bluuebunny/crossref_metadata_2025 huggingface dataset:
Consolidation Chemotherapy Is Not Beneficial for Adult Acute Lymphoblastic Leukemia Patients with Available Donor Undergoing Myeloablative Allogeneic Hematopoietic Cell Transplantation in First Complete Remission: A CIBMTR Study
Authors: Bejanyan Nelli , Lazaryan Aleksandr , Zhang Mei-Jie , Wang Hai-Lin , de Lima Marcos , Sandmaier Brenda M. , Horowitz Mary M. , Khoury H. Jean , Weisdorf Daniel J.
Published: October 2019

Allogeneic hematopoietic cell transplantation (alloHCT) is potentially curative for patients with ALL who achieve complete remission with upfront chemotherapy (CR1). However, the necessity of consolidation chemotherapy remains uncertain in patients with an available donor undergoing alloHCT, even if the goal is to reduce the detectable disease burden prior to alloHCT. We therefore compared clinical outcomes of 524 adult patients with ALL in CR1 who received ≥2 cycles (n=109) vs. 1 cycle (n=93) vs. no (n=322) consolidation chemotherapy prior to myeloabalative alloHCT from 2008-2012. The median follow up of survivors was 59 (6-78) months. All three consolidation groups had similar patient, disease and transplant characteristics, but no consolidation patients were older, took longer to achieve CR1 and less frequently received CNS prophylaxis or maintenance chemotherapy prior to alloHCT. In contrast, fewer with 1 cycle consolidation had prior comorbidities. Time to alloHCT was longer with increasing cycles of consolidation. Only a minority had either cytogenetic or molecular detectable disease in their pre-HCT CR1 assessment; and it was similar in each consolidation cohort. For ≥2 cycles, 1-cycle, no consolidation groups, the adjusted 3-year cumulative incidence of relapse was 20%, 27% and 22% and 1-year transplant-related mortality (TRM) was 16%, 18% and 23%, respectively (all p-values > 0.4). Similarly, adjusted 3-year leukemia-free survival (LFS) was 54%, 48% and 47% (Figure), while overall survival (OS) was 63%, 59% and 54%, respectively (all p-values > 0.3). Consolidation did not influence acute or chronic GVHD risks. Multivariable regression analysis adjusted for recipient age, Karnofsky performance status, time to CR1, graft source, donor type and recipient CMV serostatus, confirmed that consolidation chemotherapy was not prognostic of LFS (RR=1.20, 95% CI 0.86-1.67; p=0.28 for no consolidation; RR=1.18, 95% CI 0.79-1.76; p=0.41 for 1 cycle; ≥2 cycles=reference). Similarly, consolidation was not associated with OS, relapse, TRM or GVHD. We conclude that consolidation chemotherapy does not benefit adult ALL patients with readily available donor who undergo myeloablative alloHCT in CR1.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
A Runx1 Interactome Identifies Novel Hematopoietic Enhancers
Authors: Marsman Judith , Thomas Amarni L , Osato Motomi , O'Sullivan Justin M , Horsfield Julia A
Published: October 2019

The transcription factor Runx1 is essential for definitive hematopoiesis, and human RUNX1 is frequently translocated or mutated in leukaemia. Runx1 expression from its two promoters, the proximal P1 and distal P2, must be tightly regulated for normal hematopoiesis to occur. The mechanism(s) of promoter-specific Runx1 expression during development and differentiation of hematopoietic progenitors is poorly understood. Gene regulatory elements located in non-coding DNA, such as the previously identified +24 enhancer (Ng et al, Stem Cells 2010), are likely to be important for Runx1 transcription. Here, we identify novel cis-regulatory DNA elements that are competent to drive hematopoietic expression in zebrafish, and that interact long-range with the Runx1 gene.

Circular chromosome conformation capture sequencing (4C-seq) was used to identify long-range interactions of DNA elements with the promoters of Runx1 in the mouse hematopoietic progenitor cell line, HPC7. We used the P1 and P2 promoters and the +24 enhancer (located 24 kb downstream from the P1 promoter) as anchor points from which to view cis-interactions on mouse chromosome 16. 4C-seq reveals that high levels of chromatin interactions at Runx1 are contained within a 1 megabase domain, spanning from the upstream Setd4 gene to the downstream Clic6 gene. A comparison with HiC data in K562 cells shows that the same domain is conserved in humans.

HPC7 cells express Runx1 almost exclusively from the P1 promoter, such that DNA elements interacting with P1 are likely to activate gene expression. The active P1 promoter interacts with multiple non-coding regions both upstream and downstream of Runx1-P1. Interacting regions were overlapped with DNAse hypersensitivity peaks and transcription factor binding sites (available in the USCS genome browser) to identify putative enhancers for Runx1. The majority of putative enhancer regions function as blood specific enhancers in zebrafish (Table). Interestingly, the previously identified +24 enhancer interacted promiscuously within and outside of the 1 megabase Runx1 domain, indicating it may regulate the expression of many additional genes. Overall, our study of the Runx1 interactome identifies multiple novel hematopoietic enhancers that directly interact with the P1 promoter of Runx1, suggesting they are likely involved in regulating Runx1 expression.

Table Table.

