Howard A. Meineke

Innervation of the periarteriolar lymphatic sheath of the spleen

During the course of a neurohistochemical and two independent electron microscopic studies of the mouse spleen, unmyelinated adrenergic nerves containing numerous dense core and lucent vesicles and devoid of neurolemma were observed adjacent to reticular cells and lymphocytes in the white pulp. Some of these nerves formed an intimate relationship with these cells. Since adrenergic substances have been reported to modulate the cell cycle of lymphocytes in vitro, these findings are suggestive of a neural influence on the cell cycle of lymphocytes in vivo.

Further observations on the mechanism by which androgens and growth hormone influence erythropoiesis.

The process of erythropoiesis is controlled by a hormone, erythropoietin, and other hormones seem to have an influence on this process as well. The manner in which these other hormones influence erythropoiesis is problematical but, as can be seen in FIGURE 1, it may be simply ( 1 ) by increasing the general metabolic rate, thereby creating an anoxic condition; or more specifically, (2) by influencing the precursors of erythropoietin; ( 3 ) by the production of this substance; (4) by activating it in some manner; (5) by stimulating the stem cells directly; or ( 6 ) by enhancing maturation of the stem cells once they have been started toward the erythroid series by erythropoietin. Thyroxin has already been reported’ to influence erythropoiesis in a noncalorigenic fashion and to influence metabolism as well; and testosterone was found to increase 5gFe uptake in the polycythemic mouse by Fried et aL2 This was accomplished in the absence of any change in oxygen con~umption.~ Considerable attention has been given recently to the manner by which androgens increase the rate of erythropoiesis. Recent reports3,4 have hypothesized that androgens exert their influence by increasing production of erythropoietin by the kidney, and this hypothesis has been ~ubstantiated”~ by the finding that the plasma of animals treated with androgens alone or in combination with an0xia5.~ or cobalt7 contains an increased titer of a substance which will stimulate erythropoiesis. Naets and Wittek,8 on the other hand, have claimed that androgens act either by augmenting the action of erythropoietin on the stem cells or by influencing a later stage of erythropoiesis. In either case, the presence of erythropoietin is mandatory. Even more recently Reisnerg has found that androgens will stimulate erythropoiesis in cultures of marrow cells, and Fried and Gurneylo feel that androgens may alter the kidney in such a manner as to make it respond more vigorously to an anoxic stimulus. Growth hormone has not received as much attention as the androgens in spite of the fact that the manner in which it stimulates erythropoiesis has never been elucidated. That it does affect this process has been shown by several groups. l1 The present work was performed in an attempt to resolve the question of how androgens and growth hormone stimulate erythropoiesis.

Presence of erythropoietic factor in plasma of normal and hypophysectomized rats following bleeding.

Summary 1. Normal adult-female rats were injected intravenously with a total of 6 or 9 cc of plasma, over a 3-day period, obtained from normal rats, bled normal rats, hypophysectomized rats, or bled hypophysectomized rats. Reticulocyte response was used as criterion of erythropoietic activity and blood was removed for reticulocyte counts 24 and 48 hours after the last plasma injection. 2. Best results were obtained with the larger dose level. Erythropoietic factor was present in the plasma of the bled normal rats, the reticulocyte count being elevated 483% above normal. The plasma factor was also found to be present in bled hypophysectomized rats, the reticulocyte count of the recipient rats being elevated 138% above normal levels. This factor was also found in the plasma of unbled hypophysectomized rats, a fact which needs further investigation. Conclusions. This experiment indicates that erythropoietic factor can be demonstrated in normal bled rats and that hypophysectomy does not prevent its formation.

EVIDENCE FOR A NON-CALORIGENIC EFFECT OF THYROXIN ON ERYTHROPOIESIS AS JUDGED BY RADIOIRON UTILIZATION.

Summary Thyroxin has been shown to have an effect on erythropoiesis, as judged by Fe59 uptake, which is greater than would be expected from its influence upon oxygen consumption. This non-calorigenic effect on erythropoiesis has been shown in 4 ways—1) by observing a greater percentage increase in Fe59 incorporation than in oxygen consumption in hypophysectomized thyroxin treated rats; 2) by observing an increase in Fe59 uptake in thyroxin treated polycythemic hypophysectomized rats where the increased number of erythrocytes should take care of any erythropoietic response to oxygen need; 3) by finding that thyroxin induced a greater erythropoietic response in thyroidectomized rats than did dinitrophenol even when the latter drug induced a higher oxygen consumption, and 4) by noting that thyroxin treated thyroidectomized polycythemic rats responded by an elevation in Fe59 uptake while dinitrophenol in similar rats had no effect.

