bluuebunny/crossref_metadata_2025 huggingface dataset:
The Life Span of the Elliptocyte
Authors: MOTULSKY ARNO G. , SINGER KARL , CROSBY WILLIAM H. , SMITH VERNON
Published: October 2019

1. Red cells from three patients of different families with heteditary elliptocytosis were transfused and their survival followed by the Ashby technic of differential agglutination.

2. Life span of elliptocytes was normal in two cases.

3. A life span of forty-five days was obtained with the elliptocytes of a third patient who presented other evidence of hemolysis in the absence of anemia.

4. In two recipients with sickle cell anemia the survival of the transfused elliptocytes was also followed by counting the number of elliptocytes in sickle cell preparations. Rough agreement between the results of the Ashby and visual technics was obtained. There was no evidence of transformation from the elliptocytic into normal shape.

5. Elliptocytes from patients with hemolytic elliptocytosis were morphologically indistinguishable from elliptocytes with normal life span.

6. No hemoglobin abnormalities have yet been demonstrated in hereditary elliptocytosis. The basic defect responsible for hemolytic elliptocytosis remains unknown.

7. Symptomatic ovalocytosis occurs in many types of anemia, and must be differentiated from hereditary elliptocytosis.

8. The place of hereditary elliptocytosis in the classification of familial hemolytic diseases is discussed (table 4) and similarities to the other hereditary hemotic anemias are pointed out. It is shown that the red cell life span (and genetic) pattern of hereditary elliptocytosis resembles closely that found in thalassemia. As in thalassemia major, microspherocytosis and increased osmotic fragility due to red tell fragmentation may occur in cases of hemolytic elliptocytosis. Increased life span of these "fragmentation-spherocytes" after splenectomy may partially explain the success of the operation in hemolytic elliptocytosis.

9. No treatment is required for hereditary nonhemolytic elliptocytosis. Splenectomy is advised for "decompensated" hemolytic elliptocytosis.
Variation of the Hemoglobin Level with Age and Sex
Authors: HAWKINS W. W. , SPECK EIRLYS , LEONARD VERNA G.
Published: October 2019

During the winter and spring of 1952-53 a survey of hemoglobin values was made in Halifax among 1308 male subjects 6 to 98 years of age, and 1424 female subjects 6 to 94 years of age.

Among children 6 to 14 years old the values increased from about 13 to about 14 Gm. per 100 ml. of blood, and there were essentially no differences between the sexes. The average value for both the boys and the girls was 13.5 Gm.

In girls between 14 and 20 years of age the hemoglobin values decreased slightly, reaching about 13 Gm. per 100 ml. In boys of corresponding ages there was an increase to about 15 Gm. In both sexes these values were attained at about 20 years of age, and remained characteristic of the third decade of life. They were essentially the lowest and the highest shown respectively by the female and the male subjects of any age group.

Hemoglobin values in men between 20 and 60 years of age were typically 14.5 to 15 Gm. per 100 ml., the higher values tending to occur among the younger men. After the fifth decade there were progressive and marked decreases to an average of 12.4 in men between 80 and 90 years of age.

In women from 20 years of age onward the average hemoglobin values remained near 13 Gm. per 100 ml.
The Blood Volume of Normal Women
Authors: WADSWORTH G. R.
Published: October 2019

Measurements of red cell volume and plasma volume in eight normal women confirm that, in relation to body weight, red cell volume is distinctly lower in women than in men and plasma volume only slightly lower. The relationship between body hematocrit and venous hematocrit (0.90) was found not to be significantly different from that of men.
Some Immunohematologic Results of Large Transfusions of Group O Blood in Recipients of Other Blood Groups
Authors: CROSBY WILLIAM H. , AKEROYD JOSEPH H.
Published: October 2019

1. Following large transfusions of low titer group O blood into patients of group A, B, and AB it was not possible to demonstrate foreign isohemolysins or incomplete antibodies in the serum of recipients. Cold isoagglutinins were frequently demonstrated immediately after the transfusion, but they usually disappeared rapidly. Its several patients the titer of foreign anti-A isoagglutinin was quite high and the antibody persisted in the circulation for several days. It was suggested that the persistence of these agglutinins may have been possible because there was a relatively small amount of A substance in the body of the recipient. Where the transfused isoagglutinins persisted the patients were found to be nonsecretors or weak secretors of A substance in the saliva.

2. In most of these patients there was evidence of a selective destruction of recipient red cells after the transfusion of O blood. This was probably due to the activity of transfused isoantibodies in the plasma of the O blood. This hemolytic activity was observed where it was not possible to demonstrate the presence of foreign isoantibodies. It is suggested that there may exist forms of antibody that cannot be demonstrated by laboratory methods. These antibodies manifest themselves only by causing destruction of red cells in vivo.

3. Clinically the hemolytic disease on the basis of such transfused isoantibodies while causing destruction of native red cells did not threaten the lives or impede the recovery of these patients. No reactions were encountered and none were heard of in Korea that might have been ascribed to a dangerous universal donor. The partition of group O blood into high titer and low titer on the basis of dilution of 1:200 to 1:256 has proved in practice to be safe.

