DNA and RNA Synthesis of Circulating Atypical Lymphocytes in Infectious Mononucleosis

The percentages of mononuclear cells synthesizing DNA and RNA in serialstudies of blood from 13 patients with infectious mononucleosis were determined. Early in the disease a high percentage of atypical lymphocytes werein DNA synthesis but this percentage decreased rapidly as the disease progressed. Late in the disease many atypical lymphocytes were still presentbut few, if any, were synthesizing DNA. Similar results were found for RNAsynthesis.

Presumably active proliferation of atypical cells in the tissues is restrictedto an early period of the disease, whereas release of such atypical cells maycontinue for a considerable period.

Submitted on March 18, 1964 Accepted on May 14, 1964     

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     

Leukocyte Labeling in Rats during and after Continuous Infusion of Tritiated Thymidine: Implications for Lymphocyte Longevity and DNA Reutilization

Leukocyte labeling was studied in rats during and after continuous intravenous infusion of H³-thymidine. The radioisotope was administered forvarying periods up to 271 days. The results permit the following conclusions:

1. The median survival of small lymphocytes is about 1 month. Five to8 per cent of small lymphocytes have a life span of more than 9 months.

2. Following the administration of H³-thymidine, reutilization of the tracermarkedly delays the fall-off of labeled cells in the peripheral blood. Reutilization probably involves H³-thymidine released from labeled DNA during cell death, since suppression occurs with massive infusion of non-labeled thymidine.

3. Unlike granulocytes and large lymphocytes, small lymphocytes labelnonuniformly, and appear to be comprised of at least two populations withdifferent intensities of labeling and different turnover rates. The more heavilylabeled cells have the faster turnover.

4. The complexity of the labeling process indicated by the present observations must be considered in the interpretation of H³-thymidine data.However, the survival of unlabeled cells during continuous H³-thymidineinfusion remains a valid means of measuring the life spans of circulatingblood cells.

Submitted on October 22, 1964 Accepted on January 25, 1965     

Protein Synthesis in Rat Lymphocytes: Radioautographic Studies of Availability and Utilization of Labeled Amino Acids in Vivo

Injections of H³-methionine and H³-leucine were combined with radiochemical and radioautographic technics to study the availability time ofH³-methionine and the protein synthetic ability of rat lymphocytes in vivo.

Although 98.5 per cent of H³-methionine was removed from the serum5 minutes after injection, sufficient quantities persisted and/or re-entered theserum from tissues to cause increasing grain counts in radioautographs oflarge lymphocytes for 1 hour after isotope administration. A small amount ofadditional labeling occurred during the 2nd hour, but it is calculated thatlabeling is 97-98 per cent complete by 1 hour.

All of the large and medium lymphocytes were labeled in the thymus, lymphnode, and thoracic duct lymph at short intervals after injection of 4 µc./Gm.body weight of H³-methionine. Evidence is presented that protein synthesisoccurs in the nucleus as well as in the cytoplasm and that newly formed protein is equally distributed between daughter cells following mitosis. Previousimmunochemical studies are combined with information on generation timeand disappearance rates of radioactivity to suggest that large and mediumlymphocytes are constantly producing and releasing proteins. Large andmedium cells in lymph and lymph node are more active in this than aresimilar cells in the thymus. Evidence of reutilization of labeled metabolitesin the lymph node and especially in the thymus is discussed.

Although not all small lymphocytes were labeled by 4 µc./Gm. body weightof H³-methionine, it was shown that larger doses of isotope would label 100per cent of them. Small lymphocytes in thoracic duct lymph evidenced significant turnover of labeled protein during the 1st day after isotope administration.

Submitted on August 21, 1963 Accepted on November 9, 1963     

Platelet Levels in Infectious Mononucleosis

Platelet levels in 57 patients with infectious mononucleosis are recorded.Approximately 50 per cent of cases show some degree of thrombocytopeniaduring the first 4 weeks of the disease. Possible mechanisms for the changeare reviewed and other acute infections complicated by thrombocytopeniabriefly discussed.

Submitted on July 31, 1964 Accepted on September 29, 1964     

The Recovery of Lethally Irradiated Dogs Given Infusions of Autologous Leukocytes Preserved at -80 C

Nine normal mongrel dogs were exposed to 1200 r whole-body irradiationat 4 to 5 r per minute. They were then given intravenous infusions of 2.12 to20 x 10⁹ autologous leukocytes that had been previously stored at -80 C. in10 per cent dimethyl-sulfoxide.

