The author studied the presence and the natures of the lethal toxin of the virus isolated from the patients of hepatitis in Okayama prefecture. The results were as follows: 1) The toxin of hepatitis virus has nearly the same natures as those reported about other sorts of virus and rickettsia, and its presence is proved by the death occurring in a short time after the inoculation of the embryonated egg-culture into the caudal vein of the mouse. 2) Some difference of the toxicity of this lethal toxin was observed among the strains of virus. The toxicity generally tended to fall by the successive cultivation of the virus in embryonated eggs. When the newly isolated virus is used, the toxicity varies with the quantity of the virus and is strong when the virus multiplies very well and the inoculated embryos show the high mortality. 3) The fact that the lethal toxin comes from the virus itself is proved by the results of neutralization test and others, though definite results are not yet obtained in the neutralization test. 4) The naturs ot the toxin are common to all virus strains, and, judged from the general natures, this lethal toxin is very much alike to the virus itself. though its essential nature is still in question and needs further studies. It is considered, however, that the lethal toxin controls the natures of the virus and is greatly related with its hepatitis-causing nature.
In the present section, the author studied comparatively the death by the lethal toxin and the death by the infection of the virus itself. The caudal vein of the mouse was chosen as the site of inoculation, and the establishment of infection was judged by observing the pathological changes of the inoculated mouse. The results were as follows: 1) By the inoculation of the virus into the caudal vein of the mouse, beside the lethal toxin which killed the mouse within about 24 hours, a toxicity was observed to which the mouse succumbed a few days after the inoculation. This toxicity was proved in all tested strains, though somewhat varied according to the strains of the virus. Some cases were observed, however, in which the inoculated mice survived. 2) These two sorts of toxicity were well demonstrated in the chick embryo, particularly in the liver. The emulsion of other materials than the liver showed but a little toxicty. It is inferred, therefore, that the liver of chick-embryo plays a very great role in the manifestation of the toxicity. 3) When the multiplication of the virus was very well, the toxicity of these two kinds was very high. When the multiplication of the virus was bad, the toxicity was low and mice survived. 4) In the case of the death by the lethal toxin, the typical pathological findings could not be observed. In the case of the death by infection, however, the typical prthological changes were observed in high degree. Even in the surviving mice, the pathological changes were slightly observed.
Employing the chick-embryo method by Murakami et al., the auhtor succeeded to isolate the similar pathogenic agents by the chick-embryo and mouse from the patients of infectious hepatitis in Okayama prefecture. The results were as follows: 1) The natures of the pathogenic agents isolated by the author were nearly the same as those of the one reported by Murakami et al., and this fact proved that the method by Murakami et al. was of practical use for the isolation of the hepatitis virus. 2) The virus habituated by the successive cultivation in embryonated hens, eggs could cause the infection in the mouse. 3) The direct inoculation of the materials of the hepatitis patients into the mouse often failed in the isolation of the virus. It was proved, however, that the isolation was successful even withoul the successive cultivation in embryonated eggs if the used materials and the inoculation method were proper. The natures of the virus isolated by the mouse seemed to be the same as those of the one isolated by the chick-embryo. 4) The isolation of the virus by the direct inoculation of the liverpunctur material into mice or embryonated eggs succeeded only in one case. Theis method was considered, however, to be a useful method for the clinical diagnosis of chronic hepatitis in future.
The comparative studies on the viruses isolated from different materials and by different methods were carried out sero-immunologically. The findings are summarized as follows: 1) Using the viruses isolated from different materials and by different methods, the “Absättigunsversuch” was tried. Though the “Absättigungsversuch” was somewhat different from the original wildführ's method, unacceptable results were sometimes obtained when the sera of hepatitis patients were used. In the mouse the neutraization test was well established between the virus and the immune serum of high titer, and this was proved by the nonappearance of the pathological changes in the mouse. 2) For the inactivation of the virus, ultra-violet irradiation was more simple and certain than the addition of marzonine. It was proved. moreover, that thus inactivated virus still preserved the antigenicity. 3) In the infection-protecting test, the immunization by the inactivated virus protected the attack not only by the same living strain but also by the different strains. 4) These above-mentioned results suggest that the viruses isolated from different materials and by different methods come all from the hepatitis patients and have a pathogenic significance.
