With the purpose to elucidate the origin of phagocytes in ascites the author carried out various cytological investigations and other vital observations concerning the milky spots in the greater omentum of mice and rabbits by means of the tissue culture; and also in order to clarify still further the relationship between the milky spots in the greater omentum and the phagocytes in ascites, the auther perfcr.ned excision of the greater omentum in mice and obtained the following results: 1. Cytologically, the cells of the milky spots in the greater omentum are exactly identical with phagocytes, in ascites, and they are not histiocytes but are cells rather resembling closely to monocytes. 2. In the ascites after excision of the greater omentum number of cells are decreased, and what is more, small cells become scarce and the majority of cells are decrepit from age. 3. From these findings it is clear that the milky spots in the greater omentum are the souce supplying phagocytes in ascites.
With a view to clarify the origin of milky spots in the greater omentum the author studied the greater omentum in parallel relationship with ascites using embryos and newborns of rabbits and arrived at the following conclusions: 1. Milky spot-like cell colony can be recognized in the greater omentum of the embryo after 15 days' pregnancy, and at the same time phagocytes are found in ascites, which are cytologically identical with cells composing this cell colony. Therefore, it can be said that the origin of phagocytes in ascites is the milky spots of the greater omentum even in the embryonal stage. 2. Since a mass of erythroblasts can be recognized in the greater omentum of embryonal stage and milky spots are immature monocyte-like cell foci, it is believed that the milky spots in the greater omentum after birth have their origin in the embryonal mesenchymal hematopoiesis.
In oder to elucidate the origin of phagocytes in pleural fluid the author studied the pleura in mice and rabbits from various angles and obtained the following results: 1. Even in the pleura, there are milky spot-like cell colonies which might be called “pleural milky spots” and have the similar appearance of the milky spots in the greater omentum, and the cells composing the colony cytologically resemble closely to monocytes. 2. Cytologically cells of milky spots in the pleura are exactly identical with phagocytes in pleural fluid, and moreover, it seems that the former is the origin of the latter judging from the results obtained by cover-slip culture and by stimulation experiments by intrathoracic injection of typhoid vaccine.
With the purpose to clarify the effect of antibiotics on the hematopoietic function of the bone marrow, especially on the leucocyte series, the author observed the effects of direct addition of various antibiotics on the tissue growth area and cell density of the bone marrow by performing rabbit bone-marrow tissue culture (cover-slip method); and obtained the following results: 1. Any one of antibiotics such as penicillin, dihydrostreptomycin, chloromycetin, aureomycin, achromycin, carzinophilin, actinomycin, and sarkomycin, at a higher concentration than a certain fixed concentration acts inhibitorily on the cell growth of the bone marrow. 2. Antibiotics such as penicillin, dihydrostreptomycin, aureomycin, and achromycin at the concentration close to the maximal concentration to blood as conventionally used in clinics all have no toxic effects, but some of them give a favorable influence on the cell growth of bone marrow, though slight. 3. As for chrolomycetin, actinomycin, carzinophilin and sarkomycin, they all act as to inhibit the bone-marrow tissue growth even at a relatively low concentration.
In order to elucidate the effect of antibiotics on the hematopoietic function of the bone marrow, especially on the leucocyte series, as a follow-up study of Part 1, the author studied the effects of various antibiotics on the function of mature pseudoeosinophils in the rabbit bone marrow, especially on the wandering velocity, carbon-particle phagocytosis, and vital staining, by means of bone-marrow tissue culture; and obtained the following results. 1. Antibiotics added to the bone-marrow tissue culture at a concentration higher than a certain fixed concentration all act as to inhibit the pseudoeosinophil function, and this tendency is marked in the case of antibiotics for cancer. In addition, chloromycetin tends to inhibit the function even at a relatively low concentration. 2. At a fixed low concentration including the maximal concentration, to blood penicillin, dihydrostreptomycin, aureomycin and achromycin all act as to promote the hematopoietic function rather than inhibiting it. This seems to indicate that leucocytes play an important role in the mechanism of cure of the bacterial infection due to antibiotics.
In order to clarify the effect of antibiotics on the hematopoietic function of bone marrow, especially on the erythrocyte series, the author studied changes in the erythrocyte count and hemoglobin content in the rabbit bone-marrow tissue culture to which various antibiotics had been added directly, and obtained the following results: 1. Antibiotics such as penicillin, chloromycetin, aureomycin, carzinophilin, actinomycin, and sarkomycin, at a concentration higher than a fixed concentration all act as to inhibit the hematopeietic function of the bone marrow. 2. The effect of such antibiotics as penicillin, dihydrostreptomycin, aureomycin and achromycin at the maximal concentration to blood cenventionally used in clinics is only slight, and the use of these antibiotics at such a concentration has hardly any inhibitory action on the hematopoietic function. 3. On the other hand, chloromycetin shows a tendency to inhibit the function at a relatively lower concentration than penicillin or dihydrostreptomycin.
The incidence of myoma of vaginal portion of the cervix uteri, being less than one per cent, is rare. The present case was an unmarried nuli-para, 24 years and 4 months old, with mental weakness, and had an abrupt protrusion of the myoma outside the vulva and the disturbances of micturition and defecation. A myoma as large as a super-goose egg grown in the vagina from the posterior lip of the cervix, when left alone for one month, has grown to a size greater than a first and has protruded outside the vulva. Although she was cured after the abdominal hysterectomy, histologically it was a leio-myoma with infection. The myoma of the vaginal portion of the cervix uteri is encountered mostly in the age around 40 years and in multipara, and incidence in the anterior or the posterior lip is about the same, in size growing as large as a hen's egg to the head of an infant. The prognosis is good and the treatment of choice is surgical one.