Correlation between Oxygen Consumption and Erythropoiesis in Hypophysectomized Rats Treated with Various Doses of Thyroxin.

Summary Hypophysectomized rats were treated with 0.01, 0.05 or 0.10 mg of thyroxin daily. Oxygen consumption in the 3 groups of animals was 100, 124 and 107% of normal; total erythrocyte volumes/100 g body weight were 83, 121 and 107% of normal. Hypophysectomized controls exhibited oxygen consumption 67% of normal with total erythrocyte volume/100 g body weight, 79% of normal. These data support the oxygen need theory of post-hypophysectomy anemia.

Effect of combined thyroxin-cortisone-growth hormone therapy on hematopoiesis in hypophysectomized rats.

Summary Previous work has shown that a combined thyroxin-cortisone therapy in hypophysectomized rats will eliminate the anemia and induce a marked decrease in peripheral white cells; this was accompanied by severe hypoplasia of the bone marrow. It has also been shown that growth hormone increases peripheral white cell count in hypophysectomized rats but has no effect on peripheral anemia; the bone marrow was hyperplastic. Adding growth hormone to the thyroxin-cortisone therapy resulted in repair of post-hypophysectomy anemia, in normal histological appearance of bone marrow, in normal total number of cells/mm3 of marrow tissue, and in values for myeloid elements/mm3 of marrow and peripheral white cells slightly above normal. The values for erythroid elements/mm3 of marrow averaged 82% of normal. These hematological findings were accompanied by a normal oxygen consumption.

Presence of An Active Erythropoietic Factor (Erythropoietin) in Plasma of Rats after Prolonged Cobalt Therapy.

Summary 1) Plasma was obtained from hemorrhagic anemic rats and from rats injected with cobalt for 26 days for determination of erythropoietic activity. Cobalt concentration of plasma from cobalt treated rats was lowered to normal levels by dialysis against isotonic phosphate-cyanide solution of pH 7.5. The level of cobalt in plasma was determined by modification of the nitroso-R-salt method. 2) Plasma to be injected was concentrated 6.6 times in vacuo. The erythropoietic activity was tested, using reticulocyte changes as index of activity, in hypophysectomized rats injected subcutaneously with 1 cc of concentrated plasma for 3 consecutive days. Reticulocyte counts were made 24 and 48 hours after end of injections. Plasma from anemic animals caused increase of reticulocytes to 61.5 and 151% (24 and 48 hours respectively) above values obtained from injections of plasma from normal rats. Injections of “cobalt treated” plasma increased the level of circulating reticulocytes to 36.4 and 54.5% above values obtained from injecting plasma from normal rats for similar time intervals, and demonstrates that erythropoietic activity of plasma is increased in rats receiving prolonged cobalt therapy, and that the activity is not due to increased levels of cobalt.

Decreased Oxygen Need as a Factor in Anemia of Hypophysectomized Animals.

Summary Adult female rats were hypo-physectomized and studied 60 days later; they exhibited the usual post-hypophysec-tomy anemia. The theory that the anemia in the hypophysectomized animal is simply a reflection of a decreased need for oxygen is supported by the finding that oxygen consumption was decreased in these animals and that it remained at a low level even when the peripheral blood picture was restored to normal via transfusion of whole blood. The theory is further supported by the finding that procedures which altered the erythrocyte level caused a similar change in oxygen consumption, and by much evidence indicating that the bone marrow can function normally in the absence of the pituitary gland.

The Origin of Erythropoietin in Rats with Transplants of an Adrenal Cortical Carcinoma

Summary Rats bearing a transplantable adrenal cortical carcinoma showed the following features: elevated levels of an erythropoietic factor in the serum 2–4 weeks after tumor transplantation; no increase in erythropoietic factor in the serum 2 months after transplantation; significant levels of an erythropoietic factor in saline extracts made of both the young and the old tumor tissue; and no significant reduction of erythropoietic factor in the serum or tumor 48 hr after nephrectomy of the tumor bearing rats. This evidence indicates that the site of production of the erythropoietic factor is the tumor itself. Several features of the active tumor extracts. primarily its inactivation by neuraminidase, supports the contention that the eryth-ropoietically active material in these rats is erythropoietin.

Lack of Hematopoiesis in Tail Bones Transposed to Abdominal Cavity of Hypophysectomized Rats.

Summary Thirty-seven of 41 normal female rats, in which the ends of the tails were transposed into the abdominal cavity, showed a stimulation of hematopoiesis in the intra-abdominal vertebrae. Developing cells of all 3 types—erythroid, myeloid and megakaryocytes—were observed in these animals. In similarly treated hypophysectomized female rats only 1 of 16 animals showed any indication of response. The experiment provides another example of the depressed hematopoietic potential of the hypophysectomized rat.