bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic Applications for Transfusion Support
Authors: Chou Stella
Published: October 2019

Red blood cell (RBC) and platelet antigen determination by DNA methods (genotyping) has become an important part of the practice of transfusion medicine over the past decade.The results are reliable and highly correlated with serologic phenotyping results, and are superior in some situations. Application of RBC genotyping to provide more precise transfusion therapy for various patient populations will be discussed, with a particular emphasis on those requiring chronic RBC therapy or with complex serologic reactivity. Current DNA arrays can test for a large number of blood group antigens, but routine typing of the ABO and Rh systems is not currently feasible due to the large number of genetic mutations.

Patients with sickle cell disease (SCD) are one of the most challenging populations to safely transfuse, in part due to high rates of alloimmunization against Rh antigens despite prophylactic matching for D, C, and E antigens. Genotyping of the RH loci has revealed that RHD and RHCE variants are prevalent in African Blacks. Nearly 90% of patients with SCD have at least one altered RH gene and contributes to Rh alloimmunization in patients with SCD. The role of RH variation and alloimmunization in SCD will be reviewed, and the feasibility and approach to genetic RBC matching of patients and donors will be discussed. The use of targeted DNA arrays, whole exome sequencing, or whole genome sequencing to identify common and variant RH alleles will be addressed, including limitations of each method.

Disclosures

No relevant conflicts of interest to declare.
Transcription Factor Gene Fluorescent Reporters Track Lineage Fate in Lymphoid Commitment
Authors: Rothenberg Ellen
Published: October 2019

The transition from multipotency to lineage commitment can be followed with particular clarity for T cell precursors. In this lineage, the role of environmental signals can be clearly separated from the role of intrinsic fate programming in individual cells and the cells' developmental responses to changing conditions and can be tracked in real time. T cell precursors are still multipotent when they first enter the thymus, and if they are removed from the thymic microenvironment at this stage they can give rise to non-T cells including dendritic cells and myeloid cells. For multiple cell divisions, they preserve this multipotency and are only kept in line to become T cells conditionally, by Notch signaling from the thymic stroma. Then at a specific point of no return, the cells become unable to give rise to anything except T cells regardless of environment, and this is the point of commitment. Commitment is clearly the readout of a change in internal transcriptional regulatory state. To determine how this is controlled, we and others have charted transcription factor expression changes across this interval and changes in chromatin modification and DNA accessibility that accompany the transition, and we have been able to use functional perturbation tests to narrow down the key regulators that catalyze and enforce this transition. A particularly important commitment factor is encoded by the Bcl11b gene, which is released from previously repressed chromatin and sharply activated at the transcriptional level just as the cells become committed. The Bcl11b gene product is required in all alpha beta and most gamma delta T cells to enable the commitment process to occur. These properties make it highly illuminating as an indicator of the regulatory state in individual differentiating T-cell precursors. We have generated a series of knock-in Bcl11b fluorescent reporter alleles to probe the correlation of Bcl11b expression with changes in specific target genes, to determine the transcription factor requirements for Bcl11b gene activation in the gene regulatory network controlling commitment in single cells, and to measure the role of epigenetic modification of the Bcl11b locus on the kinetics of transition from uncommitted to committed states. These results and their implications will be presented. Importantly, the use of live-cell reporters reveals a level of all-or-none, stochastic regulation in the responses of individual cells to combinatorial transcription factor action at this developmental watershed1.

Reference:

1. Kueh HY, Yui MA, Ng KKH, et al. Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment. Nature Immunology. 2016.17, 956-965.

Disclosures

No relevant conflicts of interest to declare.
Marrow Infiltrating Lymphocytes and Vaccines in Myeloma
Authors: Borrello Ivan
Published: October 2019

Despite several decades of research with limited clinical successes, immunotherapy is now establishing a permanent presence in the clinical arena for several malignancies including multiple myeloma. The ability to understand the mechanisms mediating tumor-induced tolerance as well as the availability of reagents to target them have facilitated the clinical development of several immune-based strategies. The efficacy of adoptive cell therapies has already been demonstrated by the clinical benefit observed with allogeneic bone marrow transplantation where the immune-mediated graft vs. tumor effect accounts for the deeper and more durable clinical responses when compared to autologous stem cell transplantation. Recently, adoptive T cell approaches have focused on ex vivo manipulation of cells with the goal of increasing their tumor specificity. To date, the approaches can be divided into three groups: unmodified cells; T cells expressing transgenic T cell receptors; and cells modified with chimeric antigen receptors (CARs). Each has its own benefits. Historically, adoptive T cell therapy utilized peripheral blood lymphocytes in large part because of their ease of access. Early studies in myeloma demonstrated their ability to augment immune reconstitution and effectively transfer vaccine-specific responses. However, their lack of tumor specificity has prompted our group to evaluate the role of marrow infiltrating lymphocytes (MILs). These cells possess a broad endogenous tumor specificity and are enriched for central memory T cells that make them a more suitable source of cells for adoptive T cell approaches. The properties and potential benefits of this approach will be discussed.

Another immunotherapeutic approach with potential appeal is the use of myeloma vaccines to address the issue of minimal residual disease. In light of the fact that patients with myeloma rarely have long-term sustained remissions, it is reasonable to integrate a novel approach that is capable of eradicating the remaining disease burden by generating a broad, myeloma-specific, systemic immune response. We have developed a cellular, GM-CSF-based, myeloma vaccine which has been administered in the combination with the immunomodulatory agent, lenalidomide, to patients in a near complete remission (negative M-spike but with a positive immunofixation). We have demonstrated priming of a tumor-specific immune response which correlates with a durable clinical benefit.