Concerning the characteristics of functional uterine bleeding there many studies carried on from various angles such as from the histological, endocrinologic, and autonomic nervous systems, the blood-circulatory system, and the tissue-enzyme system. However, the actual characteristic of this bleeding has not completely been clarified, and therefore, most of therapeutic treatments that we now employ are still on the experimental stage. Histological studies of this bleeding likewise suffer from various clinically confusing problems, although thare are very few reports on the therapeutic method by the histological classification of the functional uterine bleeding. With the purpose of pursuing the relationship between the histological classification on one hand and the clinical symptoms and the aspect of treatment on the other, from the clinical viewpoint the author studied 190 tissue spccimens of the endometrium obtained from the patients with functional uterine bleeding, admitted to the Clinic of Obstetrics and Gynecology, Okayama University Medical School, and 38 tissue specimens obtained by a single scraping of curettage in the patients with functional uterine bleeding, visiting our outpatient clinic, all together 228 specimens. These tissue specimens were fixed in 10% formalin solution, embedded in paraffin, and stained in hematoxin-eosin. Since there are the proliferative phase, the secreatory phase and menstrual phase of the menstrual cycle and because these conditions or the abnormality of these conditions are reflected on the endometrial picture in functional uterine bleeding, the author classified these histological pictures into the proliferative type (Type I), the abnormal-proliferative type (Type II) (the hyperproliferative type (Type II1), the cystic glandular hyperplasia type (Type I2), the secretory phase type (Type III), the abnormal secretory type (Type IV) (mixed type (Type IV1), cystic glandular secretory type (Type IV2)), the abnormal menstruation type (Type V), and the atrophic type (Type VI). These clinical symptoms (especilly the bleeding type is classified into types I-IV) and the results of treatment have been compared statistically, and obtained the following results. 1. Although each histological type shows an individual variety of bleeding types, the abnormal proliferative type (Type II) shows abnormal persistent bleeding type (Type I) and the completely irregular bleeding type (Type IV) over 80 per cent of the atrophic type (Type VI). 2. The stature, physique, menarchal age, the question of conception and delivery, and dysmenorrhea have no significant difference according to the histological types. 3. In the adolescent stage (under 25 years) most of them belong to the abnormal proliferative type, showing a protracted bleeding type. 4. In the mature stage various histological types occur, making them difficult to differentiate from clinical findings, but the swelling of the ovaries, a high degree of anemia, and mixture of blood clots often occur in the abnormal proliferative type. In the cases of within one year's lapse of time after an abortion or premature delivery or in the case with displacement of the uterns having persistent bleeding in small amount after menstruation, they can be clasified as the abnormal menstrual type (Type V). 5. After climacteric (46-55 years) most of them belong to the abnormal proliferative type and the atrophic type; especially the persistent bleeding type is predominant, and those showing an enlargement of corpus uteri with violet coloration of the vaginal mucous membrane are of the abnormal proliferative type. Those with small and hard corpus uteri and whose vaginal mucous membrane is hyperemic, presenting an irregular bleeding type, are significantly an atrophic type. 6. The persistent bleeding type is clinically the worst bleeding type.
With the use of Staph. aureus (au), Staph. albus (al), Str. hemolyticus (hem), D. peumoniae (p), A. aerogenes (aer) and M. luteus (M), all of those were obtained from the departmental stock, the author investigated the difference in the variation of hemolytic feature of bacteria through mice passage by mean of comparison between one species inoculation of bacteria and two species mixed inoculation. The following results were obtained. 1) Mixed inoculation of au and hem into abdominal cavity of mouse showed increase of virulence, and exhibited increased asthenia of animal and higher death rate in comparison with the inoculation of either species of them. Contrarily, in the combination of au and p the virulence of bacteria was decreased, hence two species competed in their virulence. 2) After the hypodermic inoculation on the back of mouse au increased its hemolytic virulence by a short time passage of animal; however, its virulence markedlp increased when it was inoculated with hem, p, or M. Using standard strains of the microoragnisms described, the author studied the interaction between two species of bacteria on growth in blood-added or non-added fluid media and on hemolysin production. The following results were obtained. 1) Various combinations of two species of bacteria, in which one was au or al the other was taken out of hem, p, aer, or M, were made and cultured in the fluid media described above. From the results of culture, it was noticed that the growth of bacteria in combination of two species was inhibited and the hemolysin production by them was reduced compared with pure culture of either species. 2) When a species cultured in the media contained the cell free supernatant in which the other species was cultured previously, the growth of bacteria and their function of hemolysin production generally tended to inhit compared with the controll. 3) These facts presumably showed that both species of bacteria in combination produced substances that showed the inhibitory effect on the growth and the hemolysin production of other species, and that the hemolysin produced by a species was turned inactive by other species. Using the resting cells of the microorganisms described, the author observed the interaction of two species of bacteria on their hemolysin production, and obtained the following results. 1) When a suspension of restiing cells was shaken after the addition of peptone into the media, aur showed the increased production of hemolysin: hem, p, al showed only slight production; while aer, M did not produce it. 2) Hemolysin production in the mixed suspension of aur and hem, and of aur and p was increased compared with the single cell suspension. On the other hand, the hemolysin production was decreased in the combination of aur and aer and of aur and M. 3) The hemolysin produced by aur was turned inactive by shaking with the other bacteria; on the other hand the hemolysin production of aur was increased by the shaking with the cell free supernatant of the other bacteria. 4) Presumably, it was concluded that the interaction between two species of bacteria on hemolysin production included two ways, one was accelratory and the other inhibitory, and they could be observed only in over-all state depending on their environment.
On nidation of cancer cells a pure physical study of their permeability. was performed Further the irradiated lymphnodes were studied histologically and morphologically from a pure physical standpoint especially on their lymph stream way. The results were as follows: 1) After prophylactic radiations of 60 r, 600 r or 300 r×5, the little granuli (the spores of myxomycetes), the size of small cancer cells, were injected into the lymphnodes. Then their permeability was compared with that of the non-irradiated lymphondes. The spores generally decreased with the lapse of time in the latter while in the former the permeability was disturbed and the spores remained for comparatively long time. 2) Instead of the spores, Brown-Pearce tumor cells were injected. The result was the same as in the case of the spores. They remained for longer period in the marginal sinus and in the intermediate sinus of the lymphnodes which had been irradiated with 600 r and 300γ×5. 3) The lymph sinus as a way of lymphatic stream was dilated in case of irradiation by 600 r and 300 r×5 both in marginal and marrow sinus. Also a lymphatic stagnation was found. 4) It seems that the delay and slowness of lymph stream which comes from the dilation of lymph sinus is the reason of the spores or the Brown-Pearce tumor cells remaining comparatively in number when 600 r and 300 r×5 X-rays are radiated to the lymphnodes. 5) The reticular fibres which lay relatively densely at the border of the intermediated and marrow sinus are considered to hold the stream of the little granuli or tumor cells from the marginal sinus into the marrow sinus to some degree. 6) The prophylactic radiation is considered to make the nidation of cancer cells easier.