Pathological findings and rickettsia on rodent-lung are increased after inoculation into rodent which had been injected by CORTISONE used 0.3-0.4 mg per 10g of body-weight as dosis. H-antigen prepared from emulsion of lung which appeared severe pathological findings shows antigenic nature specifically as positive floculation. test for antibodies produced by Rickettsia orientalis as follows: 1. F-antigen fraction which are fresh emulsion of infected lung do not appear positive floculation against homologous antibodies produced by Rickettsia orientalis. 2. But the F-antigen appears positive reaction after the manipulation of which the material contains F-antigen fraction are frozen and disolved quickly 2-3 times continuously by dryice-Acetone. 3. H-antigen shows positive reaction against homologous antibodies whether the emulsion contains Rickettsia or not. 4. The positive serological reaction as floculation test between H-antigen and homologous antibodies appears rapidly within 4.5 minutes.
We have investigated about the nature of H-antigen comes from rodent-lung communicated by Rickettsia orientalis and have recognized that the H-antigen shows positive floculation against the homologous antibodies specifically as-follows: 1. The serum comes from guinea pig and rabit floculaes the H-antigen specifically. 2. H-antigen does not appear floculation against the serum prepared from the patients of which are Murine, typhus, Malarie, Leprosy, Scarlet fever, infectious Nephritis, Pneumonia, Tuberculosa.
Ever since 1951, the author has been experiencing of hearing numerous murmurs of the heart of the patients suffering from infectious hepatitis, the epidemics which has been rampaging both Okayama and Hiroshima Prefectures, The author at first by means of cardiotonograms and electrocardiograms has attempted to explore into cause of the disease, at the same time under the direction of Prof. Hamazaki the author has also studied from the pathological view points the hearts obtained from the corpses of infectious hepatitis patients after autopsy performed at the Department of Pathology of Okayama University Medical School, with respect to the resutls of electrocardiograms. As the results, macroscopically and pathologically no great change can he seen in the valves, but hemorrhage to a greater or less extent has been observed in the pericardium, endocardium, and in the papillary muscles. Histologically in the heart muscle, there were two cases clearly indicating viral changes, and there were some suggesting the changes caused by general hemorrhagic diathesis as well as pictures indicative of hypoalbuminemia. In two cares the electrocardiograms and the changes of the position of tissue pictures coincided electrocarocardiographically more or less with the changes of electrocardiograms. Over-all studies of the heart sound by means of cardiotonograms, and electrocardiograms, sugg-est that the murmurs of bicuspid valve are caused by the hemorrhage in the posterior papillary muscles of the left ventricle, inducing dysfunction of papillary muscle motility and consequently causing the dysfunction of valve movement. Therefore It should be necessary that clinically the murmurs of the hepatitis should be discovered at the early stage and proer treatment and advices should be given to the patients. Moreover, it is worthy of attention that the cases whose slight changes of electro cardiograms are ususally reversible in the course of treatmnt, while the majority of severe cases tend to become irreversible.
The preparation of resting cells is one of the most important and most fundamental procedures for the physiological studies on bacteria. In order to know how we should do to get the resting cells suitable to the purposes of experiments, the author studied the influence of washing and suspending on the respiratory activity of bacteria. The results are summarized as follows: 1) For the purpose to obtain the resting cells of high respiratory activity, washing with phosphate buffer is better than that with distilied water or physiological saline solution. 2) When the prepared resting cells are used in a short time after the preparation, the respiratory activity is the same regardless of the sorts of the suspending solutions, distilled water, saline solution and phosphate buffer. 3) Borate and phthalate buffers of high concentration somewhat inhibit the respiration. However, in order to make the shift of pH less, M/15 (final concentration) is good. Phosphate buffer, even of M/15, does not inhibit the respiration. 4) In borate or phthalate buffers, the fall of respiratory activity is diminished by the addition of small amount of phospate.
By devising a precipitin reaction with the use of U-tube and using Ogata's antigenantibody dilution test, ring test and mixture test have been compared, the results of which are as follows: 1) In the precipitin reaction with the use of a U-tube, antibody titer centering around the “binding zone” has been found two times more dilute than that observed in the mixture test. and the titer has almost coincided with antibody titer of the ring test. 2) This U-tube test suits convenience to manipulate tnan the ring test, and moreover, it affords relatively better observation results. 3) The U-tubes are washed out easily than capillary tubes. 4) The precipitate obtained by the precipitin reaction has presented mobility by electrophoresis equivalent to γ-globulin of original serum.
