In our laboratory, many studies on the infectious hepatitis in Okayama Prefecture, particularly on its pathogenic agent have been carried out. The infection tests on animals, of which some parts were reported, were already finished. In the present studies the author tried the serological studies, which would be the key to determine the nature of isolated virus. In these days of having no definite method for the serological diagnosis, it is not easy to appraise the value of complement fixation tests which the author tried. From the following results, however, the author was convinced that this reaction was also of practical value: 1) The antigens were prepared by Larin's or Casals's method from the liver pool of mice infected with the hepatitis virus by the abdominal inoculation. From the results of antigen titer measurement, it was certified that these antigens were worthy of practical use. 2) The complement fixation tests with the sera of convalescents gave significant results. in which even the case of positive reaction of 1. 32 of antigen titer was observed. With the sera of so-called chronic hepatitis, however, the complement fixation titer was generally low and not over 1:8.
In the preceding paper the author reported that the complement fixation test, by improvement of antigen, could be of some use for the serological diagnosis of hepatitis. In addition to this, the author tried the neutralization test and the “Absättigungsversuch” (Wildführ) with the critique of procedures and of criteria for the judgment of results. As the criteria for the judgment of results, the pathological features of animal liver were used. The results were as follows: 1) Using the sera of convalescents, the neutralization test gave appreciable results, though not acceptable as complete. 2) By the combinative use of neutralization test and modified “Absättigungsversuch”, however, it was proved that the isolated virus certainly came from the hepatitis patients and no difference was present among the strains of virus. It was approved that these neutralization test and “Absättigungsversuch” were of some diagnostic significance in the combination with complement fixation test.
In the preceding two papers the author reported that the complement fixation reaction, by improvement of antigen, and the neutralization test and “Absättigungsversuch”, by modifying its procedures, could be of use to some degree for the determination of the virus, and at the same time, could give an aid to the diagnosis of hepatitis. In the present paper the author makes reports on the antigenisity of the isolated strains and, moreover, on the immune-effect of vaccination: 1) The virus was inactivated by the addition of marzonin and preserving it in refrigerator over 3 weeks. Immunization of animals by this inactivated virus could protect them from the attack by the living virus. The fact that the infection-protection was established among the different strains suggested that the strains had no difference of antigenisity to one another but had unitary nature. 2) The modified “Absättigungsversuch” improved by the present author was serologically somewhat significant when the sera had a high titer, but was of little significance when the titer was low. It was suggested, however, that this method could be used as an aid for sero-reaction according to the purpose of tests.
By the cultivation in fertile hen's eggs, a kind of virus which could be regarded as the agent of hepatitis was isolated from the hepatitis patients in Okayama Prefectur. Under various conditions, the author carried out the experiments to study the heat-resistance of the isolated virus. The estblishment of infection was judged by the pathological changes of inoculated mouse liver. The results were as follows: 1) The isolated virus was highly resistant to heat, but was inactivated by heating at 75°C for 30 minutes. This was a very characteristic nature of the isolated virus to differentiate it from other simillar viruses. 2) In respect to the nature of heat-resistance, this virus was inferred to be very similar to the one described by Havens in his human body experiment.
In the present report the author studied the biological natures of the isolated virus, particularlv on the influence of physical and chemical factors on it. The virus, which was isolated from the infectious hepatitis patients and was preserved by the passage through embryonated hen's eggs or mice, was used throughout this work. The results were as follows: 1) The influence of the rain-water, well-water and sea-water, which were taken from the hepatitis-prevailing areas, on the isolated virus were studied. The virus survived for a long time in rain-water, and could survive for a longer time at lower temperature than at 18° to 30°C. From the results of infection test on mice, it was presumed that the virus could be preserved for 6 to 7 weeks under the usual conditions. 2) By the investigation of the influence of other physical and chemical factors on the virus, it was confirmed that this virus had the natures simillar to those described by the senior investigators, e. g. filtrability through Seitz E. K. etc. These biological natures, together with the heat and disinfectant-resisting natures, were conceived to be the important characters being useful for the identification of hepatitis virus.
The virus, which was isolated and preserved in the same way as that in the preceding report, was used in the present studies. The resistance to disinfectants was determined by observing the establishment of infection in the mice inoculated with the mixtures of virus emulsion and various sorts of disinfectants. The results were as follows: 1) The virus was severely injured by the contact with the common disinfectants, and was inactivated by the only 10 to 15 minutes' contact. 2) The inactivation of virus by the contact with disinfectants came near to completeness in about 3 weeks, though the inactivated virus still caused the pathological changes in some cases. The immunization of animals by thus inactivated virus could give rise to a sufficient immunity in them. This fact gave a important suggestion to the further studies. 3) In some cases, even the virus kept in contact with disinfectants for 4 to 6 weeks still caused some pathological changes. This fact suggested that this virus had a very strong resistance to disinfectants.