The cancer metastasis in case of X-ray irradiated lymphnodes was experimentally studied excepting the factors of the primary tumor. The results were obtained as follows and also the reasoning of the preceding part was confirmed. 1) The nidation of tumor cells in the lymphnodes irradiated with 600 r or 300 r×5 of X-ray is faster than those with 60 r or non-irradiated controls. 2) The nidation of tumor cells in the lymphnodes begins generally at the marginal sinus and they infiltrate to cortical tubercles. In the case irradiated with big dosis of X-ray, it begins at the border of the marginal and intermediated sinus and the tumor cells infiltrate to the subcortex and then the cortical tubercles. 3) After the nidation of Brown-Pearce tumor cells in the lymphnodes, they do not proliferate along the lymph sinus but mainly along the parenchym of the lymphnodes.
The growth of cancer cells in the lymphnodes was studied in two groups; one group after 24 hours from X-ray irradiation and the other after 4 weeks from X-ray irradiation. The growth of tumor and its reciprocity between surrounding connective tissue, capsule and tabecle of lymphnodes were also studied. Furthermore fibrisation of lymphnodes 4weeks after X-ray radiation and proliferation of capsule and connective tissue of parenchym were studied. 1) In case of injection after 24hours from irradiation the growth of tumor in the irradiated lymphnodes was better in those irradiated 5 times every day with 300 r or 600 r once than in those irradiated with 60 r or non-irradiated. 2) In case of injection after 4 weeks from irradiation with 300 r 10 times every other day the growth was less than the non-irradiated group. 3) Between the growth of tumor in lymphnodes and the increase of capsule and trabecle there was generally an inverse relation. The more the tumor is, the less the thickness of capsule and connective tissue of trabecle. 4) The capsule and trabecle of lymphnodes irradiated with X-ray are factors influencing on the growth of tumor. In the early stage after irradiation the anti-cancerous power of capsule and trabecle of lymphnodes was weakened, but after 4 weeks from irradiation it was accelarated.
The purpose is to study the starting points of efferent fibres from each part of nucleus lenticularis especially descending fibres reaching to nuclei of the midbrain. Dividing the cat nucleus lenticularis into five parts and making electrically cauterized lesion in each part, the secondary degeneration was studied by Marchi staining. 1) After lesion of dorsal part of the pallidum the degenerated granules were found in the nucleus thalami lateralis ventricularis at isolateral side, luys body, Forel area, substantia nigra, red nucleus at both side and nucleus interstitialis Cajali. The secondary degeneration was more marked on the isolateral side. 2) After lesion of ventral and lateral parts of the pallidum, stronger degeneration was proved though the degenerated part was almost the same as 1). No difference was seen between the both parts. 3) The fibres starting out from the pallidum and apparently communicating with the opposite side consisted of fibres passing Forel and Meynert crossing and reaching to red nucleus and nucleus interstitialis Cajali and also fibres passing the back conjunction and reaching to nucleus interstitialis Cajali. 4) After lesion of dorsal side of the putamen the degenerated fibres were found in the middle and ventral part of the pallidum. Further descending, the degenerated granules were found in substantia nigra on the isolateral side a little. 5) After lesi n of ventral inner part of the putamen only small degenerated granules were found in ventral outer side of the pallidum. 6) No degeneration was found in the fibres starting from the putamen and reaching the opposite side.
An experimental study was performed by (INOUE'S) irrigating method in the living brain of decapitated rabbits. Adding 6 phosphogluconate into the irrigating fluid, pentose was found to be produced. Then the rabbit brain was irrigated with 6-P32-G and the reactive products after irrigation were analysed with paperautoradiography. In the reactive products ribose-5-phosphate was proved.
The O2 comsumption of G 6 P, 6 P-G and R-5-P was measured in normal rabbits by Warburg manometer. G 6 P, 6 P-G and R 5-P are easily oxidated by brain cortex homogenate. The oxidation of G 6 P and 6-P-G was more accelerated by the addition of TPN. From the facts mentioned above, the Warburg Dickens scheme of the brain can not be disregarded.
The O2 consumption of G 6-P, 6-P-G and R-5-P in the brain of latent cerebral local anaphylactic rabbits was measured by Warburg's manometer and compared with that of normal rabbits. G-6-P 6-P-G and R 5-P were oxidated by the brain cortex homogenate of the latent cerebral local anaphylactic rabbits. The oxidation of G 6 P and 6-P-G was more accelrated by the addition of TPN. In the brain of the latent cerebral local anaphylactic rabbits, oxidation of G-6-P, 6-P-G and R-5-P, namely metabolism of Warburg-Dickens scheme, showed a larger tendency to be controlled than that in the brain of normal rabbits.
