A. Fundamental investigation of bacterial L-form:
1. Two kinds of media which make it possible to isolate L-form of E. coli, Proteus, Pseudomonas, Enterococcus or Staphylococcus at a high rate have been devised by improving the conventional media for L-form; first the concentration of sodium chloride was adjusted as to the problem of osmotic pressure of L-form, and next when some drugs to inhibit synthesis of bacterial cell walls were utilized essential amino acids were applied in combination with the drugs. 2. A new method to obtain rapidly L-form in comparison with an addition of penicillin has been designed by adding lysozyme in amounts of 0.1-3mg/ml to the medium in vitro. 3. The method to isolate and detect L-form from urine, tissue and secretions has been established.
B. Clinical investigation of bacterial L-form:
1. Among 313 urinalyses which were made in 114 cases of pyelonephritis and doubtful pyelonephritis, L-form could be detected in 118 examinations (the detection rate 37.70%). The cases that repetitive urinalysis in the same patient revealed L-form more than 3 times were 27 of them and the detection rate in this situation was 23.68%. No L-form could be observed in 897 urinalyses which were performed in 359 cases of various diseases except pyelonephritis.
2. In 3 of 8 patients with pyelonephritis, L-form could be demonstrated from renal tissues which were obtained by kidney biopsy. None of the biopsy specimens showed L-form when they were obtained from 147 cases of other diseases and examined with 405 times of cuture.
3. L-form could not once be detected in 174 cultures of prostate, urethral and vaginal secretions.
4. Survey of 27 patients with pyelonephritis who revealed L-form with the aid of an incubation: i) Seven cases demonstrated 3 A L-type colony, and 23 cases 3 B L-type colony. ii) Most of the patients were followed by the reduction in ability to concentrate urine, iii) No particular interrelationship could be observed among nonprotein nitrogen, creatinine and urea nitrogen. IV) The loss of the ability to hold sodium was observed in 23.1%, whereas K content was normally low value. v) Quantitative cultivation of urinary bacteria in 193 specimens revealed that 68 specimens (33.23%) showed bacteria more than 10⁵, and 11 (5.67%) presented L-form not less than 3 times. In 7 of 41 specimens which showed 10-10⁴ bacteria no L-form could be detected even by 3 times of examinations. In 79 germ-free urine specimens, 43 (54.43%) showed L-form once and 9 (11.39%) showed it 3 times by cultivation. vi) Osmotic pressure of urine was 400-500mOsm/kg H₂O in most 19 cases, and the lowest value was 230mOsm/kg H₂O. vii) The pH of urine ranged from the lowest 5.0 to the highest 7.3 and the majority were slightly acid as about 6. viii) The number of leucocytes in urine was over 10 in 9 specimens, 5 to 10 in 18 specimens, less than 5 in 7 specimens and 0 in 3 specimens. ix) Pale cells were observed in 21 cases, dark cells in 3 cases, and glitter cells in 14 cases (66.6%) x) Most of the patients had been treated with cephalosporin group or AB-PC group previous to occurrence of pyelonephritis. It was found out from the sensitivity test that exciters of pyelonephritis had frequently gained tolerance to the drugs capable of inhibiting bacterial cell wall synthesis such as PC, AB-PC, CER, CET, etc. xi) As the past illness of higher rank, pyelonephritis was recognized in 15 cases (55.5%) and artificial interruption of pregnancy in 13 cases (48.1%). xii) Basic disease was most frequently renal ptosis, and, next, passage disturbance of the urinary tract such as prostate cancer, urinary calculi, urethral caruncula, etc. xiii) In the examination of the sensitivity of 7 strains of 3 A L-type colony to antibiotics, EM, in general, showed higher sensitivity although there was little fluctuation in sensitivity due to strain difference. xiv) It was of interest that there were two cas

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Studies on experimental pyelonephritis by bacterial L-form
1. In Wistar strain rats, pyelonephritis (or pyelitis) could be experimentally produced by injecting E. coli K 12 penicillin induced L-form or E. coli K 12 lysozyme induced L-form into either vein or renal medulla. 2. In the group of intravenous injection L-form could be detected in 2.38% from renal tissues and in 0.48% from urine, whereas in the gourp of direct injection into the renal medulla renal tissue specimen showed L-form in 26.19% and urine specimen in 1.00%. 3. It has been proved that bacterial L-form is alive within the mucous membrane of the pelvis, under the mucous membrane of the pelvis or in submucous adipose tissue of the pelvis. 4. There was no correlation between normal intestinal bacteria and inoculated E. coli K 12 L-form in wistar strain rats. 5. Electron microscopic examination has disclosed that there are many round or ellipsoid large bodies with various sizes, which are surrounded by a monolayer of the unit membrane and have filament-like structure, in the mesenchyma of the pelvis and that the large bodies resembling the above ones are also present in the cells of arterioles or veins in part.