4. The persistence of foreign antibodies after a large transfusion of group O blood may make it impossible to cross match the patient’s blood with blood of his hereditary group. Severe transfusion reactions have occurred when group specific blood has been given following large transfusions of group O blood. It has been recommended that after a large transfusion of group O blood has been given, group specific blood should not be used for at least two weeks.
Changes in the Phagocytic Activity of Polymorphonuclear Leukocytes following Total Body X-Irradiation in the Rat
Authors: WILKINSON M.
Published: October 2019

The phagocytic activity of polymorphonuclear leukocytes from irradiated rats was studied over a period of thirteen days following 550 r total body x-irradiation.

Leukocytes from irradiated rats showed slightly increased phagocytosis of plague bacilli during the first six days after irradiation, but on statistical analysis this increase proved to be insignificant.

From the seventh to the thirteenth day after irradiation, these cells showed a markedly reduced capacity to phagocytose plague bacilli.

Phagocytic studies on suspensions of normal cells in plasma from irradiated animals and on cells from irradiated animals in normal plasma showed that the deficient phagocytosis during the second week following irradiation was due mainly to a defect in the polymorphonuclear leukocytes themselves, though the fact that the addition of normal plasma to cells from irradiated animals did increase their phagocytic capacity by a small but definite degree suggests that there is also a defect in the plasma of irradiated animals.
Mitogenic Responses Mediated Through the Proteinase-Activated Receptor-2 Are Induced by Expressed Forms of Mast Cell α- or β-Tryptases
Authors: Mirza Humayun , Schmidt Valentina A. , Derian Claudia K. , Jesty Jolyon , Bahou Wadie F.
Published: October 2019

The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)–dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2Arg36 → Ala36. Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3–deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of ∼80 μmol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares ∼70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of α- and β-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr ∼130, confirming the presence of secretable and functionally active homotetrameric α- and β-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at ∼3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2Arg36 → Ala36. These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.
Development of Natural Killer Cells, B Lymphocytes, Macrophages, and Mast Cells From Single Hematopoietic Progenitors in Culture of Murine Fetal Liver Cells
Authors: Aiba Yuichi , Ogawa Makio
Published: October 2019

We have recently established a clonal culture system that supports the growth of immature natural killer (NK) cells from murine fetal thymocytes. We now describe a culture system for mixed NK cell colony formation from single lymphohematopoietic progenitors. When Sca-1+c-kit+ fetal liver cells were cultured in methylcellulose media with interleukin (IL)-2, IL-7, IL-11, and steel factor (SF ), we found mixed colonies consisting of diffuse small round cells characteristic of immature NK cells and other types of cells. The single cell origin of the mixed colonies was established by micromanipulation. Individual mixed colonies derived from single cells were characterized by flow cytometric analysis and May-Grünwald Giemsa staining. All mixed colonies contained Thy-1+B220− cells, which can differentiate to mature NK cells in fetal thymus organ culture. Most of the colonies contained B220+ B-lineage cells and macrophages, and some contained mast cells. IL-1α and IL-3, which have previously been shown to inhibit the T- and B-cell potentials of blast colonies, suppressed the formation of mixed NK cell colonies. The clonal culture assay presented here may be useful in analysis of the developmental pathway and commitment of NK cells from multipotential progenitors.
Enhanced Lipid Peroxidation in Patients Positive for Antiphospholipid Antibodies
Authors: Iuliano Luigi , Praticò Domenico , Ferro Domenico , Pittoni Valerio , Valesini Guido , Lawson John , FitzGerald Garret A. , Violi Francesco
Published: October 2019

The mechanism leading to the formation of antiphospholipid antibodies (aPL) is still unknown. Because an in vitro study suggested that aPL may derive from pro-oxidant conditions, we sought a relationship between aPL and isoprostanes, indices of lipid peroxidation in vivo. Thirty patients with systemic lupus erythematosus have been studied. Seventeen (56.6%) were positive for aPL because they had lupus anticoagulant and/or high titer of anticardiolipin antibodies (aCL). Plasma levels of tumor necrosis factor (TNF ) and urinary excretion of two isoprostanes, 8-epi-PGF2α and IPF2α -I, free radical catalyzed oxidation products of arachidonic acid, were measured. Patients with systemic lupus erythematosus had higher urinary excretion of 8-epi-PGF2α and IPF2α -I than controls; urinary excretion of the two isoprostanes was highly correlated (Rho = 0.74, P < .0001). Urinary 8-epi-PGF2α was highly correlated with both aCL titer (Rho = 0.70, P < .0001) and TNF (Rho = 0.84, P < .0001), a measure of disease severity. Excretion of this isoprostane was also higher in those patients who exhibited aPL (P < .0001). Comparable correlations were observed with the isoprostane IPF2α -I. No difference of 8-epi-PGF2α was observed between patients with and without previous history of thrombosis. This study, showing the existence of a close association between aPL and increased in vivo lipid peroxidation, supports the hypothesis that these antibodies may result from pro-oxidative conditions and suggests that inflammation may play an important role.
A Coagulation Factor IX-Deficient Mouse Model for Human Hemophilia B
Authors: Lin Hui-Feng , Maeda Nobuyo , Smithies Oliver , Straight David L. , Stafford Darrel W.
Published: October 2019

