bluuebunny/crossref_metadata_2025 huggingface dataset:
SERUM PROTEIN CHANGES IN MYELOGENOUS AND LYMPHOCYTIC LEUKEMIAS AND HODGKIN’S DISEASE
Authors: NITSHE G. A. , COHEN PHILIP P.
Published: October 2019

1. Using a methyl alcohol fractionation technic, the albumin, globulin, and total protein levels were determined in a series of normal adults and compared with cases of myelogenous and lymphocytic leukemias and Hodgkin’s disease.

2. Statistically significant decreases in albumin and increases in globulin were found in the cases of Hodgkin’s disease and myelogenous leukemia, but without significant changes in total protein. Globulin levels above the highest normal value were found in 23 per cent of the former and 33 per cent of the latter group.

3. No apparent relationship was noted between the levels of the serum protein fractions and (1) the hemoglobin level, (2) the erythrocyte count, (3) the peripheral white blood cell picture, and (4) the bone marrow smears.
HEMATOPOIESIS IN PANTOTHENIC ACID-DEFICIENT RATS
Authors: ASHBURN L. L. , DAFT FLOYD S. , FAULKNER R. R.
Published: October 2019

Rats fed a purified diet low in pantothenic acid developed granulocytopenia and anemia singly or in combination. In the former, the marrow showed marked depletion of granulocytes, particularly of the more mature cells, and a slight increase in erythroid cells. In combined granulocytopenia and anemia the granulocytes of the marrow were still further reduced and the erythroid cells were also depleted. Marked reduction in the number of megakaryocytes occurred both in the granulocytopenic and in the granulocytopenic and anemic rats. Purpura was noted grossly in about 25 per cent of the rats of both groups. In anemia without accompanying granulocytopenia the marrow granulocytes showed slight to moderate depletion, whereas the erythroid index (mean) was not significantly lowered. Megakaryocytes were moderately reduced.

Lymphoid tissue—spleen, thymus, and cervical nodes—showed atrophy of variable degree, most marked in the thymus.

Adrenal glands showed marked depletion of cortical lipoids and rarely hemorrhage and necrosis.

Following treatment with combined folic acid, pantothenic acid, and niacinamide, granulocytopenic rats responded by showing a prompt rise in lymphocyte and polymorphonuclear leukocyte count, marked granulocyte response of the bone marrow, increased splenic hematopoiesis, lymphoid hyperplasia, and increased lipoid content of the adrenal glands.
THE INFLUENCE OF ANEMIA ON PHAGOCYTIC FUNCTIONS IN RATS
Authors: BERRY L. JOE , HALLER EVELYN C.
Published: October 2019

1. Young male albino rats were subjected to weekly bleedings by cardiac puncture for eleven weeks. Phagocytic activity of the granulocytes in this blood was determined at each bleeding by a whole blood in vitro technic and compared to that of the blood from a control group consisting of 5 animals. Total blood cell counts and hemoglobin determinations were also made each week with both groups. The control animals were not bled more frequently than once in six weeks.

2. Phagocytic function in the bled animals increased approximately 40 per cent above the values for the controls during the period of weekly bleeding. There was no evidence of anemia developing during this period.

3. The experimental animals were then bled three times weekly for five weeks. Phagocytosis increased about 80 per cent above control values and the anemia became severe. Macrophages obtained from a peritoneal exudate stimulated by injections of mineral oil at the end of this five week period became approximately twice as phagocytic as macrophages from control animals.

4. With the cessation of bleeding, there was a complete remission of the anemia as measured after two weeks and the phagocytic activity of the blood had dropped below normal. There was a partial return to normal within four weeks, but it was still slightly subnormal at six weeks. The macrophages, on the other hand, were normal when tested four weeks after recovery.

5. Control animals subjected to ether anesthesia thrice weekly for six weeks showed normal phagoctyic functions, as measured with blood and macrophages.

6. These results are discussed in the light of earlier experiments on the phagocytic activity of blood from anemic humans.
CYTOMORPHOLOGICAL OBSERVATIONS IN HODGKIN’S DISEASE
Authors: FORTEZA-BOVER J.
Published: October 2019

The results of cytomorphologic observations in 18 patients with Hodgkin’s disease as studied by lymph node puncture showed:

1. The origin of proliferative elements from the reticulum of the lymphatic tissue.

2. The aggressive growth and rapid evolution of a neoplastic nature in some cases of Hodgkin’s disease.

3. The value of lymph node puncture as a diagnostic method and an aid in following the course of the disease.
COMPARATIVE STUDIES OF PHAGOCYTOSIS IN NORMAL AND IN ANEMIC BLOOD
Authors: BERRY L. JOE , LEYENDECKER RUTH M. , SPIES TOM D.
Published: October 2019

1. The nature of the increased phagocytic activity of the neutrophiles in anemic blood as measured in vitro is investigated. The reliability of the method is discussed and ways of improving the accuracy are described.

2. Whole normal blood was diluted to anemic levels with physiological saline alone or with additional homologous serum in order to test the effect of mechanical interference of a variable number of cells on phagocytosis. The results are equivocal. Reducing erythrocyte numbers to anemic levels by removing the packed red cells in centrifuged blood and then resuspending the remaining cells and serum showed that no change in phagocytosis was produced by this means.