Influences of temperature, the concentration of saline solution as well as various sugars have been studied at the time when the reaction is taking place in the mixture test of precipitin reaction. 1) Temperature of 37°C has been found to be most suitable in enabling the reaction to take place to the highest degree; and the influence of temperature is most prominent on the reaction of ovoalbumin system. 2) As for the influence of the concentration of saline solution, it is most marked when antigen is diluted with distilled water; but the reaction falls behind as the concentration of saline solution more increases. Moreover, Uhlenhuth's method exerts more influence on the reaction than Ogata's antigen-antibody dilution method. 3) The precipitin reaction performed by diluting antigen antibody with the use of various sugars decline than the reaction performed by diluting antigen and antibody by physiological saline solution. 4) The reaction taking place when antigen alone is diluted by saline is stronger than the reactions observed in the case where antigen and antibody are diluted by the same kind of sugar solution. 5) The inhibitory action of sugars on the reaction has been found strong in the order of galactose>saccharose>glucose>sorbit>mannit.
1) All the ovoalbumin system and human serum system within 15 hours of low temperature complement fixation have given a similar titer as that of the conventional method (37°C a hour, cold 24 hours). 2) Two hours after the complement fixation test of ovoalbumin at 10°C and one hour after the fixation at 37°C, antibody titer has nearly coincided with that by the conventional method. 3) The complement fixation in the case of human serum system has been better at a relatively higher temperature. 4) The antibody titer of complement fixation of both ovoalbumin system and human serum system, first at 10°C for 2 hours, then at 37°C for 1 hour has been found higher than that in the case where the complement fixation has been carried out first at 37°C for an hour and then at 10°C for two hours.
Previously in our laboratory Tachibana has proven that the bone marrow like other blood-storage organs discharges a great quantity of blood in acute bleeding and that it also occupies an important position among the blood-storage organs. In order to endorse Tachibana's results still further the author has investigated the changes in the erythrocyte counts, hematocrit values, average diameter of erythrocytes, Price-Jones curve, average volume of erythrocytes. and volume index in the nutrient veins of the femur as well as the findings on the bone marrow of adult rabbits after a rapid depletion of blood in the same manner as Tachibana, and the results of the observations are as follows: 1) When a rapid depletion of blood to the amount of 15-20 c. c. /K. is performed in rabbits, an increase of erythrocyte count as well as increase in the hematocrit value can be observed in the nutrient venous blood of the femur, and these increases are especially marked 1-2 hours after depletion. However, through entire course no striking change can be recognized in the Price-Jones curves, averages of the diameter, the volume, and the volume index of erythrocytes; and from these data the increases in erythrocytes and in the hematocrit value are thought to be due to the mobilization of the blood stored in the bone marrow venous sinuses. 2) In the findings of the bone marrow tissues in the rabbit rapidly depleted of blood the decrease of blood in venous sinus is most marked or totally disappeared 1-3 hours after depletion while it later recovers gradually until finally it presents a blood repletion picture somewhat similar to that of the normal. In other words, the blood in the veins of the bone marrow is rapidly mobilized to the outside of the marrow after depletion, and later new blood corpuscles from the hematopoietic foci are recognized to be transferred and stored in the venous sinuses. 3) These results clearly support the experimental results of Tachibana of our labor atory: and therefore, it is thought that the bone marrow, being a blood-storage organ, has an important significance in the repletion of blood in general circulation.
Today anatomical studies on the bone marrow, particularly on features of vascular construction are almost completely known, and no one can refute that more than a half of the bone marrow is composed of blood vessels, thus naturally the space occupied by the vessels is quite extensive. In view of this, with the use of an isotope I131 as a tracer, the author has estimated in figures the volume of blood in the bone marrow of adult rabbits as well as that of the spleen whose vascular construction is similar to that of the bone marrow at the same time measured in the same manner the volume of blood in the bone marrow and the spleen of various experimental anemic rabbits. After comparing the results of those two groups, the following data have been obtained: 1) On the average, 0.5 c. c. of blood is contained in 1 g. of the bone marrow of the normal rabbit, the average of 0.44 c. c. in the spleen, and in 1 g. of muscle 0.07 c. c. of blood is containd on the average, thus it is clear that both the bone marrow and spleen are the organs rich in blood. Especially the voluminousness of the blood vessels in the bone marrow has been proven in figures from the peculiar vascular construction itself which enables an anatomical estimation. 2) The amounts of blood in the bone marrow and spleen decrease accompanying the decrease in the amount of blood in the circulation by bleeding, and following the recovery of the amount of circulating blood the recovery in the spleen is faster than that in the bone marrow. This due to the fact that the spleen is more elastic of the two organs. 3) As regards various experimental anemic rabbits, especially in those caused by leucocyte toxins (x-ray irradiation, injection of nitrogen mustard, or administration of benzol), the amount of blood in the bone marrow has been found to have decreased in the bone marrow whereas in the spleen it has been found to have increased. This phenomenon can be explained by the fact that in the bone marrow it is caused by disturbances of blood circulation while in the spleen by hyperemia. Again in the case of injection of phenylhydrazin which is considered to be especially toxic to erythrocytes, the amounts of blood both in the bone marrow und spleen do decrease but this is so because in this case the blood circulation is disturbed in both organs.