Coagulation factor IX deficiency causes hemophilia B in humans. We have used gene targeting to develop a coagulation factor IX-deficient (factor IX-knockout) mouse strain. Mouse embryonic stem (ES) cells were targeted by a socket-containing vector that replaces the promoter through exon 3 of the factor IX gene by neoΔHPRT, which is a functional neo gene plus a partially deleted hypoxanthine phosphoribosyl transferase minigene. Chimeric mice generated using these socket-containing ES cells transmitted the targeted factor IX gene to their female offspring. Male offspring from these females were characterized and shown to exhibit a phenotype similar to hemophilia B. This factor IX-deficient mouse strain will be useful for studying gene therapy methods and structure-function relationships of recombinant factor IX proteins in vivo.
Immunocytochemical Diagnosis of Acute Promyelocytic Leukemia (M3) With the Monoclonal Antibody PG-M3 (Anti-PML)
Authors: Falini Brunangelo , Flenghi Leonardo , Fagioli Marta , Coco Francesco Lo , Cordone Iole , Diverio Daniela , Pasqualucci Laura , Biondi Andrea , Riganelli Daniela , Orleth Annette , Liso Arcangelo , Martelli Massimo F. , Pelicci Pier-Giuseppe , Pileri Stefano
Published: October 2019

Acute promyelocytic leukemia (APL) is characterized by a reciprocal 15; 17 chromosomal translocation, which fuses the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes, leading to the expression of the PML/RARα fusion oncoprotein. Immunocytochemical labeling of the wild-type PML protein with the PG-M3 monoclonal antibody (MoAb) directed against the amino terminal portion of the human PML gene product, produces a characteristic nuclear speckled pattern that is due to localization of the protein into discrete dots (5 to 20 per nucleus), named PML nuclear bodies. The architecture of PML nuclear bodies appears to be disrupted in APL cells that bear the t(15; 17), thus resulting in a change of the nuclear staining pattern from speckled (wild-type PML protein) to microgranular (PML-RARα fusion protein). To assess whether the PG-M3 MoAb could assist in the diagnosis of APL (M3), bone marrow and/or peripheral blood samples from 100 cases of acute nonlymphoid leukemias of different subtypes were blindly immunostained with the PG-M3 MoAb, using the immunoalkaline phosphatase (APAAP) or immunofluorescence technique as detection system. Notably, the abnormal (micropunctate) pattern of the PML/RARα fusion protein (usually ≥50 small granules/per nucleus) was observed in APL (M3) samples, but not in other types of acute nonlymphoid leukemias. Immunocytochemical labeling with PG-M3 was particularly useful in the diagnosis of microgranular variant of APL (M3V) (three cases misdiagnosed as M4 and M5), and also to exclude a morphologic misdiagnosis of APL (six of 78 cases). In all cases investigated, immunocytochemical results were in agreement with those of reverse transcription-polymerase chain reaction (RT-PCR) for PML/RARα. Because the epitope identified by PG-M3 is located in the aminoterminal portion of PML (AA 37 to 51), the antibody was suitable for recognizing APL cases characterized by breakpoint occurring at different sites of PML (bcr 1, bcr 2 and bcr 3). In conclusion, immunocytochemical labeling with PG-M3 represents a rapid, sensitive, and highly-specific test for the diagnosis of APL that bears the t(15; 17). This should allow an easy and correct diagnosis of this subtype of acute leukemia to any laboratory provided with a minimal equipment for immunocytochemistry work.
A Novel Chromosomal Translocation t(4; 14)(p16.3; q32) in Multiple Myeloma Involves the Fibroblast Growth-Factor Receptor 3 Gene
Authors: Richelda Raffaella , Ronchetti Domenica , Baldini Luca , Cro Lilla , Viggiano Luigi , Marzella Rosalia , Rocchi Mariano , Otsuki Takemi , Lombardi Luigia , Maiolo Anna Teresa , Neri Antonino
Published: October 2019

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies. In multiple myeloma (MM), the most consistent chromosomal abnormality is the 14q+ marker, which originates in one third of cases through a t(11; 14)(q13; q32) chromosomal translocation; in the remaining cases, the identity of the partner chromosomes has not been well established. We used a Southern blot approach based on the linkage analysis of the joining (J) and the constant (C) μ, α, and γ regions to detect cases bearing IGH switch-mediated chromosomal translocations. We evaluated DNA of 88 nonkaryotyped patients with MM (78 cases) or plasma cell leukemia (PCL) (10 cases) and found the presence of “illegitimate” rearranged IGH fragments (no comigration between the J and C regions) in 21 cases. To confirm this analysis, we cloned the illegitimate rearranged fragments from three samples, and the molecular and fluorescent in situ hybridization (FISH) analyses indicated the presence of chromosomal translocations juxtaposing a switch IGH region to sequences from chromosomes 11q13 (one PCL case) or 4p16.3 (two MM cases). Interestingly, the breakpoints on 4p16.3 occurred about 14 kb apart in a genomic region located approximately 50 kb centromeric to the fibroblast growth-factor receptor 3 (FGFR3) gene. Moreover, Southern blot analysis using 4p16.3 genomic probes detected a rearrangement in an additional MM tumor. FISH analysis of the MM-derived KMS-11 cell line, reported to be associated with a t(4; 14)(p16.3; q32), showed that the FGFR3 gene was translocated on 14q32. High levels of FGFR3 mRNA expression were observed in the cloned MM tumors and KMS-11 cell line, but not in the cases that were apparently negative for this lesion. Furthermore, a point mutation at codon 373 in the transmembrane domain of the FGFR3 gene resulting in an amino acid substitution (Tyr → Cys) was detected in the KMS-11 cell line. These findings indicate that the t(4; 14)(p16.3; q32) represents a novel, recurrent chromosomal translocation in MM, and suggest that the FGFR3 gene may be the target of this abnormality and thus contribute to tumorigenesis in MM.
Allogeneic Versus Autologous Bone Marrow Transplantation for Refractory and Recurrent Low-Grade Non-Hodgkin's Lymphoma
Authors: Verdonck Leo F. , Dekker Adriaan W. , Lokhorst Henk M. , Petersen Eefke J. , Nieuwenhuis H. Karel
Published: October 2019