As exact knowledge of the lymph system which plays an important role in the metastasis of uterine cancer is indispensable in the diagnosis and treatment of cancer of the uterus. According to the studies on the lymphatic system of corpus uteri by various investigators, the draining lymphatic ducts of corpus uteri mainly run in two directions, namely, one toward the outside of the pelvis and the other into the pelvis, and as for the regional lymph nodes outside the pelvis the lumbar nodes are pointed out and those for inside the pelvis the hypogastric lymph nodes. Despite this, most papers deal only with the lumbar nodes. Therefore, the author performed the present experiments with the purpose to ascertain once again whether or not the lymphatic ducts running towards the pelvic lymph nodes have any great significance to corpus uteri. Of the 12 cases with cervical carcinoma operated upon in our clinic during the period from January to June 1957, by injecting 1-3 cc of specially-prepared Indian black solution into the muscle layer at the base of uterus 21 hours before the operation, we observed the sedimentation of dye in the uterus and lymph nodes after hysterectomy both macroscopically and histologically. As the result in all the cases we observed, besides the streaks of the dye sedimentation running from the base of the uterus to mesosalpinx, a marked dye sedimentation along the uterine arteries, enabling us to follow up the existence of the main vesseles flowing in the direction of the lymph nodes within the pelvis. The percentage of the dye sedimentation is 100 per cent in the lumbar nodes; 70.8 per cent in the hypogastric nodes; 40.0 per cent in the upper iliac ncdes; 23.5 per cent in the obturator nodes; and 11.7 per cent in the lower iliac nodes. In other words, apart from the clear-cut existence of main vessels of corpus uteri flowing towards the lymph nodes (lumbar nodes) outside the pelvis as is generally accepted, it has been verified that there also exist main vessels running into the lymph nodes situated in the exterior side of the pelvis. That is to say, by a high degree of the dye sedimentation along the uterine arteries in the specimens prepared from the hysterectomized materials as well as by the dye sedimentation in the pelvic lymph nodes such as the hypogastric, the upper and lower iliac ncdes and obturator nodes there are the lymphatic ducts, running from the base of uterus by way of the lateral side of uterus along the uterine artery and reaching the hypogastric nodes, which seem to have a great significance as the main vessels of the corpus uteri just as much as those lymphatic ducts going outside the pelvis. Therefore, it is necessary to give a second thought to the regional lymph nodes of corpus uteri, namely, apart from those lymph nodes (lumbar nodes) outside the pelvis, there are lymph nodes in the pelvis, the existence of which has often been ignored.
It has been believed that the cancer of corpus uteri, differing from the cervical carcinoma, progresses slowly and its metastasis is also delayed, having a relatively good prognosis, but ever since the discovery of the metastasis in the hypogastric nodes in the course of the cancer operation by Javert in 1948, the removal of pelvic lymph nodes is now recommended even in the case of cancer of corpus uteri in Europe and America. With a view to investigate whether or not the cancer of corpus uteri metastases to the lymph nodes in the pelvis, the author re-examined the method of operation. The investigations as to the cancer infiltration and metastasis have been carried on 23 cases of the cancer of corpus uteri and 2 cases of cervical carcinoma which were finally diagnosed as the cancer of corpus uteri after operation to the total of 25 cases along with the 12 cases of cervical carcinoma previously reported in Part 1, all treated in our clinic during the period ranging from January 1950 to August 1958. As the results, those who revealed the cancer infiltration to the cervical canal amount to 6 cases (24.0%), those occypying only the endometrium 12 cases (48.0%). and those who revealed the infiltration into over one half of the muscle layer amount to 4 cases (16.0%). The cancer infiltration is recognized in a much greater proportion than what has generally been believed thus far. Out of 12 cases two case (16.6%) revealed the metastasis to lymph nodes, one case showed only a slight cancer infiltration just beyond the internal os of uterus and another the intra-muscular invasion of a moderate degree. As for the metastasis of cancer of corpus uteri to lymph nodes in the pelvis in Europe and America it is said that as high a percentage of 19.5 per cent can be recognized, namely, 48 cases out of 246, and it is given just as much importance as the carcinoma of the cervix at Stage I. It is but natural that the nearer the original focus is to the lower part of the corpus the more advanced the infiltration into the muscular layer the more marked is the metastasis to the lymph nodes in the pelvis. Therefore, it is more justificable to determine the method of operation according to the degree of the infiltration, but since it is impossible to know the extent and metastasis of the cancer focus prior to the operation, the radical operation of the cancer of corpus uteri the same as in the case of the cervical carcinoma needs to be best changed to the radical extensive hysterectomy plus the removal of regional lymph nodes. However, in the case of patients of uterine cancer, who can not withstand such an extensive operation, at least after a simple hysterectomy the extent of the cancer focus must be examined, and if it proves to be near the lower part of the corpus, the follow-up removal of lymph nodes is desirable. In any case the superiority of the method mentioned above can offer an important criterion to the improvement of the operative therapy only after the evaluation on the five-year cure rate.
In an attempt to observe the variations of each fraction of serum protein in pregnancy, the serum of 6 pregnant women was analyzed by the use of paper electrophoresis, serially from the second to the tenth month in the same individual. In comparing the results obtained from the specimens taken at random from different individuals from the second to the 10th month, the same trend was seen in this series, namely, decrease of plasma protein, ratio of ablumin to globulin and converse increase of α and β-globulin proportional to the advance of gestation and no remarkable change of γ-globulin was observed. Since the change of distribution of each fraction of serum protein in pregnancy seems to be dependent upon the liver function, increase in consumption of protein in organism, and effect of hormones produced from the placenta, protection of liver function, and ample supply of protein should be an important, effective procedure against toxemia.
1. By implanting cotton pellets the weight of various granulomas has been measured. As the result it has been found that the heaviest granuloma is in the abdominal region, followed by those in the chest, the back, and the neck regions that order. 2. By implanting different substances into the dorsal subcutaneous connective tissues of rats the tissue reaction to these substances has been investigated: The reaction to raw cotton and polyvinylformal sponge is strong but it is weaker against absorbent cotton, woolen yarn, and flannel. 3. In the insertion of cotton pellets into the subcutaneous connective tissue and the abdominal cavity, the tissue reaction to the cotton pellet is stronger in the abdominal cavity than that in the subcutaneous connective tissue. 4. When sodium salicylate, hydrocortisone or prednisolone is administered to rats, the of weight increase has dropped, especially markedly in the case of prednisolone administration. 5. When the sodium salicylate is administered in a small dose, no complete anti-inflammatory action can be observed. However, with a large dose a complete anti-inflammatory action is demonstrated. No circumscribed action of sodium salicylate can be seen. 6. In the case of hydrocortisone and prednisolone even a small dose will reveal a strong anti-inflammatory action, also showing a marked local anti-inflammatory action. 7. When P32 is given in a large dose, it reveals anti-inflammatory action. 8. It has been found that the anti-inflammatory action of sodium salicylate, hydrocortisone, prednisolone, and P32 is stronger in the abdominal cavity than in the subcutaneous connective tissue.