Disclosures

No relevant conflicts of interest to declare.
Influence of Lower Dose Antithymocyteglobulin As Conditioning Regimen on Viral Infection after Haploidentical Allogeneic Stem Cell Transplantation
Authors: Lin Ren , Wang Yu , Huang Fen , Fan Zhiping , Zhang Shen , Yang Ting , Xu Yajing , Liang Xinquan , Ji Yu , Xu Na , Xuan Li , Ye Jieyu , Sun Jing , Huang Xiaojun , Liu Qifa
Published: October 2019

Background: Antithymocyteglobulin (ATG), used as conditioning regimen, can reduce graft-versus-host disease (GVHD) in haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Notwithstanding, immunosuppressive effect of ATG may increase the risk of viral infections after HSCT. To evaluate the effect of different doses of ATG on post-transplant viral infection, we conducted a multicenter prospective study to compare EBV and CMV infection in haplo-HSCT recipients receiving 7.5 mg/kg or 10 mg/kg ATG.

Methods

Between May 2013 and November 2015, 350 consecutive patients with hematological malignancies undergoing haplo-HSCT were randomized in 5 hospitals. One hundred and seventy-two patients received ATG with a total dosage of 7.5 mg/kg and 175 received 10 mg/kg ATG. Three patients did not received allocated intervention and transplantation due to leukemia relapse before transplant or toxicity of conditioning regimens.

Results

The cumulative incidence of EBV viremia on day 180 was 23.3±3.2% in 7.5 mg/kg ATG arm which was lower than that in 10 mg/kg arm (34.6±3.7%, P=0.037). CMV viremia were comparable in the two arms (7.5 mg/kg arm: 79.0±3.1% vs. 10 mg/kg arm: 76.9±3.2%, P=0.950). The incidences of CMV diseases were 0.6±0.6% and 2.4±1.2% in 7.5 mg/kg and 10 mg/kg arms, respectively (P=0.093). No difference in the incidence of post-transplant lymphoproliferative disorder (PTLD) was found between the two arms (2.4±1.2% in 7.5 mg/kg arm vs. 5.4±1.8% in 10 mg/kg arm, P=0.150). Besides, acute GVHD grade II to IV within 100 days occurred in 55 recipients with the incidence of 31.4% in 7.5mg/kg ATG arms and 45 recipients in 10 mg/kg arms with the incidence of 26.2% (P=0.279). The incidences of aGVHD grade III to IV were similar in the two arms (8.0% in 7.5 mg/kg arm: vs. 4.7% in 10 mg/kg arm, P=0.196). The 2-years overall survival were 69.5±4.7% and 69.4±3.9% for 7.5 mg/kg and 10 mg/kg group (P=0.540).

Conclusion

Compared with 10 mg/kg of ATG, the application of 7.5 mg/kg might reduce the risk of EBV infection after haplo-HSCT and not increase aGVHD.

Disclosures

Lin: National Natural Science Foundation of China 81270647: Research Funding; Science and technology planning project of Guangdong Province 2014B020226004: Research Funding; The project of health collaborative innovation of Guangzhou City 201400000003-4: Research Funding; National Natural Science Foundation of China 81400141: Research Funding.
Treatment of Myeloma Cast Nephropathy (MCN): A Randomized Trial Comparing Intensive Haemodialysis (HD) with High Cut-Off (HCO) or Standard High-Flux Dialyzer in Patients Receiving a Bortezomib-Based Regimen (the MYRE Study, by the Intergroupe Francophone du Myélome (IFM) and the French Society of Nephrology (SFNDT))
Authors: Bridoux Frank , Pegourie Brigitte , Augeul-Meunier Karine , Royer Bruno , Joly Bertrand , Lamy Thierry , Kolb Brigitte , Benboubker Lofti , Arnulf Bertrand , Jaccard Arnaud , Chevret Sylvie , Fermand Jean-Paul
Published: October 2019

Multiple myeloma (MM) is often revealed by acute kidney injury (AKI) usually related to MCN. Recovery of renal function is a key prognostic factor. With novel anti-myeloma agents, HD-independence occurs in about 30% of pts with severe AKI, advocating for the use of additional strategies to rapidly remove circulating monoclonal free light chains (FLC).

We designed a prospective randomized trial to compare the HD-independence rate in pts with inaugural severe AKI secondary to biopsy-proven MCN, treated by intensive HD (8 sessions over the first 10 days, then thrice a week) using either a HCO dialyzer or a conventional high-flux dialyzer. In both groups, pts received 21-day courses of Bortezomib and dexamethasone (BD), reinforced by cyclophosphamide after 3 cycles in the absence of haematological response (HR).

Between 2011 and 2015, 98 pts were randomised. One pt withdrew consent, and 3 had main exclusion criteria. Baseline characteristics in the control arm (n=48) and HCO arm (n=46) were close, including similarly high serum FLC levels (median 6,015mg/L).

HD independence was achieved in 33% and 43% (p= 0.31) at 3 and in 37.5% and 60 % (p=0.03) at 6 mo, in the control and HCO arms, respectively. HR rate of very good partial response or above at 3 mo based on FLC was 48% in control and 59% in HCO groups (p=0.29). Tolerance of HD schedule and of chemotherapy was acceptable in both arms. At 12 mo, 17 pts have died (10 vs 7).

This randomized trial demonstrates that in MM patients with MCN and severe AKI treated with a bortezomib-based regimen, intensive HCO-HD results in higher renal recovery rate than HD with conventional high-flux dialyzers.

Disclosures

Augeul-Meunier: janssen: Consultancy; gilead: Consultancy. Benboubker:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.
An Improved Method for Isolating Viable Human Leukocytes from Peripheral Blood
Authors: LAPIN JOSEPH H. , HORONICK ANDREW , LAPIN ROBERT H.
Published: October 2019

In an effort to isolate human leukocytes as free as possible of erythrocytes, a two-stage method has been studied. The initial step, sedimentation of the erythrocytes with Dextran results in a diminution of RBC:WBC ratio from the 1000:1 of peripheral blood, to 2.34:1. After sedimentation in isotonic saline, the RBC:WBC ratio is decreased to 0.16:1.