Patients with recurrent or refractory low-grade non-Hodgkin's lymphoma (NHL) are increasingly treated with myeloablative therapy and autologous stem cell transplantation. However, allogeneic bone marrow transplantation (BMT) is only sporadically performed in such patients. Therefore, we wish to compare treatment results of patients with recurrent or refractory low-grade NHL who underwent allogeneic BMT with those who underwent autologous BMT in our center. Twenty-eight patients were studied. The patients had received 2 to 5 lines of conventional chemotherapy before the BMT procedure. Eighteen patients, all with chemotherapy-sensitive disease at the time of transplantation, underwent autologous BMT and 10 patients, of whom 7 with chemotherapy-resistant disease at the time of transplantation, underwent allogeneic BMT. Furthermore, all allogeneic BMT patients had overt lymphoma infiltration of the BM at the time of transplantation. The conditioning regimen consisted of cyclophosphamide plus total body irradiation in all 28 patients. All allogeneic BMT patients achieved complete remission, 3 patients had a treatment-related death, and 7 patients are alive and disease-free with a median follow-up of 41 months. In contrast, none of the autologous BMT patients died of transplant-related complications. However, despite the fact that all autologous BMT patients had chemotherapy-sensitive disease and partial remission was converted to complete remission by the BMT procedure in 67% of them, only 3 of 18 patients are alive and disease-free. The probability of relapse or disease-progression among allogeneic BMT patients was 0% compared with 83% for autologous BMT patients (P = .002). Progression-free survival rates 2 years after BMT were 68% for allogeneic BMT patients and 22% for autologous BMT patients (P = .049). Although the numbers of patients are small, this study suggests that allogeneic BMT offers a better chance for cure than autologous BMT for patients with poor-prognosis low-grade lymphoma, and the difference in relapse or disease progression is strongly suggestive for the existence of a graft–versus–low-grade lymphoma effect.
T Helper 2-Dominant Antilymphoma Immune Response Is Associated With Fatal Outcome
Authors: Lee Peter P. , Zeng Defu , McCaulay Ann E. , Chen Yan-Fei , Geiler Craig , Umetsu Dale T. , Chao Nelson J.
Published: October 2019

The precise role of the endogenous immune system in modulating cancer development remains unclear. Tumor cells are generally thought to be nonimmunogenic because they are of ‘self’ origin. However, tumor-reactive lymphocytes can be isolated from patients with many types of cancer. It is unclear what role these lymphocytes play and why they fail to protect the host. Using a murine B-cell leukemia/lymphoma (BCL1) model, we showed the development of a vigorous antitumor T-cell response in the tumor-susceptible host. Specific T-cell responses against BCL1 developed as early as day 4. However, the nature of this nonprotective response is different from the protective response produced in a major histocompatibility complex–matched tumor-resistant host. Susceptible hosts developed a T helper 2 (Th2)-dominant response, whereas resistant hosts developed a Th1-dominant response to BCL1. Cytolytic activity against BCL1 developed in both resistant and susceptible hosts, but in the susceptible host, this response was weaker and delayed compared with that in the resistant host. Thus, tumor susceptibility does not necessarily mean the absence of an antitumor immune response. Rather, the nature of the antitumor immune response is critical in determining clinical outcome.
Granulocytic Differentiation of Normal Hematopoietic Precursor Cells Induced by Transcription Factor PU.1 Correlates With Negative Regulation of the c-myb Promoter
Authors: Bellon Teresa , Perrotti Danilo , Calabretta Bruno
Published: October 2019