With the ever-increasing development of immune-based approaches, combining them with various agents in different disease setting to improve the overall outcomes of myeloma patients.

Disclosures

Borrello: Celgene: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding.
The Effects of Splenectomy on the Red Blood Cells of the Dog with Particular Emphasis on the Reticulocyte Response
Authors: LORBER MORTIMER
Published: October 2019

In splenectomized dogs the following observations on the erythroid elements were made:

1. There was no unusual postoperative depression of erythrocyte, hemoglobin or hematocrit values, and regeneration occurred at the same rate as in the control dogs.

2. A significant increase in the number of circulating reticulocytes was noted, which persisted for a longer duration than the associated thrombocytosis. There is a suggestion that the reticulocytes circulating during regeneration were more immature than those of the control animals.

3. Varying numbers of target cells accompanied by increased osmotic resistance were present in all the splenectomized dogs. Most also developed normoblastemia, particularly during active regeneration, which persisted thereafter in some of the animals. Howell-Jolly bodies were rare.

4. No significant bone marrow changes were noted.

It is suggested that:

1. In nonsplenectomized animals, most erythrocytes released from the bone marrow in response to blood loss are mature red cells rather than reticulocytes.

2. Postsplenectomy reticulocytosis is not dependent on a fall in hematocrit and is not a reflection of hemolysis or even of increased erythropoiesis, except, perhaps, in part during regeneration from the blood loss incurred at surgery.

3. Postsplenectomy reticulocytosis and normoblastemia are unaccompanied by alterations in the myeloid-erythroid ratio of the bone marrow and are probably largely due to a diminution of the normal inhibition of erythroid release from the marrow, permitting their earlier entry into the circulation.

4. The aforementioned changes are due specifically to the removal of the spleen itself, rather than merely to the removal of splenic blood from the portal circulation.
Modification of X. radiation Lethality by Autologous Marrow Infusion in Dogs
Authors: ALPEN EDWARD L. , BAUM S. J.
Published: October 2019

Dogs have been treated after x-irradiation with doses of 400 r and 600 r by injection of either homologous or autologous bone marrow. The bone marrow was obtained by preirradiation biopsy and was reinjected either into the animal from which it was drawn (autologous) or into an alternate recipient (homologous). Injection of 2 x 109 autologous nucleated cells successfully protected all dogs given either 400 r or 600 r. Dogs given 1 x 109 autologous nucleated cells after 600 r failed to survive. No protection was provided by injection of 2 x 109 homologous nucleated cells after 400 r nor by injection of 2 x 109 or 5 x 109 homologous nucleated cells after 600 r. It is proposed that a differential species sensitivity to foreign cell implants accounts for the failure of homologous marrow infusions in dogs in contradistinction to the protection provided by homologous or heterologous marrow implants in rats and mice, and by homologous implants in monkeys.
Hematopoietic and Nitrogen-Sparing Effect of Preparations from Hog Gastric Mucosa with High Content of Blood Group Substances
Authors: CRESSERI ANGELO , CANTONE AURELIO , PICCININI FRANCO , CAPRARO VITTORIO
Published: October 2019

A mucin fraction with high content of A and H blood group substances was obtained from hog gastric mucosa. This fraction administered per os together with vitamin B12 in rats made anemic by phenylhydrazine injection shows a significant effect on the hematologic response in comparison with the animal treated with vitamin B12 alone.

The same fraction administered per os in cortisone-treated rats has also a significant nitrogen sparing action.

The possible mechanisms and relationships of the observed biologic effects are briefly discussed.
An In Vitro Technic for Quantitating and Studying the Dynamics of Leukocyte Agglutination
Authors: HARTMAN JOHN D.
Published: October 2019

1. An in vitro technic for quantitating leukocyte agglutination has been described.

2. With this technic the dynamics of leukocyte agglutination and the response of leukocytes to antigenic agents in both sensitive and nonsensitive systems have been studied.

3. The relationship of this study to the problem of agranulocytosis is discussed.
Enhanced Urinary Excretion of Co60 Vitamin B12 Produced by Delayed Release Capsules
Authors: JOHNSON P. C. , BERGER E. S.
Published: October 2019

1. Patients without known hematologic disease were given 8 µg. (0.2 µc. Co60 vitamin B12 as a water solution or in various types of delayed release capsules.

2. All groups showed approximately equal fecal excretion. However, the cobalt60-labeled vitamin B12 delayed release capsules showed a greater urinary excretion after a loading dose of stable B12 than did the water solution of labeled vitamin B12.
Observations on the Anemia of Cryptogenetic Splenomegaly. I. Hemolysis
Authors: MCFADZEAN A. J. S. , TODD DAVID , TSANG K. C.
Published: October 2019

Excessive hemolysis has been shown to occur in patients with cryptogenetic splenomegaly. This is corrected by splenectomy.

The survival of normal red cells transfused to such patients is greatly reduced. Following splenectomy normal red cells so transfused survive normally.

The survival of red cells from such patients upon transfusion to healthy recipients, including a splenectomized recipient, is greatly reduced. Following splenectomy the survival of red cells from such patients upon transfusion to healthy recipients is significantly increased, although still significantly less than normal. However, upon transfusion into splenectomized but otherwise healthy recipients the survival time is normal.

