The Effect of Neutropenia on the Cell Cycle of Granulocyte Precursors in an In Vivo Culture System

Proliferation of myeloid elements, macrophages,and pluripotent stem cells (CFU) in diffusionchamber cultures of normal mouse bone marrow was examined. The relative growth ofdifferentiated cells and CFU was clearly inhibited when the concentration of cells cultured was increased from 10⁵ to 20 x 10⁵ perchamber. When chambers were implanted intoneutropenic hosts the growth of CFU andgranulocytic cells was enhanced. The generativecycle of the earliest recognizable granulocyticcells, as estimated by flash labeling with tritiated thymidine, was comparable to that seenin normal bone marrow and was significantlyshortened by culture in a neutropenic environment. Growth of pluripotent stem cells andmyeloid elements in these cultures appears to beinfluenced both by short-range factors relatedto cell concentration and by long-range humoralfactors.

Submitted on March 30, 1973 Revised on June 20, 1973 Accepted on June 29, 1973     

The Use of 125I as a Membrane Protein Label for Erythrocyte Survival Studies

Radioactive iodide, incubated with erythrocytes in the presence of lactoperoxidaseand hydrogen peroxide, is covalentlybound to membrane proteins. Using thistechnique, rabbit erythrocytes were labeled with ¹²⁵I, reinjected into the donors,and erythrocyte survivals determined. Thevalues for the erythrocyte half-lives andthe shape of the decay curves were comparable to those reported using DF³²P. Following hemolysis of erythrocytes doublelabeled with ¹²⁵I and ⁵¹Cr, radioactivityfrom both these isotopes appeared mainlyin liver, lung, kidney, spleen, and urine.Enzymatic iodination provides a noneluting label of erythrocyte membraneproteins for the study of survival, sequestration and turnover of this red cellcomponent.

Submitted on June 16, 1973 Revised on September 13, 1973 Accepted on September 23, 1973     

Abnormalities of Megakaryocytes in Sl/Sld Mice

Megakaryocytopoiesis was evaluated inSl/Sl^d mice and their heterozygous andhomozygous normal (+/+) littermates.Sl/Sl^d mice had a normal concentrationof blood platelets which were of normalsize. Numbers of megakaryocytes in bonemarrow and spleen were reduced, butindividual megakaryocytes were largerthan normal.

Submitted on April 9, 1973 Revised on May 31, 1973 Accepted on June 12, 1973     

Abnormalities of Megakaryocytes in W/Wv Mice

Megakaryocytopoiesis was evaluated ingenetically anemic mice of the W/W^v genotype and was found to be abnormal.Concentration and size of blood plateletswere normal. Megakaryocytes were decreased in number in tibial marrow andspleen, and the size of mature megakaryocytes was increased.

Submitted on April 9, 1973 Revised on May 31, 1973 Accepted on June 12, 1973     

Iron in the Duodenal Mucosa of Normal, Iron-loaded and Iron-deficient Rats

Iron absorption studies with oral ⁵⁹FeSO₄were performed on 13 iron-depleted, 11iron-loaded, and 10 control rats, and nonheme iron was determined in both isolated epithelial cells and defoliated mucosa obtained from the duodenum. Meanabsorption by the control animals was18.5% of the dose. Both iron-depleted andiron-loaded groups showed significant differences in iron absorption (54.5% and2.2% respectively). Compared with thenormal controls, iron was decreased inthe epithelial cells of the iron-deficientgroup, whereas higher concentrationswere observed in the defoliated mucosaof iron-loaded animals. The latter observation was confirmed by the presence ofiron-laden macrophages seen in sectionsof the lamina propria of the iron-loadedrats.

Submitted on February 20, 1973 Revised on June 7, 1973 Accepted on June 13, 1973     

Some Properties of the Circulating Hemopoietic Stem Cells

Some properties of hemopoietic stem cells(CFU) circulating in the peripheral blood ofthe normal mouse (PCFU) were studied.Those parameters were chosen that arewell-known characteristics of bone marrow stem cell populations. Leukocytesseparated from the peripheral blood werethe source of PCFU. The radiosensitivity(D₀ value), growth curve, seeding efficiency (f number), and the turnover state(number of PCFU in S phase measured by³H-thymidine killing technique in vitro) ofPCFU were compared to the same parameters for bone marrow CFU. Several characteristic changes in these parameterswere found for PCFU: (1) Lower radiosensitivity (D₀ = 140 R), (2) Higher seedingefficiency (f number 20.9%), (3) Highersensitivity to ³H-thymidine in vitro (killingeffect 32%). The doubling time was thesame both for CFU and PCFU (21 hr), butthe logarithmic growth of the PCFU population started by approximately 36 hr laterthan that of the marrow-derived CFU population. All these findings support the ideathat PCFU represent a subpopulation ofthe heterogeneous bone marrow CFUpopulation.

