bluuebunny/crossref_metadata_2025 huggingface dataset:
Microspectrophotometric Estimation of the Desoxyribonucleic Acid (DNA) Content of Individual Normal and Leukemic Human Lymphocytes
Authors: PETRAKIS NICHOLAS L.
Published: October 2019

The DNA content, in arbitrary units, of individual circulating lymphocytes from nine normal subjects, nine patients with chronic lymphocytic leukemia, and five patients with acute lymphocytic leukemia was estimated microspectrophotometrically with the Feulgen dye. Normal circulating lymphocytes were found to contain twice the average DNA content of normal human spermatids, corroborating their diploid chromosome number. Lymphocytes from normal and leukemic lymph node and bone marrow were frequently found possessing four times the average spermatid DNA value. Three out of nine patients with chronic lymphocytic leukemia, and four of five patients with acute lymphocytic leukemia had significantly increased numbers of circulating lymphocytes containing DNA values which were elevated above the normal diploid value. The patients demonstrating cells with elevated DNA values were in a clinically exacerbated phase of their disease. The significance of these findings is discussed.
Blood Volume and Molybdenum Toxicity in Rabbits
Authors: BURKE JACK D. , ARRINGTON L. R. , DAVIS G. K.
Published: October 2019

The blood volume of normal rabbits and rabbits anemic with molybdenum toxicity was determined using the dye and P32 methods. The blood volume per unit of body weight was found to be within the normal range indicating that rabbits were able to maintain normal blood volume even though anemic as a result of molybdenum toxicity.
Case Report: A Case of Erythremic Myelosis (Di Guglielmo)
Authors: BÁEZ-VILLASEÑOR JOSÉ , SÁNCHEZ-MEDAL LUIS
Published: October 2019

A case of chronic erythremic myelosis (Di Guglielmo) is presented; the diagnosis was established by clinical, hematologic, and pathologic data. The peculiar characteristics of this particular case are discussed, as well as the differential diagnosis from hemolytic anemia and acute leukemia.
Researches on a Circulating Anticoagulant in a Hemophiliac
Authors: VAN CREVELD S. , HOORWEG P. G. , PAULSSEN M. M. P.
Published: October 2019

The influence of the administration of ACTH and cortisone on a circulating anticoagulant appearing after repeated blood transfusions in a hemophiliac was studied. It was found that the activity of the anticoagulant was diminished to a considerable degree, even though temporarily, and the clotting time was reduced by plasma transfusions more than prior to the administration of ACTH and cortisone. Antibodies to the Duffy antigen were also present together with the circulating anticoagulant, but their activity was not clearly affected by the administration of ACTH and cortisone.
Studies on Abnormal Hemoglobins
Authors: SINGER KARL , FISHER BEN
Published: October 2019

1. A family is reported in which 2 children have unmistakable sickle cell anemia, but the mother’s red cells do not sickle; the father and a third child exhibit the typical sickle cell trait. Electrophoretic studies of the hemoglobin solution prepared from the mother’s erythrocytes demonstrated, however, the presence of a small amount (5 per cent) of type S hemoglobin. This abnormal component was identified by its mobility and by addition experiments.

2. The finding that the mother’s erythrocytes contain small quantities of S hemoglobin, but are incapable of sickling, confirms the genetic concept that sickle cell anemia will only develop when both parents transmit the gene for S hemoglobin.

3. The physico-chemical and possible medico-legal aspects of the fact that S hemoglobin is sometimes only detectable by means of electrophoresis, are discussed.
The Influence of Intra-arterial Administration of Histamine upon the Circulating Leukocytes of Man
Authors: BIERMAN H. R. , BYRON R. L. , KELLY K. H. , CORDES F. , WHITE L. P. , LITTMAN A.
Published: October 2019

1. The leukocyte content of femoral arterial and venous blood was determined simultaneously following the administration of 0.1 to 0.3 mg. of histamine (as base) directly into the femoral artery in 5 patients at rates varying from 1.1 to 6.7 µg. per second.

2. A marked and prompt decrease in leukocyte number was found in the blood from the femoral vein of the same leg after the histamine infusion started; this preceded the changes in the blood from the femoral artery.

3. The discrepancy between the venous and arterial counts indicates the withdrawal of leukocytes within the circulation of the lower extremity which is generally related to the rate and amount of histamine administered.

4. In 6 patients, histamine (0.15 to 0.4 mg.) was administered directly into the aorta at levels from T3 to L2. In five of nine instances, blood from the pulmonary artery or inferior vena cava initially showed a prompt fall in leukocyte number, which exceeded the leukopenia observed in blood sampled from the aorta. In two instances, the leukocyte counts from the aorta exhibited no significant change.

5. The venous leukopenia following the administration of histamine into the aorta probably occurs as a result of sequestration of leukocytes in the peripheral capillaries which accompanies the arterial leukopenia due to a similar effect in the pulmonary circulation.
Case Report: Chronic Granulocytic (Myelogenous) Leukemia and Pregnancy
Authors: SHUB HAROLD , BLACK MAURICE M. , SPEER FRANCIS D.
Published: October 2019

1. An additional case of coexistent pregnancy and chronic granulocytic (myelogenious) leukemia is reported.

2. Coincident with pregnancy the leukemia in this patient underwent a transition from the chronic phase in remission to acute myeloblastic disease with rapid exitus.