Four cases of generalized exanthemata were observed during X-ray treatment. The cause of this ailment is not thoroughly classified yet. Studying these cases, it will be said that the disease would develop on the ground of individual idiosyncrasy toward drugs and of decreased body resistance, because of observing most of the patients to who the same dose or more of Roentgentherapy was given as those four cases showed no system reactions. It is of importance that the use of drug having various reactions should be avoided with cares to improve patient's general condition, and diuresis especially to prevent pyelonephritis, during X-ray treatment.
Activity of antigen were studied after different concentrations of formalin was added to antigen for various period of time. Experiments were performed with precipitin reactions, complement fixation tests, and anaphylaxis. Following results were obtained. There were found exaggerated reactions with increase of period during which time antigen was exposed to formalin. Activity of antigen was reduced when concentrated formalin was used as experimental reagent, while increase of activity was noticed in the case of diluted formalin. A peak of activity of antigen was found when 0.001 per-cent formalin had been reacted to antigen at 37°C for 24 hours.
Physicochemical properties of formalized proteins were studied. Decline of hydrogen ion concentration of formalized proteins was noticed in the high concentrations of formalin, while incline in the low concentrations. Colorization of formalized proteins, caused by Folin's phenol reagent, was depressed with increase of concentration of formalin. There was found increased turbidity of formalized proteins with increase of concentration of formalin. On paper electrophoresis marked changes were observed in the higher concentrations of formalin, while in the lower concentrations, slight changes were noticed compared with the controls.
Effects of formalin on the activity of antibodies were studied by means of precipitin reactions, hemagglutination with sensitized blood cells, and paper electrophoresis. No precipitin reaction occurred in the area of high concentrations of formalin, while no marked changes in reaction were observed in the area of low concentrations compared to control tests. Almost same results were noticed in hemagglutination tests with sensitized blood cells as in the case of precipitin tests. On paper electrophoresis significant changes were noticed where one per-cent formalin was used as experimental reagent, while the same results were observed in 0.001 per-cent formalin as in control tests.
One hundred and sixteen children, aged 2 to 11 years, who visited Department of Oral Surgery. Okayama University Medical School from July, 1955 to June, 1956 and had more than one deciduous carious teeth of C3 or higher grade, were investigated and summarized. Forty-three did not have any systemic symptom suspectedly due to deciduous caries, while 71 had some systemic symptoms. The latter group was analyzed accordting to their age and the seasons when they had visited. During the same period, nine cases complaining of unknown fever or some kind of systemic disturbance who visited Pediatric Department first were referred to our Department, suspecting the deciduous caries as their causes. These cases were diagnosed as nephritis, rheumatic fever or allergic sepsis in the Pediatric Department and they had many deciduous caries of C3 or higher grade. Then it could be found that the systemic symptoms disappeared after these suspicious teeth had been treated. This fact may reveal that apical lesion was frequently seen following deciduous caries and gave sometimes an onset to the severe systemic diseases.
The present experiment has been conducted with a view to study the interaction of two species of bacteria from the metabolic aspects; and as a preliminary, O2-consumption and RQ have been measured: Bacteria used: E. coli communis, A. aerogenes, Staphylococcus albus, and Staphylococcus aureus. Substrates: glucose, pyruvate, acetate, and formate. Inhibitory agents: KCN, DNP and NaN3. 1. Since the O2-consumption in the case of the substrate glucose, is accelerated most markedly, it signifies that there is an interaction. 2. The RQ in the case of the substrate glucose has shown a greater increase when two combinations of E. coli and Staph. albus and that of A. aerogenes and Staph. albus are made to act at the same time than in the cases of individual bacterium made to act separately; while the RQ in the combination of E. coli with Staph. aureus has rather tended to decrease, and moreover, its acetoin production has been found to proceed in parallel with the value of RQ. 3. In the case of the inhibitory agent KCN, a marked inhibition of the O2-consumption has been revealed, while in the cases of NaN3 and DNP, an acceleration. Especially in the case of NaN3 an acceleration of O2-consumption has been found; and in the combination of E. coli and Staph. albus with A. aerogenes and Staph. albus, the O2-consumption has been accelerated and its interaction striking. 4. In the case of an addition of the inhibitory agent NaN3, RQ has been found to give a rather similar result as that in article 2, though with a slight lowering tendency. 5. In the case without NaN3, the interaction in the acetoin production presents a similar tendency as that revealed in the case of RQ. With the addition of NaN3, the acetoin production has been inhibited. Therefore, it seems that due to an inhibition of the pyruvate to acetoin pathway by NaN3, no acetoin production can be observed while an accumulation of pyruvate is to be expected.