Numerous transcription factors allow hematopoietic cells to respond to lineage- and stage-specific cytokines and/or to act as their effectors. The transcription factors PU.1 and c-Myb are essential for hematopoiesis, most likely acting at distinct stages of differentiation, but sharing a common set of target genes. To determine whether PU.1 and c-Myb are functionally interrelated, murine bone marrow (BM) cells and 32Dcl3 murine myeloid precursor cells were infected with a retrovirus carrying a PU.1 cDNA and assessed for myeloid colony formation and for granulocytic differentiation, respectively. Compared with noninfected normal BM cells or to cells infected with an empty virus, hematopoietic precursor cells expressing PU.1 formed an increased number of interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF )–stimulated colonies. Moreover, granulocytic differentiation of 32Dcl3 cells constitutively expressing PU.1 was accelerated, as indicated by morphology and by expression of differentiation markers. Downregulation of c-Myb protein levels by expression of an antisense c-myb construct was also associated with a faster kinetics of 32Dcl3 granulocytic differentiation. Sequence analysis of the 5′ flanking region of the c-myb gene revealed a consensus PU box at position +16 to +21 able to specifically interact in electrophoretic mobility shift assays with either bacterially synthesized PU.1 protein or whole cell extracts from differentiated 32Dcl3 cells. Transient expression of PU.1 in cotransfection assays in different cell lines resulted in inhibition of chloramphenicol acetyl transferase activity driven by different segments of the c-myb promoter. Moreover, such an effect was dependent on an intact PU box. Thus, the ability of PU.1 to potentiate terminal myeloid differentiation appears to involve downregulation of c-myb expression, an essential step during differentiation of hematopoietic precursor cells.
Fas-Fas Ligand–Based Interactions Between Tumor Cells and Tumor-Specific Cytotoxic T Lymphocytes: A Lethal Two-Way Street
Authors: Zeytun Ahmet , Hassuneh Mona , Nagarkatti Mitzi , Nagarkatti Prakash S.
Published: October 2019

In the current study, we investigated the repercussions of the interaction between tumor cells (LSA) and the tumor-specific cytotoxic T lymphocyte (CTL) (PE-9) when both expressed Fas and Fas ligand (FasL). The CTL clone, PE-9, expressed high levels of Fas and FasL upon activation through the T-cell receptor (TCR). Furthermore, the activated PE-9 cells used both perforin- and FasL-based pathways to kill Fas-positive (Fas+) LSA tumor cells. Interestingly, LSA tumor cells also constitutively expressed FasL but not perforin, and killed Fas+ PE-9 CTLs and Fas+ but not Fas-negative (Fas−) activated T cells and thymocytes, as detected using the JAM test. PE-9 CTLs, cultured for 24 hours in the presence of cell lysates of FasL-bearing LSA cells but not FasL-deficient P815 cells, exhibited significant apoptosis as detected using the TUNEL method. Moreover, another FasL+ T-cell lymphoma line, EL-4, induced apoptosis in Fas+ but not in Fas− T cells in a similar fashion. The current study demonstrates for the first time that not only can the tumor-specific CTL mediate Fas-based killing of tumor cells, but FasL+ tumor cells can kill the Fas+ tumor-specific CTL. Thus, the survival of the tumor or the host may depend on which cell can accomplish this task more efficiently. The current study also suggests that FasL-based killing of CTLs by specific tumor cells may constitute a major limiting factor in successful immunotherapy.
RheothRx (Poloxamer 188) Injection for the Acute Painful Episode of Sickle Cell Disease: A Pilot Study
Authors: Adams-Graves Patricia , Kedar Amos , Koshy Mabel , Steinberg Martin , Veith Robert , Ward Daniel , Crawford Rebekah , Edwards Suzanne , Bustrack James , Emanuele Martin
Published: October 2019

RheothRx (Glaxo Wellcome Inc, Research Triangle Park, NC; poloxamer 188) Injection is a nonionic surfactant with hemorrheologic properties that suggest it may be useful in treating acute painful episodes (vasoocclusive crises) of sickle cell disease (SCD). We conducted a randomized, double-blind, placebo-controlled pilot study to evaluate the safety and efficacy of poloxamer, formulated as RheothRx Injection, in 50 patients with SCD. Patients with moderate to severe painful episodes requiring parenteral analgesics were randomized to receive a 48-hour infusion of either RheothRx or placebo. Pain was assessed every 4 hours. Efficacy endpoints included: (1) painful episode duration, (2) days of hospitalization, (3) quantity of analgesics used, and (4) pain intensity scores. Three subgroups of patients were considered for efficacy analyses based on the actual duration of the study drug infusion and the completeness of pain score data collection. Compared with placebo and depending on the subgroup, RheothRx-treated patients showed a 16% to 45% decrease in duration of painful episodes, a 1- to 2-day reduction in hospital stay, a threefold to fivefold reduction in analgesic requirements, and a 1-point reduction (using a 5-point scale) in average pain intensity scores at 72 hours. RheothRx was well tolerated; no clinically significant differences were observed between treatments with respect to adverse experiences or other safety measures. In addition, there were no differences between treatment groups in the incidence of recurrent painful episodes. In this study, RheothRx significantly reduced total analgesic use and pain intensity and showed trends to shorter duration of painful episodes and total days of hospitalization. In patients with moderate to severe vasoocclusive pain, RheothRx was safe and may offer a therapeutic benefit.
Suppression of Megakaryocyte Colony Growth by Plasma From Foals Infected With Equine Infectious Anemia Virus
Authors: Tornquist Susan J. , Crawford Timothy B.
Published: October 2019

