bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V
Authors: Lu D , Kalafatis M , Mann KG , Long GL
Published: October 2019

The proteolytic cleavage and subsequent inactivation of recombinant human factor VIII (rhFVIII) and human factor VIIIa (rhFVIIIa) by recombinant human activated protein C (rAPC) was analyzed in the presence and absence of human protein S and human factor V (FV). Membrane-bound rhFVIIIa spontaneously looses most of its initial cofactor activity after 15 minutes of incubation at pH 7.4. The remaining activity can be eliminated after incubation with rAPC. Complete inactivation of the membrane-bound rhFVIII and rhFVIIIa by APC correlates with cleavage at Arg336. The inactivation of rhFVIII and human plasma FV by rAPC were also compared. Under similar experimental conditions, complete inactivation of membrane-bound FVIII (60 nmol/L) by rAPC (10 nmol/L) requires 4 hours of incubation, in contrast to 5 minutes for FV (60 nmol/L). The presence of protein S (100 nmol/L) enhances rhFVIII inactivation by rAPC by 6.4-fold and FVa inactivation by twofold, whereas membrane-bound FV showed no protein S dependence during inactivation. The addition of human FV to the APC/protein S inactivation mixture increases by approximately twofold the rate of inactivation of rhFVIII. The effect of FV on the rhFVIII inactivation by APC is protein S-dependent, because FV alone has no effect on the inactivation rate of rhFVIII by APC. Western blotting using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of human FV showed that FV was completely cleaved by APC at the beginning of the rhFVIII inactivation process. These data suggest that FV fragments derived from the B region of the procofactor after incubation of the membrane-bound procofactor with APC, but not intact single-chain FV, stimulate APC activity in the presence of protein S. rhFVIII, FV, and rhFVIIIa were not inactivated by Glu20-- >Ala-substituted rAPC (rAPCgamma20A), and membrane-bound factor Va was only partially inactivated. Our data suggest that (1) FV and FVa are the physiologically significant substrates for APC inactivation and (2) membranes-bound APC-treated FV is a cofactor for the APC inactivation of rhFVIII only in the presence of the intact form of protein S.
Persistence of multipotent progenitors expressing AML1/ETO transcripts in long-term remission patients with t(8;21) acute myelogenous leukemia
Authors: Miyamoto T , Nagafuji K , Akashi K , Harada M , Kyo T , Akashi T , Takenaka K , Mizuno S , Gondo H , Okamura T , Dohy H , Niho Y
Published: October 2019

The leukemia-specific AML1/ETO fusion gene has been shown to be detected by reverse transcriptase polymerase chain reaction (RT-PCR) analysis in patients with t(8;21) acute myelogenous leukemia (AML) in long-term remission. In the present study, the AML1/ETO mRNA could be detected by RT-PCR in bone marrow (BM) and/or peripheral blood (PB) samples from all 18 patients who had been maintaining complete remission for 12 to 150 months (median, 45 months) following chemotherapy or PB stem cell transplantation (PBSCT), whereas it could not be detected in four patients who had been maintaining remission for more than 30 months following allogeneic BM transplantation (BMT). We surveyed the expression of AML1/ETO mRNA in clonogenic progenitors from BM in these cases. Notably, 51 of 2,469 colonies from clonogenic progenitors (2.1%) expressed the AML1/ETO mRNA in 18 cases who were RT- PCR+ in BM and/or PB samples. Expression was observed in various clonogenic progenitors, including granulocyte-macrophage colonies, mixed colonies, erythroid colonies, and megakaryocyte colonies. Furthermore, we analyzed the clonality of these progenitors by X- chromosome inactivation patterns of the phosphoglycerate kinase (PGK) gene in four female patients. The AML1/ETO mRNA+ progenitors showed the PGK allele identical to that detected in the leukemic blasts from the time of initial diagnosis. Normal constitutive hematopoiesis was sustained by polyclonal BM reconstitution in these patients. Accordingly, these committed progenitor cells that express AML1/ETO mRNA during remission likely have arisen from common t(8;21)+ pluripotent progenitor cells with at least trilineage differentiation potential. These data strongly suggest that the origin of the clonogenic leukemic progenitors of t(8;21) AML may be multipotent hematopoietic progenitors that acquired the t(8;21) chromosomal abnormality.
Molecular analysis of t(11;19) breakpoints in childhood acute leukemias
Authors: Rubnitz JE , Behm FG , Curcio-Brint AM , Pinheiro RP , Carroll AJ , Raimondi SC , Shurtleff SA , Downing JR
Published: October 2019

MLL is fused to ENL or ELL in acute leukemias that contain t(ll;19)(q23;p13). Although ENL and ELL localize to chromosome 19, bands p13.3 and p13.1, respectively, these breakpoints are not always readily distinguished by standard cytogenetics. We therefore used reverse transcriptase-polymerase chain reaction (RT-PCR) assays to analyze 26 cases of childhood acute leukemia containing t(11;19) to determine the frequencies of ENL and ELL involvement. All 17 cases of acute lymphoblastic leukemia (ALL) had MLL/ENL fusion transcripts. By contrast, of the 9 cases of acute myeloid leukemia (AML) analyzed, 6 had MLL/ENL fusions, 2 had MLL/ELL fusions, and 1 case had no RT-PCR- detectable MLL fusion mRNA. These data suggest that the majority of 11;19 translocations involve ENL, whereas involvement of ELL is relatively uncommon in childhood acute leukemia and may be restricted to AML.
Complete blockade of B7 family-mediated costimulation is necessary to induce human alloantigen-specific anergy: a method to ameliorate graft- versus-host disease and extend the donor pool
Authors: Gribben JG , Guinan EC , Boussiotis VA , Ke XY , Linsley L , Sieff C , Gray GS , Freeman GJ , Nadler LM
Published: October 2019

