Robert W. Heinle

TRANSFUSION OF LEUKOCYTES LABELED WITH RADIOACTIVE PHOSPHORUS.

Mechanisms involved in the failure to raise the white blood cell count by transfusion remain unknown. Previous studies (1) on the effect of transfusion of concentrated suspensions of leuko-cytes obtained from the peritoneal cavity of rabbits have confirmed the clinical observation that the white blood cell count cannot be effectively increased by transfusion. When concentrated sus-pensions of leukocytes were transfused into the venous system of the same rabbit from which they had been obtained or into other rabbits, not only was there no increase in the leukocyte count over a period of several hours, but a severe leuko-penia developed in nearly every animal. In over half the rabbits a subsequent marked leukocytosis developed in four to six hours. This investigation was undertaken to study the disappearance of leukocytes from the circulating blood by determining the radioactivity in various tissues following the transfusion of leukocytes labeled with P52. METHODS Leukocytes labeled with radioactive phosphorus were obtained by injecting a rabbit with approximately 1.0 millicurie of p82 intravenously prior to stimulating the production of leukocytes in the rabbits peritoneum. Two to seven days after injecting the radioactive phosphorus , leukocytes were obtained from the rabbits peri-toneal cavity by a modification of the method of Mudd and coworkers (2). Three hundred to 500 ml. of physio-logic saline were injected intraperitoneally in the evening and a similar amount about 15 hours later. Over-distension of the abdomen is poorly tolerated by the rabbits and may cause respiratory embarrassment and death. Four hours after the second injection of physio-logic saline the peritoneal fluid was removed through a 16 gauge needle using 5 mg. of heparin as an anticoagu-lant. This fluid was centrifuged at a speed of about 500-1000 R.P.M. for five minutes and the sediment re-suspended in Tyrodes solution so that the final concentration of cells was approximately 50,000 cells per cmm. The leukocytes prepared in this manner stained normally with Wrights stain, exhibited ameboid activity, were actively phagocytic for staphylococci and took up supravital stain (Janus green and neutral red). Approximately 90% of the cells were segmented and unsegmented neutrophils. The radioactive phosphorus was firmly bound within the cell, presumably in the nuclear nucleoprotein. In order to demonstrate this, samples of the leukocyte suspension were subjected to repeated washing with Tyrodes solution. Only negligible amounts of radioactivity could be removed by this procedure (Table I), indicating the chemically bound nature of the p82 in these …

Some structural requirements for the prevention of leukopenia induced by nitrogen mustard.

Administration of L-cysteine hydrochloride to animals prior to the injection of nitrogen mustard (HN2) modifies the severe leukopenia characteristically induced by HN2 (1). However, if the L-cysteine hydrochloride is administered after HN2, even immediately, the leukopenia is not modified. It has not been demonstrated whether the effect of L-cysteine depends upon the presence of certain groups or groupings in the molecule or whether it is the result of non-specific chemical inactivation of HN2. Accordingly, the specificity as well as the mechanism of L-cysteine protection was investigated by correlating the chemical structure of related compounds with their ability to prevent HN2-induced leukopenia.

Transfusion of Leukocytes and Products of Disintegrated Leukocytes.

Summary and Conclusions 1. Leukocytes were obtained in large numbers by introduction of large amounts of physiologic salt solution into the peritoneal cavity of rabbits. 2. Intravenous administration of these cells to the same or different animals was followed by very rapidly developing and severe leukopenia. A subsequent leukocytosis developed in the majority of rabbits after several hours. 3. Disintegrated leukocytes, or aqueous extract of disintegrated leukocytes produced similar results. 4. Preliminary data indicate that the leukocytes stick in the capillaries of the lung. Ether extraction of disintegrated leukocytes does not cause loss of activity but heating reduces activity. 5. These experiments indicate that unsatisfactory preservation of leukocytes in stored blood is not the reason for failure to raise leukocyte counts with transfusions, but that white blood cells contain a substance (or substances) which is capable of producing leukopenia.

Chemical Antagonism of Pteroylglutamic Acid in a Pig; Hematopoietic Effect of Extrinsic and Intrinsic Factors.

Summary Interference with the metabolism of pteroylglutamic acid in the pig, through the use of a crude chemical antagonist, interrupts growth and significantly inhibits the formation of erythrocytes and of granulo-cytes. This interference is removed, despite continued feeding of the antagonist, by administration of a crude source of extrinsic factor (essentially free of PGA), together with normal human gastric juice. This finding affords an experimental animal with which to study the mechanism of action of anti-anemic substances and their functional relation to folic acid; also, a suitable bioassay tool is offered for guiding the isolation of anti-anemic factors of unknown chemical composition. The administration of a purified diet similar to that successfully used by Cartwright et al. failed to produce a failure in growth and in hematopoiesis in swine. It is suggested that this failure may possibly be attributable to the presence of biotin in the diet employed in this study.

The treatment of acute leucemias of childhood with folic acid antagonists

Summary 1. Twenty-four children with acute leucemia were treated with the folic acid antagonists Aminopterin and A-Methopterin. Hematologic or symptomatic improvement, or both, occurred in twenty. 2. A dosage regimen is described. 3. Roentgen changes of the skeleton were present in thirteen children on the initial examination. Three children developed skeletal lesions during treatment. Four types of lesions were observed. 4. At autopsy, evidence of leucemia was sometimes minimal or absent. Severe hypoplasia of the marrow was encountered. 5. Infection and hemorrhages were frequent complications in these cases. 6. The mechanism of action of the antagonists is through production of a folic acid deficiency, although the action cannot be readily reversed by administration of folic acid. 7. Toxicity was manifested by ulcerative lesions of the oral mucous membranes. 8. While the antagonists did not constitute a satisfactory form of therapy for leucemia, they were more effective than other forms of treatment.