3. Data on varying the oxygen tension in the atmosphere above the phagocytic system showed no consistent effects except a possible elevation in phagocytosis in pure oxygen. The time of exposure to the gases varied from zero to five hours before the addition of the bacterial suspension to the system.

4. Washed leukocytes from anemic blood had the same elevated phagocytic activity when returned to either anemic serum or to normal serum, while washed normal leukocytes had the same phagocytosis in both normal and anemic serum. It was therefore concluded that the change leading to an increased phagocytosis in anemia occurs in the neutrophile rather than in the serum.

5. These findings are discussed in the light of additional data now being prepared for publication.
A STUDY OF THE BONE MARROW FROM THIRTY-SIX PATIENTS WITH IDIOPATHIC HEMORRHAGIC (THROMBOPENIC) PURPURA
Authors: DIGGS L. W. , HEWLETT J. S.
Published: October 2019

1. Observations made on the bone marrow smears of 36 patients with idiopathic hemorrhagic purpura and the correlation of the findings with the clinical picture are presented. The control series consisted of 50 patients with nonhemorrhagic conditions without blood dyscrasias.

2. The bone marrow in idiopathic hemorrhagic purpura is hyperplastic. There is a slight myeloid and erythroid immaturity and in some cases a slight eosinophilia and lymphocytosis.

3. The megakaryocyte counts ranged from 3 to 59 per 10,000 nucleated cells, with an average of 17. In the control series the megakaryocytes ranged from 1 to 54 per 10,000 nucleated cells, with an average of 16.

4. There appears to be no correlation between the number of megakaryocytes found in the marrow smears during the acute phase of the disease and the prognosis with or without splenectomy. There is also no apparent correlation between the number of megakaryocytes and the platelet response following splenectomy.

5. The megakaryocytes were classified as megakaryoblasts, immature, intermediate, or mature megakaryocytes, or as naked nuclei. The principal criteria for the differentiation of megakaryocytes are the granules and the presence or absence of granular platelets.

6. The differential megakaryocyte counts were made by examining 25 or more megakaryocytes. In the control series the mature megakaryocytes which are actively producing platelets are the predominant cells, whereas in idiopathic hemorrhagic purpura the intermediate cell without platelet production is predominant.

7. Marrow studies on 2 of our patients before and after splenectomy revealed a decrease in the relative number of megakaryocytes and an increase in the number of platelet-producing cells following operation.

8. No correlation was found between the eosinophil counts and the deaths, recurrences, or cures with or without splenectomy.

9. The principal value of the bone marrow examination in cases of suspected idiopathic hemorrhagic purpura is to exclude leukemia and aplastic anemia.
EFFECTS OF X-RAY ON LYMPHOID AND HEMOPOIETIC TISSUES OF ALBINO MICE
Authors: BRECHER G. , ENDICOTT K. M. , GUMP H. , BRAWNER H. P.
Published: October 2019

Quantitative myelograms, peripheral blood counts and the histologic appearance of lymphoid tissues were studied in albino mice of the CFW strain during a period of 4 weeks following single whole body radiation of medium dosage.

Nucleated red blood cells disappeared almost completely from the marrow within 24 hours after irradiation. Regeneration of the erythroid series commenced around the 7th day. Only mild to moderate anemia developed, presumably due to the longevity of red blood cells.

Suppression of mitotic activity and diminution of the myeloid marrow was marked during the first week after irradiation. Granulocytopenia in the peripheral blood was severe for a very short period only. In contrast, early damage to the lymphoid tissue was more pronounced and lymphopenia was of longer duration than granulocytopenia in spite of early regeneration of the lymphoid tissues. Differences in the relative amount of damage to lymphoid and myeloid tissues and in their rates of regeneration were not of sufficient magnitude to indicate a truly selective damage to the lymphoid tissues under the conditions of our expermiments.

No morphologic evidence of damage to the reticuloendothelial system was found. The possible use of irradiated animals for functional studies of the lymphoid apparatus is discussed.
THIAMIN DEFICIENCY IN THE RHESUS MONKEY
Authors: RINEHART J. F. , GREENBERG L. D. , GINZTON L. L.
Published: October 2019

Seven rhesus monkeys were subjected to one or more episodes of acute thiamin depletion. It is clear that significant metabolic inadequacies preceded demonstrable structural changes. Diminished food consumption and weight loss were manifest about two weeks after thiamin was removed from the diet. When the deficiency was prolonged the animals became apathetic, inactive and progressively weaker. This was followed by ataxia and at times ptosis and tremors. Even in such advanced states of depletion, administration of thiamin produced dramatic improvement in locomotion, appetite and reactivity. The blood thiamin content of normal monkeys ranged from 5.5 to 10γ per 100 ml. of whole blood, values which are comparable to those of healthy human beings. Following withdrawal of thiamin the blood concentration fell to values of 4γ or less. The tissue content of thiamin was correspondingly reduced in depleted animals. The minimum daily requirement for thiamin calculated on the basis of the time required to redeplete a deficient monkey following a small dose of thiamin was approximately 15γ per kilogram body weight. Characteristic degenerative changes in the heart muscle and severe retrogressive changes in the nuclear structures of the central nervous system previously reported were noted. Based on careful hematologic studies in 4 animals it is concluded that thiamin is essential for normal erythropoiesis. Acute or chronic depletion results in anemia due to suppression of red blood cell formation as indicated by severe depression or absence of reticulocytes in the blood.
THE CHEMICAL SPECIFICITY OF THE INTERACTION OF DIVERSE HUMAN PLASMA PROTEINS
Authors: COHN EDWIN J.
Published: October 2019