This method should prove valuable in preparing leukocytes for immunologic studies.
Chediak’s Anomaly of Leukocytes in Malignant Lymphoma Associated with Leukemic Manifestations: Case Report with Necropsy
Authors: EFRATI P. , JONAS W.
Published: October 2019

A case of Chediak’s anomaly of leukocytes was described, the fourth reported in the literature.

The patient was an 11-month-old boy who, in addition to Chediak’s anomaly of the leukocytes, presented a leukemic blood picture.

He died five days after admission to the hospital, and necropsy revealed a malignant lymphoma.

We consider it possible that in all the reported cases there were generalized pathologic changes in the reticulohistiocytic system.
An Electronic Blood-Cell-Counting Machine
Authors: CROSLAND-TAYLOR P. , STEWART J. W. , HAGGIS G.
Published: October 2019

An electronic blood cell counting machine operating on a flow principle is described. It is quick and easy to use; it can perform a red cell count in ten seconds with an accuracy of ± 2.1 per cent (that is, 5000 cells counted in 10 seconds for a 5 M count). Leukocyte counts can also be performed in ten seconds with an accuracy of ± 9 per cent for a single count (500 cells counted in a 5000 cu.cm. count).
Starch Block Electrophoretic Studies of Human Hemoglobin Solutions I. Technic and Results in the Normal Adult
Authors: MASRI M. S. , JOSEPHSON AARON M. , SINGER KARL
Published: October 2019

1. Erythrocyte hemolysates from hematologically normal, adult individuals were studied by means of starch block electrophoresis.

2. The electrophoretic patterns obtained indicated the presence, in addition to the main component of A hemoglobin A1, of three other hemoglobin fractions designated as A2, A3 and A4, A2 and A4 move slower, while A3 moves faster in alkaline buffer. These components were isolated by elution from the starch after separation by electrophoresis.

3. The concentrations of the various fractions in the hemolysates were estimated from a sample of normal individuals investigated.

4. In our experiments, electrophoretic patterns obtained on starch that has been re-used as a supportive medium for electrophoresis were somewhat different but comparable to those obtained on starch that was used for the first time as a supportive medium; these differences are discussed.

5. Some characterizations of the isolated components are made.
The Effects of Components of Guinea Pig Serum on Lymphosarcoma 6C3HED in C3H Mice
Authors: HERBUT PETER A. , KRAEMER WILLIAM H. , PILLEMER LOUIS
Published: October 2019

The effects of various preparations of guinea pig serum on the Gardner lymphosarcoma 6C3HED in C3H mice were studied. Complete regression of tumor as well as marked retardation of growth were noted as a result of treatment with the following sera: (1) normal; (2) depleted of complement 1 (R1); of complement 3 (R3), and of complement 4 (R4); (3) depleted of properdin (RP); (4) heated at 56 C. for 20 minutes and for 60 minutes and at 66 C. for 30 minutes; and (5) supernatant from centrifuged at 35,000 G at 2 C. for 3 hours. No tumor inhibitory effect was noted as a result of treatment with (1) serum depleted of complement 2 (R2) and (2) pellet (containing lipopolysaccharides) from serum centrifuged at 35,000 G at 2 C. for 3 hours. These results indicate that the tumor inhibitory principle (TIP) in guinea pig serum is probably not complement, properdin, or lipopolysaccharide.

Properdin titers of sera from mice treated with each of the preparations listed above disclosed low properdin levels when the tumors were growing or were large, and normal or elevated properdin levels when the tumors were regressing or disappeared completely. In addition, normal nontumor-bearing mice showed as much as a threefold increase in properdin levels when treated with normal or heated guinea pig serum.

While it is possible that the tumor inhibitory principle (TIP) in guinea pig serum may exert its effect through the animal’s own properdin system, such a relationship has not as yet been demonstrated.
The Detection of Minor Erythrocyte Populations by Mixed Agglutinates
Authors: JONES A. RICHARDSON , SILVER SHEILA
Published: October 2019

The principle of the "mixed agglutination" reaction has been applied to the development of a technic for the detection of minor populations of erythrocytes in mixtures in which the minor component is identifiable by means of one or another of the blood group iso-antigens. The method lends itself to the quantification of a minor population and provides a degree of sensitivity which compares very favorably with that which can be achieved through the use of radioactive tracers.
Selenoid (Crescent) Bodies
Authors: Cuadra Manuel
Published: October 2019

Cuadra, Manuel: Selenoid (crescent) bodies. Blood 13:258-265 (March), 1958.

On p. 260, line 7, the statement is made:

"Selenoid bodies with their inseparable components of central vacuole (‘vesicle’) and pink crescent (‘lunula’) were found in small numbers in normal blood and occurred in very large numbers in cases of acute hemolytic anemias8; they are apparently related to the physiologic and pathologic destruction of erythrocytes."