As reported in the previous paper, it has been demonstrated that the acceleration of O2-consumption is greatest in the case of the substrate glucose, while the interaction likewise seems to occur in the case of RQ. Moreover, since if being evident that the RQ value parallels with the production of acetoin, the present experimcnt has been conducted in order to trace the mechanism involved in the production of pyruvate and acetoin. Bacteria used: E. coli communis, A. aerogenes, Staphylococcus albus, and Staphylococcus aureus. Substrates: glucose, pyruvate, lactate, succinate, and acetate. Inhibiting agents: DNP and NaN3. 1. In the case of the substrate glucose, an interaction has been found to be involved in the production of acetoin, and its adequate pH seems to be 5.8-7.2. 2. The acetoin production in. the case with the use of the bacteria in the process of growth, has been nearly the same as that in the case with resting cells; and that with A. aerogenes especially pronounced. 3. In the case of the substrate pyruvate, hardly no acetoin production can be recognized. 4. In the case of the combination of E. coli with Staph. albus, which is cooperative in the acetoin production, it seems that acetoin is produced by E. coli utilizing of the decomposition products of glucose produced by albus. As for the combination of A. aerogenes with Staph. albus, it appears to be possible to assume that in the decomposition of glucose by A. aerogenes, the accumulation of pyruvate and other acids results in the lowering of pH so that acetoin production capacity of Staph. albus is activated. On the other hand, in the inhibitory combination of E. coli with Staph. anreus, it appears that E. coli might consume acetoin produced in the decomposition of glucose by Staph. aureus.
Putting great stress on the infection by the mouth, the author studied the problem of infection routes of the infectious hepatitis virus. The virus isolated from the patients of infectious hepatitis was used throughout this work. The establishment of infection was judged by the pathological changes in the inoculated mice, which were employed as the test animal. The results were as follows: 1) From the results of the experiments on mice, it was presumed that no remarkable difference of virulence was present among the isolated virus strains, 2) Of all the routes tested, the infection was most easily established by the administration by the mouth, and this caused also the remarkable pathological changes in animals. Consequently the mouth was considered to be the most favorable portal for the infection.
In the preceding part, the author reported that the alimentary canal was the most favorable and the most certain site for the establishment of infection as compared with others. In the present part, the author studied the course of infection from the administration of virus to its fixation in the liver, paying special attention to that in the stomach and intestine. Mice were used as the test animal. The results were as follows: 1) Some of the virus injected into the stomach were inactivated as the time elapsed, but the rest, not inactivated in the stomach, descended to the intestine. It was possible to recover the injected virus from the stomach. The further the environmental pH went to the acid side, the severer the virus was inactivated. 2) In about 3 to 15 hours after the injection, the virus was clearly proved in the wall of duodenum, jejunum, ileum and colon, through which the invasion of the virus seemed to be carried out.
Following the studies in part I and II, the author studied the establishment of infection by the oral administration of virus, the mechanism of infection, and viremia. These were investigated chiefly by the observation of pathological changes of the tested mice. The results were as follows: 1) The investigation of the pathological changes and the distribution of virus in the animal body revealed that the fixation and multiplication of the virus occurred in the liver, and, in parallel with these, the viremia went and reached the maximum around the 14th day after the injection of virus into the stomach. 2) As for the essential mechanism of the alimentary infection, the previously infered results were obtained. In case of the chronic infectious course, however, there remained some questionable points as to the mechanism of the establishment of infection and viremia.
The studies on the hemolytic action of pneumococci and streptococci have been conducted principally with the use of catalase carrier blood. Prof. Takahara has recently encountered the patients whose blood carried hardly any catalase. For the fundamental investigation for acatalasemia, some experiments have been attempted about actual hemolytic picture of some bacteria acting upon the catalase and non-catalase carrier blood. In the present report, blood of man, goat, guinea pig, and rabbit were used as catalase carrier, and that of domestic duck as non-catalase carrier. As for the bacteria, Pneumococcus type I, II and III, Streptococcus hemolyticus (Cook strain), and Streptococcus viridans have been employed for the experiments. And by measuring the catalase and peroxidase activity and respiration of each experimental materials, the following results have been obtained. 1. Erythrocytes and blood of man, goat, rabbit and guinea pig contains catalase, but there is practically no substantial difference in their contents; whereas that of domestic duck hardly possesses any catalase. 2. Peroxidase activities both in the catalase carrier and the non-catalase carrier erythrocytes and blood are approximately the same. 3. O2 uptake during respiration of domestic duck erythrocytes are far more than those of the catalase carriers, and this can be assumed to be dependent upon the presence or absence of nuclei as well as upon the amount of stroma. 4. Catalase activities in Pneumococcus type I, II and III are negative, and are slightly positive in St. hemolyticus and St. viridans. 5. Peroxidase activities in these bacteria nearly coincide. 6. No marked differences can be found in O2 uptake among the bacteria employed in the experiments.