3. The pertinent literature concerning pregnancy and chronic granulocytic leukemia has been reviewed and evaluated.

4. Our findings indicate that pregnancy may have a deleterious effect on the course of the patient with chronic granulocytic leukemia, and may initiate an acute exacerbation of the disease.

5. In approximately one-third of the cases of chronic granulocytic leukemia complicated by pregnancy, an exacerbation and death occurred within eight months postpartum.

6. In chronic granulocytic leukemia, pregnancy should be avoided.
Two Cases of Sickle Cell Disease Presumably Due to the Combination of the Genes for Thalassemia and Sickle Cell Hemoglobin
Authors: NEEL JAMES V. , ITANO HARVEY A. , LAWRENCE JOHN S.
Published: October 2019

A family of Greek derivation is described in which 2 out of 6 children examined exhibited a sickle cell type of anemia. The father of these children was found to have thalassemia minor and the mother the sickle cell trait. It is presumed that the anemia in the two children was due to simultaneous heterozygosity for the sickling and thalassemia genes. Biochemical studies with reference to the occurrence and amounts of normal, sickle cell, and fetal hemoglobin were carried out on the parents and the 6 children. The theoretic interpretation of the biochemical findings is discussed.
Erythrophagocytosis in a Case of Plasma Cell Leukemia
Authors: BUTTERWORTH CHARLES E. , FROMMEYER WALTER B. , RISER WILLIAM H.
Published: October 2019

A case of plasma cell leukemia is presented in which erythrophagocytosis by cells of the plasma cell series occurred. This case serves to add the cells of the plasma cell series to those of the monocytic and granulocytic series as cells capable of erythrophagocytosis under certain conditions.
Effect of Horse Serum, Adrenal Hormones, and Histamine on the Number of Eosinophils in the Blood and Peritoneal Fluid of Mice
Authors: PANZENHAGEN H. , SPEIRS R.
Published: October 2019

A technic of performing reliable eosinophil determinations on the peritoneal fluid of mice is described. This procedure was found to be rapid and did not necessitate killing the animals.

Simultaneous determinations of the blood and peritoneal eosinophils were made at 3 hour intervals throughout a 24 hour period. A marked diurnal rhythm was found in the blood eosinophils, but little change occurred in the number of eosinophils found in the peritoneal cavity.

During a period of 3 hours following stress, there was a marked blood eosinopenia, but little or no change in the peritoneal eosinophils. Repeated intraperitoneal injections of 0.1 ml. normal horse serum resulted in a great increase in the number of peritoneal eosinophils. These cells appeared to be morphologically identical with the blood eosinophils. In mice which were pretreated with horse serum, relatively large injections of cortisone, compound F, epinephrine and histamine produced a marked blood eosinopenia, but failed to reduce the number of peritoneal eosinophils within 3 hours. The response of eosinophils in blood and peritoneal fluid to cortisone was essentially the same either when it was injected subcutaneously or intraperitoneally.

Implantations of pellets of cortisone acetate in the peritoneal cavity produced a blood eosinopenia beginning within 1 hour and reaching a maximum at approximately 3 hours. The peritoneal eosinophils did not begin to drop until approximately 6 to 10 hours after the implantation.

These data were interpreted as indicating that physiologic doses of cortisone, compound F, epinephrine or histamine do not have a direct lytic action upon the eosinophils of mice.
Splenic Aspirations in Multiple Myeloma
Authors: SHAPIRO HENRY D. , WATSON R. JANET
Published: October 2019

1. Splenic aspirations were performed in ten consecutive patients with multiple myeloma, in only three of whom the spleen was palpable.

2. In nine of the ten patients there was an increase of the percentage of myeloma cells in the splenic aspirations comparable to that in the marrow.

3. It is postulated that these findings support the concept of multiple myeloma as a generalized hematopoietic disease comparable to the leukemias.
Mutations in the pyruvate kinase L gene in patients with hereditary hemolytic anemia
Authors: Lenzner C , Nurnberg P , Thiele BJ , Reis A , Brabec V , Sakalova A , Jacobasch G
Published: October 2019

We have completely sequenced the introns of the human L-type pyruvate kinase (PK) gene using the published cDNA sequence. Subsequently, DNA from 12 unrelated PK deficiency (PKD) patients of Central European origin was investigated for mutations in this gene by solid-phase sequencing. We detected 10 different mutations, 9 of which result in single amino acid alterations, whereas the tenth destroys a splice site. Eight of the 10 mutations have not been described before. We found 7 missense mutations: G994-->A (Gly-332-->Ser), G1006-->T (Ala- 336-->Ser), A1081-->G (Asn-361-->Asp), G1174-->A (Ala-392-->Thr), G1493- ->A (Arg-498-->His), G1529-->A (Arg-510-->Gln), C1594-->T (Arg-532-- >Trp), one in-frame triplet deletion (del) as well as one insertion (ins): del AAG1060–62 (del Lys-354), ins AGC after C1203 (ins Ser after Cys-401), and one splice-site mutation at the border of intron A to exon 3: g/G283-->a/G. Although the enzymatic properties are substantially changed in all PK mutations, only two affected amino acid positions are in or close to the active site. Mutations C1594-->T, G994- ->A, del AAG1060–62 and the splice-site mutation g/G283-->a/G have been detected in two different patients each. Mutation G1529-->A was found in five different alleles. Haplotype analysis with the A/C polymorphism at position 1705 gave evidence for a single origin of this most frequent mutation in PKD as suggested by Baronciani and Beutler (Proc Natl Acad Sci USA 90:4324, 1993). Carrier detection and prenatal diagnosis are now feasible for the affected families.
Increased incidence of second neoplasms in patients treated with interferon alpha 2b for hairy cell leukemia: a clinicopathologic assessment [see comments]
Authors: Kampmeier P , Spielberger R , Dickstein J , Mick R , Golomb H , Vardiman JW
Published: October 2019