Submitted on May 16, 1973 Revised on September 18, 1973 Accepted on October 18, 1973     

A Hypertransfused Mouse Assay for Thrombopoietic Factors

A method was developed for the quantitative separation of platelets from CF1mouse whole blood. This made it possibleto determine the platelet incorporation of³⁵S-sulfate without the necessity of doingplatelet counts. Daily hypertransfusions ofthe mice to three to four times normalplatelet levels for 4-5 days significantlyreduced platelet uptake of radiosulfate toan average of about 40% of the nontransfused controls. Mice rendered thrombocytopenic 48 hr earlier by antiplateletserum, had 2-day ³⁵S uptakes over 2 1/2times the controls and 6 times the hypertransfused animals. The administration of atotal of 2 ml of serum, given twice daily for3 days from a thrombocytopenic patientwith Hodgkin's disease caused a highlysignificant 103% rise in radiosulfate incorporation when compared with saline in thehypertransfused mouse. Normal humanserum from a healthy donor caused a smalland insignificant rise. The serum from a patient with Hodgkin's disease caused ahighly significant 63% rise in ³⁵S incorporation when compared to the normal serum.

Submitted on June 19, 1972 Revised on March 10, 1973 Accepted on March 10, 1973     

Sickle Hemoglobin: A Specific Radioimmunoassay

For the quantitation of hemoglobin S, aradioimmunoassay has been developedwhich is specific and highly sensitive.Hemoglobin S was purified by columnchromatography and injected with complete Freund’s adjuvant into goats. Eachgoat serum was tested for reactivityagainst hemoglobins A and S by immunodiffusion and by quantitative precipitation. Hemoglobin A reactivity was removed by absorption with hemoglobin A.One serum so treated was specific forhemoglobin S; it reacted negligibly withhemoglobins A or F. Hemoglobin S waslabeled with ¹²⁵I by the chloramine Tmethod. In the radioimmunoassay, complete precipitation of the antigen—antibody complex was insured by the additionof rabbit antigoat gamma globulin. Thisassay offers reliable and specific quantitation of as little as 1 ng of hemoglobin S.Assuming that 10% of the hemoglobin infetal blood at 16 wk gestation is of adulttype, this assay is capable of detecting theamount of hemoglobin S in 10^-7 ml ofhomozygous hemoglobin S blood. The prenatal diagnosis of sickle cell anemia byradioimmunoassay will require, in addition, a method for demonstrating theabsence of hemoglobin A and a safemethod for obtaining fetal erythrocyteswithout significant contamination by maternal erythrocytes.

Submitted on June 28, 1973 Revised on October 11, 1973 Accepted on November 1, 1973     

The Role of Peroxidase in the Bactericidal Activity of Human Blood Eosinophils

Utilizing cell preparations that containedgreater than 90% eosinophils and sodiumazide to inhibit the lysosomal enzymeperoxidase, studies of eosinophil bactericidal activity were undertaken. Eosinophils did not kill bacteria as well asneutrophils, principally because of a reduced phagocytic capacity. In contrast toneutrophils, eosinophils had high restingiodination activity but failed to increasethis activity following phagocytosis. Onemillimolar sodium azide, which inhibitsboth eosinophil and neutrophil peroxidaseas measured by the iodination of trichloroacetic acid precipitable protein, impaired neutrophil staphylocidal activity,but enhanced eosinophil killing of Staphylococcus aureus primarily by increasing phagocytic uptake. Bactericidal studiesutilizing lysostaphin suggest that neutrophil peroxidase exerts its contribution tobactericidal activity only during the earlypostphagocytic period. Eosinophil peroxidase is genetically and biochemically distinct from neutrophil peroxidase, andappears to play no role in the bactericidalactivity of intact eosinophils.