Foals infected with equine infectious anemia virus become thrombocytopenic 7 to 20 days after virus inoculation, and within a few days following the onset of detectable viremia. The thrombocytopenia is associated with suppression of platelet production. Possible mediators of suppression of thrombopoiesis include tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), cytokines that are released during inflammation. To assess effects of plasma or serum from infected foals on megakaryocyte (MK) growth and maturation in vitro, equine low-density bone marrow cells were cultured for clonogenic and ploidy assays. Neutralizing antibodies to TNF-α and TGF-β were added to cultures to determine the contribution of these cytokines to suppression of thrombopoiesis. Plasma from the immediately pre-thrombocytopenia (Pre-Tp) period significantly reduced MK colony numbers. This suppression was partially reversed upon antibody neutralization of plasma TNF-α, TGF-β, or both. There were no differences in ploidy distribution of MK grown in the presence of preinfection serum compared with those grown in the presence of Pre-Tp serum. These results indicate that TNF-α and TGF-β may contribute to suppression of MK proliferation and represent likely factors in the pathogenesis of thrombocytopenia.
Detection and Characterization of Primitive Malignant and Normal Progenitors in Patients With Acute Myelogenous Leukemia Using Long-Term Coculture With Supportive Feeder Layers and Cytokines
Authors: Ailles Laurie E. , Gerhard Brigitte , Hogge Donna E.
Published: October 2019

Analysis of the mitogenic activity of interleukin-3 (IL-3), Steel factor (SF ), and flt-3 ligand (FL) on acute myelogenous leukemia (AML) blasts using the short-term endpoints of proliferation in 3H-thymidine (3H-Tdr) incorporation assays or methylcellulose cultures (colony assays) showed that greater than 90% of samples contained cells that were responsive to one or more of these cytokines. With this information, culture conditions that were known to support normal long-term culture-initiating cells (LTC-IC) were tested, with or without supplements of one or more of these three growth factors, for their ability to support primitive progenitors from 10 cell samples from patients with AML. In all cases cytogenetically abnormal colony forming cells (CFC) were detected after 5 weeks when AML peripheral blood or marrow cells were cocultured on preestablished, normal human marrow feeders (HMF ) and/or Sl/Sl mouse fibroblast feeders and the number of CFC detected in these 5-week-old LTC maintained a linear relationship to the number of input AML cells. Limiting dilution analysis, performed on 6 of the 10 samples, showed the frequency of AML cells initiating LTC (AML LTC-IC) to be 5- to 300-fold lower than the frequency of AML-CFC in the same cell sample, whereas the average number of CFC produced per LTC-IC varied from 1 to 13. Surprisingly, in each case the concentration of cytogenetically normal LTC-IC detected in AML patient blood was at least 10-fold higher than that previously observed in the blood of normal individuals. “Mixed” mouse fibroblast feeders engineered to produce human G-CSF, IL-3, and SF did not enhance detection of AML LTC-IC but did increase the output of cytogenetically normal CFC from LTC of 3 of 4 patient samples. Supplementation of AML LTC with IL-3 and exogenously provided SF and/or FL increased the output of AML-CFC from 5-week-old LTC by greater than or equal to twofold with 5 of 9 patient samples, whereas in one case exogenous addition of FL reduced the output of malignant CFC from LTC. These studies show that conditions that support normal LTC-IC also allow a functionally analogous but rare AML progenitor cell type to be detected. In addition, differences in the responses of normal and leukemic cells to various cytokines active on normal LTC-IC were revealed. Further analysis of these differences may enhance our understanding of leukemogenesis and lead to observations that could be exploited therapeutically.
A Dinucleotide Deletion Results in Defective Membrane Anchoring and Circulating Soluble Glycoprotein Ibα in a Novel Form of Bernard-Soulier Syndrome
Authors: Kenny Dermot , Newman Peter J. , Morateck Patricia A. , Montgomery Robert R.
Published: October 2019

The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor and is composed of four membrane-spanning polypeptides: GPIbα, GPIbβ, GPIX, and GPV. A qualitative or quantitative deficiency in the GPIb-V-IX complex on the platelet membrane is the cause of the congenital platelet disorder Bernard-Soulier syndrome (BSS). We describe the molecular basis of a novel variant BSS in a patient in which GPIbα was absent from the platelet surface but present in a soluble form in the plasma. DNA sequence analysis showed a homozygous dinucleotide deletion in the codon for Tyr 508 (TAT) in GPIbα. This mutation (GPIbαΔAT) causes a frame shift that alters the amino acid sequence of GPIbα within its transmembrane region. The hydrophobic nature of the predicted transmembrane region and the cytoplasmic tail at the COOH terminal are altered before reaching a new premature stop codon 38 amino acids short of the wild-type peptide. Although GPIbαΔAT was not detectable on the platelet surface, immunoprecipitation of plasma with specific monoclonal antibodies (MoAbs) identified circulating GPIbα. Transient expression of recombinant GPIbαΔAT in 293T cells also generated a soluble form of the protein. Moreover, when a plasmid encoding GPIbαΔAT was transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GPβ-IX complex, it failed to be expressed on the cell surface. Thus, a dinucleotide deletion in the codon for Tyr 508 causes a frameshift that alters the amino acid sequence of GPIbα starting within its transmembrane region, changes the hydrophobicity of the normal transmembrane region, and truncates the cytoplasmic domain affecting binding to the cytoskeleton and cytoplasmic proteins. This mutation affects anchoring of the GPIbα polypeptide in platelets and causes the observed BSS phenotype with circulating soluble GPIbα.
Intracellular Signaling Events in CD77-Mediated Apoptosis of Burkitt's Lymphoma Cells
Authors: Taga Samir , Carlier Karine , Mishal Zohair , Capoulade Corinne , Mangeney Marianne , Lécluse Yann , Coulaud Dominique , Tétaud Cécile , Pritchard Linda Louise , Tursz Thomas , Wiels Joëlle
Published: October 2019