Three dogs survived with delayed but complete hematopoietic recovery.Three showed beginning marrow regeneration but died within 3-4 weeks ofirradiation. Three given less than 6 x 10⁹ cells died within 21 days. The numberof leukocytes infused was critical since there was no survivor among the dogsreceiving less than 9 x 10⁹ cells.

It is concluded that peripheral blood contains primitive cells capable ofrepopulating marrow spaces and restoring marrow function.

Submitted on May 8, 1963 Accepted on June 27, 1963     

Granulocytopoiesis. II. Emergence and Pattern of Labeling of Neutrophilic Granulocytes in Humans

1. Following administration of H³-thymidine to 15 patients with a variety ofhemopoietic conditions, the emergence and the pattern of labeling of neutrophilic granulocytes were studied in peripheral blood leukocytic concentrates. The hematologic diagnosis included five in which the hemopoiesis appeared to be in a steady state equilibrium at the time of study, three withvarious types of leukemia, one with lymphosarcoma, two with multiple myeloma, one with myelofibrosis, two with pernicious anemia (once before andonce after therapy) and two with bacterial infections.

2. The emergence time of neutrophilic segmented granulocytes (time fromH³-thymidine injection to the first appearance of labeled segmented formsin the peripheral blood) was found to vary in steady state equilibrium from96 to 144 hours. It was shortened to 48 hours in two instances with bacterialinfection. This was interpreted as indicating a faster than normal nuclearmaturation with normal or delayed cytoplasmic maturation (dissociation innuclear and cytoplasmic maturation).

3. The number of segments of neutrophilic granulocytes was found to beunrelated to cell age as had been hypothesized by Arneth many years ago.However, bandforms were found in the circulation about 24 hours earlier thansegmented forms, suggesting that they are younger and that some are acceptable to the blood while others continue to mature to segmented forms. Pelgeroid cells with round or bilobed nuclei found in one case of subleukemicmyelocytic leukemia were found to emerge simultaneously 132 hours afterH³-thymidine injection. This suggests that both types are identical in theirdegree of maturation. Thus the cells with round nuclei are not band forms butresult possibly from a delayed nuclear maturation.

4. In patients studied for at least 2 weeks, characteristic undulations of thelabeling indices of the segmented granulocytes were found. If the samplingintervals were 24 hours, peaks were found 6 days apart, the second peak beingabout half of the labeling index of the first. If the sample interval was shorter,a finer structure was observed with undulations showing peak intervals of2-3 days. Although the significance is obscure at present, the constancy of thefindings suggest that there may be a constant input of cells with the index oflabeling varying due to some synchrony of the precursor population(s). Alternative explanations are discussed.

Submitted on January 29, 1964 Accepted on March 31, 1964     

Increased Activity of Some Folic Acid Enzyme Systems in Infectious Mononucleosis

Increased levels of activity for the formate activating enzyme and N⁵,N¹⁰-methylene FH₄ dehydrogenase have been found in the leukocytes from patients with infectious mononucleosis; in addition dihydrofolic reductase, anenzyme not found in mature circulating leukocytes, has been detected in infectious mononucleosis cells. Glucose-6-phosphate dehydrogenase activity isless in infectious mononucleosis leukocytes than in normal leukocytes.

These findings are similar to the results obtained in acute and chronic myelogenous leukemia and indicate that the leukocytes seen in infectious mononucleosis have the enzymatic apparatus associated with synthesis of DNA.

Submitted on November 6, 1961 Accepted on January 12, 1962     

Granulomatous lesions in the bone marrow in infectious mononucleosis; a comparison of the changes in the bone marrow in infectious mononucleosis with those in brucellosis,tuberculosis, sarcoidosis and lymphatic leukemia

1. The findings in the blood and in aspirated bone marrow in 23 cases of infectious mononucleosis have been described.

2. Unequivocal evidence of involvement of the bone marrow has been found in70 per cent of the cases.

3. Evidence of granulomatous inflammation of the marrow was found in 48 percent of the cases.

4. Epithelioid cells were found in the films of bone marrow in 48 per cent of thecases. These cells appear morphologically identical with those seen in imprints oflymph nodes from infectious mononucleosis and sarcoidosis and with the epithelioid cells seen in films of the marrow in brucellosis, sarcoidosis and tuberculosis.

5. The granulomatous lesions of infectious mononucleosis seem most similar tothose of brucellosis, but they also resemble the small granulomatous lesions ofsarcoidosis and tuberculosis.

