THE DEMONSTRATION OF A TUMOR GROWTH-INHIBITING FACTOR FROM NORMAL HUMAN CONNECTIVE TISSUE

Extracts from fresh normal human connective tissue (rectus sheath) exhibited a decided inhibiting action on grafts of Rat Tumor 256 transplanted into test animals. There was complete inhibition of tumor growth in 66 per cent of the animals; there was a marked retardation in the rate of growth in another 15 per cent, the tumors in these instances being much smaller than the controls transplanted at the same time. In only one experiment was there failure to obtain a lasting growth-inhibitory effect. The tumors in these animals (19 per cent) although showing an initial retardation in growth, apparently overcame the restraint and at the end of 5 weeks were larger than the control series. Extracts from fresh normal human muscle tissue, on the other hand, showed no such inhibiting action on grafts of Rat Tumor 256.     

FURTHER STUDIES ON EFFECTS OF TISSUE EXTRACTS ON STAPHYLOCOCCUS AUREUS

1. The ability of alcoholic-precipitated extracts of beef tissue—brain, spleen, heart, and kidney—to stimulate the growth of Staphylococcus aureus, in vitro, and to convert the yellow S form to a white R variant with altered biochemical characteristics conforming to those of an avirulent organism, has been confirmed. 2. The avirulence of the white R variant has been established by tests in vivo on mice. 3. Staphylococcus aureus infections induced subcutaneously, intraperitoneally, and intravenously in mice responded favorably to brain extract following subcutaneous or oral administration. The mortality was 2 per cent in 444 experimental animals and 81 per cent in 448 control animals. 4. The extracts appeared equally efficient when used therapeutically (mortality 2 per cent of 162 experimental animals and 90 per cent in the control series) or prophylactically (mortality 2 per cent of 282 experimental animals and 76 per cent in 286 control mice). Extracts of brain and spleen were more effective than those of either heart or kidney. 5. Studies concerning the mechanism of action of the tissue extracts indicate that they prevented the formation of toxin by Staphylococcus aureus, and had but little effect on toxin actions. 6. Toxicity tests revealed that the brain and spleen extracts were relatively non-toxic, dosages equivalent to 2 per cent of the body weight being well tolerated. Kidney and heart extracts were much more toxic, producing mortality in dosages as low as 0.3 per cent of the body weight.     

STUDIES BASED ON A MALIGNANT TUMOR OF THE RABBIT : IV. THE RESULTS OF MISCELLANEOUS METHODS OF TRANSPLANTATION,WITH A DISCUSSION OF FACTORS INFLUENCING TRANSPLANTATION IN GENERAL.

The results of seven methods of transplantation of a malignant tumor of the rabbit are reported and contrasted with results obtained by intratesticular inoculation. It was found that inoculations made into the brain, the anterior chamber of the eye, and the muscles gave an almost uniform series of takes with the production of comparatively large and vigorously growing tumors. Intracutaneous inoculations were less favorable, while with subcutaneous, intravenous, and intraperitoneal inoculations, a growth was obtained in only 20 to 25 per cent of the animals. In contrast with the malignant course of the disease produced by testicular transplantation, the tumors produced by methods other than intracerebral inoculation were relatively benign. In all cases, the growth was of a purely local character; in some instances, there was invasion of the surrounding tissues with local or regional extension of the growth, but no metastases developed in distant organs. Tumors in the brain soon caused death as a result of pressure, and the ultimate fate of tumors in the eye was not determined, but the growth produced by other methods of local inoculation was of relatively short duration and terminated with spontaneous resolution. It was found that while a good growth could be obtained with material which had been subjected to repeated freezing, thawing, and grinding, the use of cell-free filtrates or desiccated tumor tissue gave negative results. In discussing the experiments reported, it was pointed out that there were three groups of factors which had played important parts in determining the results of transplantation. These were the capacity for growth of the tumor cells, animal resistance, and the advantage given the tumor or the animal by the use of a particular method of inoculation. From an analysis of the results obtained with reference to the operation of these factors, the conclusion was reached that while the quality of malignancy displayed by the tumor was conditioned upon the capacity for growth of the tumor cells, the degree of malignancy exhibited in any given instance was a function of animal resistance which was not entirely specific but corresponded closely with the ability of the animal to meet excess demands arising from other causes and, to this extent, represented an expression of the functional capacity of a mechanism which is concerned primarily with the maintenance of the general animal economy.     