The author desires it to read:

"The stromata of the erythrocytes appeared in the shape of a red disc and were clearly differentiated from erythrocytes; they were found in small numbers in normal blood and occurred in very large numbers in cases of acute hemolytic anemias8; they are apparently related to the physiologic and pathologic destruction of erythrocytes."
Ribosome Biosynthesis in Cultured Lymphocytes: II. The Role of Ribosomal RNA Production in the Initiation and Maintenance of Lymphocyte Growth
Authors: RUBIN ARNOLD D.
Published: October 2019

Ribosomal RNA production in cultured lymphocytes appeared to be regulated by the rate of 45S rRNA precursor transcription and also by the rate of 45S processing to the 28S and 18S components found in mature ribosomes. Cultures of resting lymphocytes restricted ribosome formation by maintaining both transcription and processing of 45S RNA at low levels. Still about 50 per cent of the RNA finally processed to 28S lacked corresponding 18S components as a result of selective degradation in resting lymphocytes. After PHA treatment, the rates of 45S rRNA precursor transcription and cleavage to 28S products, detectably increased within one hour, rose to a maximum at 48 hours. However, conservation of 18S components reached maximal efficiency between one to five hours after PHA treatment. By 48 hours, at the height of the growth response, PHA-stimulated cultures once again began to selectively degrade 18S RNA. At 168 hours, after the proliferative process had ceased, 45S processing slowed considerably despite a brisk rate of 45S RNA transcription —approximately 50-75 per cent of maximum.

Thus, the initiation of lymphocyte growth and the maintenance of the growing condition may depend on the rate of ribosome biosynthesis which, in turn, may be regulated by the efficiency of 45S rRNA precursor utilization for ribosome assembly.
Increased in vitro and in vivo generation of procoagulant activity (tissue factor) by mononuclear phagocytes after intralipid infusion in rabbits
Authors: Montemurro P , Lattanzio A , Chetta G , Lupo L , Caputi-Iambrenghi L , Rubino M , Giordano D , Semeraro N
Published: October 2019

Intralipid, a fat emulsion widely used in parenteral nutrition, can produce marked functional changes of the mononuclear phagocyte system. We investigated the effect of Intralipid administration on the generation of procoagulant activity by rabbit mononuclear phagocytes. Two groups of ten rabbits given either a single infusion of Intralipid 10% or a similar volume of sterile saline were studied before and after infusion. Procoagulant activity was measured on isolated blood mononuclear cells after incubation with and without endotoxin, using a one-stage clotting assay. Cells from animals infused with Intralipid produced significantly more procoagulant activity than controls (P less than .01). Results were similar when freshly collected whole blood was incubated with and without endotoxin, and procoagulant activity was measured on subsequently isolated mononuclear cells (P less than .01). In addition, when rabbits were given a single injection of endotoxin, blood and spleen mononuclear cells harvested 50 to 60 minutes after the injection from animals pretreated with Intralipid expressed five to seven times more procoagulant activity than did cells from animals pretreated with saline. In all instances, procoagulant activity was identified as tissue factor. These findings suggest that Intralipid may cause functional changes in mononuclear phagocytes, resulting in increased production of tissue factor on incubation in short-term culture in vitro and in response to endotoxin in vivo.
Adult T cell leukemia: a potential target for ricin A chain immunotoxins
Authors: Kronke M , Depper JM , Leonard WJ , Vitetta ES , Waldmann TA , Greene WC
Published: October 2019

Adult T cell leukemia (ATL) is an almost uniformly fatal malignancy of mature T cells associated with human T cell leukemia/lymphoma virus type 1 (HTLV-1) infection. Cells from this leukemia are characterized by the expression of large numbers of receptors for interleukin 2 (IL- 2). In an attempt to prepare an immunotoxin with selective cytotoxicity for ATL cells, we conjugated anti-Tac, a monoclonal anti-IL-2 receptor antibody, to purified ricin A chains. Although unmodified anti-Tac had no effect on the protein synthesis of these cells, anti-Tac-ricin A chain conjugates produced half-maximal inhibition of protein synthesis in HTLV-1-infected leukemic T cell lines at concentrations of 2 to 6 X 10(-10) mol/L (ID50). An essentially identical ID50 was obtained with leukemic peripheral blood T lymphocytes isolated from two patients with ATL. In contrast, half-maximal inhibition of protein synthesis in HTLV- uninfected, IL-2 receptor-negative T and B cell lines required 200- to 1,000-fold higher concentrations of anti-Tac-ricin A chain conjugates. Both unconjugated anti-Tac and immunoaffinity-purified IL-2 completely inhibited the toxic effects of anti-Tac-ricin A, confirming the specificity of the conjugate-IL-2 receptor interaction. Clonogenic assays demonstrated that anti-Tac-ricin A chain was able to eliminate greater than 99.9% of an HTLV-1-infected T cell population at concentrations only marginally affecting IL-2 receptor-negative cells. The data presented demonstrate that anti-Tac-ricin A is selectively cytotoxic for HTLV-1-infected leukemic T cells in vitro and raises the future possibility of specific therapeutic intervention with immunotoxins in this disease.
Expression of vimentin intermediate filament cytoskeleton in acute nonlymphoblastic leukemias
Authors: Dellagi K , Tabilio A , Portier MM , Vainchenker W , Castaigne S , Guichard J , Breton-Gorius J , Brouet JC
Published: October 2019

Since vimentin intermediate filament (IF) expression in hemopoietic cells varies with the cell lineage as well as the state of differentiation of the cells, we studied the vimentin cytoskeleton by direct immunofluorescence and electron microscopy in 50 cases of acute nonlymphocytic leukemias. We found that malignant cells tend to reproduce the vimentin organization characteristic of their normal cellular counterpart. Thus, in M2 and M3 leukemias (French-American- British classification), vimentin was often reduced to a juxtanuclear bundle of filaments contrasting with the rich filamentous network expressed by M4 or M5 leukemias. In erythroblastic leukemias (M6) and megakaryoblastic leukemias, both identified by the expression of lineage-specific antigens, the absence of vimentin IFs could be correlated with the level of differentiation reached by the blasts. M1 leukemias displayed an abnormal pattern of vimentin organization with aggregated filaments giving a ring-like structure. However, no abnormality of the vimentin polypeptide could be detected by two- dimensional electrophoresis. These results show that the expression of the vimentin IF cytoskeleton may be a useful marker of differentiation in the study of leukemic cells.
Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets
Authors: Kinlough-Rathbone RL , Rand ML , Packham MA
Published: October 2019