Graft-versus-host disease (GVHD) is initiated by adoptively transferred donor T cells that recognize host alloantigens. Whereas the absence of donor T-cell proliferation to host alloantigens in a mixed-leukocyte reaction does not predict freedom from GVHD, the frequency of alloreactive precursor helper T lymphocytes (pHTL) is predictive. Complete blockade of 87 family-mediated costimulation, but not of major histocompatibility complex recognition or adhesion, induces host alloantigenic-specific energy by reducing cytokine production below threshold levels necessary for common gamma chain signaling. The associated reduction of alloreactive pHTL frequency below that predictive for GVHD, without depletion of either nonallospecific T cells or hematopoietic progenitors, has led us to embark upon human clinical trials of haplomismatched allogeneic bone marrow transplantation.
Morphogenesis of the bone marrow: fractal structures and diffusion- limited growth
Authors: Naeim F , Moatamed F , Sahimi M
Published: October 2019

Bone marrow (BM) provides a particular spatial organization that allows interaction between its various components. Characterization of the spatial patterns in the BM and understanding the mechanisms that give rise to them may play a role in better understanding of the BM pathologic processes. Morphometric analyses were performed in BM biopsy samples from 30 patients (16 men and 14 women) with an average age of 46 years, ranging from 17 to 77 years. The biopsies were obtained during the course of patient care to rule out BM involvement in a variety of hematologic disorders before or after therapy. Three different, but structurally interrelated, parameters were measured: (A) cellular area, (B) nuclear area, and (C) cell numbers. All three methods, in all cases, showed that the spatial structure of the BM is fractal. The average values of the fractal dimensions (Df) were 1.7 +/- 0.08, 1.64 +/- 0.1, and 1.69 +/- 0.04 for categories A, B, and C, respectively. The overall value of Df for the cellularity in the range of 40% to 60% was about 1.67 +/- 0.09. Fractal dimensions of 1.6 to 1.7 represent configurations that correspond to two-dimensional diffusion limited aggregation structures, suggesting that the structural configuration of hematopoietic cells is dependent on the diffusion of regulatory cytokines in the BM.
The proinflammatory cytokine response to coagulation and endotoxin in whole blood
Authors: Johnson K , Aarden L , Choi Y , De Groot E , Creasey A
Published: October 2019

Acute inflammatory illnesses, including the sepsis syndrome, often include a component of coagulation. A human whole blood culture system was developed so that the relationship between coagulation activation and cytokine responses in the presence or absence of lipopolysaccharide (LPS) could be evaluated. In the absence of LPS stimulation, coagulation activation resulted in a novel pattern of cytokine production. During a 4-hour culture of coagulating blood, significant production of interleukin-8 (IL-8; >2,000 pg/mL) was observed, whereas other proinflammatory cytokines including IL-1 beta, IL-6, or tumor necrosis factor a were undetectable or less than 35 pg/mL. The cytokine profile was distinct from that of fully anticoagulated, LPS-stimulated blood, which showed levels of all the indicated proinflammatory cytokines > or = 2,000 pg/mL over the same time period. Over 24 to 48 hours, the coagulation-induced cytokine response was characterized by marked and sustained IL-8 production, limited IL-6 generation (with kinetics delayed relative to IL-8), and minimal or undetectable tumor necrosis factor alpha levels. The magnitude of the whole blood IL-8 response correlated with the level of coagulation activation as determined by measurement of thrombin-antithrombin III complex formation. The combined stimuli of coagulation activation and LPS challenge induced a synergistic enhancement of IL-8 production but not of IL-6. Coagulation-induced cytokine production and the synergistic production of IL-8 by coagulation and LPS could be attenuated by hirudin or tissue factor pathway inhibitor (TFPI). Studies to elucidate mechanisms implicated (1) the TFPI third Kunitz and carboxy-terminus as important structural components for TFPI regulation of coagulation activation and (2) thrombin as a candidate mediator of the mononuclear cell cytokine response to coagulation activation. In summary, a unique aspect of the crosstalk between the coagulation and cytokine cascades in whole blood is shown with the identification of IL-8 as a key proinflammatory participant.
Regulation of human heme oxygenase-1 gene expression under thermal stress
Authors: Okinaga S , Takahashi K , Takeda K , Yoshizawa M , Fujita H , Sasaki H , Shibahara S
Published: October 2019

Heme oxygenase-1 is an essential enzyme in heme catabolism, and its human gene promoter contains a putative heat shock element (HHO-HSE). This study was designed to analyze the regulation of human heme oxygenase-1 gene expression under thermal stress. The amounts of heme oxygenase-1 protein were not increased by heat shock (incubation at 42 degrees C) in human alveolar macrophages and in a human erythroblastic cell line, YN-1–0-A, whereas heat shock protein 70 (HSP70) was noticeably induced. However, heat shock factor does bind in vitro to HHO-HSE and the synthetic HHO-HSE by itself is sufficient to confer the increase in the transient expression of a reporter gene upon heat shock. The deletion of the sequence, located downstream from HHO-HSE, resulted in the activation of a reporter gene by heat shock. These results suggest that HHO-HSE is potentially functional but is repressed in vivo. Interestingly, heat shock abolished the remarkable increase in the levels of heme oxygenase-1 mRNA in YN-1–0-A cells treated with hemin or cadmium, in which HSP70 mRNA was noticeably induced. Furthermore, transient expression assays showed that heat shock inhibits the cadmium-mediated activation of the heme oxygenase-1 promoter, whereas the HSP70 gene promoter was activated upon heat shock. Such regulation of heme oxygenase-1 under thermal stress may be of physiologic significance in erythroid cells.
Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B
Authors: Hortelano G , Al-Hendy A , Ofosu FA , Chang PL
Published: October 2019