The chemical methods that have been developed for the separation, concentration and purification of the protein, glycoprotein, mucoprotein, and lipoprotein components of any biologic system, by fractionation in alcohol-water mixtures at controlled pH, salt, and protein concentration, at the subzero temperatures necessary to prevent denaturation, have thus far led to the recognition and concentration of over twenty-five different protein components of human plasma. These include albumins of more than one kind; immune globulins which differ in their physical properties and interactions with antigens: lipoproteins which differ in their physical properties and interactions with steroids; enzymes with protease, peptidase, lipase, phosphatase and esterase activity; thrombin, fibrinogen, and the antihemophilic globulin concerned with blood coagulation; iodoprotein and the recently crystallized metal-combining protein which interacts with both copper and iron and is presumably concerned with transport in the plasma.

This number of plasma proteins is far greater than can be detected electrophoretically or in the ultracentrifuge. Chemical fractionation has yielded at least four β1-globulins and at least two α1-and three α2-globulins. The α2-globulins include a mucoprotein and glycoproteins of more than one kind; the α1-globulins, the bilirubin-containing globulin in Fraction V-I and the lipoprotein in Fraction IV-I. The β1-globulins include the carotene-rich euglobulin which combines with three times its weight of lipid as well as a high molecular weight lipid-free β1-globulin, both of which are concentrated in Fraction III-o. Fraction III also contains β1-globulins of different molecular properties. The iron-binding component of the plasma, crystal ized from fraction IV-7, is a lipid-free β1-globulin and is more closely related to the albumins than to other globulins from the point of view of osmotic activity. Electrophoretically indistinguishable, these different β1-globulins have no other common property. The lipid-binding plasma component is a β1-euglobulin, the iron-binding β1-component a pseudoglobulin. They differ in size, shape, in solubility, in chemical composition and interaction, and in physiological function.

The separation and concentration of the various proteins of human plasma was undertaken during this war in order to render as many as possible available as therapeutic agents and thus to increase our knowledge and control of the composition of the blood in health and in disease. Many more have been separated and are being studied chemically than have thus far been brought to clinical trial. Their study renders possible the further investigation of the chemical specificity of the interactions of the plasma proteins, which are responsible for many of the tests that have in the past, or may in the future, prove of value in the clinic in the study of pathological sera and the understanding of the specific protein component that is either elevated or deficient in this condition.
THE HEMOGLOBIN OF HEALTHY COLLEGE UNDERGRADUATES AND COMPARISONS WITH VARIOUS MEDICAL, SOCIAL, PHYSIOLOGIC AND OTHER FACTORS
Authors: HEATH CLARK W.
Published: October 2019

1. The distribution of hemoglobins (obtained by the Sahli method on venous blood and by the O2 capacity method of Van Slyke) and the red blood cell counts of 153 to 259 college men, selected for participation in the Grant Study, are reported.

2. There is a statistically significant relationship between the hemoglobin and the pulse rate (those subjects having higher pulse rates tending to have higher hemoglobins). This relationship is apparently associated with emotional factors.

3. No significant relationship could be established between hemoglobin level and a variety of other factors, including socio-economic, dietary, physiologic, medical, body-build and others.
HISTOCHEMICAL METHODS APPLIED TO HEMATOLOGY
Authors: RHEINGOLD JACK J. , WISLOCKI GEORGE B.
Published: October 2019

In table 1 we have attempted briefly to characterize some of the principal blood cell types by means of the various histochemical methods. The data assembled in the table are derived from the present study as well as from the previous papers from this laboratory. The condensed phrases of the table should be referred back to the observations and discussions presented in the text. The spaces which are left blank do not indicate negative reactions but signify merely that critical data are still outstanding.

Besides offering new observations, the present paper summarizes some recent literature on the chemical histology of the cells of blood and bone marrow. The investigation concerns the demonstration and characterization of lipids, nucleo-proteins, mucopolysaccharides, acid and alkaline phosphatases and glycogen by a variety of recently developed technics. It serves to illustrate how histochemical procedures can be utilized to explore the chemical composition and functional activities of cells.
THE POSSIBILITY OF PRECIPITATING THE LEUKEMIC STATE BY EMOTIONAL FACTORS
Authors: MILLER F. R. , JONES H. W.
Published: October 2019

The emotional background of 6 cases of chronic myeloid leukemia are reviewed after psychiatric interviews. In 4, emotional instability was of long standing and in these 4, chronic worry and anxiety seemed to be an integral part of the pre-leukemic history. One of the other 2 suffered emotional trauma early in life and one had lived a life of misery as a child and an adult. Further study of emotional factors in relation to pre-leukemic histories should be made.
THE SIGNIFICANCE OF MEGAKARYOCYTES IN THE PERIPHERAL CIRCULATION
Authors: WHITBY LIONEL
Published: October 2019

1. Leuko-erythroblastic anemia, with leuko-erythroblastosis, rather than anemia, when associated either with the presence of megakaryocytes or giant platelets in the circulation, is very suggestive of the myeloid metaplasia so commonly found with myelofibrosis.