In the hematopoietic system CD77, a glycolipid surface antigen, is restricted to group I Burkitt's lymphoma (BL) cell lines and a subset of germinal center B lymphocytes. Recently, we have reported that recombinant B subunits of Verotoxin, which specifically binds to CD77, induce programmed cell death of CD77+ BL cells. Here, we show that an anti-CD77 monoclonal antibody (38.13) immobilized on tissue culture dishes also induces apoptosis, and we have explored the signal transducing events leading to this cell death. We show that ligation of CD77 antigen causes an increase of the intracellular Ca2+ concentration owing to an influx of extracellular Ca2+ through calcium channels. Chelation of extracellular Ca2+ with EGTA partially prevents anti-CD77–induced apoptosis, indicating that this process is probably Ca2+ dependent. We show that the cross-linking of CD77 provokes an increase of intracellular cAMP levels followed by cAMP-dependent protein kinase activation. We report that BL cells produce ceramide when they are exposed to 38.13 but, unexpectedly, without a concomitant decrease in sphingomyelin or CD77 content. Finally, we provide evidence that C2-ceramide, calcium ionophore, and forskolin (which increases intracellular levels of cAMP) independently induce apoptosis of CD77+ BL cells and, moreover, that C2-ceramide and forskolin strongly synergize to cause cell death. The possible role of CD77-mediated apoptosis in the B cell selection that occurs in germinal centers is discussed.
Transforming Growth Factor-β1 Abrogates Fas-Induced Growth Suppression and Apoptosis of Murine Bone Marrow Progenitor Cells
Authors: Dybedal Ingunn , Guan Fengan , Borge Ole Johan , Veiby Ole Petter , Ramsfjell Veslemøy , Nagata Shigekazu , Jacobsen Sten Eirik W.
Published: October 2019

Fas, a member of the tumor necrosis factor (TNF ) receptor superfamily is a critical downregulator of cellular immune responses. Proinflammatory cytokines like interferon-γ (IFN-γ) and TNF-α can induce Fas expression and render hematopoietic progenitor cells susceptible to Fas-induced growth suppression and apoptosis. Transforming growth factor-β1 (TGF-β1 ) is an essential anti-inflammatory cytokine, thought to play a key role in regulating hematopoiesis. In the present studies we investigated whether TGF-β1 might regulate growth suppression and apoptosis of murine hematopoietic progenitor cells signaled through Fas. In the presence of TNF, activation of Fas almost completely blocked clonogenic growth of lineage-depleted (Lin−) bone marrow (BM) progenitor cells in response to granulocyte-macrophage colony-stimulating factor (GM-CSF ), CSF-1, or a combination of multiple cytokines. Whereas TGF-β1 alone had no effect or stimulated growth in response to these cytokines, it abrogated Fas-induced growth suppression. Single-cell studies and delayed addition of TGF-β1 showed that the ability of TGF-β1 to inhibit Fas-induced growth suppression was directly mediated on the progenitor cells and not indirect through potentially contaminating accessory cells. Furthermore, TGF-β1 blocked Fas-induced apoptosis of Lin− BM cells, but did not affect Fas-induced apoptosis of thymocytes. TGF-β1 also downregulated the expression of Fas on Lin− BM cells. Thus, TGF-β1 potently and directly inhibits activation-dependent and Fas-mediated growth suppression and apoptosis of murine BM progenitor cells, an effect that appears to be distinct from its ability to induce progenitor cell-cycle arrest. Consequently, TGF-β1 might act to protect hematopoietic progenitor cells from enhanced Fas expression and function associated with proinflammatory responses.
Normal Platelets and Megakaryocytes Are Produced In Vivo in the Absence of Thrombopoietin
Authors: Bunting Stuart , Widmer Ramon , Lipari Terry , Rangell Linda , Steinmetz Hope , Carver-Moore Karen , Moore Mark W. , Keller Gilbert-Andre , de Sauvage Frederic J.
Published: October 2019

Thrombopoietin (TPO) has been established as the major regulator of megakaryocyte and platelet production. In vitro and in vivo studies have demonstrated that TPO affects both megakaryocyte proliferation and maturation. In vitro, TPO has been reported to be essential for full development of megakaryocytes and platelets. These studies are in contrast to results observed in vivo in mice deficient in the TPO or c-mpl gene (TPO-/- and c-mpl-/-). Both TPO-/- and c-mpl-/- mice exhibit a 90% reduction in megakaryocyte and platelet levels. But even with this small number of circulating platelets, these mice do not have any excessive bleeding. Ultrastructural analysis indicates that platelets and megakaryocytes present in the knockout mice are morphologically normal. Characterization of platelet function shows that platelets from knockout mice are functionally identical to the wild-type platelets as measured by upregulation of 125I-fibrinogen binding to platelets in response to adenosine diphosphate (ADP) stimulation and by platelet attachment to the immobilized extracellular matrix proteins, collagen and von Willebrand factor (vWF). These results demonstrate that in vivo, TPO is required for the control of megakaryocyte and platelet number but not for their maturation. Other factors with megakaryocytopoietic activity may be able to compensate for the maturational role of TPO and lead to the formation of normal megakaryocytes and platelets in TPO-/- and c-mpl-/- mice.
Anti-CD43 Inhibits Monocyte-Endothelial Adhesion in Inflammation and Atherogenesis
Authors: McEvoy Leslie M. , Jutila Mark A. , Tsao Philip S. , Cooke John P. , Butcher Eugene C.
Published: October 2019