In the present study the author determined the appearance of Heinz's body in erythrocytes of patients with gastric cancer, and gastroduodenal ulcers by Yoshida-Kawamura's method, and then in vitro determinations of Heinz's body in erythrocytes of normal person loaded with serum of patients were carried out. In addition, the relationship between Hainz's body and erythrocyte resistance, and comparison of Heinz's body with blood transfusion as well as of Heinz's body before and after gastric resection were pursued. Also by determining Heinz's body in blood of rabbits injected with extract of gastric cancer tissue or extract of gastroduodenal ulcers tissue, the following results were obtained. 1) In the erythrocytes of patients, more Heinz's bodies appeared in cancer but less in ulcer. 2) In the determinations of Heinz's bodies in blood of normal person left floating in the serum of patient for three hours, the serum of cancer patient gave higher values. 3) As for the relationship between Heinz's body appearance and erythrocyte resistance, in cancer more Heinz's bodies appeared along with the dilution by physiological saline solution. Namely, it has been proven that erythrocyte resistance in patient is diminished by the formation of Heinz's body. 4) In comparing Heinz's bodies before and after blood transfusion, the rate of decrease is high in ulcers but it is lower in. cancer. In other words, these value gave evidences that blood transfusion in cancer was not so effective as in the case of ulcers. 5) In the comparison of Heinz's bodies before and after resection of the stomach, a greater decrease is found in cancer. Namely, this proves that removal of tumor by gastric resections is significant. 6) In the cases of rabbits given intravenous injection of extract of fresh cancer tissue or rabbits administered subcutaneously with a large amount of cancer tissue extract, both showed more Heinz's bodies than in the rabbits injected with ulcers tissue extract. From these clinical and experimental data it has become clear that there is cancer toxin in patients, which helps to promote the formation of Heinz's bodies, and it has been verified that the determination of Heinz's body plays an important and useful rôle in establishing clinically the severity of disease, the diagnosis, and prognosis.
By labelling erythrocytes of normal person with radio active isotope 32p and adding the serum of gastric-cancer patient with the same type of blood to this, in vitro observations were carried on the gradual hemolytic phenomena, and thus the migration of 32p to the serum was periodically determined. As the control similarly sera of gastroduodenal ulcers patient with the same type of blood was added to the labelled erythrocytes. By comparing the results of periodical determinations in the two groups with a view to discover difference, the following were obtained. 1) Cases in which serum of patient was added to erythrocytes labelled with 32p: immediately after addition of serum cancer or ulcer patient almost no migration of 32p to the serum can be observable in either case, but abcut, two hours afterward 32p quickly migrate to the patient's serum. Thereafter 32p invariably migrate more to the serum of patient, and by 24 hours hemolysis is completed and by that time the rate of migration of 32p becomes equal and no more difference can be recognized. 2) Cases in which patient's serum and 10 mg/dl of hydrochloric hydroxylamine were added to the erythrocytes labelled with 32p immediately after addition extremely little of 32p is observed to migrate rapidly to the patient's serum. Thereafter 32p migrates more to the serum of cancer patient, but it is characteristic that from the standpoint of time element 32p migrates rapidly both to sera of cancer and ulcer patients. Eighteen hours later the 32p migration becomes the same in the two. 3) Cases in which patient's serum and 50 mg/dl of hydrochloric hydroxylamine were loaded to the 32p-labelled erythrocytes: immediately after addition a considerable amount of 32p is already recognized to migrate to the serum. In this instance also more of 32p migrates to the serum of cancer patient. Because the 32p migration starts immediately after addition, there is no tendency to migrate rapidly from the standpoint of time element at first as abserved in the case where 10 mg/dl of hydrochloric hydroxylamine was added. About 16 hours later hemolysis is completed and the rate of migration becomes the same in the two cases. Even in this experiment from the macroscopic hemolytic phenomena, the completion of hemolysis is observable earlier in the serum of cancer patient. 4) Erythrocte resistance of the 32p-labelled erythrocytes: By labelling erythrocytes of cancer and ulcerpatients each with 32p, and by adding diluted physiological saline solution at various concentrations to each erythrocyte, one hour after addition supernatants are obtained from respective group by centrifugation and amount of 32p in each supernatant is determined. The rate of migration of 32p to physiological saline solution shows a very little difference in the concentrations of 0.6% and 0.5% solutions, but with concentrations of 0.35% and 0.3% saline solutions in cancer patient a greater amount of 32p migrate than in the case of the control. 5) Cases in which patient's serum after resection of the stomach for gastric cancer was added to erythrocytes labelled with 32p: In addition of the patient's serum which coursed 2 weeks after resection of the stomach, migration of 32p to the serum of cancer patient decreased and the rate of migration of 32p becomes no mofe difference can be recognized between serum of cancer and ulcer patients. Cases iu cancer patient who underwent merely exploratory laparotomy because radical operation was impossible to be performed, migration of 32p to the serum becomes more clearly in comparison with ulcer patient.
The author has performed iron loading tests by mouth on healthy, peptic ulcer and gastric carcinoma cases pre- and postoperatively, and also determined liver iron levels on the cases with gastric carcinoma injected ferric gluconate preoperatevely, obtaining following results. 1) There were no significant differences in mean value for serum iron between healthy men and patients with peptic ulcer, but the level in gastric carcinoma was less than one half of that in healthy men. 2) The fluctuation curve of serum iron by iron loading tests by mouth in healthy, patients with peptic ulcer or gastric carcinoma: In healthy, female showed higher value than male, and same value was observed in both healthy and peptic ulcer cases. The value in gastric carcinoma was markedly low compared with former two. 3) In the gastric carcinoma giving sulfuric iron with hydrochloric acid, folic acid and V. B12, highest value of serum iron was slightly higher than in cases giving reduced iron, without noticeable difference in those fluctuation curves. 4) In postgastrectomy cases, on discharge, the fluctuation curve of serum iron in the cases with peptic ulcer was markedly low as in the cases with gastric carcinoma compared with preoperative cases. in reduced iron loading test. Also in those cases operated for peptic ulcer and gastric carcinoma passed over two and one half years after gastrectomy, restoration of the fluctuation curve was slight. It is, clinically, of importance to consider postgastrectomy iron deficiency. 5) Liver storage iron was less in gastric carcinoma than in peptic ulcer, and in gastric carcinoma the iron level was not improved even after the injection of ferric gluconate, though serum iron level was slightly elevated.