Human platelets are aggregated and induced to release their granule contents and form thromboxane by peptides as short as 6-amino acid residues (SFLLRN) corresponding to the newly released N-terminus of the thrombin receptor that is cleaved by thrombin. Using washed platelets, we found that these responses to SFLLRN (2 to 6 mumol/L) were enhanced by fibrinogen. However, neither SFLLRN nor SFLLRNPNDKYEPF had any effect on washed rabbit or rat platelets, although they were fully responsive to human thrombin. Concentrations of the peptides as high as 100 mumol/L did not cause the platelets of rabbits or rats to change shape, aggregate, release granule contents, or form thromboxane. SFLLRN did not affect the extent of aggregation induced by adenosine diphosphate (ADP) or a low concentration of thrombin. Pig platelets responded to 50 mumol/L SFLLRN with reversible aggregation, which was enhanced by fibrinogen, but not accompanied by the release of dense granule contents. Guinea pig platelets aggregated and released granule contents in response to 25 or 50 mumol/L of SFLLRN, but responded with only shape change to lower concentrations. Thus, these experiments indicate that rabbit and rat platelets lack a functional response to human thrombin receptor peptides that fully activate the previously described human thrombin receptor, despite a full response of both rabbit and rat platelets to human thrombin, and that pig and guinea pig platelets have incomplete responses to these human thrombin receptor peptides. The results suggest that platelets of rabbits and rats, and perhaps guinea pigs and pigs, respond to thrombin through an alternative receptor that has also been suggested to be present on human platelets.
Differential usage of an Ig heavy chain variable region gene by human B- cell tumors
Authors: Stevenson FK , Spellerberg MB , Treasure J , Chapman CJ , Silberstein LE , Hamblin TJ , Jones DB
Published: October 2019

A monoclonal anti-idiotypic antibody has been raised that recognizes Igs with heavy chains encoded by a member of the VH4 family, the VH4–21 gene segment. The idiotope (Id) is detectable on a high percentage of early B cells in fetal spleen, and is expressed by certain autoantibodies, particularly cold-reactive anti-red blood cell antibodies. Therefore, it was of interest to investigate usage of this VH gene by neoplastic B cells; 81 chronic lymphocytic leukemias (CLLs) involving CD5+ B cells and 62 B-cell lymphomas of varying histologic type have been analyzed. The Id was expressed by only 3 of 81 (3.7%) of the CLLs, indicating a relatively low usage by these tumors. In contrast, the Id was expressed by 9 of 62 (14.5%) of the lymphomas across a range of histologic types, indicating a differential use of the VH4–21 gene among B-cell neoplasms. For three of the Id-positive lymphomas, each of a different histologic class, the nucleotide sequence of the tumor-derived VH gene was determined; the VH4–21 gene was identified, as expected. The sequence from the CLL was identical to the germline sequence, and the marginal zone lymphoma showed only 3 nucleotide changes, 2 of which gave rise to amino acid substitutions. In contrast, the sequence from the follicular lymphoma showed 29 nucleotide changes giving rise to 14 amino acid substitutions, which were scattered among the CDR and FW regions.
Localization and quantitation of the vitamin D binding protein (Gc- globulin) in human neutrophils
Authors: Kew RR , Sibug MA , Liuzzo JP , Webster RO
Published: October 2019

Plasma-derived vitamin D binding protein (DBP) is an important physiologic regulator of the neutrophil chemotactic response to activated complement. A cell-associated form of DBP has been observed in numerous cell types. We now report that mature, circulating human neutrophils also contain cell-associated DBP. Immunofluorescence studies of normal untreated neutrophils showed the presence of DBP on the cell surface. Western blotting of detergent-soluble neutrophil lysates with a polyclonal anti-DBP showed two major immunoreactive bands, one with an apparent molecular weight of 56 Kd (identical to purified plasma-derived DBP) and a second less prominent band at 12 to 14 Kd. Quantitation of the immunoreactive bands by video densitometry indicated that normal human neutrophils contain 1.5 +/- 0.8 ng DBP/10(6) cells (n = 9). Immunoprecipitation of detergent-soluble lysates with the polyclonal anti-DBP showed only the 56-Kd form by Western blotting. In contrast, a monoclonal anti-DBP immunoprecipitated the 12 to 14 Kd form of DBP from lysates of surface-radioiodinated cells. Western blots of subcellular fractions showed that immunoreactive bands were found in the specific (secondary) granule and plasma-membrane fractions. In addition, pretreatment of neutrophils with 10 nmol/L phorbol myristate acetate (PMA) resulted in approximately a 50% reduction in the amount of DBP in both the specific granule and plasma-membrane fractions. Finally, analysis of the cell- free supernates showed that DBP was spontaneously released into the extracellular milieu: moreover, this release was enhanced if the cells were first stimulated with C5a, formyl-norleucyl-leucyl-phenylalanine (fNLP) or PMA.
Connexin-43-type gap junctions mediate communication between bone marrow stromal cells
Authors: Dorshkind K , Green L , Godwin A , Fletcher WH
Published: October 2019