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Studies on the synergistic action of derivatives of penicillin or cephalosporin were investigated in vitro, employing four strains of gram negative rod bacteria which were isolated from urinary tract infections and resistant against cephaloridine (CER) and ampicillin (AB-PC). The strains tested were Citrobacter 205, Klebsiella 343, E. coli K-12/R 743 (received R-factor carrying penicillin resistant marker from. Rettgerella 743) and Pseudomonas 1050. The patterns of these strains were different from each other. The combinations of drugs in the studies presented were CER-methicillin (DMP-PC) and AB-PC-DMP-PC. The inhibitory effect of the combination on bacterial growth was investigated by turbidmetric assay of growth in broth, plate dilution test and disc test. Enzymological studies were performed employing DMP-PC as an inhibitor of hydrolytic β-lactamase action. Kinetic studies were done also. Synergistic action of CER and DMP-PC was well demonstrated in Citrobacter 205, which was moderately resistant against CER and showed weak cephalosporinase activity. Klebsiella 343 was highly resistant against CER and AB-PC and showed high cephalosporinase activity. On this strain the synergistic action of CER and DMP-PC was clearly demonstrated. However, the growth inhibitory effect of AB-PC and DMP-PC on the strain was only additive. It might be explained by lower affinity of CER to the enzyme and higher affinity of penicillin from the results of kinetic analysis of the enzyme produced by Klebsiella. 343. On E. coli K-12/R 743, no synergistic action was observed even at a high concentration (4mg/ml) of each drug. On Pseudomonas 1050, inspite of very low activity of β-lactamase, synergistic action was demonstrated in the combination of CER and DMP-PC. Generally, synergistic action was observed. more clearly in the combination of CER and DMP-PC than in that of AB-PC and DMP-PC, The mechanism of synergistic action has been thought to be chiefly due to inhibitory effect of DMP-PC on β-lactamase action, but as in the case of Pseudomonas 1050, the above explanation might be incomplete. Further investigations are necessary

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For clinical evaluations of the combination therapy, ten patients suffering from complicated urinary tract infections were selected. In two chronic pyelonephritis patients, the bacteriological studies of pathogens, the determination of blood and urine levels of drugs (preceding cephaloridine therapy, employing larger amount of drug) were performed. Though no bacteriological response was observed by preceding administration of larger amount of cephaloridine, prompt sterilization of urine was achieved by the combination of lesser amounts of cephaloridine and methicillin. Satisfactory correlation of clinical course and laboratory study was observed in these cases. Among other eight patients prompt sterilization was observed in five, significant decrease in bacterial count in urine in one. Relapse of bacteriuria was noticed in two, one of which had neurogenic bladder and the other had indwelled uretero-cutaneostomy tubes. In one case, acute renal failure has developed during the course, however, prompt recovery was noticed by discontinuance of the medication. In one Pseudomonas bacteremia case, the combination of carbenicillin and methicillin was unsucecssful. The preventive employment of the combination of cephaloridine and methicillin for wound infection was satisfactory in one. On the data of laboratory investigations and clinical findings, problems of the combination therapy were discussed.

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It was shown that autoantibodies to the tissue of the rabbit prostate and the male accessory glands could be produced in the albino rabbits by intensive immunization with soluble protein of tissue preparation. Production of antibodies was detected by tanned cell hemagglutination or gel-diffusion technique. It was shown that antibodies produced reacted with tissue preparation from prostate and accessory glands, namely coagulating gland, bulbourethral glands and seminal vesicles. Two precipitation lines were seen on gel-diffusion plate, showing two reactive components in prostatic tissue preparation as reported by Shulman et al. No precipitation line was formed between serum and testis, kidney, liver, lung and pancreas. Chromatography on DEAE cellulose column separated tissue antigens in six fractions. 70% of total activity was recovered in the 2nd fractions. Disc electrophoresis on acrylamido-gel revealed six bands which located at the different site from that of acid phosphatase. However, precipitation band was visualized only faintly at the site of α₂-globulin and γ-globulin by immunoelectrophresis. Histlogical examination of the prostatic tissue from some immunized rabbits showed atrophy and eosinophilic stain reaction of epithelial cells suggesting diminished secretory activity. The direct immunofluorescent technique performed on prostatic tissue revealed some fluorescent spots at epithelium.

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The physico-chemical properties of acid phosphatase isozymes were investigated in the normal human prostate, prostatic tumor, adrenal gland, kidney, seminal vesicle, semen and dog kidney, using pevikon zone electrophoretic and DEAF-Sephadex A-50 column chromatographic techniques. The existence of acid phosphatase isozymes (API and APII) were demonstrated in normal human prostate and prostatic tumor (adenoma and carcinoma). The two acid phosphatase active fractions were obtained on DEAE-Sephadex column chromatography of prostate extract while another active fraction (APK) was obtained in kidney tissue, showing these are isozymes each other. The characteristics of acid phosphatase isozymes of prostate were as follows: The optimum pH of AP I was pH 4.0, and that of AP II was pH 5.0. The Km values of AP I and AP II were 0.166×10^-6M and 0.250×10^-6M, respectively.
These two enzyme proteins were not resistant against heat treatment, although AP II was more stable than AP I. The inhibitory effects of various compounds, such as EDTA, magnesium chloride, sodium fluoride, formaldehyde solution, tartrate, diethylstilbesterol (Honvan), testosterone propionate (Enarmone) were tested. All those affected both isozymes to the same extent.
The characteristics of kidney acid phosphatase were as follows:
The optimum pH was pH 4.25 and the Km value was 0.570×10^-6M. The isozyme (APK) was found to be more resistant than AP I and AP II against heat treatment. APK was strongly inhibited by formaldehyde solution more than AP I and AP II were, and less inhibited by sodium fluoride and tartrate than AP I and AP II were.
The isozyme patterns of the bladder, seminal vesicles and semen were similar to those of the prostate, and those of the adrenal gland and dog kidney were similar to those of the human kidney.

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