2. Associated splenomegaly with subsequent slow progress to hepatomegaly and a tendency to hypertension are confirmatory clinical features.

3. Sternal puncture may or may not confirm the diagnosis, but if the specimen contains megakaryocytes, the fact is highly significant. Difficulty in piercing the bone or in obtaining a satisfactory marrow sample are points in favor of a myelofibrosis, which should be confirmed by the histologic examination of a trephined specimen.

4. Controlled radiologic examination of the bones is sometimes of value in establishing a diagnosis in the idiopathic disease.

5. A similar blood picture may occur with polycythemia vera, with myeloid leukemia and, occasionally, with other conditions in which invasion of the bonemarrow occurs. In polycythemia vera the finding suggests a terminal phase of exhaustion; in myeloid leukemia, likewise, the megakaryocytic phenomenon is usually an ominous sign of the terminal phase.

6. When megakaryocytes are found in the circulation a diagnosis of myelofibrosis should always be considered.
BONE MARROW OF NORMAL GUINEA PIGS
Authors: SAWITSKY ARTHUR , MEYER LEO M.
Published: October 2019

1. A method is presented describing the technic for obtaining bone marrow from the iliac crest of the guinea pig by aspiration.

2. The cellular constituents in the peripheral blood and bone marrow of normal guinea pigs are presented.

3. Peripheral blood and bone marrow findings in guinea pigs are compared with those of other observers and with other animal species where the aspiration technic was used.
ON CERTAIN VARIATIONS IN ERYTHROBLASTOSIS FETALIS
Authors: CHOWN BRUCE
Published: October 2019

We have attempted first, to show that raw, mass statistics on erythroblastosis, however valuable they may be in some respects, can lead to serious error. Results will vary with the mode of collection, and with the care with which every family included in the statistics is studied. The inclusion of mild, often subclinical, cases will make the prognosis appear better. The inclusion of families in which unrecorded abortions or transfusions have taken place, will make the prognosis appear worse. Abortion and transfusion are most active agents in producing disease in later born children.

We have further set out our observations on certain blood group relationships and their possible effect on the development of Rh-erythroblastosis. These observations we do not regard as more than indications for further study.

Finally we have given, so far as we have them, the pregnancy summaries of 13 families in which anomalous results occurred, namely 9 in which normal children succeeded diseased children; 4 in which normal children were born to mothers with anti-Rh antibodies in their blood, of whom 2 had received Rh-incompatible transfusions, one had antibodies at the sixth week of a first pregnancy, and was therefore probably presensitized, and 1 was sensitized by multiple pregnancies only. While it is possible that the blood groups may be a factor in the last 4, they do not seem to play a part in the first 9. To us, this is a group of great importance, suggesting as it does, that other factors than blood incompatibility play an important, at times a deciding, role in the development or non-development of erythroblastosis.
Rh ISOSENSITIZATION IN THE AMERICAN NEGRO
Authors: SACKS MILTON S. , GUILBEAU JOSEPH A. , BRADFORD GEORGE T. , JAHN ELSA F.
Published: October 2019

1. Studies of the Rh factor in 11,486 pregnant female American Negroes revealed 1,302 who were Rh0 negative (8.4 per cent). Sixty-four cases of isosensitization were encountered which gave an incidence of 4.9 per cent in the Rh-negative patients. In comparison, among 8,889 Rh0-negative Caucasians, 460 cases of isosensitization (5.2 per cent) were encountered. The difference was found to be statistically insignificant.

2. Two typical examples of erythroblastosis fetalis in American Negro infants are presented.
EXTRAMEDULLARY BLOOD PRODUCTION
Authors: PLUM CLAUS MUNK
Published: October 2019

Investigations of extramedullary foci of hematopoieses demonstrate that, in human beings, the extramedullary hematopoiesis in the fetus at term is more pronounced in the mammary glands of the female than in those of the male. The author suggests the possibility that there may be a connection between the location of the foci, and the liability of metastasis in cancer, especially in mammary and prostatic cancer.
SOME OBSERVATIONS ON THE EFFECT OF FOLIC ACID ANTAGONISTS ON ACUTE LEUKEMIA AND OTHER FORMS OF INCURABLE CANCER
Authors: FARBER SIDNEY
Published: October 2019

A general discussion is presented of the present status of folic acid antagonist therapy in acute leukemia in children and in other forms of incurable cancer. Conclusions reached in our initial report have been supported by a far greater experience. Temporary remissions in acute leukemia as marked as those caused by aminopterin have been produced by the use of two compounds closely related chemically to aminopterin—amethopterin and amino-an-fol, both of which, however, are also toxic compounds. Despite the increasing number of patients in whom temporary remissions have been produced, with survival in some far beyond the usual course of the disease, no evidence has been presented which would justify the use of the word "cure" of acute leukemia. A carcinolytic action on related and on certain unrelated forms of incurable cancer has been observed. Further research for less toxic related compounds with even greater effectiveness is not only justified by these studies but is imperative. The value of this direction of research in cancer has been established.