STUDIES ON EASTERN EQUINE ENCEPHALOMYELITIS : IV. INFECTION IN THE MOUSE WITH FRESH AND FIXED VIRUS

A fresh strain of equine encephalomyelitis virus is infectious for adult mice in high dilutions by all modes of peripheral inoculation. A fixed strain has very limited invasive power when injected peripherally unless virus is placed in fairly close contact with nerve cell bodies, as in the intranasal or intraocular routes. For fixed virus the effectiveness of the mode of inoculation may be graded in the following descending order: intracerebral, intraocular and intranasal, intravenous, intraperitoneal, intramuscular, subcutaneous. Fixed virus has a very limited power of invading the central nervous system along the axones of peripheral nerves even when injected directly into the nerve. Infants are more susceptible to infection than are adults. But even for infants, intraperitoneal inoculation with fixed virus is significantly less effective than similar inoculations with fresh virus. Brain trauma does not increase the effective titer of fresh or fixed viruses but may shorten the incubation period for fresh virus. With fixed virus injected intramuscularly, a pronounced facilitating effect may be produced by the simultaneous intraperitoneal injection of 0.20 to 0.25 cc. of 50 per cent glycerine. Other irritants tried are without effect.     

Storage of human platelets in liquid nitrogen isotopic studies

Concentrated platelet suspensions in 5% DMSO —5% PVP (MW 17,500) in plasma were frozen in sterile, slliconized glass vials at a rate of 1-3C per minute to — 40C They were then immersed in liquid nitrogen and stored for a period of six to 12 months. After rapid thawing, platelets were labeled with ⁵¹Cr and injected into hematologically normal and ABO- and RH- compatible subjects. The radioactive recovery of frozen platelets, two hours after injection, was 11 per cent (3.9-22). The recovery of fresh platelets labelled under the same conditions was 36 per cent (30-50%). The platelet survival time was found to be four to six days for frozen platelets and six to nine days for fresh platelets. These results suggest that platelets frozen and stored in liquid nitrogen have a definite but reduced therapeutic efficiency. The method looks valuable for some blood banks.     

THE EFFECT OF EXTRACTS OF CERTAIN ORGANS FROM NORMAL AND IMMUNIZED ANIMALS ON THE INFECTING POWER OF VACCINE VIRUS

Brain and testicle tissue from immune rabbits brought in contact with the Levaditi or Noguchi strains of vaccine virus will fix or inactivate the virus. Extracts of normal testicles from susceptible animals enhance to an extraordinary degree the infectivity of both the neuro- and dermal strains of vaccine virus. The Noguchi virus is not affected by testicle extracts when injected into the skin, but kidney extract has a definite enhancing power on the strain when injected into either skin or testicle. The effect of tissue extracts seems to be on the cells of the host rather than on the virus. This is indicated by the fact that virus injected intravenously localizes most readily in an area of skin previously injected with testicle extract. Furthermore an enhanced lesion results if the virus is injected into an area as long as 3 days after the area has been injected with testicle extract. The augmenting substance of the tissue extracts is little affected by high dilutions, passes through a Berkefeld V candle and is carried down with the proteins precipitated by weak acids. Rabbits with enhanced lesions show general symptoms and about 25 per cent die with generalized vaccinia. Kidney, and probably skin, brain and liver extracts possess enhancing properties, but to a much less degree than the testicle. On the other hand, spleen, blood and probably lymph nodes and bone marrow not only fail to produce enhancement, but actually restrain or even suppress entirely the vaccinal skin infection.     