Submitted on June 8, 1973 Revised on October 2, 1973 Accepted on October 25, 1973     

A Comparison of the Behavior of 111In and 59Fe-labeled Transferrin on Incubation With Human and Rat Reticulocytes

The uptake by human and rat reticulocytesof ¹¹¹In and ⁵⁹Fe bound to transferrin hasbeen studied. The results indicate a significant difference between the behaviorof the two isotopes in both human and ratincubation mixtures. Reticulocyte uptakeof ¹¹¹In from human and rat serum was30% and 12% that of the ⁵⁹Fe after a30-min incubation. The process was temperature dependent, inhibited by sodiumarsenite, and related to the reticulocytepercentage of the cells in the reactionmixture. Washed reticulocytes, previouslyincubated for 30 min with either ⁵⁹Fe or¹¹¹In bound to serum were reincubated inunlabeled serum. Up to 85% of the ¹¹¹Inlabel and less than 10% of the ⁵⁹Fe on thereticulocytes were released on reincubation, indicating that, in contrast to ⁵⁹Fe,the majority of the ¹¹¹In label remainedmembrane bound. Specific binding of unlabeled In transferrin was demonstratedby its inhibitory effect on ⁵⁹Fe-transferrinuptake by rat reticulocytes. Both In- andFe-transferrin were found to have similarbinding affinities for the receptor sites.The ¹¹¹In remaining in lysates obtainedfrom washed reticulocytes after incubation with ¹¹¹In-labeled serum and reincubation in unlabeled serum did not appearto be associated with either the hemoglobin or heme molecules.

Submitted on October 2, 1973 Revised on October 28, 1973 Accepted on October 30, 1973     

Hemoglobin Rush [beta101 (G3) Glutamine]: A New Unstable Hemoglobin Causing Mild Hemolytic Anemia

A mild hemolytic anemia in a 43-yr-old blackwoman was attributed to the presence of an abnormal hemoglobin (Hb Rush) which migratedcathodically to Hb A at pH 8.0. Its structuralabnormality was found to be in the -chain,101 (G3) glu gln. Another electrophoreticband at pH 8.0 proved to be a hybrid tetramer(^A₂^ARush). Hb Rush is heat unstable. Alikely explanation of the instability is thepresence of an uncovered positive charge in thecentral cavity where normally glutamic acid inposition 101 neutralizes arginine in position104 contributing to the net neutrality in thisregion. This neutrality is disturbed by the substitution of glutamic acid by glutamine in HbRush.

Submitted on May 10, 1973 Revised on June 25, 1973 Accepted on July 16, 1973     

Erythropoiesis in Carotid Body Resected Cats

It has been reported that removal of carotid bodies in cats results in a brief, profuse reticulocytosis followed by depressionto below-normal levels of reticulocytesand a progressively severe anemia. Injection of cat carotid body extract was reported to increase erythrocyte ⁵⁹Fe incorporation in polycythemic rats. Otherstudies could find no hematological abnormality in humans after bilateral carotidbody resection. We reexamined the effectof carotid body resection in the cat withserial bone marrow aspirations, hematocrit determination, and reticulocyte determination. T_1/2 for plasma ⁵⁹Fe removal,erythrocyte ⁵⁹Fe incorporation, and ⁵¹Crlifespan determinations were performedon five operated and three control cats.No significant differences were found. Weconclude that the carotid body has nodirect effect on erythropoiesis and thatthe anemia reported in a prior study wassecondary to sepsis resulting from an indwelling femoral vein catheter.

Submitted on February 15, 1973 Revised on April 20, 1973 Accepted on April 25, 1973     

Cytochemical and Radioautographic Identification of Cells Induced to Synthesize Hemoglobin

In order to facilitate the identification ofbone marrow cells in which hemoglobinsynthesis is initiated, erythropoiesis wasfirst suppressed in guinea pigs through theinduction of posthypoxic polycythemia,and then it was restimulated by bleedingand reexposure to hypoxia. Hemoglobinsynthesis was detected with ⁵⁵Fe incorporation on radioautographs, and itspresence was demonstrated in the lightmicroscope with the benzidine reactionand absorption of monochromatic light at 4046 Å. In the electron microscope,hemoglobin was detected in the cytoplasm by a general increase in electrondensity after treating the tissue withdiaminobenzidine (DAB) and OsO₄. Densitometric measurements were carried outon electronmicroscopic negatives, usingreticular cell cytoplasm as a base line. Innormal marrow, proerythroblasts were theearliest cells in which hemoglobin couldbe detected, but during the early phase oferythropoietic stimulation, hemoglobinwas demonstrated in transitional cellswith all the methods employed. Withoutthe specific demonstration of hemoglobin,these cells could not be recognized morphologically as erythroblasts nor couldthey be distinguished from the precursorsof bone marrow small lymphocytes.Transitional cells were numerous in themarrow at the time of stimulation, and40 hr later a small number of them werelabeled with ⁵⁵Fe and synthesized hemoglobin in detectable amounts. Proerythroblasts were absent at the time of the stimulus, and when they reappeared themajority were benzidine or DAB positiveand had incorporated ⁵⁵Fe. The findingssuggest that progenitor cells of erythroblasts are among the basophilic membersof the transitional cell population, anderythropoietic stimulation induces hemoglobin synthesis in them. The relationshipof these cells to the progenitors of otherhemopoietic cells, as well as to the pluripotent stem cell, is discussed.