Cancer extract and gastric wall extract as control were injected intravenously on adult dogs, and examined iron absorption through intestine, resulting anemia, and liver iron by reduced iron loadinh test. 1) On reduced iron loading, the fluctuation curve of serum iron was highest in dogs injected gastric wall extract, slightly less in normal dogs, lowest in dogs injected cancer extract. 2) on pouring of sulfuric iron into the intestinal canal iron absorption value was highest after 30 minutes. in dogs injected cancer extract the value was lower than in normal dogs. 3) In dogs after subsequent injection of cancer or gastric wall extract, R. B. C., Hb and serum iron level were reduced day after day, especially marked in dogs injected cancer extract. Total liver non-hemin iron and ferritin were increased 3 to 6 days after injection and decreased later. The fluctuation was marked in cancer extract, increasing significantly. On 9th day those values ware restored to normal in the control group, though remained lower in cancer extract.
On the frozen-dried tissues prepared by using the apparatus designed by SENO a scrutiny has been carried out for the freezing, embedding and the fixation. Observations on those embedded in paraffin reveald that different results can be obtained by the different treatment followed; the treatment with peteroleum-ether for the deparaffinization with the simultaneous fixation of tissues often causes the destruction of the cell structure, prefixation of the sections by floating on 40% formol or 80% alcohol prior to the deparaffinization gives a good result. However, the tissues embedded in paraffin often yield some crevices at sectioning. Embedding in methacrylate in vacuum, proved to be the best by which the tissues can be preserved in a good condition, sectioned and stained without removing the methacrylate. If needed, the methacrylate can be removed by amyl-acetate or acetone after the fixation by the method just described. Thin sections for the electron microscopy is also available in the material embedded in methacrylate.
Some histochemical observations on the cells frozen, dried, and embedded in methacrylate have been carried out. In the hematoxylin-eosin staining the cells appeared somewhat larger than those fixed in formol and embedded in paraffin Chromatin nets and nucleoli appeared distinct in the nuclei but the cytoplasm entirely homogeneous excepting the granules and vacuoles which are mainly distinguishable under phase-contrast microscope, no spaces can be seen between the cells in any tissue and between Bowman's capsule and glomerulus or in the tubules of the kidney. Alkaline phoshatase reaction by Gomori's method appears localizing strictly at the brusb border of the tubules of kidney, but a slight positive reaction of the cytoplasm and the nucleus can be seen by the reaction after removing methacrylate or on the section embedded in paraffin. Glycogen in the liver parenchymal cells appear gross and granular but not diffuse on the cells embedded in methacrylate. Removing of the methacrylate with xylol gives no effect on the picture of glycogen granules, excepting the rapid staining in the latter. The staining of Unna-Pappenheim before or after the treatment with ribonuclease gives almost the same picture as in the cases of conventional fixation with formol. These observations show that the embedding in methacrylate of frozen-dried cells is an exellent one for the histochemical demonstration of some substances.
From the theoretical and experimental view points the author has improved the method for freezing-drying and succeeded in establishing a method suitable for the electron microscopy by which the frozen-dried cells show their detailed fine structures under electron microscope without staining or using any fixative. The method is as follows: a piece of fresh tissue smaller than 0.5 mm3 in size is frozen promptly in liquid propane, cooled at about -180°C by liquid nitrogen, and dried at -60°C for 60 hours with the vapor trap of liquid nitrogen set at as short distance as less than 0.5 cm. and in vacuum of less than 10-4 mm. Hg. After 60 hours the temperature is raised gradually to the room temperature during the subsequent 15 hours The tissue is dipped in methacrylate without breaking vacuum and embedded by polymerizing with benzol-peroxide at 40°C for 12 hours. Thin sections can be obtained and observed under electron microscope without staining or affecting with any fixative. Successive photographs of pancreatic exocrine cells are seen in the last two figures.
By means of the method discribed in the third report the author observed the frozen-dried pancreatic exocrine cells under electron microscope, revealing several new findings. The well-preserved cells through the freezing-drying and embedding show their detailed structures in cytoplasm. The double-layered endoplasmic reticulum (ER) having Palade's granules can be seen clearly. The Palade's granules are originally electron dense ones. Treatment with ribonuclease results in the disappearance of the Palade's granules remaining only the wall of endoplasmic reticulnm. Mitochondria appear as homogeneous electron dense structure but sometimes crevices like cristae can be seen. Osmic staining make it possible to distinguish the existence of cristae. Golgi apparatus appear as vacuolated structure. Zymogen granules appear round and electron dense masses. Cytoplasmic membrane can be seen as less electron dense lines. Nuclear membrane is of double layer having some pores; the outer membrane is one half of the endoplasmic reticulum and the inner membrane is smooth one on which the chromatin nets are densely adhered. In the nucleus the fibrous chromonema can be distinguished between the electron dense chromatin masses. Nucleolus, which is composed of fibrous structure, is seen to be sccrrounded by the nucleolus associated chromatin. These observations show that the osmic fixation is an exellcnt one for the preservation of the detailed structures of cytoplasm of living cell, though some swelling and osmic staining result.
With the purpose to reveal the relationship between hemoglobin (Hb) synthesis and lipid synthesis, the author studied the intra-cellular appearance of Hb and lipids cytochemically. The absorption of heme at 4, 060 Å in the cells appears as early as in the stage of basophilic erythroblast, both in the cytoplasm and the nucleus. In the nucleus it appears in the interchromatin spaces and diffusely in cytoplasm. With the progress of maturation of the cells heme-absorption increases in its intensity, and in the stages of orthchromatic erythroblast it reaches to the level of that of reticulocyte. The absorption in the nucleus generally disappears with the pycnosis of the nucleus and the absorption reaches the maximum in matured red cells. The globin synthesis observed by Giemsa staining proves that this protein appears in the same stage and site as heme. The peroxydase reaction which will be given by Hb also shows the same tendency as in the case of heme and globin. These results show that the site of the heme absorption seems to indicate the site of Hb synthesis. By observing erythroid cells in each maturation stage, stained with Sudan Black B, a quantity of lipids has been detected always in the site where Hb is detected, showing that the Hb synthesis is always accompanied by lipid synthesis.