Several morphologic studies have suggested that gap junctions exist between bone marrow stromal cells. This possibility was examined by analysis of stromal cells present in the adherent layer of primary long- term lymphoid bone marrow cultures and in additional studies using a stromal cell line. Results showing that the fluorescent dye lucifer yellow, when microinjected into a single stromal cell, transferred between most other contacting stroma and that stromal cells were electronically coupled provided support that cell-cell communication occurs between these microenvironmental elements. Additional studies showed that transcripts for connexin (Cx) 43, but not for Cx26 or Cx32, were present in a stromal cell line. To examine the potential for regulated cell-cell communication between the stroma, cells were treated with interleukin-1 (IL-1), a cytokine known to affect stromal cell function, and the effects on dye transfer were examined. IL-1 treatment resulted in a reversible decrease in the ability of dye to transfer between stromal cells in contact. Taken together, these studies show that gap junctions exist between stromal cells and that their permeability can be regulated. However, gap junction-mediated cell-cell communication could not be shown between the stroma and developing lymphoid cells.
Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway
Authors: Galmiche MC , Koteliansky VE , Briere J , Herve P , Charbord P
Published: October 2019

In human long-term marrow cultures connective tissue-forming stromal cells are an essential cellular component of the adherent layer where granulomonocytic progenitors are generated from week 2 onward. We have previously found that most stromal cells in confluent cultures were stained by monoclonal antibodies directed against smooth muscle- specific actin isoforms. The present study was carried out to evaluate the time course of alpha-SM-positive stromal cells and to search for other cytoskeletal proteins specific for smooth muscle cells. It was found that the expression of alpha-SM in stromal cells was time dependent. Most of the adherent spindle-shaped, vimentin-positive stromal cells observed during the first 2 weeks of culture were alpha- SM negative. On the contrary, from week 3 to week 7, most interdigitated stromal cells contained stress fibers whose backbone was made of alpha-SM-positive microfilaments. In addition, in confluent cultures, other proteins specific for smooth muscle were detected: metavinculin, h-caldesmon, smooth muscle myosin heavy chains, and calponin. This study confirms the similarity between stromal cells and smooth muscle cells. Moreover, our results reveal that cells in vivo with the phenotype closest to that of stromal cells are immature fetal smooth muscle cells and subendothelial intimal smooth muscle cells; a cell subset with limited development following birth but extensively recruited in atherosclerotic lesions. Stromal cells very probably derive from mesenchymal cells that differentiate along this distinctive vascular smooth muscle cell pathway. In humans, this differentiation seems crucial for the maintenance of granulomonopoiesis. These in vitro studies were completed by examination of trephine bone marrow biopsies from adults without hematologic abnormalities. These studies revealed the presence of alpha-SM-positive cells at diverse locations: vascular smooth muscle cells in the media of arteries and arterioles, pericytes lining capillaries, myoid cells lining sinuses at the abluminal side of endothelial cells or found within the hematopoietic logettes, and endosteal cells lining bone trabeculae. More or less mature cells of the granulocytic series were in intimate contact with the thin cytoplasmic extensions of myoid cells. Myoid cells may be the in vivo counterpart of stromal cells with the above-described vascular smooth muscle phenotype.
Development of antitissue factor antibodies in patients after liver surgery
Authors: Tsuda H , Higashi S , Iwanaga S , Kubota T , Morita T , Yanaga K
Published: October 2019

After liver surgery, two patients developed unexplainable prolonged prothrombin times (PT) that were not associated with bleeding tendency. The substitution of rabbit thromboplastin for either human or monkey thromboplastin in performing PT tests resulted in a normal clotting time. Tissue factor (TF) procoagulant activity assays and an immunoblotting analysis showed that these patients had developed IgG lambda-type immediate anticoagulants directed against both rabbit and bovine TF that did not crossreact with human or monkey TF. In a chromogenic assay, the patient IgG caused a decrease in both the Km and the Vmax of the factor X activation by rabbit TF-factor VIIa complex. The lack of reactivity of the patient IgG with human TF presumably explained why there was no clinical bleeding. Both patients had been treated earlier with a topical hemostatic agent prepared from bovine corium, microfibrillar collagen hemostat, while undergoing previous surgery. In an immunoblotting analysis, the patient IgG stained a 42-Kd band in the Triton extract of the collagen preparation under either reducing or nonreducing conditions. The Triton extract of the collagen preparation blocked the binding of the patient IgG to bovine TF. Thus, it is suggested that the iatrogenic immunization by intraoperative exposure of bovine TF retained in the collagen preparation may be responsible for the development of anti-TF antibodies in these patients. The anti-TF antibodies resulted in a clinical error in the evaluation of coagulation status after the use of rabbit thromboplastin.
Heterogeneous chromosomal aberrations generate 3' truncations of the NFKB2/lyt-10 gene in lymphoid malignancies
Authors: Migliazza A , Lombardi L , Rocchi M , Trecca D , Chang CC , Antonacci R , Fracchiolla NS , Ciana P , Maiolo AT , Neri A
Published: October 2019