Two of the most pressing problems demanding solution are concerned with the nature, the prevention, and the treatment of toxic changes, including hemorrhage, produced by these folic acid antagonists and the causes, prevention and mechanism of hemorrhage in acute leukemia. The use of the folic acid antagonists in the treatment of incurable cancer including leukemia must remain in the realm of research until answers to these questions are found.
CONCOMITANT INFECTIOUS MONONUCLEOSIS AND HEMOLYTIC ICTERUS
Authors: APPELMAN DAVID H. , MORRISON MAURICE M.
Published: October 2019

Hemolytic anemia concomitant with infectious mononucleosis, was observed in a 22 year old white male whose history indicates no pre-existing hemolytic disease. Recovery occurred spontaneously.
HEMOLYSINS IN ACQUIRED HEMOLYTIC ANEMIA
Authors: DACIE J. V.
Published: October 2019

The presence is recorded of an abnormal hemolysin in the serum of a patient with severe acquired hemolytic anemia. Its activity in vitro was determined by the pH of the corpuscle-serum suspension; the optimum pH was about 6.8 to 7.0 and there was inhibition above pH 8 and below pH 6. This pH range is contrasted with that of other human serum hemolytic systems; it is similar to that found in nocturnal hemoglobinuria.
F-cells in the adult: normal values and levels in individuals with hereditary and acquired elevations of Hb F
Authors: Wood WG , Stamatoyannopoulos G , Lim G , Nute PE
Published: October 2019

Specific antibodies to human fetal hemoglobin were prepared and, after conjugation with a fluorescent dye, were used to determine the distribution of Hb F-containing cells in blood smears from normal adults and individuals with hereditary and acquired conditions associated with abnormal levels of Hb F. The mean proportion of F-cells in normal persons was 2.7% +/- 1.4%, with a range of 0.5%-7.0%. Proportions of F-cells were increased in persons with several acquired and inherited disorders that are associated with an increased percentage of Hb F in hemolysates. A strong linear correlation between the amount of Hb F and proportion of F-cells was observed. This technique may prove useful in studies of a variety of disorders associated with Hb F elevations and also in investigations of the mechanisms controlling the transition from fetal to adult hemoglobin during the course of human development.
Two genetic defects in a patient with complete deficiency of the b- subunit for coagulation factor XIII
Authors: Hashiguchi T , Saito M , Morishita E , Matsuda T , Ichinose A
Published: October 2019

A genomic DNA obtained from a female patient with complete b-subunit deficiency was examined by Southern blotting analysis and in vitro amplification. Nucleotide sequence analysis showed that adenosine-4161 at the acceptor splice junction of intron A/exon II was deleted in half of the amplified DNAs, resulting in a loss of the obligatory AG splicing sequence. The absence of adenosine-4161 was confirmed by cleavage with TaqI endonuclease of the amplified DNAs. Moreover, sequence analysis showed that guanosine-11499 coding for Cys 430 (TGC) in exon VIII was replaced by thymidine in half of the amplified DNAs, resulting in an amino acid change to Phe (TTC) and the destruction of a disulfide bond in the seventh Sushi domain. This mutation was also confirmed by cleavage with MboII endonuclease. Thus, the proband turned out to be a compound heterozygote of two separate defective alleles. Although half of the amplified DNAs for exon VIII of her daughter or son were cleaved by MboII, those for intron A were not cleaved by TaqI. The replacement of guanosine-11499 by thymidine in their exon VIII has also been confirmed by sequence analysis, indicating that they are heterozygous for one normal and one defective allele.
The VLA-2 (alpha 2 beta 1) I domain functions as a ligand-specific recognition sequence for endothelial cell attachment and spreading: molecular and functional characterization
Authors: Bahou WF , Potter CL , Mirza H
Published: October 2019

The integrin VLA-2 (alpha 2 beta 1), generally considered to represent the specific collagen receptor on human endothelial cells, contains an alpha 2-subunit inserted I domain with structural similarity to the type A domains found within the recently described superfamily of receptor-ligand recognition proteins. This region of the cDNA has now been isolated and used for molecular and functional characterization of this heterodimeric receptor complex. Comparative sequence analysis with the porcine homologue revealed 93% amino acid sequence identity, suggestive of a developmentally conserved function. To complete structure/function studies, this region of the human cDNA was expressed as a chimeric protein in Escherichia coli, and a rabbit polyclonal antibody (anti-I domain) was used to study determinants of endothelial cell attachment and spreading in vitro. Quantifiable and visual disruption of endothelial cell attachment to gelatin, type I collagen, and laminin was evident using the specific anti-I domain antibody, with minimal inhibitory effects demonstrable using fibronectin or fibrinogen matrices. Therefore, these data would suggest that the alpha 2 beta 1 I domain confers ligand-binding specificity for both known alpha 2 beta 1 substrates (laminin and collagen), and that this region subserves a regulatory function in the molecular processes controlling endothelial cell attachment and spreading in vitro.
Effect of n-3 and n-6 fatty acids on proliferation and differentiation of promyelocytic leukemic HL-60 cells
Authors: Finstad HS , Kolset SO , Holme JA , Wiger R , Farrants AK , Blomhoff R , Drevon CA
Published: October 2019