6. Lymphocytosis of the marrow as well as of the blood was demonstrated in allcases. Evidence of formation of lymphocytes in the marrow was presented, and thealtered lymphocytes of infectious mononucleosis were found in films of the marrow.The degree of lymphocytosis of the marrow in infectious mononucleosis was shownto be less than that in lymphatic leukemia. Lymphocytosis of the marrow was notfound in brucellosis, sarcoidosis or tuberculosis. The lymphocytic reaction demonstrable in the marrow in infectious mononucleosis is believed to be of value in differential diagnosis.

Note: ACKNOWLEDGMENTSWe wish to acknowledge the generous cooperation of Dr. Ruth E. Boynton and the Staff of theStudent’s Health Service of the University of Minnesota throughout the course of this year long studyof infectious mononucleosis. We are indebted to Dr. T. Edward Bell and Dr. James Cardy for performingthe sternal aspirations. The photomicrographs were made by Mr. Henry Morris.

     

The Intravascular Lifespan of Monocytes

Monocytes, defined as peroxidase positive mononuclear cells in the peripheral blood of healthy rats, were labeled by frequent intermittent injections oftritiated thymidine. About 25 per cent of the monocytes were labeled within 1day and 82 per cent in 8 days. Both labeled and unlabeled monocytes disappeared from the circulation in accordance with an exponential function with ahalf-time of about 3 days. Mean grain counts increased asymptotically towarda limit reached in 4 or 5 days. The monocyte turnover rate in the rat is in theneighbourhood of 3.6 x 10⁶ cells per day.

It is concluded that monocytes leave the circulation at random and not as aconsequence of senescence. It is probable that they are the product of a celllineage consisting of about 3 generations from the primitive precursor to themature form, and that the average generation time is about 24 hours. Becauseof the rapid appearance of large numbers of labeled cells, it is unlikely thatthey are derived from lymphocytes which acquire label much more slowly.

Submitted on October 27, 1965 Accepted on January 31, 1966     

Perturbation of Generation Cycle of Human Leukemic Blast Cells by Cytostatic Therapy In Vivo: Effect of Corticosteroids

The in vivo cell cycle effects of intensiveshort-term corticosteroid treatment onbone marrow lymphoblasts have beenstudied in five patients with acute lymphoblastic leukemia. In repeated bonemarrow samples, the following parameters were determined: mitotic index,stathmokinetic index after vincristine,³H-thymidine labeling index and singlecell DNA content. It is concluded thatprednisone has a general destructive effect both on actively proliferating and onquiescent leukemic lymphoblasts. Besides, prednisone has an additional effecton proliferating lymphoblasts whichmanifests itself by a decreased influxfrom G₁ phase into DNA synthesis phase.From the present data it cannot be decided whether this is due to a depopulation of the G₁ pool or results from anarrest of cells in G₁. No evidence hasbeen found for selective cell death during DNA synthesis. Some implications ofthis cytokinetic perturbation for therapeutic combination of corticosteroidswith methotrexate and vincristine arediscussed.

Submitted on May 26, 1970 Revised on July 13, 1970 Accepted on July 19, 1970     

The Distribution of the Philadelphia Chromosome in Patients with Chronic Myelogenous Leukemia

Thirteen patients with chronic myelogenous leukemia continued to havethe Ph¹ chromosomes in 90-100 per cent dividing marrow cells during drug-induced clinical remissions. The Ph¹ chromosome was present in erythroidas well as granulocytic marrow cells, and possibly in megakaryocytes.

The presence of Ph¹ chromosomes was also studied in cultures of peripheralblood. In six patients in relapse, 40 per cent of metaphases contained the Ph¹chromosome, and the percentage of these cells corresponded roughly to therelative frequency of immature granulocytes in the blood. In contrast, duringremission, few or no Ph¹ chromosomes were found in peripheral blood cultures, presumably because in the absence of immature granulocytes the dividing cells in the cultures originate from lymphocytes, as they do in normalblood.

It is suggested that the Ph¹ chromosome usually arises in a precursor cellcommon to the erythroid, granulocytic, and megakaryocytic, but not thelymphoid series of hemopoietic cells.

Submitted on April 5, 1963 Accepted on June 30, 1963     

Sternberg-Reed Cells in the Peripheral Blood of Patients with Hodgkin's Disease

Our observations of 135 patients indicate that 37 per cent of those sufferingfrom Hodgkin’s disease exhibit abnormal cells in the leukocyte concentrates ofthe peripheral blood during the course of their illness. Typical Sternberg-Reedcells were found in 18.5 per cent of patients and were present only in theadvanced stages of generalized Hodgkin’s disease.