We report that there is an unexpectedly high incidence of second neoplasms in patients after treatment of hairy cell leukemia (HCL) with interferon alpha 2b (IFN). In a cohort of 69 patients with HCL entered in a protocol using IFN as the primary treatment, and followed thereafter for a median of 91 months (range, 0.2 to 109 months), 13 patients (19%) developed a second neoplasm. Six neoplasms were of hematopoietic origin, whereas the remaining seven were adenocarcinomas. The expected number of second tumors in this cohort is three (based on calculations from the National Cancer Institute's SEER data), so the excess frequency (observed:expected) is 4.33. However, the excess frequency is even greater for the hematopoietic neoplasms; the expected frequency is 0.15, whereas six hematopoietic tumors occurred, for an observed:expected ratio of 40. In general, the second neoplasms have behaved aggressively, and the median survival after diagnosis of the second neoplasm was only 8.8 months. Although we cannot entirely exclude the possibility that IFN therapy has some direct oncogenic effect, we suspect that increased frequency of second tumors is related to prolonged survival of patients who are immunocompromised because of HCL and thus prone to develop second tumors. If so, the frequency of second neoplasms in patients with HCL may be even greater in the future with continued improvements in therapy.
Granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), and IL-5 greatly enhance the interaction of human eosinophils with opsonized particles by changing the affinity of complement receptor type 3
Authors: Blom M , Tool AT , Kok PT , Koenderman L , Roos D , Verhoeven AJ
Published: October 2019

Eosinophil functions can be modulated by several cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 3 (IL-3), and IL-5. We have investigated the modulatory role of these cytokines on the interaction of human eosinophils with opsonized particles (serum-treated zymosan [STZ]). Addition of STZ to eosinophils isolated from the peripheral blood of normal human donors resulted in an interaction of the STZ particles with only 15% to 25% of the cells. Treatment of the eosinophils with GM-CSF, IL-3, or IL-5 strongly enhanced both the rate of particle binding and the percentage of eosinophils binding STZ. The effect of the cytokines is most likely mediated by a change in affinity of the complement receptor type 3 (CR3) on the eosinophils for the complement fragment iC3b on the STZ particles. This is indicated by the observation that (1) the effect of the cytokines on STZ binding was prevented by a monoclonal antibody against the iC3b-binding site on CR3 and (2) the enhanced binding was already apparent before upregulation of CR3 on the cell surface was observed. In a previous study, similar results were obtained with platelet-activating factor (PAF)-primed eosinophils. Because we found that the cytokines strongly enhanced the STZ-induced PAF synthesis, we investigated the role of both released PAF and cell-associated PAF in the priming phenomenon by the cytokines. Cytokine priming appeared to be largely independent of the synthesis of PAF.
Regulation by interleukin-2 (IL-2) and interferon gamma of IL-2 receptor gamma chain gene expression in human monocytes
Authors: Bosco MC , Espinoza-Delgado I , Schwabe M , Gusella GL , Longo DL , Sugamura K , Varesio L
Published: October 2019

The interleukin-2 receptor gamma chain (IL-2R gamma) gene codes for a subunit of the IL-2R and is expressed in human lymphoid cells. The present study was undertaken to determine whether human monocytes expressed the IL-2R gamma gene constitutively or after activation by IL- 2 or interferon gamma (IFN gamma). Fresh human monocytes constitutively expressed low but significant levels of IL-2R gamma mRNA, and nuclear run-on experiments showed that IL-2R gamma gene was transcriptionally active. Stimulation with IL-2 or IFN gamma induced a major increase of IL-2R gamma mRNA in a time- and a dose-dependent manner. However, neither cytokine increased the transcriptional activity of the gene. The enhancement of IL-2R gamma mRNA expression by either IL-2 or IFN gamma was concomitant with the stabilization of the mRNA, suggesting a postranscriptional level of control. Finally, the augmented expression of IL-2R gamma in IL-2- and IFN gamma-treated monocytes was associated with an increased IL-2-binding activity, compared with that of unstimulated cells. These results provide the first evidence of the expression of the IL-2R gamma gene in nonlymphoid cells and of its modulation by IL-2 and IFN gamma through posttranscriptional mechanisms.
Persistence of host-type hematopoiesis after allogeneic bone marrow transplantation for leukemia is significantly related to the recipient's age and/or the conditioning regimen, but it is not associated with an increased risk of relapse
Authors: van Leeuwen JE , van Tol MJ , Joosten AM , Wijnen JT , Verweij PJ , Khan PM , Vossen JM
Published: October 2019