The NFKB2(lyt-10) gene codes for a protein that is a member of the NK- kappa B/rel family of transcription factors containing a DNA-binding rel domain and a carboxy-terminal ankyrin-like domain. The NFKB2 gene represents a candidate proto-oncogene, since it has been found to be involved in a chromosomal translocation t(10;14)(q24;q32) in one case of B-cell lymphoma and in gene rearrangements in various types of lymphoid malignancies. To elucidate the structural and functional consequences of NFKB2 rearrangements, we report the molecular characterization of three novel rearranged NFKB2 genes in lymphoid tumors. In one case of multiple myeloma (MM), cloning and sequencing analysis of reciprocal breakpoint sites showed that they occurred within intron 15 of the NFKB2 gene and led to the complete deletion of the 32 portion of the gene coding for the ankyrin domain. Fluorescent in situ hybridization (FISH) analysis showed that the novel regions involved in the NFKB2 rearrangement originated from chromosome 7q34, thus implying the occurrence of a t(7;10)(q34;q24) reciprocal chromosomal translocation. In one case of T-cell cutaneous lymphoma (CTCL) and in one of B-cell chronic lymphocytic leukemia (B-CLL), NFKB2 rearrangements occurred, respectively, within exons 18 and 20 of the gene and involved recombinations with distinct regions of chromosome 10q24. Molecular analysis suggested that these rearrangements may occur as a consequence of small internal chromosomal deletions. In both of these cases, the rearrangements led to specific carboxy-terminal truncations of NFKB2 generating abnormal transcripts that coded for proteins lacking portions of the ankyrin domain. These proteins localize in the nucleus, suggesting their constitutive activation in vivo. Overall, our results indicate that NFKB2 rearrangements in lymphoid neoplasia may occur by heterogeneous mechanisms, including internal chromosomal deletion or chromosomal translocation. The common consequence of these rearrangements appears to be the deletion of 32 sequences of NFKB2 leading to the production of carboxy-truncated constitutively nuclear proteins that may be involved in tumorigenesis.
A donor-derived asialo-GM1+ cell induces depression of B-cell genesis during systemic graft-versus-host disease
Authors: Xenocostas A , Ghayur T , Setrakian JC , Lapp WS , Osmond DG
Published: October 2019

The nature of the effector cell(s) responsible for the depression of B- cell genesis in the bone marrow of mice undergoing systemic graft- versus-host disease (GVHD) has been examined. Donor C57BL/6 (B6) mice were treated in vivo with either a single injection of anti-asialo GM1 antibody (anti-ASGM1) to eliminate naturally occurring (endogenous) ASGM1+ cells or B6xAF1 (B6AF1) lymphoid cells followed by anti-ASGM1 to eliminate both endogenous and “induced” ASGM1+ cells. Lymphoid cells from donor mice after the elimination of endogenous ASGM1+ cells produced severe GVHD and concomitant depression of B-cell genesis when injected into B6AF1 recipients. In contrast, cells from donors depleted of both the endogenous and inducible ASGM1+ populations did not cause GVHD or depletion of B lineage cells in B6AF1 recipients but did depress B-cell genesis in B6C3F1 mice. The “induced” ASGM1+ cells were Thy 1+, but their elimination did not significantly alter either overall T-cell function or specific cytotoxic T-cell (CTL) reactivity against the sensitizing (B6AF1) strain. The results suggest that the effector cell responsible for the depression of B-cell genesis during systemic GVHD can be induced to express ASGM1, is strain-specific and Thy 1+; but is not a conventional CTL.
Identification of potential hot spots in the carboxy-terminal part of the Epstein-Barr virus (EBV) BNLF-1 gene in both malignant and benign EBV-associated diseases: high frequency of a 30-bp deletion in Malaysian and Danish peripheral T-cell lymphomas
Authors: Sandvej K , Peh SC , Andresen BS , Pallesen G
Published: October 2019

In this study, we have sequenced the C-terminal part of the Epstein- Barr virus (EBV)-BNLF-1 gene encoding for the latent membrane protein-1 from tissues of EBV-positive Danish Hodgkin's disease (HD) and of Danish and Malaysian peripheral T-cell lymphomas (PTLs) and from tonsils of Danish infectious mononucleosis (IM). Our study showed that some of the 7 single-base mutations and the 30-bp deletion previously detected between codons of amino acid 322 and 366 in the BNLF-1 gene of the nasopharyngeal carcinoma cell line CAO were present in all Malaysian PTLs and in 60% of the Danish PTLs. In HD and the IM cases, the mutations were present in about 30%. The 30-bp deletion and the single base mutations occurred independently, and mutations were detectable in the majority of EBV type B-positive cases. These findings suggest that the 30-bp deletion and the 7 single-base mutations in the C-terminal part of the CAO-BNLF-1 gene do not characterize a new EBV type A substrain. Rather, some of the positions of single base mutations and the 30-bp deletion are hot spots that may have mutated independently through the evolution of EBV strains.
Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of CD34++/CD38- long-term culture-initiating cells
Authors: Croisille L , Auffray I , Katz A , Izac B , Vainchenker W , Coulombel L
Published: October 2019

Very primitive human hematopoietic progenitor cells are identified indirectly by their ability to give rise to clonogenic progenitors in the presence of either human or murine stromal cells. These long-term culture-initiating cell (LTC-IC) assays are usually performed in the presence of hydrocortisone based on the initial observation that hydrocortisone was required for prolonged hematopoiesis in standard long-term bone marrow cultures. In this report, we investigated the role of hydrocortisone in LTC-IC assays initiated with CD34++/CD38- cells seeded onto either human bone marrow LTC-derived adherent cells or a murine marrow-derived stromal cell line, MS-5. It was found that weekly addition of hydrocortisone to the cultures reduced the frequency of LTC-IC (from 1/5 to 1/20) calculated from limiting dilution experiments and also reduced fivefold to 10-fold the number of their progeny clonogenic cells detected after 4 to 5 weeks. In contrast, the frequency and differentiative potential of CD34++/CD38- grown in the presence of human marrow feeders was unaltered by the addition of glucocorticoids. Data are consistent with the hypothesis that hydrocortisone inhibited LTC-IC differentiation by downregulating the expression of a synergistic factor produced by MS-5 cells. (1) In the absence of hydrocortisone, the number of clonogenic progenitors generated by LTC-IC was much higher in cultures seeded on MS-5 than in cultures seeded on human marrow adherent cells, which was also true when cytokines were added to the cocultures. However, based on the phenotype of the colonies, progenitors produced in MS-5 cocultures were more mature than those generated on human marrow adherent cells. (2) Hydrocortisone counteracted the stimulatory effect of recombinant human cytokines (interleukin-3, interleukin-6, and steel factor) in assays performed on MS-5 but not on human marrow feeders. (3) Hydrocortisone led to a 50% decrease in the numbers of colony-forming units- granulocyte-macrophage found in methycellulose colony assays of CD34++/CD38- cells performed in the presence of MS-5 cells. Taken together, our results indicate that hydrocortisone acts differently on a murine stromal cell line and on marrow-derived human stromal cells and may suppress the expression by MS-5 cells of an activity selectively promoting amplification of clonogenic cells derived from primitive LTC-IC.
Hereditary antithrombin deficiency: heterogeneity of the molecular basis and mortality in Dutch families
Authors: van Boven HH , Olds RJ , Thein SL , Reitsma PH , Lane DA , Briet E , Vandenbroucke JP , Rosendaal FR
Published: October 2019