Study of osmotic fragility before splenectomy demonstrates the existence of a mixed population showing both increased and decreased fragility to hypotonic saline. Following splenectomy there emerges a population with increased resistance to hypotonic saline.

It is concluded that prior to splenectomy the pathologic spleen exerts an effect upon circulating erythrocytes including transfused normal red cells which results in marked reduction in their survival. Following splenectomy a second defect in the erythrocytes becomes apparent and this also results in reduction in life-span, although the presence of the spleen would appear necessary for this reduction to occur. It is suggested that this second defect develops as the result of liver damage.
Calcium-Lipid Complexes in Human Platelets
Authors: WALLACH DONALD F. HOELZL , SURGENOR DOUGLAS M. , STEELE BARBARA B.
Published: October 2019

Carefully washed human platelets contain calcium in an unusual state of chemical combination. Platelet calcium is non-ionic and is not exchangeable with ionic Ca45. On extraction of platelets with lipid solvents, the calcium separates with the phospholipids. The calcium-lipid complex thus obtained does not undergo any exchange with radioactive calcium. Study of other properties of platelets reveals that the clot-promoting activity of platelets (activated by antihemophilic factor and plasma thromboplastin component) also separates into the lipid fraction. Preliminary observations are reported on the nature of platelet lipids as revealed by paper chromatography and paper electrophoresis in solvents of low dielectric constant. Small amounts of proteolipid have been isolated from the lipid extracts. The unique calciumlipid complex is thought to originate in the cell membrane of the platelet.
The Effect of Heparin on Components of the Human Fibrinolytic System
Authors: VON KAULLA KURT N. , MCDONALD TOM S.
Published: October 2019

It was demonstrated with human urokinase, human fibrinolytic euglobulins and human pyrogen plasma as enzyme source, and with bovine fibrin plates and human plasma plates as substrate, that high heparin concentrations inhibit fibrinolysis of preformed fibrin, in contrast to small ones which may enhance this phenomenon, provided albumin is present.

No interference could be observed in the absence of plasma, serum or albumin fraction. Albumin or substances associated with the albumin fraction are required for interference of heparin with fibrinolysis. This interference probably takes place with the action of plasmin on fibrin.
Acquired hypoprothrombinemia due to non-neutralizing antibodies to prothrombin: mechanism and management
Authors: Bajaj SP , Rapaport SI , Barclay S , Herbst KD
Published: October 2019

A patient developed bleeding due to an acute acquired specific prothrombin deficiency. Unlike previously described patients, this patient had no evidence of an associated lupus anticoagulant. Prothrombin activity and antigen were decreased concordantly and the patient's plasma did not neutralize the activity of added prothrombin or interfere with its measurement by electroimmunoassay. Nevertheless, immunoelectrophoresis and experiments using 125I-prothrombin revealed a prothrombin-binding antibody. The residual prothrombin in the patient's plasma was in the form of a prothrombin-antibody complex. Administration of adrenal corticosteroids was associated with a rise in prothrombin activity and cessation of bleeding, but circulating prothrombin was still bound to the antibody. This suggests that non- neutralizing antibodies to prothrombin cause plasma prothrombin deficiency because of a rapid clearance of prothrombin-antibody complexes, which is slowed by adrenal corticosteroids. The antibody had a relatively low affinity for prothrombin (Kd 5 to 8 X 10(-7)) and was transient. It is possible, therefore, that the antibody arose not to prothrombin itself, but to an antigen sharing an epitope with prothrombin.
Pretransplant detection of human minor histocompatibility antigen- specific naive and memory interleukin-2-secreting T cells within class I major histocompatibility complex (MHC)-restricted CD8+ and class II MHC-restricted CD4+ T-cell subsets
Authors: Theobald M , Bunjes D
Published: October 2019

Recent studies have shown that host-reactive interleukin-2 (IL-2)- secreting donor T lymphocytes (TI) are critically involved in the development of acute graft-versus-host disease (GVHD) after allogeneic HLA-identical sibling bone marrow transplantation (BMT). To further characterize the responding TI, we determined the frequency of pretransplant IL-2-secreting TI-precursors (TI-p) between eight HLA-A, - B, -C, -DR, and -DQ-identical sibling donor-host pairs in both the graft-versus-host (GVH) and the host-versus-graft (HVG) direction. High frequencies of pretransplant host-reactive donor TI-p (1/18,000 to 1/49,000) were detectable in five patients with grade II acute GVHD. Donor-reactive host TI-p (1/3,700 to 1/31,000) were observed in previously in vivo primed (n = 5) and unprimed (n = 1) patients. In two pairs tested after previous in vivo priming, pretransplant donor- reactive host TI-p were highly enriched within the CD45RO+ memory T- cell subset. Previously unprimed host-reactive donor TI-p occurred in almost equal frequencies within CD45RO+ and CD45RO- T cells. Both CD4+ and CD8+ T-cell subsets contributed in comparable frequencies to host- and donor-reactive TI-p. Recognition of minor histocompatibility (mH) antigens by CD8+ TI-p appeared to be class I major histocompatibility complex (MHC)-restricted, whereas CD4+ TI-p operated in a class II (HLA- DR) MHC-restricted fashion. Even between oligonucleotide-defined HLA- DPB1-disparate sibling donor-host pairs (n = 3), either responding T- cell subset was found to recognize cellularly defined mH antigens. These data indicate that various T-cell subsets contribute to host- and donor-reactive IL-2-secreting TI in allogeneic sibling BMT.
Effects of the recombinant hematopoietic growth factors interleukin-3, interleukin-6, stem cell factor, and leukemia inhibitory factor on the megakaryocytic differentiation of CD34+ cells
Authors: Debili N , Masse JM , Katz A , Guichard J , Breton-Gorius J , Vainchenker W
Published: October 2019