The presence of Sternberg-Reed cells in the peripheral blood indicates anadvanced stage of the disease but does not necessarily predict an immediatelyfatal outcome.

Comparative studies, searching for Sternberg-Reed cells in the splenic circulation, showed no Sternberg-Reed cells to be present in the splenic arteries ofpatients with Hodgkin’s disease; but numerous Sternberg-Reed cells were present in the splenic vein, particularly after mechanical squeezing of the spleen.A possible hypothesis is given to support the evidence for the circulation ofSternberg-Reed cells and an explanation for their lower incidence in the peripheral blood.

Our observations support the hematogenous metastasis of Hodgkin’s disease.

Submitted on June 2, 1965 Accepted on August 2, 1965     

Subcellular Distribution of 51Cr and Characterization of its Binding Sites in Human Platelets

Subcellular fractionation of human platelets labeled with radioactive sodiumchromate was utilized to study distribution and characterization of the ⁵¹Crbinding sites in these cells. The cytoplasmic fraction of the platelets containedthe major portion of the radioactivity while microsomal and mitochondrialfractions played a minor role in the binding of chromate. The stromal sediment had approximately one third of the total radioactivity. Only about 20per cent of the ⁵¹Cr taken up by the platelets was in a free ionic form andwas in the cytoplasmic fraction. The major portion of the ⁵¹Cr in the plateletswas bound to nucleotides in the platelet cytoplasm. Nucleotides of the-granules had no radioactive chromate. Hypertonic media and platelet antiserum produced marked release of ⁵¹Cr from the platelets, whereas aggregatingagents produced none.

In physiologic conditions, elution of ⁵¹Cr from labeled platelets is verylimited because there is only little ⁵¹Cr in free ionic form in the cells. Releaseof considerable amount of ⁵¹Cr from the platelets can occur only in case ofcell damage with release of nucleotides from the cytoplasmic pool. This wasseen with hypertonic solutions and with platelet antibody. Platelet degranulation, as it occurs with aggregating agents, is not associated with release of ⁵¹Crsince the granular pool of nucleotides is not labeled by this compound.

     

Lymphocyte Production and Life-span in the Bone Marrow of the Guinea Pig

Guinea pigs were given 14 daily injections of ³H-thymidine to label a proportion of cells with a slow rate of turnoverin addition to rapidly proliferating cells.In the bone marrow the only unlabeledcells were some reticular, endothelial, andplasma cells, damaged cells, and 14.1%of small lymphocytes. Six weeks afterdiscontinuation of ³H-thymidine 7% ofthe marrow lymphocytes remained labeled. In guinea pigs injected every 4 hrwith ³H-thymidine for 4 days to label allcells entering DNA synthesis, 14.4% ofsmall lymphocytes remained unlabeledalong with some reticular, endothelial,phagocytic, monocytoid, damaged, andplasma cells. The pattern of appearanceof labeled lymphocytes was consistentwith the kinetics of transitional cells thatfunction as their precursors. Thus, in thebone marrow of the guinea pig the majority of lymphocytes have a short lifespan and a rapid turnover, whereas about14% turn over more slowly and 7% havea life-span exceeding 4 wk. In this respect the kinetics of marrow lymphocyteproduction differs from that of the rat.

Submitted on March 11, 1971 Revised on April 9, 1971 Accepted on April 14, 1971     

An Attempt to Produce Hypersplenism in the Dog, Using Methylcellulose

(1) Hematological and histopathologic changes were studied in dogs andrats after infusions of solutions of methylcellulose.

(2) After 4 to 8 weeks of daily intravenous infusions of 0.6 Gm. of 400 centipoise methylcellulose, the dogs developed moderate anemia with a reductionof the mean red cell volume to 27 ± 4 cc./Kg., compared with 38.6 ± 3 cc./Kg.in the controls, and a reduction in the apparent red cell life span studied withCr⁵¹ from 24 ± 3 days in the controls to 18 ± 3 in the treated animals.

(3) Following the infusions in the dog there was an increase of the meanblood urea nitrogen from 15 mg. per cent to 96 mg. per cent, with only a smallincrease in the rat.

(4) At necropsy there was foamy cytoplasmic vacuolization throughout thereticuloendothelial systems of both rat and dog. In the dog, the spleens werepale and moderately enlarged. Large cells with vacuolized cytoplasm largelyreplaced the normal structures of this organ. In the markedly enlarged ratspleen there were islands of methylcellulose-filled cells, surrounded by a rimof lymphocytes and strikingly engorged red pulp.