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.
Antiphosphatidylserine antibodies in human immunodeficiency virus-1 patients with evidence of T-cell apoptosis and mediate antibody- dependent cellular cytotoxicity [see comments]
Authors: Silvestris F , Frassanito MA , Cafforio P , Potenza D , Di Loreto M , Tucci M , Grizzuti MA , Nico B , Dammacco F
Published: October 2019

Serum reactivities to a panel of phospholipid antigens, including cardiolipin (CL), phosphatidylserine (PS), sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine, were measured by enzyme-linked immunosorbent assay in 196 human immunodeficiency virus- l+ (HIV-1+) patients with CDC II to IVC clinical disease. Significant levels of IgG to CL, PS, or both were observed in 23 patients lacking evidence of thrombophilic events or any peculiar clinical feature of HIV-1 infection. Fluorescence-activated cell sorting analyses showed that in vitro apoptosis of T cells was increased in patients with high serum anti-PS IgG, whereas the overexpression of Fas/Apo-1 marker was detected in all patients regardless of their antiphospholipid reactivities. Macrophages from patients with significant titers of anti- PS IgG antibodies were not activated by the presence of apoptotic CEM lymphoblasts or by purified anti-PS IgG from the same patients. By contrast, these antibodies greatly improved the effector functions of autologous macrophages in antibody-dependent cellular cytotoxicity (ADCC) assays using 51Cr-labeled CEM cells, whereas polyspecific IgG were unable to induce an equivalent cytotoxicity in all instances. An increasing effect on ADCC was also observed in tests using macrophages from healthy controls to CEM coated with anti-PS IgG. These results support a potential correlation of anti-PS specificity with T-cell apoptosis in HIV-1 infection. Because PS is exteriorized by apoptotic lymphocytes, its persistence may stimulate antibodies which cooperate with macrophages in the clearance of dead cells by an enhanced ADCC mechanism. This interpretation could explain the absence of thrombophilia in HIV-1+ patients with serum elevations of antiphospholipid reactivities.
Linkage of a familial platelet disorder with a propensity to develop myeloid malignancies to human chromosome 21q22.1-22.2
Authors: Ho CY , Otterud B , Legare RD , Varvil T , Saxena R , DeHart DB , Kohler SE , Aster JC , Dowton SB , Li FP , Leppert M , Gilliland DG
Published: October 2019

Linkage analysis was performed on a large pedigree with an autosomal dominant platelet disorder and a striking propensity in affected family members to develop hematologic malignancy, predominantly acute myelogenous leukemia. We report the linkage of the autosomal dominant platelet disorder to markers on chromosome 21q22. Four genetic markers completely cosegregate with the trait and yield maximum logarithm of difference scores ranging from 4.9 to 10.5 (theta = .001). Two flanking markers, D21S1265 and D21S167, define a critical region for the disease locus of 15.2 centimorgan. Further analysis of this locus may identify a gene product that affects platelet production and function and contributes to the molecular evolution of hematologic malignancy.
A single dose of granulocyte-macrophage colony-stimulating factor induces systemic interleukin-8 release and neutrophil activation in healthy volunteers
Authors: van Pelt LJ , Huisman MV , Weening RS , von dem Borne AE , Roos D , van Oers RH
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are frequently used in the clinical management of neutropenia. These cytokines not only enhance the proliferation of myeloid precursor cells but also influence the function of mature leukocytes. In a previous study, we found that the in vivo effects of G-CSF on neutrophils differed from those in vitro. In the present study, we investigated the effects of a single dose of recombinant GM-CSF (7.5 microg/kg, subcutaneously) on neutrophils, eosinophils, and monocytes in healthy volunteers. We analyzed leukocyte kinetics, phenotypical changes, neutrophil degranulation, and systemic cytokine production. After GM-CSF injection, phenotypical changes included upregulation of CD11b on all three cell types and a decreased expression of L-selectin and Fc(gamma)RIII on neutrophils. Neutrophil degranulation was evident from the increased plasma concentrations of lactoferrin and elastase. GM-CSF induced the release of interleukin-8 (IL-8), but not of IL-6 or tumor necrosis factor alpha. In comparison to the results from our previous study with G-CSF in healthy volunteers, GM-CSF induced a stronger activation of mature neutrophils but had a much less pronounced effect on the production and maturation of neutrophil precursors. These data may help to guide the choice between the two cytokines in different clinical situations.
The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria
Authors: Hall SE , Rosse WF
Published: October 2019