THE INFLUENCE OF BILE ON METABOLISM

1. Bile increases the digestion of fat when given by the mouth in pill form. The percentage of fat lost in the stools of our patient with a complete biliary fistula was 63 per cent in the first period and 57 per cent in the third. This closely corresponds to the results that Müller obtained in human beings and dogs with complete obstruction of the common duct. Under bile medication the stools contained 23 per cent less fat than in the first period, and 17 per cent less than in the third. This represents an actual diminution of the amount of fat lost in the stools. Looking at the result in another way, it may be said that the average digestion of fat in the periods without bile was 40 per cent; in the periods with bile, 60 per cent, i. e., bile increased the digestion of fat relatively by 50 per cent. 2. The digestion of nitrogenous food is improved by the use of bile pills when the amount of fat in the stools is large. Instead of an average of 15 per cent being lost in the fæces, but 7 per cent escaped digestion during the four days the patient took bile. The reason for this, perhaps, lies in the better digestion of fat at this time, in consequence of which the proteid elements of the food were more thoroughly exposed to the digestive juices. 3. Ox bile is a cholagogue. The amount of bile-solids secreted in the bile period was 47 per cent greater than in the periods before and after. This confirms the work of Pfaff and Balch, here in Boston, on a human being, and that of Stadelmann and his pupils, in Germany, on dogs. 4. The effect of the bile on the bowels in this case was not remarkable, although they moved more satisfactorily during the bile period. In my experiments with dogs I usually obtained diarrhœa when giving bile. I do not feel sure, however, that this should be attributed wholly to the medication, for the diarrhœas as a rule appeared six or more days after the beginning of the experiment and the animals were then in poor condition. Dr. Pfaff, who has had more experience with the administration of bile than I, tells me that he has found its action variable in patients. In some cases it is a laxative; in others, in which there is diarrhœa, due apparently to large amounts of fat in the food, it has the opposite effect. 5. As to the general effect of bile on body metabolism, it was observed that the urea and nitrogen were excreted in greater amount in the bile period than in either of the others. No definite conclusions can be drawn from this fact, because more nitrogen was ingested during these four days; moreover, it must be borne in mind that in these results the salol may have been a factor. 6. The amount of urine was increased by more than 50 per cent in the bile period. It is interesting to note that the amount was about the same during this bile period as in the second experiment when the bile was again taking its natural course. Von Noorden has recorded a similar increase in the amount of urine following the removal of the obstruction in acute catarrhal jaundice. The salol coating of the bile pills, which amounted to one and a quarter grammes a day, is not sufficient to account for this effect. This is evident from the work of Kumagawa, who gave two grammes of sodium salicylate daily to a dog of 25 kilos without essentially changing the amount of urine secreted. On the other hand, in taking the 30 pills daily the patient drank several extra glasses of water, and in the second experiment her general condition was naturally better than at any other time.     

THE RELATION OF THE SPLEEN TO BLOOD DESTRUCTION AND REGENERATION AND TO HEMOLYTIC JAUNDICE : X. CONCERNING THE SUPPOSED REGULATORY INFLUENCE OF THE SPLEEN IN THE FORMATION AND DESTRUCTION OF ERYTHROCYTES.

1. The blood of the splenic artery and vein shows either no differences, or only such slight irregular variations as may be due to the errors inherent in hematologic methods, or are common to arterial and venous blood of the general circulation. 2. The observation of Banti and Furno that free hemoglobin occurs in the blood of the splenic vein is not confirmed. 3. Extracts of the spleen have no definite hemolytic action in vitro. 4. Intraperitoneal injection of fresh saline extracts of the spleen causes in the dog a sharp increase in the number of red cells and the hemoglobin content which lasts for one or two days and may recur on a second injection. Extracts of liver, kidney, and erythrocytes similarly prepared do not give this effect. This observation supports Danilewsky''s theory that the spleen may exert a stimulating effect upon the formation of red cells in the bone marrow. 5. On the other hand, the feeding of raw beef spleen to splenectomized dogs over long periods of time has no clearly defined influence in preventing the anemia which usually follows splenectomy.     