We investigated the chimerism pattern within flow-sorted peripheral blood- or bone marrow-derived cell populations after allogeneic bone marrow transplantation (BMT) for the treatment of leukemia in children. This study was performed to define the identity of persistent host-type cells, to identify prognostic variables for the persistence of host- type hematopoiesis, and to determine the prognostic significance of the chimerism pattern on the duration of the leukemia-free interval, the overall survival, and the leukemia-free survival. The patients received either HLA-identical non-T-cell-depleted (n = 46) or HLA nonidentical T- cell-depleted (n = 7) BMT. In the peripheral blood, the children showed either stable mixed chimerism (SMC; ie, persistent host-type hematopoiesis; n = 14), (transient) mixed T-lymphoid chimerism (MTLC; n = 9), or complete chimerism (CC; n = 30). In the bone marrow, only donor-type cells were found in children with either CC (n = 8) or MTLC (n = 2), and a mixture of donor- and recipient-type cells was found in children with SMC (n = 7). The persistence of host-type hematopoiesis (SMC) was significantly related to a lower age of the recipient, the type of conditioning regimen, a lower total body irradiation dose, T- cell depletion of the bone marrow graft, and the use of cyclosporine A for acute graft-versus-host disease prophylaxis. No significant differences were found between patients with (SMC) or without (CC/MTLC) persistent host-type hematopoiesis with respect to the duration of the leukemia-free interval, the overall survival, or the leukemia-free survival. We conclude that ablation of host-type hematopoiesis is not compulsory for long-term leukemia-free survival after allogeneic BMT for various hematologic malignancies.
Unrelated donor or autologous marrow transplantation for treatment of acute leukemia
Authors: Busca A , Anasetti C , Anderson G , Appelbaum FR , Buckner CD , Doney K , Martin PJ , Petersdorf E , Sanders JE , Hansen JA
Published: October 2019

High-dose chemoradiotherapy followed by marrow transplantation from an HLA-matched sibling donor is curative for patients with acute leukemia. Autologous marrow transplantation has been used with success for some patients without such a sibling. Alternatively, the option of performing a transplant from an HLA-matched unrelated donor has been made possible by the recent development of large registries of HLA- typed volunteers. The purpose of this study was to compare the outcomes for patients with advanced leukemia treated by unrelated or autologous marrow transplantation. Forty-three patients with acute myeloid or lymphoid leukemia were transplanted from a closely HLA-matched unrelated donor. Results were compared with those of a disease-, disease-stage-, and age-matched cohort of 77 patients treated with autologous marrow transplantation at the same institution during the same period. Myeloid reconstitution with peripheral granulocyte counts greater than 10(9)/L was achieved in 93% of unrelated recipients and 70% of autologous recipients at a median of 24 and 36 days after transplantation, respectively (P = .0001). The cumulative proportions of patients discharged alive (79% v 77%) and times from transplant to first hospital discharge (35 v 34 days) were not different between unrelated and autologous recipients (P = .65). For patients transplanted in complete remission, relapse occurred after transplantation in 27% of the unrelated and in 55% of the autologous recipients (P = .08). For patients transplanted in relapse, the corresponding posttransplant relapse rates were 48% and 63%, respectively (P = .72). Forty percent of unrelated recipients and 28% of autologous recipients died in remission. Leukemia-free survivals were 33% for unrelated and 25% for autologous recipients transplanted in remission (P = .45), and 12% for unrelated and 5% for autologous recipients transplanted in relapse (P = .75). Unrelated donor transplants appear no less effective than autologous transplants to achieve long-term survival and may be more effective in eradicating leukemia in patients who have failed conventional chemotherapy. Further studies are warranted to assess the relative effectiveness of unrelated and autologous transplantation performed earlier in the course of the disease.
Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay
Authors: Wang Jean C.Y. , Doedens Monica , Dick John E.
Published: October 2019

We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.
Cytokine Treatment of Endothelial Cells Increases Glycoprotein Ibα-Dependent Adhesion to von Willebrand Factor
Authors: Beacham Dorothy A. , Tran Linh-Phuong , Shapiro Sandor S.
Published: October 2019

Endothelial cells (EC) possess at least two membrane receptors for von Willebrand factor (vWF ), the vitronectin receptor (VNR, αvβ3 ), which recognizes an Arg-Gly-Asp (RGD) sequence in the C-terminus of vWF, and glycoprotein Ibα (GP Ibα), which interacts with a region in the N-terminal A1 domain of vWF. In the absence of added cytokines, EC attachment to a vWF substratum is mediated largely through the αvβ3 , with a smaller contribution by GP Ibα. In the present study, we have examined the effect of cytokines on the receptor specificity of EC attachment to wild-type vWF (WT-vWF ) and to vWF, which had been mutated in the C-terminal RGDS sequence (RADS-vWF ). Exposure of human umbilical vein EC (HUVEC) to tumor necrosis factor-α (TNF-α) or to TNF-α in combination with interferon-γ (IFN-γ), but not to interleukin-1β (IL-1), increased attachment to RADS-vWF by about twofold. The TNF-α–induced increase in EC attachment was accompanied by an increase in cell surface GP Ibα expression; GP Ibα surface expression was not increased by IL-1. Attachment of untreated HUVEC to WT-vWF could be inhibited 60% to 70% by a monoclonal antibody (MoAb) (LM609) to the VNR and 30% to 40% by the A1 fragment of vWF (containing the GP Ibα binding domain). The pattern of inhibition of attachment to WT-vWF was largely unchanged after TNF-α treatment of HUVEC. In contrast, the attachment to WT-vWF of HUVEC, treated with TNF-α +IFN-γ was completely inhibited by vWF-A1 and inhibited only 35% by the anti-VNR antibody LM609. Two MoAbs to GP Ibα produced similar, but incomplete, inhibition. Pretreatment of HUVEC with the combination of TNF-α +IFN-γ produced a dramatic decrease in VNR expression, confirming previous findings of Defilippi et al. These results suggest that in the presence of the inflammatory cytokines TNF-α +IFN-γ, the endothelial GP Ib complex is a major determinant of HUVEC adhesion to surface-bound vWF.
Control of Cell Cycle Progression in Human Natural Killer Cells Through Redox Regulation of Expression and Phosphorylation of Retinoblastoma Gene Product Protein
Authors: Yamauchi Akira , Bloom Eda T.
Published: October 2019