We have characterized the erythrocytes, granulocytes, and platelets of 54 patients with paroxysmal nocturnal hemoglobinuria (PNH) with antibodies to glycosylphosphatidylinositol-anchored proteins (anti- CD55, anti-CD59, and anti-CD16) and flow cytometry to establish the usefulness of this technique in the diagnosis of this disorder. All patients demonstrated either completely (PNH III) or partially (PNH II) deficient red cells and granulocytes. Anti-CD59 best demonstrated PNH II red cells, which were present in 50% of the patients. The proportion of abnormal granulocytes was usually greater than the proportion of abnormal red cells; 37% of the patients had >80% abnormal granulocytes. Anti-CD55 did not delineate the erythrocyte populations as well as did anti-CD59. Either anti-CD55 or anti-CD59 could be used equally well to analyze granulocytes; anti-CD16 did not demonstrate cells of partial deficiency. Platelets could not be used for detailed analysis as the normal and abnormal populations were not well distinguished. Flow cytometry of erythrocytes using anti-CD59 or of granulocytes using either anti-CD55 or anti-CD59 provides the most accurate technique for the diagnosis of paroxysmal nocturnal hemoglobinuria; it is clearly more specific, more quantitative, and more sensitive than the tests for PNH that depend upon hemolysis by complement (the acidified serum lysis [Ham] test, the sucrose lysis test, and the complement lysis sensitivity [CLS] test).
Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets
Authors: Miyakawa Y , Oda A , Druker BJ , Miyazaki H , Handa M , Ohashi H , Ikeda Y
Published: October 2019

Thrombopoietin is known to be essential for megakaryocytopoiesis and thrombopoiesis. Recently, we and others have shown that thrombopoietin induces rapid tyrosine phosphorylation of Jak2 and other proteins in human platelets and BaF3 cells, genetically engineered to express c- Mpl, a receptor for thrombopoietin. The Jak family of tyrosine kinases are known to mediate some of the effects of cytokines or hematopoietic growth factors by recruitment and tyrosine phosphorylation of a variety of Stat (signal transducers and activators of transcription) proteins. Hence, we have investigated whether Stat proteins are present in platelets and, if so, whether they become tyrosine phosphorylated in response to thrombopoietin. We immunologically identified Stat1, Stat2, Stat3, and Stat5 in human platelet lysates. Thrombopoietin induced tyrosine phosphorylation of Stat3 and Stat5 in these cells. Thrombopoietin also induced tyrosine phosphorylation of Stat3 and Stat5 in FDCP-2 cells genetically engineered to constitutively express human c-Mpl. Thus, our data indicate that Stat3 and Stat5 may be involved in signal transduction after ligand binding to c-Mpl and that this event may have a role in megakaryopoiesis/thrombopoiesis or possibly a mature platelet function such as aggregation.
Interleukin-13 inhibits interleukin-2-induced proliferation and protects chronic lymphocytic leukemia B cells from in vitro apoptosis
Authors: Chaouchi N , Wallon C , Goujard C , Tertian G , Rudent A , Caput D , Ferrera P , Minty A , Vazquez A , Delfraissy JF
Published: October 2019

Human interleukin-13 (IL-13) acts at different stages of the normal B- cell maturation pathway with a spectrum of biologic activities overlapping those of IL-4. B chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of slow-dividing and long-lived monoclonal B cells, arrested at the intermediate stage of their differentiation. In vitro, B-CLL cells exhibit a spontaneous apoptosis regulated by different cytokines. In this report, we show that IL-13 (10 to 200 ng/mL) acts directly on monoclonal B-CLL cells from 12 patients. (1) IL-13 enhances CD23 expression and induces soluble CD23 secretion by B-CLL cells but does not exhibit a growth factor activity. (2) IL-13 inhibits IL-2 responsiveness of B-CLL cells, activated either with IL-2 alone or through crosslinking of lgs or ligation of CD40 antigen. (3) IL-13 protects B-CLL cells from in vitro spontaneous apoptosis. The effects of IL-13 on neoplasic B cells were slightly less than those of IL-4 and occurred independently of the presence of IL-4. The present observations show that IL-13 may exhibit a negative regulatory effect on neoplasic B cells in contrast with that observed in normal B cells, and suggest that IL-13 could be an important factor in the pathogenesis of CLL by preventing the death of monoclonal B cells. Moreover, B-CLL may be an interesting model to study the regulation of the expression of IL-13 receptor and/or signal transduction pathways.
Expression of the hematopoietic growth factor receptor FLT3 (STK- 1/Flk2) in human leukemias
Authors: Carow CE , Levenstein M , Kaufmann SH , Chen J , Amin S , Rockwell P , Witte L , Borowitz MJ , Civin CI , Small D
Published: October 2019

Normal expression of the hematopoietic growth factor receptor FLT3 (STK- 1@Flk2) is limited to CD34+ stem/progenitor cells. We have evaluated the expression of FLT3 by RNase protection assay and Western blotting in 161 primary bone marrow (BM) samples from patients with leukemia. FLT3 RNA was found to be expressed at a higher level than in normal BM controls in 33 of 33 B-lineage acute leukemias, 11 of 12 acute myeloid leukemias (AMLs), and 3 of 11 T-cell acute leukemias (T-ALLs). Expression of FLT3 RNA was also observed in some cases of blast crisis CML. The FLT3 signal resulted from expression on the leukemic blasts, and was not caused by increased FLT3 expression on normal CD34+ stem/progenitor cells in the leukemic samples. To determine if FLT3 protein was also overexpressed, proteins were extracted from leukemic BM samples and screened by Western blotting with anti-FLT3 antisera. FLT3 protein was not detected in normal BM controls, but was found in 14 of 14 B-lineage ALLs, 36 of 41 AMLs, and 1 of 4 T-ALLs. Stimulation of patient samples with FLT3 ligand resulted in autophosphorylation of the FLT3 receptor, suggesting the receptor is functional in these cells. These data show that FLT3 RNA and protein are aberrantly expressed by AML and ALL cells in that CD34 expression and FLT3 expression are no longer synchronous, and suggest the possibility that overexpression of FLT3 could play a role in the survival and/or proliferation of malignant clones in acute myeloid and lymphoid leukemias.
Fas ligation induces apoptosis and Jun kinase activation independently of CD45 and Lck in human T cells
Authors: Latinis KM , Koretzky GA
Published: October 2019