Submitted on May 29, 1973 Revised on November 12, 1973 Accepted on November 15, 1973     

The Effect of Erythropoietin on Hematopoietic Organs of the Polycythemic Mouse

Increased RNA-P³²/DNA-P³² ratio, iron incorporation and heme synthesisin spleens of polycythemic mice was demonstrated following an injection oferythropoietin. These changes were correlated with the appearance of erythroblasts. These findings suggest an intimate relation between RNA synthesisand stem cell differentiation.

Submitted on June 8, 1965 Accepted on July 25, 1965     

PHA Responsiveness and Subpopulations of Circulating Lymphocytes in Pernicious Anemia

Lymphocyte transformation responses tothe mitogen phytohemagglutinin (PHA)were measured in 20 patients with provenpernicious anemia (PA) and 20 matchedcontrols using ³H-thymidine label. The patients with PA showed significant depression of lymphocyte transformation to thethree doses of PHA employed, as judgedby beta counting; however, radioautographic examination of PHA-stimulatedcells indicated that the results were due toa failure of intranuclear incorporation of³H-thymidine by PA lymphocytes, ratherthan a failure of PHA to induce blastogenesis. The percentages and numbers ofT and B lymphocytes in peripheral bloodwere measured in 30 patients and controlsby rosette and immunofluorescence techniques, respectively. There was no significant difference in the B cell subpopulations between patients and controls; theT cell subpopulation was slightly lower inthe PA patients (mean 62.4%) than in thecontrols (mean 65.5%), but the differencewas not statistically significant. The depressed uptake of ³H-thymidine by stimulated lymphocytes in PA would seem toreflect a chemical defect rather thaninherent immunologic abnormality.

Submitted on November 13, 1973 Accepted on June 12, 1974     

The Metabolism of Transferrin-bound 111In and 59Fe in the Rat

The metabolism of transferrin-bound indium and iron were compared by in vivostudies in the rat. Rats were injected withserum transferrin labeled with ¹¹¹In and⁵⁹Fe, and, at intervals ranging from 20min to 5 days after injection, they werekilled. They were perfused through theportal vein with 200 ml of 0.9% saline,and the residual radioactivity, expressedas a percentage of the injected dose, wasmeasured in liver, spleen, kidney, muscle,washed red cells, red marrow, and femur.At 5 days, 76% of the injected ⁵⁹Fe wasrecovered in the red cell mass; only1%-2% of ¹¹¹In could then be recovered.Uptake and release of the ¹¹¹In label bythe femur was markedly less than that ofthe ⁵⁹Fe. Whereas 85% of the injected⁵⁹Fe could be recovered from the circulating red cells, liver, and spleen, only about15% of the injected ¹¹¹In could be so recovered. Approximately 35% of the injected ¹¹¹In was excreted, and 43% wasrecoverable from the carcass. The subcellular distribution of the two isotopes in theliver at timed intervals following intravenous injection was studied. While 35%of the ⁵⁹Fe activity in the homogenatewas associated with ferritin, only 4% ofthe ¹¹¹In could be so identified. The results indicate a significant difference between the metabolism of ¹¹¹In and ⁵⁹Fein the rat and make it unlikely that themetabolism of ^In in man bears muchsimilarity to that of iron.