Many studies on the hemolytic actions of Pneumonococci and streptococci have been carried out up to date, but they are all on the catalasemic blood while those on the acatalasemic blood are scarce. And ever since the discovery of acatalasemic patients in the department of oto-rhino-laryngology of our school, various clinical studies have been conducted. Prompted by the urgency of studying the latter, the author have conducted the experiments to clarify the hemolytic action of each bacterium mentioned above on the acatalasemic blood. As for the bacteria of the present experiment, Pneumococci type I, II and III, St. hemolyticus (Cook's strain), and St. viridans have been selected. As for the catalase carrier, the blood of man, domestic rabbit, goat and guinea pig have been employed, and for the acatalase carrier, the blood of domestic duck or that of goose. Thereupon the following results have been obtained: 1. In the case of the 4% blood agar medium, the presence or the absence of catalase in the culture medium makes almost no difference in the growth of each bacterium. 2. PII, PIII, St. hemolyticus and St. viridans form the larger hemolytic and the Met Hb rings in the acatalasemic medium than in the catalase medium. As for PI, it scarcely forms any hemolytic ring in either medium, but its MetHb ring is the largest of all. 3. In the acatalase carrier medium, the large discolored rings are formed, and this can be construed to be due to a pentdyopent reaction. 4. In the 1%-glucose medium, the growth of bacteria is better and the hemolytic and the discolored rings are far larger. It seems that the formation of MetHb is somewhat inhibited in this medium. 5. In the anaerobic medium, the formation of MetHb is far less than that in the aerobic medium.
The experiments have been chonducted in order to investigate the effects of hydroxylamin and KCN upon Pneumococci, St. hemolytics, and St. viridans in the catalase carrier and the acatalasemic blood; and the following results have been otained: 1. Hydroxylamine (HXA) and KCN at the concentration of 10-5 Mol. inhibit the catalase reaction of blood cells without interfering the blood respiration. Furthermore, hardly any influence is exerted on the respiration of bacteria at this concentration. 2. When HXA at the above-mentioned concentration is added to K blood, the Met-Hb rings of PIII, H, and Vi become markedly larger than those of the control; and they approach to those of the AK medium. Moreover, the hemolytic rings of each colony become discolorized. 3. In the case when KCN has been added, they give results rather similar to those of HXA, on the other hand, the size of each ring is smaller. 4. HXA and KCN little affect AK blood, and almost no qualitative changes can be recognized. 5. Even in the case when HXA and KCN are added, the Met-Hb formation in the anaerobic medium as compared with that in the aerobic case can hardly be recognizable or extremely little if any. 6. The results obtained on the observation of the Met-Hb formation in the liquid medium, with the aid of automatic spectrophotometer, coincide rather closely to those revealed in the solid medium.
Bone changes in residents in the high fluorine zone, which were described by Prof. Dr. Hamamoto, called general attention in the medical world. This study was made based on clinical experiences that sclerotic changes of vertebrae found in patients complaining of lumbago were possibly caused by taking water from well which contained abnormally high concentration of fluorine. 1) Slight degree of mottled teeth and weight loss, averaging 20 gms, were found in rats fed on water containing 13 p. p. m. fluorine for one year. Bone changes, however, were not noticed and no changes were found in levels of serum calcium and inorganic phosphor. 2) Marked mottled teeth developed in rats fed on water containing 100 p. p. m. fluorine for same period, though general condition were not involved. Upper incisor overgrew, though fragile. No marked changes were found in skeletal system and level of serum calcium. Slight increase in serum inorganic phosphor was noticed with concomitant degeneration of renal parenchym suggesting close relation between them. Slight parenchymal degeneration were found in liver, heart, lung, spleen and other organs.
1) Originally oxyphil cells are absent in parathyroid of normal rat. It was found that dark chief cells increased and light chief cells decreased in parathyroid of rats in chronic fluorine poisoning. These changes were considered to show retarded function. There were, however, no changes in weight, weighting about one mg. There were found no changes in thyroid gland, thymus and submaxillary gland. 2) Extirpation of parathyroid glands gave no effect in development of fluorine poisoning. 3) Parotin could not prevent development of fluorine poisoning though occurrence of poisoning was seemed to be somewhat retarded by alleviating interference of ossification of epiphyseal cartilage. 4) General condition of intoxicated rats were deteriorated in most cases by fracturestress and callus formation were not found in such cases. Callus formation were noticed, though slightly poor compaired with in control groups, in poisoned rats whose general condition were not so involved. Histologically, disturbance in osteogenesis and retarded absorption of cartilagenous callus were found.