To observe the relationship between the hemoglobin and lipid synthesis in the erythroid cells in each ripening stage the quantitative analyses have been carried out on the cells of those smeared and fixed with methanol at 4, 060 Å and those stained with Sudan Black B at 5, 800 Å, respectively by the microspectrophotometry. The results showed that the hemoglobin synthesis goes on keeping a constant ratio with the lipids synthesis in the sense of the strict quantitative scale, suggesting the possibility of the chemical binding between lipids and hemoglobin molecules.
For the purpose to know the components of lipids appearing with hemoglobin the crystalline horse Hb has been obtained by the method of SUZUKI. By the test of this crystalline horse Hb, extending the solution electrophoretically on paper and staining with Sudan Black B with or without pretreatment by various nonpolar solvent, the existence of the phospholipids has been found. Further, the crystalline Hb has been digested with pepsin and tripsin and extracted with acetone and ethanol. These extracts have been extended on paper using acetone and ethanol as the solvents respectively, and stained wih rodamine. The result shows that the Hb crystals contain a certain amount of cholesterol, lecitnin and other unidentified lipids.
The major complain of chronic fluorine poisoning in man is lumbago and sclerosis of the vertebrae is usually found. While in the experimental animals, however, various change can be demonstrated. In order to clarify the mechanism of the poisoning, the author performed the experimental studies in rats. Oral administration of fluorine water (100 p. p. m. F.) was performed for more than three months and then glycogen (PAS stain) and alkaliphosphatase in the vertebrae, the intervertebral disc and the femur were examined histochemically. 1) The disturbance of enchondral ossification of the vertebrae and the femur with the decrease of width of the epiphyseal cartilage was recognized. The localized degenerative necrosis of the vertel rae and the intervertebral discs was more frequently appeared in poisoning rats than in the normal. 2) In the epiphyseal cartilage of the femur and the vertebrae, the decrease of the positive zone of alkali-phosphatase and that of glycogen were found. Alkali-phosphatase in the intervertebral discs could not be demonstrated both in poisonig and normal rats. Glycogen in that parts was found to be decreased in poisoning rats.
The author determined the Ca-content in the vertebrae, femur, and blood of rats with chronic fluorine poisoning, and obtained the following results: 1) In both the experimental animals and the control no difference can be recognized in the Ca-content of blood. 2) The Ca-content in the femur and the vertebrae of the test-animals is somewhat decreased as compared with that in the control. After successive intravenous injections of 2% NaF for 2 months into rabbits, and by injecting radio isotope Ca45 into these rabbits. the author measured the amounts of Ca45 in the vertebrae and femur of these rabbits. 1) The Ca45-content in the vertebrae, the head and the trunk of the femur of the test animals is found to have somewhat less than that in the control. 2) As for the distribution of Ca45, it has been found in the descending order of the vertebrae, the head and the trunk of femur in content, and the Ca45-content in the vertebrae is two-folds of that in the trunk of femur.
By examining 25cases of chronic fluorine poisoning that occurred in the high fluorine zones of Okayama Prefecture the author obtained the following results 1) The onset of this disease is rather mild and usually it is realized at the later age of 40 years old. The clinical symptoms are lumbago, the disturbances of joint movement, and rheumatic pain, and in the moderate cases they are lumbago during working hours, and in far advanced cases the patient can not move about and is therefore unable to leave bed. As for the X-ray findings, there is a tendency of osteo sclerosis of vertebrae, spur formation and fusion of two adjacent vertebrae, and the malformation or the neoplastic periosteal swelling can be recognized in the bones of arms and legs. 2) In the cases other than those in far advance stage the general conditions are fairly mild, and no anemia or no increase in the blood count can be observed, 3) Likewise no change can be recognized in the Ca-content or alkali-phosphatase in blood.
By means of the precipitation reaction, the author studied the antigenic significance of various fractions extracted from cholera vibro and, in addition to this, the variation of the cellular composition present between the newly isolated strain and the previously isolated and preserved ones. The results are briefly summarzed as follows. 1) Of all the extrats from cholera vibrio, Boivin's was concluded to be the only one which was of use as the precipitinogen, and Julianelle's substance, crude nucleoprotein and others had not only low antigenic titers but also little type specificity. 2) Boivin's substance, which is significant as the antigen, shows a specific precipitation reaction; manifestations of antibody titers in the range of low and high dilutions had an aspect of reaction with two sorts of antigen. Designated the former as X antigen and the latter as Y antigen, X antigen reacted upon the homologous serum and Y antigen upon the heterologous one, and antigenic titers seemed to be higher upon the homologous serum and lower upon the heterologous one. 3) From manifestation of precipitation reaction, some difference was proved to be present between the newly isolated and preserved ones.
By pathologic findings, the author studied the infection of purified hepatitis virus by oral administration, and compared it with those by the other infectious routes. The results are briefly summarized as follow: 1) The establishment of infection by oral administration of the purified virus could be well proved by observing the pathologic findings. Compared with those caused by oral administration of the crude virus, the purified virus caused severer pathologic findings and mecroid or necrotic parts were widely observed without exception. 2) The infection by the purified virus through the other routes was also well established, though it was often weaker than that through the mouth. 3) Compared with that of the crued virus, the inoculation of the purified virus produced more marked pathologic findings which made the determination of infection easier. These results suggests that the application of the purified virus made the infectious exeriment a step of advance.
In the present part. by observing the pathologic findings, the author studied the course of infection by oral administration of the purified virus. 1) Of the purified viruses orally administered, some are inactivated in the stomach, but the rest descends to the intestine. The regain of the viruses from the stomach inpossible. By pathologic findings, it was well proved that the dilution of the viruses with acid solution inactivated the viruses. 2) About 3 hours after oral administration of the viruses, the viruses were well proved from the wall of the duodenum, the jejunum, the ileum and the colon. The absorption of the viruses through the intestinal wall was inferred to be more marked in the jejunum and the ileum. In the case of the purified viruses, the absorption of the viruses was faster and the pathologic changes were more marked than those in case of the crued viruses. 3) From the fact that the virologic changes in the liver are proved with certainty 96 to 12) hours after administration, it is inferred that the viruses reach the liver and multiply in pretty earlier stage. 4) When the viruses are inoculated into the caudal vein, the pathologic changes appear not only in the liver prior to the lung but also more marked in the liver. 5) The viruses which settled and multiplied in the liver then appear in the blood and reach the other organs. In the case of purified viruses, the movement of the viruses is markedly speedier than that in the case of crude virus material.