Using thiol deprivation, we have previously shown that the response of natural killer (NK) cells to interleukin-2 (IL-2) is subject to redox regulation downstream of IL-2 binding and internalization. We have now used the IL-2–dependent cell line, NK3.3 to study redox regulation of NK cells further, and found that NK3.3 cells neither incorporated [3H]-thymidine nor completed the G1-S phase transition in medium lacking the thiol-related compounds, L-cystine, and glutathione, despite the presence of sufficient IL-2. Thiol deprivation did not alter the induction of DNA interferon-γ activated sequence (GAS)-binding activity in response to IL-2. However, the retinoblastoma gene product (RB), a cyclin-dependent kinase (CDK) substrate, was phosphorylated within 24 hours after IL-2 stimulation in standard medium, but its expression and phosphorylation were reduced in thiol-depleted medium in both NK3.3 cells and freshly isolated NK cells. These reductions were not associated with an increased level of p27Kip1, an inhibitor of CDKs CDK6/2 in association with G1 cyclins. Reducing agents, N-acetylcysteine, reduced glutathione or 2-ME restored both RB phosphorylation and DNA synthesis in thiol-deprived NK3.3 cells. The in vitro kinase activities of CDK6 and CDK2 were prematurely increased by thiol deprivation. This enhancement was associated with CDK hyperphosphorylation and prolonged phosphorylation, and could be observed before and beyond IL-2 stimulation. The data suggest the possibility that the premature and prolonged enhancement of CDK activity in thiol-deprived NK cells is associated with, and therefore may contribute to, the reduced expression and phosphorylation of RB, and the associated cell cycle arrest.
Augmented Intracellular Glutathione Inhibits Fas-Triggered Apoptosis of Activated Human Neutrophils
Authors: Watson R. William G. , Rotstein Ori D. , Jimenez Maria , Parodo Jean , Marshall John C.
Published: October 2019

Agonist signals delivered through cell surface Fas induce apoptosis. However, the apoptotic program can be modulated by signals from the environment, and in particular, by signals delivered through adhesion molecules. Because neutrophil functional activity in inflammation is contingent on cell survival, and because circulating neutrophils normally die rapidly through a constitutively expressed apoptotic program, we evaluated Fas-mediated apoptosis in resting and inflammatory human neutrophils. We show that normal neutrophils respond to Fas engagement with accelerated rates of apoptosis, but cross-linking of β2 integrins or priming with bacterial lipopolysaccharide (LPS) prevents this increase. Adhesion molecule cross-linking results in increased intracellular glutathione (GSH). Augmentation of intracellular GSH with exogenous GSH or N-acetylcysteine is sufficient to reduce the Fas-triggered increase in apoptotic rates. Prevention of the activation induced GSH increase by buthionine sulfoximine, a cell permeable inhibitor of GSH biosynthesis, restored Fas responsiveness in activated neutrophils, an effect that could be blocked with exogenous GSH. Taken together, these data show that Fas-induced signaling for neutrophil apoptosis is blocked in a redox sensitive manner by costimulatory signals delivered through β2 integrins or activation by LPS, and provide a biologic explanation for sustained neutrophil survival in the inflammatory environment.
Fibronectin Enhances In Vitro Lipopolysaccharide Priming of Polymorphonuclear Leukocytes
Authors: Bortolussi Robert , Rajaraman Kausalya , Qing Gefei , Rajaraman Rengaswami
Published: October 2019

We investigated the role of humoral factors in lipopolysaccharide (LPS) priming of polymorphonuclear leukocytes (PMN) using cells isolated from adults and from neonates. Plasma from newborn infants had decreased priming activity of adult plasma when mixed with LPS in studies measuring oxidative radical production of PMN after stimulation with a formyl bacterial oligopeptide (fMLP). This marked difference was not caused by LPS binding protein (LBP) because the LBP concentration in newborn and adult plasma were similar (138.4 ± 12.9 U for adults, and 126.9 ± 12.1 U for neonates, P = .53). Therefore, we attempted to identify other plasma factors that may contribute to LPS priming of PMN. We identified an LPS priming factor for PMN that is present in plasma, heat stable (56°C for 30 minutes), enhanced by heparin, and concentrated in cold precipitates of plasma. Because these properties resemble those of plasma fibronectin, we assessed the role of fibronectin in LPS priming of PMN. Although fibronectin in phosphate-buffered saline (PBS) had little effect on LPS priming of PMN, fibronectin in combination with other plasma factors appeared to play a role in LPS priming of PMN because (1) removing fibronectin from adult plasma dramatically decreased LPS priming activity from plasma (P < .005), (2) addition of fibronectin to fibronectin-depleted plasma restored its LPS plasma priming activity (P < .05), and (3) neutralizing fibronectin with antibody decreased the LPS priming activity of plasma (60.3 ± 1.3 v 30.2 ± 2.2, P < .01). Thus, plasma fibronectin plays a role in LPS priming of PMN in the presence of other factors in plasma.
A Retinoid-Resistant Acute Promyelocytic Leukemia Subclone Expresses a Dominant Negative PML-RARα Mutation
Authors: Shao Wenlin , Benedetti Laura , Lamph William W. , Nervi Clara , Miller Wilson H.
Published: October 2019