STUDIES ON THE BIOCHEMICAL,BIOPHYSICAL, AND IMMUNOGENIC PROPERTIES OF JAPANESE B TYPE ENCEPHALITIS VIRUS AND VACCINES

Studies on the biochemical, biophysical, and immunogenic properties of Japanese B type encephalitis virus and vaccines have been made in order to determine whether a purified vaccine suitable for human use could be obtained by means of differential centrifugation of extracts of infected mouse brains. Studies were also made on extracts of normal mouse brains, it was found that extracts of normal as well as of infected mouse brains contained fairly large amounts of several components of high molecular weight. Components having sedimentation constants near 5 and 40 Svedberg units were found in extracts of infected brains. However the rates of sedimentation of the different components were so similar that it was found impossible, from a practical standpoint, to secure a vaccine consisting largely of virus by means of differential centrifugation. It was also found that a considerable portion of virus was lost or destroyed in the centrifugation process so that it was impossible to secure an effective degree of concentration of immunogenic potency. Although vaccines possessing about twice the immunogenic potency of the starting material were obtained, it was concluded that it was not practical to purify and concentrate Japanese B type encephalitis virus in infected mouse brain extracts by means of differential centrifugation for the production of a vaccine on a large scale. The optimum pH stability range of Japanese B type encephalitis virus activity was found to be near pH 8.5. The virus is inactivated fairly rapidly at pH 7 and very rapidly at more acid reactions. The virus is inactivated rapidly near pH 10. Extracts of infected mouse brains with buffers near pH 8 containing disodium phosphate were found to possess slightly higher titers than saline extracts near pH 7. However vaccines prepared from such extracts were found to possess essentially the same immunogenic potency, hence, although extraction at the more alkaline reaction may perhaps remove more active virus, there was no indication that more immunogenic material was removed at the more alkaline reaction. The use of different diluents in the titration of virus activity and the use of different agents in the preparation and storage of virus suspensions were investigated. It was found that low titers were obtained when Ringer''s solution, phosphate buffer at pH 7, or saline-phosphate buffer at pH 8.2 were used as diluents but that high titers were obtained when 10 per cent rabbit serum in saline or in phosphate buffer, 10 per cent skim milk in saline or in phosphate buffer, or 1 per cent arginine at pH 8.3 were used. Undiluted skim milk adjusted to pH 8.4 was found to be as satisfactory as undiluted rabbit serum for the preparation of infected brain suspensions for storage at –70° C. A satisfactory neutralization test was conducted with virus stored in undiluted skim milk at –70° C. and subsequently diluted with 10 per cent skim milk in saline. The demonstration that skim milk can be substituted for rabbit serum in the storage, titration, and neutralization tests of Japanese B type encephalitis virus is of practical importance, for skim milk is more convenient to prepare and more readily available in many localities.     

STUDIES ON BACILLUS WELCHII WITH SPECIAL REFERENCE TO GAS GANGRENE

1. Spores of the Bacillus welchii group of bacteria were found on 100 per cent of the uniforms of Belgian soldiers who had come directly from the trenches, and in the meshes of all the samples examined of the new cloth from which the uniforms were made. 2. In fifteen out of twenty fresh war wounds members of this group of bacteria were found. Of the fifteen patients, only three later developed gas gangrene. Once the spores of Bacillus welchii have been carried into a wound the deep-lying lacerated muscle tissue appears to be the most important factor in the onset of gas gangrene. 3. Bacillus welchii is able to grow and produce gas in broth containing up to 40 per cent saccharose. Some strains were able to multiply and produce gas in 50 per cent saccharose broth; but none of those examined were able to grow when the concentration of the sugar reached 60 per cent. 4. The bubbling of pure oxygen through milk or dextrose broth cultures of Bacillus welchii has a definite depressor action on the production of gas. This does not appear to be-due to a reduced number of organisms in the culture.     

The natural history of gastric ulcer in a community: four-year study.

