were performed on 150 inpatients with primary urinary bladder tumors admitted to the department of Nara Medical University from January 1, 1963, to December 31, 1968. During the period of 6 years number of inpatients with urinary bladder tumors increased almost every year. Of the 150 inpatients, 126 were male and 24 were female, and the male-to-female ratio was 5.25 with a marked male predominance. The age distribution of the inpatients with urinary bladder tumors ranged from 23 year-to 84 year-old, and the maximum incidence lies in the 6th decade of both sexes, 33.3% in males 7.3% in females, with the total of over 40 year-old age groups occupying 96.7%.
Occupational distribution of 89 inpatients with urinary bladder in Nara revealed a remarkably frequent occurrence in workers in agriculture and forestry. However, a patient was exposed to aniline dye for 7 years as a socks-maker. A correlation between cigarette smoking and the development of urinary bladder tumors was significantly noticed in the present studies. In male, smoking habit was found in 47 patients (90.2%) of 52 cases and in 71 patients (72.4%) of 98 controls. X²-test showed a significant difference between these two groups at risk of below 2.5% (X²=6.22, 0.025>p>0.01). In females, smoking habit was found in 7 patients (63.3%) of 11 cases and in 9 patients (16.7%) of 54 controls (X²=8.48, p<0.01). Data on the serological test for syphilis or on distribution of ABO blood type showed no relationship between 86 patients with urinary bladder tumors and 120 patients of stomach cancer. The in itial symptoms in 83 cases of 119 patients (69.7%) with urinary bladder tumors were asymptomatic gross hematuria. A significant difference on initial symptoms was observed between superficial tumors and deep infiltrative tumors.
Morphologically, the single tumors of urinary bladder were noticed in 93 of 150 patients (62.0%). In 50 of 93 tumors (53.8%) of urinary bladder were located on the lateral wall, in 19 (20.4%) on the trigone, in 9 (9.7%) on the posttrigone, in 8 (8.6%) on the bladder neck, in 6 (6.4%) on the vault, and in 1 (1.1%) on the anterior wall. Histologically, the tumors of 98 cases was classified according to the grade of malignancy and the stage of infiltration. Classification of 98 patients by cell type of tumors in ninety six of 98 cases showed transitional cell papilloma and carcinoma, and in others showed squamous cell carcinoma. Distribution of 96 cases of transitional cell tumors classified according to the grade of malignancy showed papilloma: 3, grade I:18, grade II:34, grade III:31 and grade IV:10, according to the stage of infiltration showed stage O:20, stage A:24, stage B₁:19, stage B₂:14, stage C:8, stage D₁:5 and stage D₂:6.
In 10 cases who were given no essential treatment, the survival rates from onset of initial symptoms were, 1 year survival rate: 30.0%; 2 year survival rate: 20.0%; 3 year and 4 year survival rates: 10.0%; and 5 year survival rate: 0.0%. The survival rates from the time of diagnosis were, 6 months survival rate: 60%, 1 year survival rate: 10.0%, and 2 year survival rate: 0%. In 5 cases treated by transurethral fulgulation, the 5 year survival rate was 80.0%. In 12 cases treated by suprapubic local excision of the tumors, the 5 year survival rate was 50.0%. However, the 5 year survival rate with noninfiltrative tumors was 71.4%. In 5 cases treated by transurethral resection of the tumors, the 4 year survival rate was 80.0%. In 11 cases treated by segmental resection of bladder, the 5 year survival rate was 63.6%. However, the 5 year sur- vival rate with superficial tumors was 87.1%. In 6 cases treated by total cystectomy, the 5 year survival rate was 50.0%.

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1. Culture cells originated from human bladder carcinoma tissue, (transitional cell carcinoma Grade 3) have continued active proliferation over 920 days passing 30 times of subcultures since the preliminary report which described the successful culture of the cells as they passed 12 months.
2. The cells are stable and tend to be more monomorphic by passing subcultures.
3. Colonies of the cells are larger and thinner than that of HeLa cells cultured in the same conditions.
4. Mean generation time of the cells was 36 hours by the growth curve.
5. Activities of β-glucuronidase determined by histochemistry on the cells showed high in the primary cultures but gradually decreased by passing subcultures. The enzyme activities in HeLa cells were lower and diffuse. There was no remarkable differences in lactic dehydrogenase and succinic dehydrogenase activities.
6. Diacetylglucarodilactone, an inhibitor of β-glucuronidase affects on the primary culture cells but not on the cells after passing subcultures. The degree of degeneration was severer than on the HeLa cells in the same conditions.
7. Activities of β-glucuronidase in culture with chemically determined media remained high in the primary cultures but lowered by passing subcultures. The enzyme activities in HeLa cells were lower than in the cells.
8. By electron microscopy several morphological characteristics of transitional cells, microvilli on the surface, intercellular connections, and intracellular compressive vesicles were observed in the cells.
9. Tetracycline fluorescence of the cells was strongly observed than the HeLa cells under ultraviolet fluorescence microscope.

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