The unique t(15; 17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor α (RARα) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RARα associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RARα protein. In contrast to mutations in RARα found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RARα remains wild-type. In vitro expression of a cloned PML-RARα with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RARα protein retains the ability to heterodimerize with RXRα and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RARα engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RARα selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXRα and RARβ and the RA-induced loss of PML-RARα protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RARα-mediated pathway that is independent from gene expression induced or repressed by PML-RARα. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically.
Defensin Stimulates the Binding of Lipoprotein (a) to Human Vascular Endothelial and Smooth Muscle Cells
Authors: Higazi Abd Al-Roof , Lavi Ehud , Bdeir Khalil , Ulrich Anthony M. , Jamieson Dara G. , Rader Daniel J. , Usher David C. , Kane William , Ganz Tomas , Cines Douglas B.
Published: October 2019

There is evidence to suggest that elevated plasma levels of lipoprotein (a) [Lp(a)] represent a risk factor for the development of atherosclerotic vascular disease, but the mechanism by which this lipoprotein localizes to involved vessels is only partially understood. In view of studies suggesting a link between inflammation and atherosclerosis and our previous finding that leukocyte defensin modulates the interaction of plasminogen and tissue-type plasminogen activator with cultured human endothelial cells, we examined the effect of this peptide on the binding of Lp(a) to cultured vascular endothelium and vascular smooth muscle cells. Defensin increased the binding of Lp(a) to endothelial cells approximately fourfold and to smooth muscle cells approximately sixfold. Defensin caused a comparable increase in the amount of Lp(a) internalized by each cell type, but Lp(a) internalized as a consequence of defensin being present was not degraded, resulting in a marked increase in the total amount of cell-associated lipoprotein. Abundant defensin was found in endothelium and in intimal smooth muscle cells of atherosclerotic human cerebral arteries, regions also invested with Lp(a). These studies suggest that defensin released from activated or senescent neutrophils may contribute to the localization and persistence of Lp(a) in human vessels and thereby predispose to the development of atherosclerosis.
Kinetic Evidence of the Regeneration of Multilineage Hematopoiesis From Primitive Cells in Normal Human Bone Marrow Transplanted Into Immunodeficient Mice
Authors: Cashman Johanne D. , Lapidot Tsvee , Wang Jean C.Y. , Doedens Monica , Shultz Leonard D. , Lansdorp Peter , Dick John E. , Eaves Connie J.
Published: October 2019

Based on initial observations of human CD34+ Thy-1+ cells and long-term culture-initiating cells (LTC-IC) in the bone marrow of some sublethally irradiated severe combined immunodeficient (SCID) mice transplanted intravenously with normal human marrow cells, and the subsequent finding that the NOD/LtSz-scid/scid (NOD/SCID) mouse supports higher levels of human cell engraftment, we undertook a series of time course experiments to examine posttransplant changes in the number, tissue distribution, cycling activity, and in vivo differentiation pattern of various human hematopoietic progenitor cell populations in this latter mouse model. These studies showed typical rapid posttransplant recovery curves for human CD34− CD19+ (B-lineage) cells, CD34+ granulopoietic, erythroid, and multilineage colony-forming cells (CFC), LTC-IC, and CD34+ Thy-1+ cells from a small initial population representing <0.1% of the original transplant. The most primitive human cell populations reached maximum values at 5 weeks posttransplant, after which they declined. More mature cell types peaked after another 5 weeks and then declined. A 2-week course of thrice weekly injections of human Steel factor, interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (administered just before the mice were killed for analysis) did not alter the pace of regeneration of either primitive or mature human hematopoietic cells, or their predominantly granulopoietic and B-lymphoid pattern of differentiation, although a significant enhancing effect on the level of human cell engraftment sustained after 3 months was noted. Cycling studies showed the human CFC present at 4 to 5 weeks posttransplant to be rapidly proliferating even in mice not given human growth factors. However, by 10 weeks and thereafter, only quiescent human CFC were detected; interestingly, even in mice that were given the 2-week course of growth factor injections. These studies indicate the use of this model for future analysis of the properties and in vivo regulation of primitive human hematopoietic cells that possess in vivo repopulating ability.
Thrombin Generation by Apoptotic Vascular Smooth Muscle Cells
Authors: Flynn Paul D. , Byrne Christopher D. , Baglin Trevor P. , Weissberg Peter L. , Bennett Martin R.
Published: October 2019