All (151) adult patients in north-east Scotland newly diagnosed in one year (1967) as having a supposedly benign definite gastric ulcer crater were studied after a minimum of four years, with a 98 per cent follow-up. Incidence and male to female ratio both increase with advancing age. Ulcers of the body of the stomach were twice as common as ulcers of the antral region. Major coincidental disease was present in 59 per cent of all patients and in 97 per cent of those dying, and was the chief cause of death. Twenty-one patients presented with bleeding but only one other bled subsequently. One patient presented with perforation of gastric ulcer. Two ulcers proved to be malignant. The effect of associated duodenal ulceration on presentation and course was not great. There was a direct correlation between short durations of symptoms and increasing age and mortality. Fifty-five per cent of the medically treated and 60 per cent of the surgically treated patients were alive and entirely free from gastric ulcer symptoms at the end of four years; the respective mortalities were 18-2 per cent and 20-3 per cent. For the whole series, life expectation was significantly reduced in the females, but not in the males.     

The Natural History of Gastric Ulcer in a Community: A Four-year Study

All (151) adult patients in north-east Scotland newly diagnosedin one year (1967) as having a supposedly benign definite gastriculcer crater were studied after a minimum of four years, witha 98 per cent follow-up. Incidence and male to female ratioboth increased with advancing age. Ulcers of the body of thestomach were twice as common as ulcers of the antral region.Major coincidental disease was present in 59 per cent of allpatients and in 97 per cent of those dying, and was the chiefcause of death. Twenty-one patients presented with bleedingbut only one other bled subsequently. One patient presentedwith perforation of gastric ulcer. Two ulcers proved to be malignant.The effect of associated duodenal ulceration on presentationand course was not great. There was a direct correlation betweenshort duration of symptoms and increasing age and mortality.Fifty-five per cent of the medically treated and 60 per centof the surgically treated patients were alive and entirely freefrom gastric ulcer symptoms at the end of the four years; therespective mortalities were 18.2 per cent and 20.3 per cent.For the whole series, life expectation was significantly reducedin the females, but not in the males.     

STUDIES ON PNEUMOCOCCUS GROWTH INHIBITION : I. THE PROTECTIVE ACTION OF GELATIN FOR PNEUMOCOCCI IN SUSPENSION.

Experiments were undertaken with the purpose of determining the type of non-nutrient fluid most suitable for the suspension of pneumococci with a minimum amount of injury. For comparative studies, dilutions in the various fluids tested were made from a standard suspension containing approximately 1,000 million pneumococci per cc. The protective properties of each solution were judged by the length of time viable organisms could be recovered (in culture) from a suspension containing 0.0000001 cc. of the standard suspension. The hydrogen ion concentration of the solutions, the temperature at which the suspensions were kept, and the culture media were carefully controlled. Furthermore, plates were made at the beginning of the experiment to determine the number of organisms present in a unit of suspension. It was found that pneumococci suspended in Locke''s solution, 0.5 and 0.9 per cent NaCl solutions, and water, remained alive for only a few hours at most. Salt solution was shown to be the least suitable of these three; not infrequently by the time the 0.000001 cc. dilution in this fluid had been reached, the suspension was sterile. The addition, however, of 0.1 per cent gelatin to the above fluids transformed all, except 0.9 per cent NaCl, into highly favorable solutions. Pneumococci suspended in gelatin-water and gelatin-Locke''s solution remained alive at room temperature for 6 to 7 days; in gelatin-salt 0.5 per cent solution, for at least 2 days. It was possible to vary the H ion concentration of the suspension fluids from pH 7.0 to 8.2 without any marked effect on the results. However, a pH of 7.4 to 8.0 appeared to be the most suitable. The nature of the protective action of gelatin was investigated. Series of plates made at frequent intervals failed to reveal any growth in suspensions of pneumococci containing this small concentration of gelatin. The beneficial effect of gelatin in the above solutions was found to lie largely in its protection of the pneumococci against the mechanical injury which occurs during the process of dilution in crystalloid solutions or water. Gelatin shows in addition a well marked preservative action, that is to say, it protected organisms against early dissolution. The nature of this preservation is uncertain. The presence of 0.1 per cent gelatin in 0.9 per cent NaCl failed to protect pneumococci against the toxic action of Na. But it was found possible to neutralize to a considerable degree this toxic effect of sodium by the addition of a small quantity (2 per cent) of a M/15 balanced phosphate mixture or sodium phosphate. In a gelatin-salt 0.9 per cent solution buffered with phosphate, pneumococci survived for 24 to 48 hours. Even in gelatin-salt, 0.5 per cent the presence of phosphate had a definite beneficial effect.     

PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : VIII. THE EFFECT OF TESTICLE EXTRACT ON THE RATE OF GROWTH OF CHICKEN TUMOR I

Extracts prepared from the testicle tissue of the rat, rabbit, fowl or bull, injected together with extracts of Chicken Tumor I or with cells of this tumor, showed no definite effect of either enhancement or inhibition as concerned the resulting tumors.     

Preservation of the Clot-Retracting Activity of Platelets by Freezing in Dimethylsulfoxide and Plasma

The clot retracting activity (CRA) of frozen platelets was studied as the in vitro test best representing the functional "integrity" of platelets. Dimethylsulfoxide as an additive in the preservation by freezing of platelets acts most effectively in the presence of plasma. Freezing of the platelets in a suspending medium consisting of 50 per cent plasma in saline with 15 per cent DMS, resulted in a 10 to 15 per cent loss over the fresh control CRA values. Freezing rates ranging from 3 C. to 30 C. per minute can be used without modifying the final result. Storage in liquid nitrogen for 38 days resulted in a 34 per cent CRA reduction over the frozen but not stored platelets. These results seem to justify trials of the in vivo survival of platelets preserved by these methods.     

JENNERIAN PROPHYLAXIS BY MEANS OF INTRADERMAL INJECTIONS OF CULTURE VACCINE VIRUS

The second revived strain of culture vaccine virus has been propagated through 130 culture passages during a period of 3 years. It seems to be adapted to in vitro cultivation and still has an intradermal titer (rabbits) of 1 to 100,000 or 1 to 1,000,000. Intradermal inoculations in human beings of 0.1 cc. amounts of culture virus diluted from 2.5 to 10 times result in primary takes in unvaccinated people and immune reactions or accelerated takes in individuals previously successfully vaccinated. Primary takes produce an immunity to standard strains of calf lymph. Culture virus mixed with purified gum acacia (2.5 per cent), frozen, desiccated, and sealed in vacuo retains its activity for a month at 37°C., and when the dried virus is resuspended in saline solution it is suitable for intradermal vaccination of human beings.     

STUDIES BASED ON A MALIGNANT TUMOR OF THE RABBIT : II. PRIMARY TRANSPLANTATION AND ELIMINATION OF A COEXISTING SYPHILITIC INFECTION.

Several attempts were made to transplant a spontaneous malignant tumor in a syphilitic rabbit before a method was devised which proved successful. Subcutaneous and intracutaneous inoculations were unsuccessful on account of the vigorous granulomatous reaction which was aroused by the introduction of tumor fragments into these tissues. This difficulty was overcome by the use of intratesticular inoculations. With this method, a good growth was obtained in practically all animals. It appeared possible, however, that a syphilitic infection had been transmitted along with the tumor, and hence treatment with arsphenamine was instituted as a means of eliminating this infection. A study of the transplanted tumor in the first and second generations showed that it possessed a high degree of malignancy. Metastases occurred at an early period in the majority of animals, and while some of them appeared to recover, the indications were that more than 50 per cent might be expected to succumb to the effects of the tumor growth within a period of 6 to 7 months. The method of intratesticular inoculation now in use is described in detail.     