Stimulation through the Fas/APO-1 receptor results in apoptosis through an incompletely characterized signaling pathway. More is known regarding signal transduction events that occur after ligation of the T- cell antigen receptor (TCR). It has been shown that TCR stimulation requires both the membrane tyrosine phosphatase, CD45, and the Src- family kinase, Lck, to result in cellular activation. Although prior studies suggest a role for protein tyrosine kinases and phosphatases in Fas signaling, we report here that Fas ligation induces apoptosis in T cells deficient in either CD45 or Lck. Further, in normal and CD45- or Lck-deficient cell lines, Fas stimulation results in activation of Jun kinase (JNK), a proposed mediator of stress activation pathways. Previous studies have also demonstrated a role for endogenous ceramide release in Fas-mediated apoptosis. We show that stimulation with a synthetic ceramide analog results in JNK activation as well as apoptosis, suggesting ceramide release occurs proximal to JNK activation in Fas signaling. Our data suggest that although CD45 and Lck are not required for Fas signaling, JNK activation may play an important role transducing distal signals that lead to apoptosis after Fas ligation.
Role of different hematologic variables in defining the risk of malignant transformation in monoclonal gammopathy
Authors: Baldini L , Guffanti A , Cesana BM , Colombi M , Chiorboli O , Damilano I , Maiolo AT
Published: October 2019

The presenting clinico-hematologic features of 386 patients with nonmyelomatous monoclonal gammopathy (MG) were correlated with the frequency of malignant transformation to evaluate the most important variables conditioning its evolution into multiple myeloma (MM) or Waldenstrom macroglobulinemia (WM). Most of the patients (335) had monoclonal gammopathy of undetermined significance (MGUS: 39 IgA, 242 IgG, 54 IgM): the remaining 51 patients (12 IgA, 39 IgG) fulfilled all of the MGUS diagnostic criteria (according to Durie) except that bone marrow plasma cell (BMPC) content was 10% to 30%, and so they were defined as having monoclonal gammopathy of borderline significance (MGBS). There were no significant differences between the MGUS and MGBS groups in terms of age, sex, or median follow-up. After a median follow- up of 70 and 53 months, respectively, 23 of 335 MGUS and 19 of 51 MGBS patients had undergone a malignant evolution. Univariate analysis of the IgA and IgG patients showed that the cumulative probability of the disease evolving into MM correlated with diagnostic definition (MGBS v MGUS), BMPC content (> or = 10% v < 5% and < or = 5% v > 5%) and reduced serum polyclonal Ig. In the IgG cases, there was also a significant correlation with detectable Bence Jones proteinuria, serum monoclonal component (MC) levels and age at diagnosis (> 70 v < = or 55 years). In the IgG cases as a whole, the same variables remained in the Cox model where the BMPC percentage was considered after natural logarithmic transformation and the monoclonal component as g/dL value. The relative risks of developing MM are the following: 2.4 for each 1 g/dL increase of IgG, serum MC, 3.5 for detectable light chain proteinuria, 4.4 for the increase of 1 unit in log. BMPC percentage, 6.1 for age > 70, 3.6 and 13.1 for a reduction in one or two polyclonal Ig. In conclusion, our study allows the identification of a particular subset of MGUS patients (MC < = or 1.5 g/dL, BMPC < 5%, no reduction in polyclonal Ig and no detectable light chain proteinuria) at very low- risk of evolution, who can be considered as having benign monoclonal gammopathies. We also describe a previously undefined group of MG patients (with monoclonal gammopathy of borderline significance) who are at high-risk of malignant evolution. These findings could have a considerable impact on the cost/benefit ratio of monitoring programs in these patients.
Measurement of endogenous plasma granulocyte colony-stimulating factor in patients with acquired aplastic anemia by a sensitive chemiluminescent immunoassay
Authors: Kojima S , Matsuyama T , Kodera Y , Nishihira H , Ueda K , Shimbo T , Nakahata T
Published: October 2019

Endogenous plasma granulocyte colony-stimulating factor (G-CSF) concentrations were serially measured in 68 patients with acquired aplastic anemia (AA). A very sensitive chemiluminescent immunoassay (CLEIA) was used to measure the plasma G-CSF concentration. The minimum detection limit of this assay (0.5 pg/mL) was sufficient for the determination of G-CSF concentrations in patients and normal subjects. The plasma G-CSF concentrations were significantly higher in 51 AA patients without signs of infection as compared with healthy control subjects. In AA patients with signs of infection, the G-CSF concentrations appeared to increase during the acute phase. There was a significant negative correlation between plasma G-CSF concentrations and absolute neutrophil counts (ANC) in AA patients without signs of infection. Although a decrease in the plasma G-CSF concentration was observed in all patients who achieved self-sustaining hematopoiesis following bone marrow transplantation (BMT) or immunosuppressive (IS) therapy, it was lower in patients undergoing BMT as compared with those receiving IS therapy for any given degree of neutropenia. Plasma G-CSF concentrations were higher in patients responding to IS therapy than in nonresponders. This study demonstrated the negative feedback regulation of G-CSF that plasma G-CSF concentrations may be useful in predicting the clinical response to IS therapy.
The membrane-distal cytoplasmic region of human granulocyte colony- stimulating factor receptor is required for STAT3 but not STAT1 homodimer formation
Authors: de Koning JP , Dong F , Smith L , Schelen AM , Barge RM , van der Plas DC , Hoefsloot LH , Lowenberg B , Touw IP
Published: October 2019

Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) involves the activation of the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway. G-CSF induces tyrosine phosphorylation of Jak1, Jak2, STAT1, and STAT3. The membrane-proximal region of G-CSF-R is sufficient for activation of Jaks. It is still unclear how STAT proteins are activated by G-CSF-R. We investigated the possible involvement of the C-terminal region of G-CSF-R in the recruitment of STAT proteins using BAF3 cell transfectants expressing wild type (WT) G-CSF-R, C-terminal deletion mutants and tyrosine-to-phenylalanine substitution mutants. Electrophoretic mobility shift assays with STAT-binding oligonucleotides (m67) showed that activation of WT G-CSF-R induces three distinct STAT complexes, namely STAT3 homodimers, STAT1-STAT3 heterodimers, and STAT1 homodimers. However, STAT1 homodimers and STAT1- STAT3 heterodimers were predominantly formed after activation of a C- terminal deletion mutant d685, which lacks all four conserved cytoplasmic tyrosine residues, located at positions 704, 729, 744, and 764. Antiphosphotyrosine immunoblots of STAT3 immunoprecipitates showed that activation of WT G-CSF-R induced phosphorylation of STAT3. In contrast, no phosphorylation of STAT3 was observed after activation of deletion mutant d685. These findings establish that the C-terminal region of G-CSF-R plays a major role in the activation of STAT3. By using tyrosine-to-phenylalanine substitution mutants of G-CSF-R, we further show that tyrosine 704, present in a YXXQ consensus sequence shown to be essential for STAT3 binding to gp130, is not exclusively involved in the activation of STAT3 by G-CSF-R.
Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation
Authors: Gabbeta J , Yang X , Sun L , McLane MA , Niewiarowski S , Rao AK
Published: October 2019

Platelet-agonist interaction results in activation of glycoprotein (GP) IIb-IIIa complex and fibrinogen binding, a prerequisite for platelet aggregation. Fibrinogen binding exposes new antibody binding sites on GPIIb-IIIa (ligand-induced binding sites: LIBS). Signal transduction events, including pleckstrin phosphorylation by protein kinase C (PKC), are considered to regulate GPIIb-IIIa activation. We studied a 16-year- old white male with lifelong mucocutaneous bleeding manifestations and abnormal platelet aggregation and secretion in response to multiple agonists. Pleckstrin phosphorylation was diminished in response to platelet-activating factor (PAF; 4 and 400 nmol/L) and thrombin (0.05 U/mL). Binding of monoclonal antibodies (MoAbs) 10E5 and A2A9, which bind to both resting and activated GPIIb-IIIa, was normal. Binding of MoAb PAC1, which binds to only activated GPIIb-IIIa, was diminished upon activation with PAF, adenosine diphosphate (ADP), thrombin receptor agonist peptide (SFLLRN), A23187, and 1,2-dioctonylglycerol (DiC8). Signal transduction-dependent LIBS expression (studied using MoAb 62) induced by ADP, SFLLRN, and DiC8 and signal transduction- independent LIBS expression induced by RGDS peptide or disintegrin albolabrin were normal or minimally decreased, indicating the presence of intact ligand binding sites. We conclude that the patient's platelets have a defect in inside-out signal transduction-dependent GPIIb-IIIa activation due to an upstream defect in the signal transduction mechanisms rather than in the GPIIb-IIIa complex itself. Our findings extend the spectrum of congenital mechanisms leading to impaired aggregation from defects in GPIIb-IIIa per se to aberrations in signaling mechanisms.
Autoimmune manifestations in the transforming growth factor-beta 1 knockout mouse
Authors: Yaswen L , Kulkarni AB , Fredrickson T , Mittleman B , Schiffman R , Payne S , Longenecker G , Mozes E , Karlsson S
Published: October 2019

Targeted disruption of the transforming growth factor-beta 1 (TGF-beta 1) gene in mice results in the development of a massive multifocal inflammatory disease in many tissues. Because no detectable pathogen was identified, we examined whether autoimmune mechanisms played a role in initiating or maintaining the inflammatory disease. The serum of TGF- beta 1 knockout mice contained elevated titers of antibodies to nuclear antigens (ssDNA, dsDNA, Sm, and RNP) as well as reactivity against the 16/6 idiotype (16/6 Id). In addition, Ig deposits were detected in renal glomeruli of TGF- beta 1 knockout mice. Transplantation of TGF- beta 1 knockout hematopoietic cells into normal irradiated recipients resulted in a similar profile of autoantibody production as well as in the induction of inflammatory lesions. Our results describe autoimmune activity that ensues when the TGF-beta 1 cytokine is absent.
CD5-CD56+ T-cell receptor silent peripheral T-cell lymphomas are natural killer cell lymphomas [see comments]
Authors: Emile JF , Boulland ML , Haioun C , Kanavaros P , Petrella T , Delfau-Larue MH , Bensussan A , Farcet JP , Gaulard P
Published: October 2019