Using a liquid culture system and human CD34+ marrow cells, we examined the effects of recombinant interleukin (IL)-3, IL-6, stem cell factor (SCF), and leukemia inhibitory factor (LIF) on megakaryocyte (MK) growth, endoreplication, and maturation. MK proliferation, ploidy distribution, and volume were studied by flow cytometry. IL-3 was the only cytokine that, alone, induced a marked increase in MK proliferation. At a high CD34+ cell concentration, addition of IL-6, SCF, and LIF to IL-3--containing medium increased the number of MK (approximately 20%). At a low CD34+ cell concentration, IL-3 alone was a less potent inducer of MK growth, but IL-6, SCF, and their combination had a marked effect, increasing the number of MK by a factor 1.7, 2.9, and 4.4, respectively. These differences may be related to the endogenous release of cytokines in the culture. The effects of these cytokines were subsequently tested on a more mature type of MK progenitor (CD34+ cells isolated after 6 days of incubation in liquid culture). IL-3 remained the most potent cytokine, but IL-6 or SCF alone also increased MK number in comparison to unstimulated cultures. The ploidy distribution of MKs grown with IL-3 was not markedly changed by the addition of the other cytokines, with the exception of SCF, which induced a significant increase in the mean ploidy. However, in all cultures, glycoprotein (GP)IIIa+ 2N and 4N cells were present in large but variable numbers (35% to 75%). The number of these low-ploidy MKs directly correlated with MK proliferation. Therefore, we subsequently explored the absolute number of polyploid MK produced in culture. SCF, IL-6, or their combination, in association with IL-3, increased the number of polyploid MK up to fourfold. In addition, they improved the maturation of MK grown in the presence of IL-3, leading to the synthesis of demarcation membranes and platelet shedding. A similar effect of growth factors on the maturation of day 6 CD34+ cells was observed. We conclude that IL-6 and SCF have a broad range of activities on megakaryocytopoiesis, acting both on the early and late stages. However, the proliferative properties of these cytokines largely predominate in our cultures. Therefore, in the absence of a specific MK regulator, this study further extends the need for a combination of growth factors to maximize megakaryocytopoiesis.
Prevalence and clinical correlations of MLL gene rearrangements in AML- M4/5
Authors: Bower M , Parry P , Carter M , Lillington DM , Amess J , Lister TA , Evans G , Young BD
Published: October 2019

Rearrangements of the human trithorax gene (MLL, HRX, Htrx-1, All-1) were studied by Southern blotting in blast cells stored at presentation from 65 adults with de novo acute myelomonocytic (AML-M4) and acute monocytic leukemia (AML-M5). MLL rearrangements were demonstrated in 15 (23%) cases, including eight patients in whom karyotype analysis had failed to detect abnormalities of chromosome band 11q23. The patients with MLL rearrangements did not differ significantly from those with germline configurations in terms of the sex and age of the patients, the presence of lymphadenopathy, hepatosplenomegaly, or central nervous system involvement, and the absolute blast count at diagnosis. Kaplan- Meier analysis of the treated patients demonstrated no difference in survival for patients with MLL rearrangements compared with those without rearrangements. Therefore, in contrast to infantile acute leukemia, in adults with AML-M4 and AML-M5, MLL rearrangements do not identify a subgroup of patients with different clinical features or prognosis.
Changes in G protein pattern and in G protein-dependent signaling during erythropoietin- and dimethylsulfoxide-induced differentiation of murine erythroleukemia cells
Authors: Kesselring F , Spicher K , Porzig H
Published: October 2019

We have studied the expression of G protein subtypes and the role of G protein-dependent signaling in two subclones of RED-1 cells, an erythropoetin(Epo)-sensitive, murine erythroleukemia cell line. Clone 6C8 showed terminal erythroid differentiation in response to a combined treatment with Epo and dimethylsulfoxide. Clone G3 was resistant to these inducers, but responded to Epo with enhanced proliferation. We measured G protein alpha subunit levels by toxin-catalyzed adenosine diphosphate (ADP)-ribosylation with [32P]-nicotinamide adenine dinucleotide (NAD) and by semiquantitative immunoblotting with specific antisera. Native RED-1 cells expressed G alpha i2, alpha i3, alpha s, and alpha q/11, but not alpha i1 and alpha o. Terminal differentiation was associated with a selective loss (approximately 80%) of G alpha i3 and an increase in a truncated cytosolic form of G alpha i2, while the membrane levels of alpha i2, alpha q/11, and alpha s did not change significantly. Treatment of G3 cells with the inducers was without effect on G protein abundance. However, except for alpha s, G3 cells contained significantly higher levels of the different G protein alpha subunits tested. Stimulation of G protein-coupled receptors by thrombin and ADP caused a pertussis toxin (PTX)-inhibitable transient increase in intracellular Ca2+ that was markedly reduced in differentiated cells. In G3 cells, but not in 6C8 cells, thrombin also caused a PTX- sensitive inhibition of isoprenaline-stimulated cyclic 3',5'-adenosine monophosphate (cAMP) formation. Our results show that specific alterations in G protein expression and function are associated with erythroid differentiation of erythroleukemia cells but do not prove a causal relationship. The loss of G alpha i3 may affect cellular responses that are mediated via P2T purine or thrombin receptors.
Interleukin-1 induces interleukin-8 secretion from endothelial cells by a juxtacrine mechanism
Authors: Kaplanski G , Farnarier C , Kaplanski S , Porat R , Shapiro L , Bongrand P , Dinarello CA
Published: October 2019