Submitted on October 2, 1973 Revised on October 28, 1973 Accepted on October 30, 1973     

Correlation Between Cytokinetically Resting Lymphocytes and Bone Marrow Restoration: Experiments Using a Discontinuous Albumin Gradient

Rats were labeled with ³H-thymidine(³H-TdR) in utero and for 6 wk after birthin order to obtain 100% labeling of all bonemarrow cells. Six weeks after the last³H-TdR injection only cytokinetically resting cells were still labeled. At this time, theregenerative capacity of fractions obtainedafter centrifugation on a discontinuous albumin gradient was tested in 1200-Rx-irradiated recipients, and the results werecompared with the effect observed aftertransplantation of the same number ofunfractionated bone marrow cells. One ofthe fractions obtained had a regeneratorycapacity tenfold that of unfractionatedcells. In contrast, when the response to aPHA stimulation test was evaluated, thisfraction showed a decreased incorporationof ¹⁴C-TdR as compared to other fractions.In a second test system, fractions of bonemarrow from ³H-TdR-labeled donors madehypoplastic by repeated injections of hydroxyurea were transfused into 1200-Rx-irradiated recipients. Regenerative capacity was similar to that seen in the firstexperiment. The findings indicate a correlation between cytokinetically resting,small mononuclear cells and the regeneratory process in lethally x-irradiatedrecipients.

Submitted on January 17, 1972 Revised on January 19, 1973 Accepted on January 24, 1973     

Kinetics of Human Monocytopoiesis

Information on the structure of humanmonocytopoiesis was derived from thechronologies of labeled monocytes entering the blood following injection of ³H-thymidine (³H-TDR) and ³H-diisopropylfluorophosphate (³H-DFP). It was foundthat monocytes are released into the blooddirectly from a proliferating precursorpool. The marrow cell egress was almostcompletely restricted to G₁-phase monocytes. The egress rate increased withmaturation time of promonocytes. The majority of the cells was released at maturation times between 50 and 60 hr. Undernormal conditions promonocytes underwent a mean of two catenated cell cycles.The cell cycle time averaged 29 hr andDNA-synthesis time 10 hr. Direct investigations of the promonocytes demonstratedthat the medullary promonocyte pool averages 6 x 10⁸ cells/kg body weight inhealthy adults. This pool produces a meanof 7 x 10⁶ cells/kg-hr. Proliferation capacity of promonocytes is only partiallyutilized in normal monocytopoiesis. Induction of acute inflammatory reactions gaverise to an immediate enhancement ofpromonocyte proliferation activity, thusaffording short-term adaptation of monocyte production to monocyte demand. Increase in monocyte production was associated with a shift of marrow release infavor of immature cells.

Submitted on November 14, 1973 Accepted on June 10, 1974     

Hematopoietic dynamics in grey collies.

Using Lomb periodogram analysis we have quantified variations in the peripheral neutrophil and platelet counts of the cyclical neutropenia animal model-the grey collie. We found that the amplitudes of the oscillations in these two cell lineages vary concomitantly. Further, the power spectrum and the shape of the oscillations in the absolute neutrophil counts vary together with the amplitude of the oscillations. As the amplitude of the oscillations increases, the height of the second subharmonic increases, giving rise to a distorted oscillation with two peaks per cycle. The particular dynamics of the absolute neutrophil counts can be reproduced by a combination of a delayed peripheral feedback, representing the peripheral control of granulopoiesis through granulocyte colony stimulating factor, together with a sinusoidal input representing an oscillatory input from the pluripotential stem cells to the granulocytic lineage. The same pluripotential stem cell input is probably responsible for the sinusoidal oscillations observed in the other cell lineages.     

Protein - Quinone Interaction: In Vitro Induction of Indirect Antiglobulin Reactions With Methyldopa

Methyldopa-treated gamma globulin can bedemonstrated serologically on either the redcell surface or on latex beads by the indirectantiglobulin reaction. The development of apositive antiglobulin reaction was related tomethyldopa concentration and the length andtemperature of incubation of methyldopa withprotein and could be partially inhibited by theaddition of albumin to the incubation mixtures.After more prolonged incubation, antiglobulinpositivity also developed with plasma-treatedwith methyldopa. ¹⁴C-methyldopa was covalently bound to gamma globulin. Aggregation ofgamma globulin following treatment withmethyldopa could be demonstrated by bothsedimentation velocity and molecular weightdeterminations employing low-speed equilibrium centrifugation. Protein aggregation was afunction of time, temperature, and methyldopaconcentration. Detectability by the antiglobulinreaction, the darkening noted in solutions towhich methyldopa or hydroquinone had beenadded, as well as the aggregation of protein wasinhibited by a reducing agent which preventedformation of a quinone from the hydroquinone.Some of the immunologically atypical featuresof the sensitization of red cells by methyldopaor its structural analogues are explicable by theadherence, in vivo, of chemically modified, nonantibody gamma globulin which renders the redcell directly antiglobulin positive.

Submitted on December 28, 1972 Revised on May 29, 1973 Accepted on June 29, 1973