Recruitment of blood monocytes into tissues is a central event in the inflammatory response and in atherogenesis. The mechanisms leading to monocyte adhesion and migration through endothelium are not completely defined. We recently reported that MAb L11, against the leukocyte sialomucin CD43, blocks T-lymphocyte binding to lymph node and Peyer's patch high endothelial venules (HEV) and inhibits T-cell extravasation from the blood into organized secondary lymphoid tissues. We have now assessed the ability of L11 to inhibit monocyte-endothelial (EC) interactions and trafficking. L11 blocks binding of WEHI78/24 cells, a murine monocytoid cell line, to inflamed lymph node HEV and inhibits recruitment of monocytes and neutrophils to thioglycollate-inflamed peritoneum. Because monocyte adhesion to the endothelium and diapedesis in lesion-prone regions of the vasculature is among the earliest events in atherogenesis, leading to formation of lipid-laden foam cells, the ability of L11 to block monocyte recognition of aortic endothelial cells was assessed in a novel ex vivo assay of monocyte binding to intact rabbit aortic endothelium. Cholesterol feeding of rabbits induces enhanced aortic adhesiveness for monocytes and WEHI78/24 monocytoid cells, and this adhesion is inhibited by L11. The inhibitory effect of L11 is additive with that of a cocktail of anti–L-selectin and anti-α4 and β2 integrin monoclonal antibodies. Thus, CD43 represents a novel target for manipulation of monocyte recruitment in inflammation and atherogenesis.
A Monocyte Conditioned Medium Is More Effective Than Defined Cytokines in Mediating the Terminal Maturation of Human Dendritic Cells
Authors: Reddy Anita , Sapp Mark , Feldman Mary , Subklewe Marion , Bhardwaj Nina
Published: October 2019

Mature human dendritic cells can be generated in substantial numbers from nonproliferating progenitors in human blood using a two-step protocol. T cell–depleted mononuclear cells are first cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) and then exposed to monocyte conditioned medium (MCM). The dendritic cells generated using this approach are rendered terminally mature and are the most potent antigen presenting cells identified to date in humans. We sought to characterize factors in MCM that induce the terminal differentiation of dendritic cells. MCM contained substantial, although varying, quantities of several factors including tumor necrosis factor-α, IL-1β, IL-6, and interferon-α. However, none of the four factors, individually or in various combinations, could fully substitute for the MCM to generate irreversibly differentiated dendritic cells. The yields, percentage of cells expressing the mature phase marker CD83, and mixed leukocyte reaction–stimulatory function were lower when defined cytokines were used in the place of MCM. Therefore, the full maturation of dendritic cells, because it entails changes in many known cell and molecular properties, requires a number of different cytokines that are released in tandem from appropriately stimulated monocytes. We propose that MCM-matured dendritic cells will be the most effective adjuvants for immunotherapy in vivo.
Primary Anaplastic Large-Cell Lymphoma in Adults: Clinical Presentation, Immunophenotype, and Outcome
Authors: Tilly Hervé , Gaulard Philippe , Lepage Eric , Dumontet Charles , Diebold Jacques , Plantier Isabelle , Berger Françoise , Symann Michel , Petrella Tony , Lederlin Pierre , Brière Josette
Published: October 2019

Anaplastic, CD30+, large-cell lymphoma is now a well-recognized pathologic entity that accounts for 2% to 8% of all lymphomas. Recent progress has been made in the understanding of certain biologic features found in anaplastic large-cell lymphoma, but information about its clinical behavior, in comparison to other large-cell lymphomas, is limited. The pathologic review of a large multicenter study of the treatment of aggressive lymphoma identified 146 cases of anaplastic large-cell lymphoma (ALCL) on the basis of morphology and CD30 expression. We compared initial presentation, immunophenotype, and clinical outcome of these cases with those of the 1,695 nonanaplastic diffuse large-cell lymphomas (non-ALCL) included in the same trial. Patients with ALCL were more likely to be male (P = .018) and were younger (P < .0001) than those with non-ALCL. B symptoms were more frequent in ALCL (P = .006). Skin (P < .0001) and lung (P < .05) involvement was also more frequent in ALCL, but frequency of bone marrow involvement was identical (P = .5). Tumor cell phenotype was B in 56 cases (38%), T in 49 cases (34%), and null in 33 cases (22%). Response to chemotherapy (P = .001), event-free survival (P = .006), and overall survival (P = .0004) were better for ALCL than for non-ALCL. Multivariate analyses identified anaplastic character as an independent factor that predicted a longer survival. Tumor cell phenotype did not influence event-free survival (P = .72) or overall survival (P = .83). ALCL in adults is a clinicopathologic entity which, independent of its phenotypic characteristics, has a better outcome than other diffuse large-cell lymphomas.