Inflammation is characterized by migration of neutrophils through the endothelium, and the chemokine interleukin-8 (IL-8) appears to be involved. We asked whether adherence of cells bearing a membrane-form of interleukin 1 (IL-1) induces IL-8 secretion from human umbilical vein endothelial cells (HUVEC) and fibroblasts. Human peripheral blood mononuclear cells (PBMC) were stimulated with endotoxin for 12 hours and then fixed for 4 hours with paraformaldehyde. When these cells were added to HUVEC or fibroblasts, IL-8 production was induced. This stimulation by fixed PBMC was attributed to IL-1, because pretreatment of HUVEC or fibroblasts with IL-1 receptor antagonist (IL-1Ra) reduced the induction by 95% and 80%, respectively, P < .005. Using anti-IL-1 alpha monoclonal antibodies, reduction was complete, whereas anti-IL-1 beta had no effect. IL-1 alpha was shown on the surface of monocytes by fluorescence-activated cell sorter (FACS) analysis. Blockade of IL-1 receptors on PBMC did not affect the activity of membrane-associated IL- 1 alpha, indicating that IL-1 is not anchored to the membrane through its receptors. However, PBMC treated with D-mannose before fixation resulted in a loss of activity; this loss of activity was associated with release of IL-1 alpha, not IL-1 beta, into the supernatant. Thus, anchoring of IL-1 alpha to the membrane may be via a lectin or mannose receptor-like interaction. Blockade of membrane IL-1 alpha required a 30-fold and fivefold excess of IL-1Ra compared with the amount required to block soluble IL-1 beta and IL-1 alpha, respectively. We conclude that the fixed PBMC IL-8 inducing activity is almost entirely caused by IL-1, that this represents IL-1 alpha bound to a surface lectin or mannose receptor on the monocyte, and that it functions in inflammation via juxtacrine interactions.
Early myeloablative therapy for multiple myeloma
Authors: Alexanian R , Dimopoulos MA , Hester J , Delasalle K , Champlin R
Published: October 2019

The value of early myeloablative therapy supported by autologous bone marrow or blood progenitor cells was assessed in 72 patients with multiple myeloma who were treated within 1 year of initial therapy. Forty-five patients were consolidated during remission, and 27 patients were treated for primary refractory disease. Outcomes were compared with those of similar patients who did not receive intensive treatment primarily for socioeconomic reasons. Among patients who had responded previously, myeloablative therapy increased the rate of complete remission from 5% to 45% (P < .01) but did not prolong progression-free intervals or survival times. The same treatment controlled the myeloma in 70% of patients with primary resistant disease and prolonged the median survival from 37 to 83 months (P = .03). Intensive treatment for primary resistant myeloma administered later in the disease course resulted in significantly lower response rates and shorter progression- free intervals. Current myeloablative regimens supported by autologous stem cells appeared useful primarily in patients with primary resistant disease during the first year of therapy.
Identification of 15 different candidate causal point mutations and three polymorphisms in 19 patients with protein S deficiency using a scanning method for the analysis of the protein S active gene
Authors: Gandrille S , Borgel D , Eschwege-Gufflet V , Aillaud M , Dreyfus M , Matheron C , Gaussem P , Abgrall JF , Jude B , Sie P
Published: October 2019

To screen for point mutations causing protein S deficiency, we used a sequence of techniques specifically for the study of the protein S active gene, PS alpha. This strategy comprises amplification of exons and intron/exon junctions by means of the polymerase chain reaction (PCR) and electrophoresis of the amplified fragments in polyacrylamide gel containing a gradient of denaturing agents (denaturing gradient gel electrophoresis). Only fragments with altered melting behavior are sequenced after asymmetric PCR. Beside the frequent polymorphism already described on Pro 626, we detected 18 different sequence variations by studying exons II, IV, V, VIII, X, and XV in 19 of 100 consecutive patients with protein S deficiency. Fifteen were candidate causal mutations, 4 of which were associated with a qualitative deficiency (type IIa or IIb). The remaining three sequence variations were probably polymorphisms.
Heterotypic adherence between human B-lymphoblastic and pre-B- lymphoblastic cells and marrow stromal cells is a biphasic event: integrin very late antigen-4 alpha mediates only the early phase of the heterotypic adhesion
Authors: Patrick CW Jr , Juneja HS , Lee S , Schmalstieg FC , McIntire LV
Published: October 2019