Non-Hodgkin's lymphomas are divided into B- and T-cell neoplasms. The existence and the clinical relevance of lymphomas derived from the third lymphocyte lineage, ie, natural killer (NK) cells are still controversial. NK cells are lymphocytes that mediate cytotoxicity without prior sensitization. NK cells also have phenotypic and genotypic characteristics: they express the NK-related antigen CD56, T- cell markers such as CD2 and CD7, but do not express CD5 and T-cell receptor (TCR) proteins, and their TCR locus is not rearranged. Therefore, if NK cell lymphomas exist, they should express some T-cell markers, but not alpha beta or gamma delta TCR proteins. Such lymphomas are actually called TCR silent peripheral T cell lymphomas (PTCL). To detect and characterize NK cell lymphomas, we investigated the immunophenotype and immunogenotype of 35 patients with TCR silent PTCL. The first group included 16 patients with a lymphoma of CD5-CD56+ phenotype, which is identical to normal NK cells. These patients had either a nasal/nasopharyngeal lymphoma (11 cases) or a lymphoma with predominant non-nasal/non-nodal initial involvement (five cases). Eight of the nine cases for which immunogenotypic data were available lacked clonal rearrangement of the TCR gamma genes. Thus, these tumors are likely to be NK cell lymphomas. The second group of 15 cases had a CD5+ phenotype (14 were CD56-, and 1 was CD56+) and clonal rearrangement of TCR gamma genes, indicating that they were true PTCL with unproductive TCR rearrangement. The four remaining cases were CD5- CD56- lymphomas and disclosed either a clonal (two cases) or no clonal (two cases) rearrangements of the TCR gamma genes. Altogether these findings show that CD5-CD56+ so-called “TCR silent PTCL” bear the immunophenotype and immunogenotype of normal NK cells and display peculiar clinical features distinct from true PTCL.
Development of an in vivo model of human multiple myeloma bone disease
Authors: Alsina M , Boyce B , Devlin RD , Anderson JL , Craig F , Mundy GR , Roodman GD
Published: October 2019

Osteolytic bone destruction and its complications, bone pain, pathologic fractures, and hypercalcemia, are a major source of morbidity and mortality in patients with multiple myeloma. The bone destruction in multiple myeloma is due to increased osteoclast (OCL) activity and decreased bone formation in areas of bone adjacent to myeloma cells. The mechanisms underlying osteolysis in multiple myeloma in vivo are unclear. We used a human plasma cell leukemia cell line, ARH-77, that has disseminated growth in mice with severe combined immunodeficiency (SCID) and expresses IgG kappa, as a model for human multiple myeloma, SCID mice were irradiated with 400 rads and mice were injected either with 10(6) ARH-77 cells intravenously (ARH-77 mice) or vehicle 24 hours after irradiation. Development of bone disease was assessed by blood ionized calcium levels, x-rays, and histology. All ARH-77, but none of control mice that survived irradiation, developed hind limb paralysis 28 to 35 days after injection and developed hypercalcemia (1.35 to 1.46 mmol/L) a mean of 5 days after becoming paraplegic. Lytic bone lesions were detected using x-rays in all the hypercalcemic mice examined. No lytic lesions or hypercalcemia developed in the controls. Controls or ARH-77 mice, after developing hypercalcemia, were then killed and bone marrow plasma from the long bones were obtained, concentrated, and assayed for bone-resorbing activity. Bone marrow plasma from ARH-77 mice induced significant bone resorption in the fetal rat long bone resorption assay when compared with controls (percentage of total 45Ca released = 35% +/- 4% v 11% +/- 1%). Histologic examination of tissues from the ARH-77 mice showed infiltration of myeloma cells in the liver and spleen and marked infiltration in vertebrae and long bones, with loss of bony trabeculae and increased OCL numbers. Interestingly, cultures of ARH-77 mouse bone marrow for early OCL precursors (colony-forming unit-granulocyte- macrophage [CFU-GM]) showed a threefold increase in CFU-GM from ARH-77 marrow versus controls (185 +/- 32 v 40 +/- 3 per 2 x 10(5) cell plated). Bone-resorbing human and murine cytokines such as interleukin- 6 (IL-6), IL-1 alpha or beta, TGF-alpha, lymphotoxin, and TNF alpha were not significantly increased in ARH-77 mouse sera or marrow plasma, compared with control mice, although ARH-77 cells produce IL-6 and lymphotoxin in vitro. Conditioned media from ARH-77 cells induced significant bone resorption in the fetal rat long bone resorption assay when compared with untreated media (percentage of total 45Ca released = 22% +/- 2% v 11% +/- 1%). This effect was not blocked by anti-IL-6 or antilymphotoxin (percentage of total 45Ca released = 19% +/- 1% and 22% +/- 1%, respectively). Thus, we have developed a model of human multiple myeloma bone disease that should be very useful to dissect the pathogenesis of the bone destruction in multiple myeloma.
Detection of 11q13 rearrangements in hematologic neoplasias by double- color fluorescence in situ hybridization
Authors: Coignet LJ , Schuuring E , Kibbelaar RE , Raap TK , Kleiverda KK , Bertheas MF , Wiegant J , Beverstock G , Kluin PM
Published: October 2019