Thrombin activation requires assembly of a prothrombinase complex of activated coagulation factors on an anionic phospholipid surface, classically provided by activated platelets. We have previously shown that anionic phosphatidylserine is exposed by rat vascular smooth muscle cells (VSMCs) undergoing apoptosis after serum withdrawal. In this study, using a chromogenic assay, we have shown thrombin generation by apoptotic VSMCs expressing c-myc (VSMC-myc) with an area under the thrombin-generation curve (AUC) of 305 ± 17 nmol⋅min/L and a peak thrombin (PT) of 154 ± 9 nmol/L. The thrombin-generating potential of the apoptotic VSMC-myc cells was greater than that of unactivated platelets (P = .003 for AUC; P = .0002 for PT) and similar to calcium-ionophore activated platelets (AUC of 332 ± 15 nmol⋅min/L, P = .3; PT of 172 ± 8 nmol/L, P = .2). Thrombin activation was also seen with apoptotic human VSMCs (AUC of 211 ± 8 nmol⋅min/L; PT of 103 ± 4 nmol/L) and was inhibited by annexin V (P < .0001 for AUC and PT). VSMC-myc cells maintained in serum generated less thrombin than after serum withdrawal (P = .0002 for AUC and PT). VSMCs derived from human coronary atherosclerotic plaques that apoptose even in serum also generated thrombin (AUC of 260 ± 2 nmol⋅min/L; PT of 128 ± 4 nmol/L). We conclude that apoptotic VSMCs possess a significant thrombin-generating capacity secondary to phosphatidylserine exposure. Apoptotic cells within atherosclerotic plaques may allow local thrombin activation, thereby contributing to disease progression.
Frequency of Immune Thrombocytopenia in Newborns: A Prospective Study
Authors: Dreyfus Marie , Kaplan Cécile , Verdy Elizabeth , Schlegel Nicole , Durand-Zaleski Isabelle , Tchernia Gil ,   the Immune Thrombocytopenia Working Group
Published: October 2019

Thrombocytopenia is a common condition in distressed newborns, but little is known about thrombocytopenia in an unselected cohort of neonates. In an attempt to address this issue, a multicenter prospective study was conducted in three obstetrical wards of AP-HP in Paris. We found the frequency of neonatal thrombocytopenia (<150 × 109/L) to approximate 0.9% (48 of 5,632 appropriate samples). An immune mechanism was likely to be the cause of thrombocytopenia in 10 of the 33 cases studied, implying an incidence of 0.3% of immune neonatal thrombocytopenia in the general population. The frequency of alloimmune thrombocytopenia was 1.5/1,000 liveborn neonates, and 1/1,000 when considering anti–HPA-1a allo-immunization. Because thrombocytopenia, whatever its cause, was often silent and delayed, it appears that the only way to detect neonatal thrombocytopenia in time to prevent its potential disastrous complications would be to perform a systematic neonatal blood sampling for platelet count. All cases of ascertained thrombocytopenia should then be screened for an immune mechanism to enable early detection of autoimmune diseases in mothers and careful monitoring of subsequent pregnancies and deliveries, leading to appropriate prevention of potential severe deleterious effects in the offspring.
Tumor Dormancy and Cell Signaling. V. Regrowth of the BCL1 Tumor After Dormancy Is Established
Authors: Vitetta Ellen S. , Tucker Thomas F. , Racila Emilian , Huang Yi-Wu , Marches Radu , Lane Nancy , Scheuermann Richard H. , Street Nancy E. , Watanabe Takeshi , Uhr Jonathan W.
Published: October 2019

The majority of BALB/c mice immunized with the BCL1 lymphoma-derived idiotype (Id+) IgM and subsequently challenged with BCL1 tumor cells develop a state of tumor dormancy. The vast majority of dormant lymphoma cells are in cell cycle arrest, but there are also residual replicating cells. In the present studies, we attempted to define features of both the dormant lymphoma cells and the host that lead to escape from dormancy. Escape from dormancy occurs at a steady rate over a 2-year period, suggesting that it is a stochastic process. We found that, in the majority of mice, escape was due to the emergence of genetic variants that were no longer susceptible to the anti-Id–mediated induction of dormancy. Ten percent of these variants were Id−; the remainder were Id+ but could grow in the presence of anti-Id antibodies, suggesting that there were mutations in molecules involved in one or more mIg-mediated negative-signaling pathways. In two of five such escapees, alterations in either Syk, HS1, and/or Lyn were observed. In a small percentage of mice, a low titer of circulating anti-Id antibody before tumor challenge correlated with a subsequent, more rapid loss of dormancy.
Distribution of ABL and BCR Genes in Cell Nuclei of Normal and Irradiated Lymphocytes
Authors: Kozubek S. , Lukášová E. , Rýznar L. , Kozubek M. , Lišková A. , Govorun R.D. , Krasavin E.A. , Horneck G.
Published: October 2019