The author studied the influence of ultra-violet irradiation on R. tsutsugamushi which had been isolated from the patients of Umayado disease in Kagawa Prefecture. The liver and spleen emulsions of mice infected with the human strain (Mitani) and the rat strain (XXII) were irradiated with ultra-violet ray. The results are briefly summarized as follows: 1) Ultra-violet irradiation for 60 seconds was not sufficient for inactivation of rickettsiae, but that for 120 to 160 seconds inactivated rickettsiae nearly completely. 2) The infectious protection index by thus inactivated rickettsiae was below 1.5, and the immune effect was low and indefinite; this is considered due to the low and indefinite quantity of rickettsiae in the liver and spleen emulsion. From this fact and the results of the experiments with Formalin-inactivated vaccine, it was inferred that ultra-violet irradiation has some effect on rickettsiae themselves. 3) For dilution of rickettsial emulsion, PGS (Snyder), 30% sucrose and saline (pH 7.6) solutions were used, but the sorts of solutions had no noticeable influence on the inactivation time by ultra-violet irradiation or on the antigenicity of the inactivated one. 4) The immune effect by inoculation of thus inactivated emulsion was determined by log LD50 in mice and that in the controle group. The fatal cases meant the infection of rickettsiae, but, even in non-fatal cases, the infection of rickettsiae was proved by staining and serial passage.
In order to study the immune effect of the rickettsiae inactivated by ultra-violet irradiation, the author performed immune experiments in rabbits. The results are briefly summarized as follows: 1) The liver and spleen emulsion of mice infected with Kagawa strain of R. tsutsugamushi was inactivated by ultra-violet irradiation as reported in part I. Inoculation of thus inactivated rikettsiae into rabbits produced neutralization antibody to some extent, but the neutralization effect was low and could not prevent the attack of ricketsiae. 2) Filling up of the reticuloendotherial system by injection of indian ink or by addition of adjuvant could not raise the neutralization effect. 3) In the neutralization test by inoculation of the mixture of R. tsutsugamushi antisera, inactivated rickettsiae and active rickettsiae, the virulence of active rickettsiae was enhanced, and the neutralization effect was lowered. In the neutralization test with the antisera, which had been previously absorbed by inactivated rickettsiae, and active rickettsiae, the virulence of active rickettsiae was enhancedand the neutralization effect was lowered; this fact suggests the absorption of antibody by the inactivated rickettsiae.
In acatalasemia we often encounter a specific oral gangrene. With the purpose to elucidate the causative factor of this gangrene the author performed animal experiments. Namely, ducks and geese having only a trace of catalase in their blood were selected as experimental animals and for the control group rabbits and hens were used. By injecting 1% hydrogen peroxide solution under the mucous membrane of the palate in respective animals histopathological investigations were carried out at a fixed time after the injection, and the following results were obtained. 1. Macroscopically in the ducks 24 hours after three injections of 1% hydrogen peroxide solution at intervals of 24 hours there appeared gangrene resembling the one observable in acatalasemia cases, and microscopically marked changes were recognized in blood vessel wall and muscles. 2. The pathological findings induced by experiments in geese were marked and almost identical with those in ducks. 3. The induced changes in rabbits were superficial and milder than those in ducks, being circumscribed at the site in injection. 4. The changes in hens resembled those in rabbits. From these results it is assumed that the fact that the changes in the muscles and blood vessel wall in the ducks and geese with almost no catalase were more marked than those in the rabbits and hens proves that the iron-carrying chromoproteins possessing a strong affinity to hydrogen peroxides such as myoglobin and cytochromes contained in muscles, are affected more strongly by the oxidative action of hydrogen peroxide than in the case of rabbits and hens, thus bringing about the retrogressive degeneration. Therefore, it seems that the causative factor of the gangrene in acatalasemia cases lies in the fact that the so-called respiratory enzyme group such as cytochromes, myoglobin as well as yellow enzymes and SH enzymes contained in tissue cells are disturbed by the accumulation of hydrogen peroxide with resultant retrogressive degeneration of tissues, as well as that the circulatory disturbances brought about by the marked changes in blood vessel wall (especially the wall of arteries) seem to evoke definitively the occurrence of gangrene.
About one half of acatalasemia cases showed the specific oral gangrene, and two cases of them had unilateral tonsillar gangrene furthermore. The author conducted clinical observations on this gingival gangrene and tonsillar gangrene, and at the same time made a comparative study of these two types of gangrene histopathologically. As for the peculiarities of the gingival gangrene the following may be pointed out. 1. These cases can be divided into three groups by the degree of the progress in symptoms. Namely, the minimal type of an extremely early stage, showing a highly advanced alveolar pyorrhea accompanied by ulcer of the gingiva; the moderately advanced type with the atrophy and sinking of the gingival bone and the spontaneous falling off of teeth due to the still further advanced gangrene; and the severe type presenting a progressive gangrene in the superior maxillary or the inferior maxillary bone, and or in the mucous membrane tissue of tongue base and cheek due to a still more violent advance. 2. As for the histopathological characteristics the cell infiltration is slight in despite of the tissue necrosis is vigorous. The boundary of the tissue necrosis is distinct and the infiltration of eosinophilis can be recognized around the boundary line. Next, as for the characteristics of tonsillar gangrene, the following results have been obtained. 1. Macroscopically the inflammatory reaction is slight, and ulceration tends to start from the tonsillar lacuna and spread from there, and the area surrounding the lesion is like sawteeth and undermined but not so deep, presenting the shape like a sack. 2. Histopathologically lacunar tonsillitis and the necrosis commencing from tonsillar lacuna can be observed. The boundary line of the necrotic region is clear-cut, and the infiltration of eosinophilis can be seen around the necrosis. In addition, marked changes can be recognized in the wall of the blood vessels and muscle surrounding the necrosis.