Promyelocytic leukemic HL-60 cells were incubated with different fatty acids. Arachidonic acid (AA; 20:4, n-6) and eicosapentaenoic acid (EPA; 20:5, n-3) were the most potent inhibitors of proliferation in a dose- dependent way. Retinoic acid (RA) was used as a positive control. Inhibitors of cyclooxygenase and lipoxygenase or addition of antioxidants did not influence the effect of EPA or AA on cell proliferation. Increased capacity to generate superoxide anions after phorbol ester treatment and a reduced serglycin messenger RNA level in cells treated with AA or EPA indicated that these fatty acids induced differentiation in HL-60 cells similar to that induced by RA. However, down-regulation of the c-myc mRNA level, also typical for differentiation with RA in HL-60 cells, was not observed in cells incubated with AA or EPA. Flow cytometric analyses showed that in cultures incubated with AA or EPA, the proportion of cells in the G1 phase of the cell cycle increased. Similar effects were observed with RA. By flow cytometry and light scatter analyses it could be shown that AA made 8% of the cells apoptotic and 7% necrotic. The corresponding numbers were 21% and 10% for RA-treated cells, and 19% and 32% for EPA- treated cells. The present study shows that AA and EPA reduce the proliferation rate of HL-60 cells. This is mediated by mechanisms independent of eicosanoids or lipid peroxidation products and is due to effects both on apoptosis/necrosis and cell differentiation.
Granulocyte colony-stimulating factor administration to healthy volunteers: analysis of the immediate activating effects on circulating neutrophils
Authors: de Haas M , Kerst JM , van der Schoot CE , Calafat J , Hack CE , Nuijens JH , Roos D , van Oers RH , von dem Borne AE
Published: October 2019

In four healthy volunteers, we analyzed in detail the immediate in vivo effects on circulating neutrophils of subcutaneous administration of 300 micrograms of granulocyte colony-stimulating factor (G-CSF). Neutrophil activation was assessed by measurement of degranulation. Mobilization of secretory vesicles was shown by a decrease in leukocyte alkaline phosphatase content of the circulating neutrophils. Furthermore, shortly postinjection, Fc gamma RIII was found to be upregulated from an intracellular pool that we identified by immunoelectron microscopy as secretory vesicles. Intravascular release of specific granules was shown by increased plasma levels of lactoferrin and by upregulation of the expression of CD66b and CD11b on circulating neutrophils. Moreover, measurement of fourfold elevated plasma levels of elastase, bound to its physiologic inhibitor alpha 1- antitrypsin, indicated mobilization of azurophil granules. However, no expression of CD63, a marker of azurophil granules, was observed on circulating neutrophils. G-CSF--induced mobilization of secretory vesicles and specific granules could be mimicked in whole blood cultures in vitro, in contrast to release of azurophil granules. Therefore, we postulate that the most activated neutrophils leave the circulation, as observed shortly postinjection, and undergo subsequent stimulation in the endothelial microenvironment, resulting in mobilization of azurophil granules. Our data demonstrate that G-CSF should be regarded as a potent immediate activator of neutrophils in vivo.
X-linked sideroblastic anemia: identification of the mutation in the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) in the original family described by Cooley
Authors: Cotter PD , Rucknagel DL , Bishop DF
Published: October 2019

In 1945, Thomas Cooley described the first cases of X-linked sideroblastic anemia (XLSA) in two brothers from a large family in which the inheritance of the disease was documented through six generations. Almost 40 years later the enzymatic defect in XLSA was identified as the deficient activity of the erythroid-specific form of delta-aminolevulinate synthase (ALAS2), the first enzyme in the heme biosynthetic pathway. To determine the nature of the mutation in the ALAS2 gene causing XLSA in Cooley's original family, genomic DNAs were isolated from two affected hemizygotes, and each ALAS2 exon was PCR amplified and sequenced. A single transversion (A to C) was identified in exon 5. The mutation predicted the substitution of leucine for phenylalanine at residue 165 (F165L) in the first highly conserved domain of the ALAS2 catalytic core shared by all species. No other nucleotide changes were found by sequencing each of the 11 exons, including intron/exon boundaries, 1 kb of 52-flanking and 350 nucleotides of 32-flanking sequence. The mutation introduced an Mse I site and restriction analysis of PCR-amplified genomic DNA confirmed the presence of the lesion in the two affected brothers and in three obligate heterozygotes from three generations of this family. Carrier diagnosis of additional family members identified the mutation in one of the proband's sisters. After prokaryotic expression and affinity purification of both mutant and normal ALAS2 fusion proteins, the specific activity of the F165L mutant enzyme was about 26% of normal. The cofactor, pyridoxal 52-phosphate, activated and/or stabilized the purified mutant recombinant enzyme in vitro, consistent with the pyridoxine-responsive anemia in affected hemizygotes from this family.
Synergistic actions of stem cell factor and other burst-promoting activities on proliferation of CD34+ highly purified blood progenitors expressing HLA-DR or different levels of c-kit protein
Authors: Sonoda Y , Sakabe H , Ohmisono Y , Tanimukai S , Yokota S , Nakagawa S , Clark SC , Abe T
Published: October 2019