Rearrangements within the chromosome 11q13 region are frequent in hematologic malignancies. 50% of 75% of mantle cell lymphomas (MCLs) carry a translocation t(11;14) (q13;q32). Using Southern blot analysis, a BCL1 breakpoint can be detected in approximately 50% of MCLs. It is not known whether other MCLs harbor also breakpoints at 11q13. Breakpoints in this region not involved in t(11;14), are detected in chronic lymphocytic leukemia and acute myeloid leukemia. To detect and localize breakpoints at 11q13 more accurately, we have developed fluorescence in situ hybridization using two probe sets of differently labeled cosmids, symmetrically localized at either side of the major translocation cluster of BCL1. These probes span a region of 450 to 750 kb. We applied this assay to a series of hematologic malignancies with 11q13 abnormalities identified by classical cytogenetics. All four samples with a t(11;14) (q13;q32) showed dissociation of the differently colored signals in metaphase and interphase cells, thereby indicating a chromosomal break in the region defined by the probe sets. The frequency of abnormal metaphase and interphase cells was comparable with that observed in any of the 13 malignancies with other chromosomal 11q13 abnormalities, indicating that these chromosomal breaks occurred outside the 450- to 750-kb region covered by the probes. One patient showed triplication and one patient showed monoallelic loss of this region. The current data show that double-color fluorescence in situ hybridization is a simple and reliable method for detection of the t(11;14)(q13;q32) in interphase cell nuclei and that is can be used to distinguish this translocation from other 11q13 rearrangements in hematologic malignancies.
Effect of ultraviolet-B light on lymphocyte activity at doses at which normal bone marrow stem cells are preserved
Authors: Gowing H , Lawler M , Hagenbeek A , McCann SR , Pamphilon DH , Hudson J , van Weelden H , Braakman E , Martens AC
Published: October 2019

Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) involves the activation of the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway. G-CSF induces tyrosine phosphorylation of Jak1, Jak2, STAT1, and STAT3. The membrane-proximal region of G-CSF-R is sufficient for activation of Jaks. It is still unclear how STAT proteins are activated by G-CSF-R. We investigated the possible involvement of the C-terminal region of G-CSF-R in the recruitment of STAT proteins using BAF3 cell transfectants expressing wild type (WT) G-CSF-R, C-terminal deletion mutants and tyrosine-to-phenylalanine substitution mutants. Electrophoretic mobility shift assays with STAT-binding oligonucleotides (m67) showed that activation of WT G-CSF-R induces three distinct STAT complexes, namely STAT3 homodimers, STAT1-STAT3 heterodimers, and STAT1 homodimers. However, STAT1 homodimers and STAT1- STAT3 heterodimers were predominantly formed after activation of a C- terminal deletion mutant d685, which lacks all four conserved cytoplasmic tyrosine residues, located at positions 704, 729, 744, and 764. Antiphosphotyrosine immunoblots of STAT3 immunoprecipitates showed that activation of WT G-CSF-R induced phosphorylation of STAT3. In contrast, no phosphorylation of STAT3 was observed after activation of deletion mutant d685. These findings establish that the C-terminal region of G-CSF-R plays a major role in the activation of STAT3. By using tyrosine-to-phenylalanine substitution mutants of G-CSF-R, we further show that tyrosine 704, present in a YXXQ consensus sequence shown to be essential for STAT3 binding to gp130, is not exclusively involved in the activation of STAT3 by G-CSF-R.
Peripheral blood CD34+ cells differ from bone marrow CD34+ cells in Thy- 1 expression and cell cycle status in nonhuman primates mobilized or not mobilized with granulocyte colony-stimulating factor and/or stem cell factor
Authors: Donahue RE , Kirby MR , Metzger ME , Agricola BA , Sellers SE , Cullis HM
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) have been shown to stimulate the circulation of hematopoietic progenitor cells in both mice and nonhuman primates. We evaluated the immunophenotype and cell cycle status of CD34+ cells isolated from the bone marrow (BM) and leukapheresis product of cytokine-mobilized nonhuman primates. CD34+ cells were isolated from rhesus macaques that had received no cytokine therapy, 100 micrograms/kg/d G-CSF, 200 micrograms/kg/d SCF, or a combination of both 100 micrograms/kg/d G-CSF and 200 micrograms/kg/d SCF as a subcutaneous injection for 5 days. BM was aspirated before (day 0) and on the last day (day 5) of cytokine administration. On days 4 and 5, peripheral blood (PB) mononuclear cells were collected using a novel method of leukapheresis. Threefold more PB mononuclear cells were collected from animals receiving G-CSF alone or G-CSF and SCF than from animals that had received either SCF alone or no cytokine therapy. CD34+ cells were positively selected using an immunoadsorptive system from the BM, PB, and/or leukapheresis product. Threefold and 10-fold more CD34+ cells were isolated from the leukapheresis product of animals receiving G-CSF or G-CSF and SCF, respectively, than from animals receiving no cytokine therapy or SCF alone. The isolated CD34+ cells were immunophenotyped using CD34- allophycocyanin, CD38-fluorescein isothiocyanate, and Thy-1- phycoerythrin. These cells were later stained with 4′, 6-diamidino-2- phenylindole for simultaneous DNA analysis and immunophenotyping. BM- derived CD34+ cells did not differ significantly in cell cycle status and Thy-1 or CD38 phenotype before or after G-CSF and/or SCF administration. Similarly, CD34+ cells isolated from the leukapheresis product did not differ significantly in immunophenotype or cell cycle status before or after G-CSF and/or SCF administration. However, there were consistent differences in both immunophenotype and cell cycle status between BM- and PB-derived CD34+ cells. CD34+ cells isolated from the PB consistently had a smaller percentage of cells in the S+G2/M phase of the cell cycle and had a higher percentage of cells expressing Thy-1 than did CD34+ cells isolated from the BM. A greater proportion of PB-derived CD34+ cells were in the S+G2/M phase of the cell cycle after culture in media supplemented with interleukin-6 and SCF, However, culturing decreased the proportion of CD34+ cells expressing Thy-1.