Fujii, Fujita, and Soejima of our laboratory, after a series of experiments with adult rabbits, have already made it clear that retension or mobilization of the marrow blood by the marrow circulation of active substances in the marrow is controlled by contraction or dilatation of the marrow blood vessels, namely, by the vascular actions: and they further have mentioned that no such retension or mobilization is observable in the rabbits blocked of the reticul-endothelial system by indian ink. Therefore, in order to investigate by what mechanism the blocking of the reticulo-endothelial system inhibits vascular actions, the author has performed the following experiment. Namely, by pouring 1% Patent blue, Tripan blue, or Berlin blue dissolved in saline in intact adult rabbits and in the rabbits blocked of the reticulo-endothelial system by indial ink. Splteholz's preparates and tissue specimens of the femur have been made, and the permeability through blood vessels of the dyes mentioned above has been observed: and the following interesting results have been obtained: 1) Any one of these dyes can readily pemeate from venous sinuses of the bone marrow if intact rabbits while none permeates in the case of the rabbits blocked of the reticulo-endothelial system by indian ink. 2) From the above fact, it seems that all the active substances of the bone marrow permeate through venous sinusess into the marrow parenchyma on which they act, at the sametime exerting influences on the marrow nerves; while in the cases whose reticuloendothelial system is blocked by indian ink, that is, when the walls of venous sinus are blocked with indian ink, the permeability of such active substances is inhibited and consequently no action takes place.
Relative to the determination of O2-consumption and glycolysis with the use of Warburg's apparatus, as one of the methods in tracing bone marrow functions, the authors have carried out comparative studies on the following three methods by using the same normal domestic rabbits. METHODS 1. Slicing method is a method in which two Graefe's knife are used as to obtain the slices of bone marrow in the thickness, the socalled limit slice. 2. Homogenate method is a method in which the precipitate of bone marrow is obtained after homogenization and centrifugation. 3. Boring method is a method in which punctuate is obtained from a given portion of the tibia by aspirating it as rapidly as possible at the pressure of -10 mg. Hg. RESULTS 1. The bone marrow pictures observed both in the slicing and in the homogenate methods presented rather similar findings, while in the case of the boring method it revealed the increases of the pseudo-eosinophiles, the mononuclear, and the polymorphonuclear leucocytes as well as a marked proliferation of lymphocytes, mixed with some peripheral blood. 2. The impairment of cells has been found least in the case of the slicing, followed by tho homogenate method, and the geatest in the boring. The boring method, however, has advantages of making over-all course observations of the same individual possible and it is simpler to manipulate, but it also has disadvantages of giving lower values of O2-consumption and glycolysis, and a greater error in the evaluation. As for the determination of O2-consumption values in the slicing method, the values yielded are highest and the lowering rate of value during the observation period is least. For the determination of anaerobic glycolysis, the values obtained by the homogenate method have been found to be highest; while in the case of the slicing method, more constant values with respect to the elapse of time during observation has been obtainable. From these results I have reached a conclusion that the slicing method is the best of the three methods for the determination of O2-consumption and glycolysis in the rabbit bone marrow.