Heterotypic adherence between marrow stromal cells (MSC) and lymphoblastic cells is essential for normal lymphopoiesis and malignant lymphoblastic development. However, the detailed molecular mechanisms by which this heterotypic adherence occurs are poorly understood. The cell-cell interactions between a B-lymphoblastic cell line (UTMB-460) and a pre-B-cell line (NALM-6) with MSC were chosen as models to investigate potential mechanisms and adhesion molecules involved in the apposition between normal and malignant lymphoblastic cells and MSC. A parallel-flow detachment assay (PFDA) and a 51Cr detachment assay, coupled with monoclonal antibody (MoAb) blocking experiments, were used to quantify the attachment of lymphoblastic cells to confluent monolayers of MSC. The apposition between MSC and B-lymphoblastic cells (UTMB-460 cells) was investigated for variable time periods, ranging from 1 minute to 4 hours. Results from the temporal study suggest that the heterotypic adherence of the B-lymphoblastic cells to MSC is a biphasic event and the interactions occur rapidly (< or = 1 minute) after the two cells come into contact. More specifically, the early phase of adherence (< or = 15 minutes) solely involves very late antigen-4 alpha (VLA-4 alpha)/vascular cell adhesion molecule 1 (VCAM- 1) interactions, as evidenced by the nearly complete inhibition (93%) of UTMB-460 cell adherence in the presence of anti-VLA-4 alpha. The late phase (> or = 30 minutes) proceeds despite the continuous presence of anti-VLA-4 alpha. In addition, the late-phase adherence is not affected by MoAbs to LFA-1, CD44, VCAM-1, E-selectin, or L-selectin, which suggests the possible involvement of other adhesion molecules. Adherence of pre-B-lymphoblastic cells (NALM-6) to MSC is also biphasic. Integrin VLA-4 is again a major player in the early phase of pre-B-lymphoblastic cell/MSC interactions. The early phase of adherence may be important in homing of the malignant lymphoblastic cells to the MSC and the late phase in retention of malignant lymphoblastic cells in the bone marrow.
Soluble kit receptor in human serum
Authors: Wypych J , Bennett LG , Schwartz MG , Clogston CL , Lu HS , Broudy VC , Bartley TD , Parker VP , Langley KE
Published: October 2019

c-kit encodes the transmembrane receptor tyrosine kinase (Kit) for the recently described ligand stem cell factor (SCF). We have developed an enzyme-linked immunosorbent assay for measuring soluble human Kit and we have used the assay to show high levels of soluble Kit in human serum. The distribution of soluble Kit levels was investigated among 112 normal human serum donors. The mean serum level (+/- SD) was found to be 324 +/- 105 ng/mL with the values falling between 163 ng/mL and 788 ng/mL. No correlation between soluble Kit levels and the sexes or ages of the donors was found. Partial purification using immunoaffinity chromatography allowed us to characterize the soluble Kit from pooled human serum. Antibodies generated to a 497-amino acid recombinant human soluble Kit corresponding to the N-terminal extracellular domain of the receptor recognized the serum-derived soluble Kit by immunoblotting. We found that the serum-derived soluble Kit is glycosylated, with mostly N- linked but also O-linked carbohydrate, and with terminal sialic acid residues. When compared with the recombinant human soluble Kit, the serum-derived material was similar both in size and glycosylation pattern. CNBr cleavage of the isolated serum-derived material followed by amino terminal sequencing confirmed the presence of five peptides expected for the extracellular portion of the Kit molecule. The immunoaffinity purified serum-derived soluble Kit inhibited binding of [125I]SCF to membrane-bound receptor in an in vitro assay. These results indicate that soluble Kit could modulate the activity and functions of SCF in vivo.
Granulocyte-macrophage colony-stimulating factor expression by human fibroblasts is both upregulated and subsequently downregulated by interleukin-1
Authors: Patil RR , Borch RF
Published: October 2019

Interleukin-1 (IL-1) treatment of human WI-38 lung fibroblasts results in granulocyte-macrophage colony-stimulating factor (GM-CSF) expression as well as a delayed increase in prostaglandin E2 (PGE2) production that closely correlates with the decline of GM-CSF mRNA levels. Pretreatment with PGE2 reduces the IL-1 induced GM-CSF mRNA and protein expression to 10% to 15% of control values at concentrations of PGE2 that are endogenously produced after IL-1 stimulation. Inhibition of PGE2 synthesis by indomethacin prolongs the IL-1 induced GM-CSF mRNA expression and increases the cumulative GM-CSF protein secretion. Exposure of WI-38 fibroblasts to PGE2 results in an increase in intracellular cyclic adenosine monophosphate (cAMP) levels. The inhibition of GM-CSF expression by PGE2 can be mimicked by stable cAMP analogs as well as cAMP elevating agents such as cholera toxin, forskolin, and isobutylmethylxanthine. Thus the inhibition exerted by PGE2 is mediated via cAMP. Taken together, these results suggest that IL-1 stimulation of human fibroblasts provides not only the upregulatory signal for GM-CSF expression but also a delayed and indirect downregulatory signal that serves to limit GM-CSF expression in the continued presence of IL-1.
Detection of homozygous deletions of the cyclin-dependent kinase 4 inhibitor (p16) gene in acute lymphoblastic leukemia and association with adverse prognostic features
Authors: Fizzotti M , Cimino G , Pisegna S , Alimena G , Quartarone C , Mandelli F , Pelicci PG , Lo Coco F
Published: October 2019