A STUDY OF THE THERAPEUTIC MECHANISM OF ANTI-PNEUMOCOCCIC SERUM ON THE EXPERIMENTAL DERMAL PNEUMOCOCCUS INFECTION IN RABBITS : III. THE INFLUENCE OF NON-SPECIFIC FACTORS

The purpose of the present investigation was to determine whether or not non-specific agents were capable of exerting any influence on the response of pneumococcus-infected animals to specific serum therapy. It has been demonstrated in these experiments that whereas gold (empirically chosen) by itself had very little effect either on the course or the outcome of the experimental pneumococcus infection, it is nevertheless capable of exerting a definite and marked beneficial effect in rabbits treated with a subeffective dose of the specific antiserum. Of the rabbits treated with the subeffective dose of serum alone, 71 per cent died and only 29 per cent survived; the additional administration of gold reversed this death-survival ratio with the result that of a large group of rabbits which received the combined therapy, 77 per cent survived and only 23 per cent died.     

IN VITRO STUDIES OF ULCERATIVE COLITIS : II. CYTOTOXIC ACTION OF WHITE BLOOD CELLS FROM PATIENTS ON HUMAN FETAL COLON CELLS

Freshly isolated fetal human colon cells were labeled with ³²P-orthophosphate or ¹⁴C-amino acids and exposed to white blood cells from children with ulcerative colitis or from healthy controls. Exposure of the colon cells to patients' white cells led to a rapid isotope release, significantly higher than that obtained with normal white cells. After 150 minutes of incubation, 75 per cent of the total isotope present was found in the media of the colitis samples but only 40 per cent in those of the controls. Consistent results were obtained with white blood cells from 14 patients and 18 healthy individuals. Similar results were obtained with either fresh white cells or with white cells aged for 12 to 18 hours and consisting to 60 to 70 per cent of lymphocytes and to 20 to 30 per cent of large mononuclear cells. No specific cytotoxic activity could be conferred onto normal white cells by pretreating them with patients' serum containing antibodies against colon antigen. The cytotoxic action of the patients' white cells was immunologically specific, since no difference from the controls was found in the isotope release when cells from other organs or animals were similarly treated. Preliminary experiments suggested that the patients' white cells could be desensitized by pretreating them with colon extract. For obtaining a significant cytotoxic effect of the patients' white cells, the presence of 10 to 20 per cent of fresh guinea pig or human serum in the incubation medium was required.     

IN VITRO STUDIES OF ULCERATIVE COLITIS : I. REACTIONS OF PATIENTS' SERUM WITH HUMAN FETAL COLON CELLS IN TISSUE CULTURES

By means of immunofluorescent methods it has been shown that sera from children with ulcerative colitis contain antibodies which react with fetal colon cells in tissue culture. 5 out of 13 sera from patients reacted positively when tested for staining antibodies while 12 sera from healthy individuals yielded negative results. The specificity of the staining reactions was confirmed by inhibition experiments. The staining capacity of various sera was correlated to their hemagglutinating titer when tested against phenol-water extracts of human colon. The presence of blood group substances of the ABO system on fetal colon cells in tissue culture could be demonstrated by application of fluorescent H agglutinins from eel. Cross-inhibition experiments indicated that the H agglutinins stained colon antigens which were different from those reacting with the antibodies of ulcerative colitis sera. The reactivity of cultured fetal colon cells with the antibodies in ulcerative colitis sera was retained for up to 12 days, with optimal staining at 4 to 5 days. Reactivity with H agglutinins was present for a longer period, sometimes more than 20 days. Although antigen could be shown to be present on fetal colon cells in tissue culture, exposure of the culture, in the presence of fresh guinea pig serum, to sera from patients with ulcerative colitis did not lead to any visible cytotoxic damage. In order to investigate the possible cytotoxic effect of the sera with a more sensitive technique, freshly explanted fetal colon was dispersed by trypsinization and the cells labeled with ³²P-orthophosphate. Subsequently, these cells were exposed to sera, in a final concentration of 30 per cent, from patients or healthy controls in the presence of fresh guinea pig serum (final concentration 15 per cent). Approximately 20 per cent of the cellular isotope was released into the medium within 150 minutes of incubation, but the release was the same in the samples treated either with patients' sera or normal control sera. Thus, under the present conditions, the patients' sera did not exert any specific cytotoxic action on colon cells.