By determining periodically the O2-consumption and the anaerobic glycolysis in the bone marrow of the rabbits depleted successively 4 times the rate of 10 c.c. /kg., with the slicing and the homogenate methods mentioned in the previous report, and by tracing the effects of the sera of these depleted rabbits on the O2-consumption in the bone marrow of normal rabbits, the authors have obtained the following results: 1. As regards the rate of O2-consumption in the case where the slicing method had been used or that in the homogenate method, the rates in both cases tended to increase 24 hours after a single depletion, and it later showed a continuous rise. Whereas in those rabbits subjected to four successive depletions, the O2-consumption reached the highest point 24 hours afterwards, and on cessation of depletion and following the gradual recovery of anemia, the O2-consumption decreased concomitantly; and 9 days after the cessation of depletion the rate returned more or less to normal. 2. The rate of O2-consumption paralleled with the percentage of immature cells of the erythroblastic system as well as of erythroblasts in the bone marrow and with the increase of mitotic figures. 3. A slight increase of the anaerobic glycolysis QMN2 could be observed only during the depleting periods in the case where either of the two methods had been employed; and the periodic fluctuations of QMN2 tended to be similar to those of the O2-consumption. The rate, however, returned to normal 3 days after the cessation of depletion; and later lowering slightly, it finally returned to normal. 4. A parallel relationship existed between the lowering of respiratory and glycolytic ration (QMN2/QO2) and the percentage of the myeloid cells in the bone marrow. 5. Some substances promoting the O2-consumption of the bone marrow of normal rabbits were found to exist in the serum of the depleted rabbits previously mentioned; and the time of the appearance or the disappearance of these substances as well as their effects seemed to coincide with the amounts of O2-consumption of the bone marrow itself. 6. These substances found in the above-mentioned serum were found to be so heatresistant that they could not be destroyed even when heated at the temperature of 100°C for 30 minutes.
Investigating the bone-marrow pictures and the O2-consumption as well as the glycolysis in the bone marrow both of the hook-worm anemics and of the hook-worm dogs, the author, in comparison with those of the bleeding anemia reported in Part II, arrived at the following conclusions: 1. On puncturing the sternum of hook-worm anemics, a picture of maturity arrest was revealed in their bone-marrow picture. On the other hand, in the bone marrow of hook-worm dogs (oral infection with 500 larvae), a bleeding-anemia picture had been revealed at the early stage of anemia, but along with the aggravation of the disease, a picture of maturity arrest was recognized. 2. The slight increase of the O2-consumption and an increase of the anaerobic glycolysis were observed in the punctuate of the hook-worm anemics. 3. In the bone marrow of dogs orally infected with 1, 000 larvae, the rate of the O2-consumption had increased at the early stage while it decreased along with the aggravation of anemia. 4. In the bone marrow of the dogs receiving 500 oral larvae, the rate of the O2-consumption also increases at the early stage, and though lowering gradually along with the aggravation of the disease, it never goes below the normal rate. Whereas the rate of anaerobic glycolysis has been seen to rise by degrees in proportion to the degree of anemia. 5. On comparative observations, the effects on the O2-consumption of the normal-rabbit bone marrow injected with the serum of the hook-worm anemics differ not in any great extent from those of the normal-rabbit bone marrow injected with the serum of normal person. 6. The oxygen consumption and glycolysis of the bone marrow of the hook-worm anemia differ from those of the hemorrhagic anemia; and of the causative factors of this disease, not only the hemorrhagic factor but also the toxic factor seem to play very important roles.
At first with sternum-puncture of the patients suffering from essential chloranemias (idiophathic hypochlomic anemia), the observations on the bone-marrow picture and the determinations of the O2-consumption and glycolysis had been conducted; and then, after adding the serum of the patient to the bone marrow of normal rabbit, in vitro, again the rates of the O2-consumption as well as of glycolysis of the bone marrow of the rabbits receiving continuously for one week injections of the same serum were determined. With these results and with comparative studies on the case of bleeding anemia with that of hook-worm anemia, the author have drawn the following conclusions: 1. The number of nucleated cells in the punctuate of the essential-chlorosis patient as well as the percentage of the erythroblasts in the bone-marrow picture increased; and the majority of the latter were basophilic; and furthermore, of those that were neutrophilic, promyelocytes and or metamyelocytes showed a slight increase in number while mature cells revealed a decrease. 2. The O2-consumption and glucolysis of the punctuate drawn from the patient showed an increase in the rate, the latter especially markedly; whereas the fat-free dry weight of the punctuate had been light but after the treatment it showed a tendency to return to normal. 3. On comapring the O2 consumption and glycolysis of the bone marrow of the rabbits receiving continuous injections of the patient's serum with those of the bone marrow of the rabbits injected with the serum of the normal, no marked difference whatever could be recognized. 4. Similar comparative observations made with regard to the effects on the O2-consumption both in the case where the patient's serum had been added to the normal rabbit bone-marrow and where the normal person's serum to the rabbit's bone marrow, revealed no substantial difference. 5. A marked rise of the rates of both the O2-consumption and glycolysis of the punctuate from the patient is in a strange contrast to the case of hemorrhagic anemia where there is no change at all; and furthermore, the rise has a certain similarity to the rising picture of hookworm anemia but the degree of the rise in the case of this desease is far greater than that of the latter.