Using dual-color fluorescence in situ hybridization (FISH) combined with two-dimensional (2D) image analysis, the locations of ABL and BCR genes in cell nuclei were studied. The center of nucleus-to-gene and mutual distances of ABL and BCR genes in interphase nuclei of nonstimulated and stimulated lymphocytes as well as in lymphocytes stimulated after irradiation were determined. We found that, after stimulation, the ABL and BCR genes move towards the membrane, their mutual distances increase, and the shortest distance between heterologous ABL and BCR genes increases. The distribution of the shortest distances between ABL and BCR genes in the G0 phase of lymphocytes corresponds to the theoretical distribution calculated by the Monte-Carlo simulation. Interestingly, the shortest ABL-BCR distances in G1 and S(G2 ) nuclei are greater in experiment as compared with theory. This result suggests the existence of a certain regularity in the gene arrangement in the G1 and S(G2 ) nuclei that keeps ABL and BCR genes at longer than random distances. On the other hand, in about 2% to 8% of lymphocytes, the ABL and BCR genes are very close to each other (the distance is less than ∼0.2 to 0.3 μm). For comparison, we studied another pair of genes, c-MYC and IgH, that are critical for the induction of t(8; 14) translocation that occurs in the Burkitt's lymphoma. We found that in about 8% of lymphocytes, c-MYC and IgH are very close to each other. Similar results were obtained for human fibroblasts. γ-Radiation leads to substantial changes in the chromatin structure of stimulated lymphocytes: ABL and BCR genes are shifted to the nuclear center, and mutual ABL-BCR distances become much shorter in the G1 and S(G2 ) nuclei. Therefore, we hypothesize that the changes of chromatin structure in the irradiated lymphocytes might increase the probability of a translocation during G1 and S(G2 ) stages of the cell cycle. The fact that the genes involved in the t(8; 14) translocation are also located close together in a certain fraction of cells substantiates the hypothesis that physical distance plays an important role in the processes leading to the translocations that are responsible for oncogenic transformation of cells.
Cyanocobalamin [c-lactam] Inhibits Vitamin B12 and Causes Cytotoxicity in HL60 Cells: Methionine Protects Cells Completely
Authors: Matthews J.H.
Published: October 2019

The [c-lactam] derivative of cobalamin antagonizes vitamin B12 in vivo. Therefore, we investigated its effects in tissue culture to develop a model in which to study vitamin B12-deficient hemopoiesis. HL60 cells were cultured in medium containing either methionine or L-homocysteine thiolactone, and various concentrations of 5-methyltetrahydrofolate or pteroylglutamic acid. In medium with L-homocysteine thiolactone, 5-methyltetrahydrofolate, and dialyzed serum, cyanocobalamin [c-lactam] caused cell death, reversible by additional vitamin B12 . Pteroylglutamic acid did not prevent this cytotoxic effect. Methionine completely protected cells against cyanocobalamin [c-lactam] for periods of up to 4 months of culture, irrespective of the folate source. Cyanocobalamin [c-lactam] reversibly impaired the incorporation of 5-[14CH3]-tetrahydrofolate and [1-14C] propionic acid by intact cells, consistent with inhibition of methionine synthase and methylmalonyl-CoA mutase. A substantial proportion of 5-[14CH3]-tetrahydrofolate uptake could not be suppressed by methionine and may, therefore, have occurred outside of the methionine synthase pathway. These findings are the first indication that cyanocobalamin [c-lactam] antagonizes vitamin B12 in vitro and causes cell death from methionine deficiency. The model should be valuable for investigating the biochemical pathology of vitamin B12-deficient hemopoiesis. The results suggest that methylfolate is not trapped when methionine synthase is inhibited in HL60 cells, but they do not disprove the methylfolate trap hypothesis as applied to normal blood cells.
Organization of the Human LU Gene and Molecular Basis of the Lua/Lub Blood Group Polymorphism
Authors: El Nemer Wassim , Rahuel Cecile , Colin Yves , Gane Pierre , Cartron Jean Pierre , Le Van Kim Caroline
Published: October 2019

The Lutheran (Lu) blood group antigens and the B-cell adhesion molecule (B-CAM) epithelial cancer antigen are carried by recently cloned integral glycoproteins that belong to the Ig superfamily. We have previously shown that the Lu and B-CAM antigens are encoded by the same gene, LU, and that alternative splicing of the primary transcript most likely accounts for the presence of both antigens on two isoforms that differ by the length of their cytoplasmic tails. In the present report, we isolated the human LU gene by cloning a 20-kb HindIII fragment from Lu(a − b+) genomic DNA. The LU gene is organized into 15 exons distributed over 12.5 kb. Alternative splicing of intron 13 generates the 2.5- and 4.0-kb transcript spliceoforms encoding the long tail and the short tail Lu polypeptides, respectively. Sequencing of the major mRNA species (2.5 kb) amplified from human bone marrow, kidney, placenta, and skeletal muscle did not suggest the presence of tissue-specific Lu glycoprotein isoforms. The same transcription initiation point, located 22 bp upstream from the initiation codon, was characterized in several tissues. In agreement with the wide tissue distribution of the Lu messengers, the GC-rich proximal 5′ flanking region of the LU gene does not contain TATA or CAAT boxes, but includes several potential binding sites for the ubiquitous Sp1 transcription factor. In addition, the distal 5′ region, encompassing nucleotides −673 to −764, contains clustered binding sequences for the GATA, CACCC, and Ets transcription factors. Analysis of the coding sequences amplified from genomic DNA of Lu(a + b−) or Lu(a − b+) donors showed a single nucleotide change in exon 3 (A229G) that correlates with an Aci I restriction site polymorphism and results in a His77Arg amino-acid substitution. Polymerase chain reaction/restriction fragment length polymorphism analysis indicated that the A229G mutation is associated with the Lua/Lub blood group polymorphism. When expressed in Chinese hamster ovary (CHO) cells, Lu cDNAs carrying the A229 or the G229 produced cell surface proteins that reacted with anti-Lua or anti-Lub antibodies, respectively, showing that these nucleotides specify the Lua and Lub alleles of the Lutheran blood group locus. CHO cells expressing recombinant short-tail or long-tail Lu glycoproteins reacted as well with anti-Lu as with anti–B-CAM antibodies, providing the definitive proof that the Lu blood group and B-CAM antigens are carried by the same molecules.