In the rat there was vacuolization of the renal glomerular cells but no significant change in the tubules or interstitial tissue. In the dogs sacrificed oneweek after the termination of the methylcellulose infusions, foamy cytoplasmicdistention of the glomeruli and interstitial cells and tubular dilatation and cystformation were noted. Progressive fibrosis and hyalinization of the glomerulusand fibrosis and calcification and lymphocytic infiltration of the interstitialtissue were seen in the kidneys of dogs sacrificed from 3 months to 3 years aftertermination of the methylcellulose infusions.

(5) Although methylcellulose infusions produce splenomegaly and anemiain the dog, the associated uremia precludes this preparation as a model forthe study of hypersplenism.

Accepted on February 23, 1961     

The Red Cell Chromium Elution Rate in Patients with Some Hematologic Diseases

1. The erythrocyte Cr⁵¹ elution rate was determined in 38 patients withhematologic diseases.

2. In four patients with finite red cell life spans, two exponential Cr⁵¹elution rate constants could be calculated. In the remaining 34 patients, thedata were consistent with a single exponential elution rate constant from day1 to day 30-40 following Cr⁵¹ administration.

3. The single elution rate varied from 0.62 to 2.27 per cent per day.

4. In two patients, the chromium elution rates determined on two separateoccasions were not significantly different. In a third individual, the chromiumelution rate constant was 0.75 per cent per day when the red cell life span was66 days and 1.07 per cent per day when red cell life span was 79 days.

Submitted on April 23, 1962     

Studies on Agranulocytosis. IV. Effects of Chlorpromazine on Nucleic Acid Synthesis of Bone Marrow Cells in Vitro

1. When viable marrow cells were incubated with tritiated thymidine anduridine, the cells became radioactive as nucleic acid synthesis proceeded.

2. Excessive concentrations of chlorpromazine (CPZ) partially inhibitedthe influx of thymidine and uridine into granulocytes of all patients who hadhad agranulocytosis due to this drug, and of about 75 per cent of randomhospital patients. This drug did not effect nucleic acid synthesis in nine individuals who had been treated with a large amount of CPZ, with no alterationin their leukocyte count.

3. A comparison of the effects of dilution of CPZ upon influx of H³ thymidine into granulocytes showed significant dose-dependent depression of granulocyte labeling in CPZ sensitive persons, and no depression in granulocytes ofpersons on large doses of CPZ who did not develop leukopenia. Random hospital patients not on CPZ showed a degree of depression between these twogroups.

4. The number of radioactive normoblasts in the marrow culture preparations diminished slightly, but not significantly, following incubation with CPZ.

5. Non-myelotoxic drugs, such as Phenergan, aspirin, penicillin, and tetracyclene had no significant effect on the degree of influx of tritated thymidineand uridine.

6. There was no effect of CPZ upon incorporation of H³ DL-leucine intomarrow cells.

Submitted on March 5, 1962 Accepted on June 3, 1962     

The binding of Cr 51 to hemoglobin. II. In vivo elution rates of CR 51 from Hb CC, Hb CS, and placental red cells

In vivo elution rates of Cr⁵¹ from red cells containing Hb C and fromplacental red cells containing large amounts of Hb F have been determined.These were found to be 1.8 and 0.85 per cent per day, respectively, and do notdiffer greatly from those of normal adult red cells. Therefore, no special correction factors for elution need be used in evaluating Cr⁵¹ survival curves inthese clinical situations. The in vivo elution rate of 3.5 per cent/day observedfor Hb C-S is significantly greater than that of normal red cells.

Submitted on December 1, 1965 Accepted on January 16, 1966     

Hematopoietic Graft Detected by a Change in ABO Group

A change in blood group was discovered in a group O boy with acutelymphocytic leukemia. Fifty-six per cent of his red cells were of group AB6 weeks after he received white cell-rich plasma from a group AB donor withchronic myelogenous leukemia. It is concluded that hematopoietic cells inthe peripheral blood of the donor survived and divided in the recipient, andwere then rejected 6 weeks after transfusion. Factors favoring the acceptanceof the graft, consideration of the type of cell or cells that proliferated, and therelation of this finding to recently published reports of survival of cells containing the Ph¹ chromosome are discussed.

Submitted on June 28, 1963 Accepted on August 14, 1963