We studied the synergistic effects of stem cell factor (SCF) and other burst-promoting activities (BPAs) such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-9 on proliferation of human peripheral blood-derived highly purified progenitors. SCF, IL-3, GM-CSF, and IL-9 showed significant BPA when CD34+HLA-DR+ cells were used as the target population. IL-3 exerted the most potent BPA, and GM-CSF supported approximately 40% to 70% of the erythroid burst-forming units that are responsive to IL-3. SCF and IL-9 showed much weaker BPA than that of IL-3 or GM-CSF. Combinations of IL- 3 with other BPAs did not show synergistic actions supporting erythroid- burst formation. However, GM-CSF showed a significant additive effect with IL-9 or SCF. When CD34+c-kithigh cells were used as the target, SCF showed a much stronger BPA. Also, a distinct additive effect between SCF and IL-3 or GM-CSF on erythrocyte-containing mixed colony formation was observed. On the other hand, when CD34+c-kitlow cells were used as the target, SCF, IL-3, and GM-CSF could express BPA. In contrast, IL-9 alone failed to support erythroid-burst formation. Because CD34+c-kithigh cells weakly expressed CD34 antigen, these cells appeared to be more mature progenitors than CD34+c-kitlow cells. These results suggest that IL-9 acts on more mature progenitors than those of SCF, IL-3, or GM-CSF and that the primary target of SCF is multipotential progenitors at the very early stage of development.
Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular N-terminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes
Authors: Smythe JS , Spring FA , Gardner B , Parsons SF , Judson PA , Anstee DJ
Published: October 2019

This report describes the production and characterization of 13 rodent monoclonal antibodies to the human erythrocyte anion transport protein AE1 (syn. band 3). Eleven antibodies (4 murine and 7 rat) recognize epitopes dependent on the integrity of the third extracellular loop of the protein. Two antibodies (1 murine and 1 rat) recognize epitopes on the N-terminal cytoplasmic domain. Quantitative binding studies using radioiodinated IgG and Fab fragments of antibodies to extracellular epitopes on AE1 ranged from 77,000 to 313,000 (IgG) and from 241,000 to 772,000 (Fab) molecules bound at saturation. The results indicate that the epitopes recognized by different antibodies vary in their accessibility and suggest that there is heterogeneity in the organization of individual AE1 molecules in the red blood cell membrane. Quantitative binding studies on South East Asian ovalocytes using several antibodies to AE1 and an anti-Wrb show a marked reduction in the number of antibody molecules bound at saturation. These results are consistent with the existence of highly cooperative interactions between transmembrane domains of AE1 in normal erythrocytes and the disruption of these interactions in the variant AE1 found in South East Asian ovalocytes.
Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation
Authors: O'Malley CJ , Rasko JE , Basser RL , McGrath KM , Cebon J , Grigg AP , Hopkins W , Cohen B , O'Byrne J , Green MD , Fox RM , Berndt MC , Begley CG
Published: October 2019

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.
Increased numbers of long-term culture-initiating cells in the apheresis product of patients randomized to receive increasing doses of stem cell factor administered in combination with chemotherapy and a standard dose of granulocyte colony-stimulating factor
Authors: Weaver A , Ryder D , Crowther D , Dexter TM , Testa NG
Published: October 2019

Long-term culture-initiating cells (LTC-IC) are arguably the most primitive human hematopoietic cells detectable by in vitro functional assays. We have investigated the mobilization of these cells into the blood of patients with ovarian carcinoma randomized to receive granulocyte colony-stimulating factor (G-CSF; 5 micrograms/kg) plus different doses of stem cell factor (SCF; c-kit ligand) after chemotherapy or G-CSF alone after chemotherapy. We have shown a significant SCF dose response for the mobilization of LTC-IC, with a 5.8-fold increase in LTC-IC mobilization in those patients receiving chemotherapy, G-CSF, and 20 micrograms/kg of SCF, the highest dose used, compared with the patients receiving chemotherapy and G-CSF alone. We have shown a threefold increase in CD34+ cells and up to a 64-fold increase in CD34+/33- cells was seen in patients treated with chemotherapy, G-CSF, and 20 micrograms/kg of SCF compared with those patients treated with chemotherapy and G-CSF alone. However, significant numbers of CD34+/38- cells were only found in the patients receiving 20 micrograms/kg of SCF as part of their mobilization regimen. Patients receiving chemotherapy plus G-CSF and SCF have enhanced mobilization of primitive cells and of the more committed progenitor cells compared with those patients receiving chemotherapy followed by G-CSF alone.
High-dose multidrug resistance in primary human hematopoietic progenitor cells transduced with optimized retroviral vectors
Authors: Eckert HG , Stockschlader M , Just U , Hegewisch-Becker S , Grez M , Uhde A , Zander A , Ostertag W , Baum C
Published: October 2019