A recently described putative tumor suppressor gene, the cyclin-dependent kinase 4 inhibitor (p16), has been shown to be altered by deletions and/or point mutations in various human cancers. To assess the incidence and clinico-biologic correlations of p16 homozygous deletion in hemopoietic tumors, we studied a panel of 244 DNA samples representative of distinct acute (99 cases) and chronic (57 cases) leukemia subtypes, myelodysplastic (22 cases) and myeloproliferative (15 cases) syndromes, and lymphomas (51 cases). A 361-bp probe complementary to the p16 exon 2 gene sequences was generated by polymerase chain reaction and used in Southern blot hybridization against these tumor DNAs. Homozygous deletions of p16 (p16-/-) were detected in 10 of 58 (17%) cases of acute lymphoblastic leukemia (ALL) of either B or T lineage and in no other tumors. Single-strand conformation polymorphism analysis of p16 exons 1 and 2 was also performed in 40 of the 58 ALL cases and in 16 lymphomas. In no cases were point mutations detected. The comparison of clinical features at presentation in p16-/- and in p16 germline ALL cases showed a greater leukemic cell mass (P = .001) and higher white blood cell counts (P = .01) in the former group. Two ALL cases in which diagnostic and relapse DNA samples were available showed p16-/- in both specimens. We conclude that homozygous p16 gene deletions characterize a subset of ALL with features of aggressive disease.
The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit
Authors: Kuter DJ , Rosenberg RD
Published: October 2019

Thrombopoietin (c-Mpl ligand) has recently been purified and is considered to be the humoral regulator of platelet production. To see whether this molecule possessed the physiologic characteristics necessary to mediate the feed-back loop between blood platelets and the bone marrow megakaryocytes, we determined the relationship between blood levels of thrombopoietin and changes in the circulating platelet mass. We developed a model of nonimmune thrombocytopenia in rabbits by the subcutaneous administration of busulfan. Compared with pretreatment plasma, plasma taken from all thrombocytopenic rabbits at their platelet nadir contained increased amounts of thrombopoietin. All of this activity was neutralized by soluble c-Mpl receptor. We subsequently measured the level of thrombopoietin in the circulation over the entire time course after the administration of busulfan. As the platelet mass declined, levels of thrombopoietin increased inversely and proportionally and peaked during the platelet nadir. With return of the platelet mass toward normal, thrombopoietin levels decreased accordingly. When platelets were transfused into thrombocytopenic rabbits near the time of their platelet count nadir, the elevated levels of thrombopoietin decreased. In addition, platelets were observed to remove thrombopoietin from thrombocytopenic plasma in vitro. These results confirm that thrombopoietin is the humoral mediator of megakaryocytopoiesis and suggest that the platelet mass may directly play a role in regulating the circulating levels of this factor.
Autologous stem cell transplantation after first remission induction treatment in multiple myeloma: a report of the French Registry on autologous transplantation in multiple myeloma
Authors: Harousseau JL , Attal M , Divine M , Marit G , Leblond V , Stoppa AM , Bourhis JH , Caillot D , Boasson M , Abgrall JF
Published: October 2019

Eighteen French centers reported 133 autologous stem cell transplantations performed after first remission induction in multiple myeloma. The source of stem cell was marrow (81 cases), blood (51 cases) or marrow plus blood (1 case). The immediate outcome after transplantation was 49 (37%) complete remissions (CRs) (13 maintained, 36 achieved), 61 (46%) partial remissions, 17 failures and 5 toxic deaths. With a median follow-up of 35 months, the median remission duration was 33 months, the median time to treatment failure was 22 months. The median survival was 46 months overall, 54 months for the 103 patients responding to primary treatment, and 30 months for the 30 nonresponders. In univariate analysis, the outcome was influenced by age, Ig isotype, initial beta 2 microglobulin level, response to initial chemotherapy, plasma cell marrow involvement at the time of harvest, albumin and beta 2 microglobulin level at the time of transplantation, and CR achievement after transplantation. In multivariate analysis, the most important prognostic factor was the quality of response after transplantation. The conditioning regimen and the source of stem cell had no significant impact on immediate and long-term results. Maintenance therapy with interferon alpha did not appear to prolong remission duration or survival. Autologous stem cell transplantation is an effective consolidation for patients responding to primary treatment and a salvage therapy for some nonresponding patients. This approach has to be compared to conventional chemotherapy in prospective randomized studies. The critical impact of CR achievement on survival implies new strategies to increase the CR rate.
A four-hour infusion of recombinant hirudin results in long-term inhibition of arterial-type thrombosis in baboons
Authors: Kotze HF , Lamprecht S , Badenhorst PN
Published: October 2019

Intravenous recombinant (r)-hirudin has a potent antithrombotic effect in aspirin- and heparin-resistant platelet-dependent thrombus formation in baboon models. However, these thrombi reform when therapy is stopped after 60 minutes. To determine if 4 hours of therapy can produce a lasting antithrombotic effect, we investigated the extent of deposition of 111In-labeled platelets onto 0.5-cm2 segments of Dacron vascular grafts for 53 hours. These grafts had been incorporated into exteriorized permanent femoral arteriovenous shunts in baboons. Platelet deposition in eight untreated animals was generally sigmoidal. Maximum platelet deposition, 1.7% +/- 0.9% of injected labeled platelets, was reached after approximately 4 hours. Deposition then gradually decreased to 0.4% +/- 0.2% of injected labeled platelets after 53 hours. After a thrombus was allowed to form for 15 minutes in six animals, intravenous treatment with r-hirudin at a dose of 20 nmol (0.14 mg)/kg-min-1 (aPTT > 300 seconds) was started and maintained for 4 hours. Platelet deposition was interrupted during treatment. After infusion was stopped, platelets accumulated again, but not as much as in the untreated animals. Maximum platelet deposition, 0.7% +/- 0.2% of injected labeled platelets, was significantly less (P < .01), and was reached after approximately 23 hours. Thereafter, deposition decreased to 0.4% +/- 0.2% at 53 hours. The shunts in all of the untreated animals occluded at some stage during the study, while only one shunt occluded in the treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Selective inhibition of spontaneous IgE and IgG4 production by interleukin-8 in atopic patients
Authors: Kimata H , Lindley I , Furusho K
Published: October 2019