We studied the molecular basis and genetic heterogeneity of hereditary antithrombin (III) deficiency in nine Dutch families. Polymerase chain reaction (PCR) amplification and direct sequencing of all antithrombin gene exons and flanking intronic regions identified mutations in eight families. Given the opportunity to correlate the molecular basis with survival, we addressed the relevance of molecular defects to mortality in inherited antithrombin deficiency. The defects included single nucleotide deletions (7671 del G, 7768–69 del G) and insertions (5501 ins A, 2463 G-->TC) that lead to frameshifts, a single base substitution [5381 C-->T (129Arg-->stop)] leading to a premature termination codon, and single base substitutions resulting in amino acid substitutions [2652 A-->C (63Tyr-->Ser), 13380 T-->C (421Ile-->Thr), and 13407 G-->T (430Cys-->Phe)]. All affected individuals were heterozygous for the defects. Previously we found in Dutch families that antithrombin deficiency did not lead to higher mortality compared with the general population. In accordance with these findings, we observed no excess mortality in the nine families [Observed:Expected, 52:52.6; standardised mortality ratio (SMR) 1.0, 95% confidence interval (CI), 0.7–1.3]. Our findings confirmed a considerable genetic heterogeneity underlying antithrombin deficiency. We therefore concluded that the lack of excess mortality in these families is not caused by a Dutch mild defect. We suggest that the longevity is not affected by molecular defects in the antithrombin gene and hypothesize that differences in mortality or natural history between families most likely result from other (genetic) risk factors.
A defect in the early phase of T-cell receptor-mediated T-cell activation in patients with common variable immunodeficiency
Authors: Fischer MB , Hauber I , Eggenbauer H , Thon V , Vogel E , Schaffer E , Lokaj J , Litzman J , Wolf HM , Mannhalter JW
Published: October 2019

Common variable immunodeficiency (CVID) is characterized by an impairment of specific antibody production and a decrease in all or selected Ig isotypes. Abnormalities at the level of the B cells, T cells, and antigen-presenting cells have been described. In the present study, we have focused our attention on T-cell activation in CVID. T cells from 15 of 24 patients failed to respond to recall antigens (eg, tetanus toxoid, Escherichia coli). Of these 15 patients, 11 were studied in detail and showed significantly decreased T-cell proliferative responses and/or decreased interleukin-2 and interferon- gamma production on T-cell receptor-mediated stimulation with recall antigens and superantigens (staphylococcal enterotoxins [SE]); however, T-cell response to mitogens (anti-CD3 monoclonal antibody, phytohemagglutinin) was normal. The defect in interleukin-2 and interferon-gamma release on tetanus toxoid stimulation could also be documented in purified CD4 T cells of the patients and was present in patients with high and normal CD8 counts alike. Furthermore, patients' T cells failed to mount a significant elevation in free intracellular calcium (Ca++ flux) in response to superantigen, whereas the response to phorbol myristate acetate and ionomycin, bypassing receptor-mediated signaling, was unimpaired. These results indicate a defect in the early phase of T-cell activation after triggering of the T-cell receptor in a significant subgroup of CVID patients.
Abnormal expression of the p53-binding protein MDM2 in Hodgkin's disease
Authors: Chilosi M , Doglioni C , Menestrina F , Montagna L , Rigo A , Lestani M , Barbareschi M , Scarpa A , Mariuzzi GM , Pizzolo G
Published: October 2019

The possible involvement of p53 tumor suppressor gene in the pathogenesis of Hodgkin's disease (HD) is suggested by the frequent finding of abnormal accumulation of p53 protein in the nuclei of Reed- Sternberg cells and their variants (H-RS) in a large proportion of cases. This finding, besides being consistent with the presence of p53 gene mutations, might represent a consequence of the inactivating interaction between p53 and p53-binding proteins such as the product of the MDM2 cellular oncogene. We have examined an unselected series of 77 HD cases of different histologic patterns for the expression of p53 and MDM2 proteins, using specific monoclonal antibodies and sensitive immunohistochemical techniques in single- and double-marker combination. In the large majority of cases (66/77), a variable proportion of H-RS cells expressed MDM2 that was strictly confined to the nuclei. Coexpression of both MDM2 and p53 was common in the same cells. The abnormal nuclear expression of p53 and MDM2 did not seem to correlate with the presence of Epstein-Barr virus infection, as shown by the results of LMP-1 antigen expression and EBER in situ hybridization analysis. Our data suggest that the abnormal accumulation of MDM2 and p53 proteins in HD might reflect a derangement of molecular mechanisms that could play a pathogenetic role in this disease.