Retroviral transfer of the multidrug-resistance 1 (mdr1) cDNA into primary human hematopoietic progenitor cells (HPC) of cancer patients undergoing high-dose chemotherapy has been proposed to protect the bone marrow from the dose-limiting cytotoxicity of cytostatic agents. Preclinical studies performed with vectors derived from the Moloney murine leukemia virus (MoMuLV) or the related Harvey murine sarcoma virus have established that chemoprotection of HPC is feasible. The efficacy of vector-mediated multidrug-resistance under high doses of cytostatic agents, however, remained unclear. We report here that this goal can only be achieved with improved vector design. Novel vectors termed SF-MDR and MP-MDR, which are based on the spleen focus-forming virus or the myeloproliferative sarcoma virus for the enhancer and the murine embryonic stem cell virus for the leader, significantly elevate survival of transduced primary human HPC under moderate doses of colchicine and paclitaxel in vitro when compared with a conventional MoMuLV-based vector. Importantly, SF-MDR and also MP-MDR confer an absolute advantage at high doses of paclitaxel in vitro corresponding to peak plasma levels achieved in patients during chemotherapy. This observation has important consequences for a variety of ongoing and planned gene therapy trials.
VH and VL gene analysis in sporadic Burkitt's lymphoma shows somatic hypermutation, intraclonal heterogeneity, and a role for antigen selection
Authors: Chapman CJ , Zhou JX , Gregory C , Rickinson AB , Stevenson FK
Published: October 2019

Tumor cell lines and one tumor biopsy from seven cases of Epstein-Barr virus (EBV) genome-negative sporadic Burkitt's lymphoma (BL) have been investigated for usage and mutational pattern of Ig variable region genes. The VH genes were derived from the VH 3 (one) and VH4 (six) families and both the IgM-positive (six) and the IgA-positive (one) were all mutated from their germline counterparts. The VL genes were derived from V kappa 1 (one), V kappa 3 (one), V lambda 1 (four), and V lambda 2 (one) families and were also somatically hypermutated. Biopsy material from one of the IgM-positive cases showed VH and VL sequences that matched the derived cell line, with additional intraclonal sequence heterogeneity, indicating that the tumor cells had undergone posttranformation somatic mutation. Mutational patterns in V(H) genes did not show a conventional role for antigen in selecting tumor cell sequences. In contrast, patterns in VL sequences were consistent with a role for antigen in five of seven cases. The pattern of extensive scattered somatic hypermutation and intraclonal variation is similar to that in VH sequences of EBV genome-positive endemic BL, although the degree of mutational activity is less. These common features indicate that B cells involved in the two variants of BL may share a common clonal history.
Oxidative stress and antioxidant status in beta-thalassemia major: iron overload and depletion of lipid-soluble antioxidants
Authors: Livrea MA , Tesoriere L , Pintaudi AM , Calabrese A , Maggio A , Freisleben HJ , D'Arpa D , D'Anna R , Bongiorno A
Published: October 2019

Because of continuous blood transfusions, thalassemia patients are subjected to peroxidative tissue injury by the secondary iron overload. In accordance, analysis of serum from 42 beta-thalassemia patients, aged 4 to 40 years, showed that the mean concentrations of conjugated diene lipid hydroperoxides (CD), lipoperoxides evaluated as malondialdehyde/ thiobarbituric acid (MDA/TBA) adducts, and protein carbonyls increased about twofold with respect to control. Ferritin levels were positively correlated with the amount of MDA (r = .41; P = .007) and showed a positive trend with CD (r = .31; P = .07) and protein carbonyls (r = .35; P = .054), as further evidence of the deleterious effects of high tissue iron levels. Marked changes in the antioxidant pattern were also observed in all patients. Evidence is presented of a net drop in the concentration of ascorbate (-44%), vitamin E (-42%), vitamin A(-44%), beta-carotene (-29%), and lycopene (-67%). On the other hand, an increase of uric acid and bilirubin was observed, whereas serum albumin and glutathione were in the normal range in all patients. As a result, the total serum antioxidant potential, measured as trolox equivalent antioxidant capacity appeared significantly decreased by 14%. Serum levels of vitamin E were inversely correlated with ferritin (r = -.45; P = .003), suggesting a major consumption of this antioxidant under iron overload. Nontransferrin bound iron (NTBI) was in the range 4.5 to 54.8 micrograms/dL (mean, 21.8 +/- 13.9). Although NTBI had a positive trend with ferritin (r = .37, P = .03), no clear correlation was found with either MDA or vitamin E. A mild to severe hepatic damage, as assessed by serum transaminases, was shown in 24 of 42 patients. Serum levels of vitamin E (r = -.49, P = .015), vitamin A (r = -.48, P = .016) and lycopene (r = -.47, P = .020), were inversely correlated with the levels of transminases. On the other hand, lipid-soluble antioxidants in thalassemia patients were depleted to the same extent in hepatitis C virus (HCV)-infected (31 subjects) and in HCV-uninfected (10 subjects), while in the normal range in serum from 30 nonthalassemic patients with HCV-related chronic hepatitis. These results point out that the iron-induced liver damage in thalassemia may play a major role in the depletion of lipid-soluble antioxidants. The variations of the parameters evaluated in the present study were not correlated with the age of the patients. Our results suggest that the measurement of peroxidation products, matched with evaluation of antioxidants, may be a simple measure of iron toxicity in thalessemia, in addition to the conventional indices of iron status.