The effects of interleukin (IL)-8 on spontaneous IgE and IgG4 production in atopic patients were studied. IL-8 inhibited IgE and IgG4 production by purified surface (s) IgE+ and sIgG4+ B cells, respectively, while it had no effect on IgG1, IgG2, IgG3, IgM, IgA1, and IgA2 production by corresponding sIg+ B cells. The IL-8-induced inhibition was counteracted by IL-6 and tumor necrosis factor-alpha (TNF-alpha) and was blocked by anti-IL-8 monoclonal antibody (MoAb). Conversely, the addition of anti-IL-6 MoAb and anti-TNF-alpha MoAb, in the absence of IL-8, inhibited IgE and IgG4 production by sIgE+ and sIgG4+ B cells, respectively. Purified sIgE+ and sIgG4+ B cells expressed IL-6 receptors (R), TNF-alpha R, and IL-8R, and they produced IL-6 and TNF-alpha, but not IL-8. IL-8 had no effect on IL-6R or TNF- alpha R, while it abrogated IL-6 and TNF-alpha production in these cells. In contrast, slgG1+, slgG2+, slgG2+, slgG3+, slgM+, slgA1+, and slgA2+ B cells expressed IL-6R and TNF-alpha R but not IL-8R, and they produced IL-6 and TNF-alpha. IL-8 had no effect on IL-6R and TNF-alpha R, or on TNF-alpha and IL-6 production in these cells. These results indicate that IL-8 inhibits spontaneous IgE and IgG4 production in slgE+ and slgG4+ B cells, respectively, by inhibiting the endogenous production of IL-6 and TNF-alpha.
Regulation of cell adhesion molecule expression and function associated with neutrophil apoptosis
Authors: Dransfield I , Stocks SC , Haslett C
Published: October 2019

We have investigated the adhesive capacity of neutrophils after spontaneous apoptosis, which occurs during in vitro culture. Apoptotic neutrophils show reduced adhesion to E selectin and the CD18 integrin ligand fibrinogen. Neutrophil apoptosis is associated with changes in the levels of surface expression of key receptors that mediate neutrophil adhesion events. Notably, apoptotic neutrophils show reduced expression of L-selectin/selectin ligand. In contrast, CD11b/CD18 and CD11c/CD18 integrins are expressed at increased levels. The reduced capacity for adhesion of apoptotic neutrophils may be achieved by very different mechanisms. Regulation of the levels of surface expression of receptors/ligand may control selectin-mediated adhesion, possibly as a result of protease/sialidase activity. In contrast, modulation of integrin-mediated adhesion may involve functional uncoupling of receptors present on the surface of the apoptotic cell without alteration in levels of surface expression. The altered adhesive potential of the apoptotic neutrophil may serve to limit release of its histotoxic contents and reduce inappropriate tissue injury.
Dysregulated Fas and Bcl-2 expression leading to enhanced apoptosis in T cells of multiple myeloma patients
Authors: Massaia M , Borrione P , Attisano C , Barral P , Beggiato E , Montacchini L , Bianchi A , Boccadoro M , Pileri A
Published: October 2019

We have previously reported the presence of activated (HLA-DR+) T cells in multiple myeloma (MM) patients. These cells produce high amounts of interleukin (IL)-2 and interferon (IFN)-gamma and generate a potent antiplasma cell activity after appropriate in vitro stimulation, but they are unable in vivo to hold in check the disease. Activated T cells are highly susceptible to apoptosis, a form of programmed cell death involved in the modulation of immune responses and regulated by molecules such as Fas (CD95) and bcl-2. The aim of this study was to determine the expression of Fas and bcl-2 antigens and the susceptibility to apoptosis in T cells of MM patients. Fas+ cells were significantly higher, whereas bcl-2+ cells were significantly lower in MM patients than in the controls. MM patients with the highest number of HLA-DR+ T cells showed the highest Fas and the lowest bcl-2 expression. Two-color cytofluorometric analysis confirmed in individual cells that HLA-DR+ T cells coexpressed Fas and lacked bcl-2. Susceptibility to apoptosis was then investigated to evaluate the consequence of dysregulated Fas and bcl-2 expression. The percentage of apoptotic cells after incubation in medium alone (spontaneous apoptosis) or in the presence of methylprednisolone (MP) or anti-Fas monoclonal antibody (triggered apoptosis) was significantly higher in MM and mainly restricted to HLA-DR+ T cells. Spontaneous apoptotosis was reverted by exogenous IL-2. In conclusion, MM T cells have a dysregulated expression of Fas and bcl-2 antigens that is associated with an enhanced susceptibility to apoptosis. These data may unravel a novel mechanism by which activated MM T cells are weakened in their ability to exert an effective antitumor activity in vivo.