THE ROLE OF CONJUGATED AND FREE FORMS OF FOLIC ACID IN THE CONTROL OF PERNICIOUS ANEMIA: II. BIOCHEMICAL ASPECTS

In addition to the failure of conjugated folic acid of yeast to produce a clinical response in pernicious anemia, there is other evidence of a biochemical defect in the utilization of this material. Observations on the excretion of the L. casei factor have disclosed that only traces appear in the urine of normal individuals on ordinary diets; this excretion approximates 2 to 4 micrograms per day. When synthetic folic acid (L. casei factor) is administered parenterally, from 15 to 75% of the dose usually appears in the urine within 24 hours. In the patient given daily intramuscular injections of 2.5 mg. of vitaMin B, conjugate, there was no appreciable augmentation of the basal urinary elimination of the L. casei factor; of the injected doses, in terms of L, casei factor, only l%, on the average, appeared in the urine daily. On the previous dosage regime, 0.85 mg. of synthetic folk acid per day, the patient had excreted approximately 15% of the daily dose. Following the administration of the single intramuscular dose of 30 mg. of conjugate, none of the material was found in the urine, either in the free or in the conjugated form. However, a dose of the equivalent amount (11 mg.) of synthetic folic acid not only caused a clinical response, but also resulted in the excretion of approximately 4.1 mg. of the L. easei factor during the following 48 hours. It is clear, therefore, that, in these patients with pernicious anemia, the conjugated folic acid of yeast is not broken down appreciably to release L. casei factor, and its failure to appear as such in the urine suggests that it is either destroyed or, more probably, stored in the tissues. Although conjugated folic acid appears to be utilized effectively by chicks, rats, and monkeys, it is necessary to offer proof that human subjects without pernicious anemia can make use of such materials. Since a patient with a purely nutritional deficiency of folic acid was not available, the ability of a normal subject to metabolize the yeast conjugate was demonstrated by a study of the urinary excretion of the L. casei factor, following dosage with the conjugate. A normal human subject, whose output of L. casei factor in the urine was known consistently to

THE ROLE OF CONJUGATED AND FREE FORMS OF FOLIC ACID IN THE CONTROL OF PERNICIOUS ANEMIA: I. CLINICAL OBSERVATIONS

The ability of synthetic folic acid to produce remissions in patients with macrocytic anemia associated with megaloblastic arrest in the bone marrow has been established. This raises the question of why such patients should develop a deficiency of this substance. Foiic acid is present in natural forms in many items of the daily diet, although in amounts unknown at the present time. Bacterial synthesis of folic acid, also, has been demonstrated to occur in the intestinal tract of animals, but whether this occurs in man has not been established. That folic acid is synthesized by normal human tissues has not been disproved, but no evidence for such a synthesis is available. It seemed possible, therefore, that some of the dietary forms of folic acid might not be effectively utilized in pernicious anemia. This problem presented the necessity for studying naturally occurring derivatives of folic acid, including Streptococcus lactis R factor, “fermentation” folic acid, yeast conjugate, and other natural forms of folic acid which occur in the diet. Streptococcus Eactis R factor has been shown to have no folic acid activity in any animals studied to date. Goldsmith has reported that “fermentation” folic acid was effective in one patient with macrocytic anemia. Castle,.on the other hand, was unable to elicit a response in two patients with pernicious anemia who received 2.3 mg. and 3.6 mg., respectively, of “fermentation” folic acid, administered with normal human gastric juice. The yeast conjugate has been found to relieve folic acid deficiency in monkeys, chicks, and rats. It also occurred to us that other members of the vitamin B complex might contribute to the effect of folic acid. The following brief case reports are presented to demonstrate the response of patients to folic acid and the failure of other components of the vitamin €3 complex to augment the effect of folic acid. (See TABLE 1 for summary of blood findings a t beginning of treatment.)

Proliferation of Myeloid and Lymphoid Cells Induced by Extracts of Urine from Leucemic Patients.

Summary Myeloid metaplasia in liver, spleen, adrenal, and other organs, as well as myeloid white cell hyperplasia of the bone marrow, was produced in 33 guinea pigs by injections with extracts of urines obtained from patients with chronic myeloid leucemia. One animal, similarly treated, showed no change. Twenty-eight guinea pigs, injected with extracts of the urine from normal individuals and individuals with diseases other than myeloid leucemia, showed no myeloid change. The animals which received the extracts of urine from patients with chronic lymphoid leucemia and multiple myeloma showed lymphoid hyperplasia. Two animals showed hyperplasia which has not been classified.

Negative Effect of Gastric Juice Administered Intravenously to Patients with Pernicious Anemia

Conclusion Normal, whole, neutralized gastric juice which had been passed through a Berkefeld V filter and administered intravenously to patients with pernicious anemia does not produce a hematologic response probably because there is no extrinsic factor as such in the parenteral tissues to provide a substrate for interaction.

A Characterization of a Urinary Fraction Capable of Producing Myeloid Metaplasia in Guinea Pigs

Heinle, Wearn, Weir, and Rose1 reported myeloid hyperplasia and metaplasia in guinea pigs following the injection of extracts and adsorbates of human urine. The majority of potent preparations had been obtained from patients with chronic myeloid leukemia.