bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Gamma Globin Gene Regulation in Knock-In Mouse Models of Anemia.
Authors: McConnell Sean C. , Huo Yongliang , Liu Shan-Run , Zhang Ting-Ting , Ulrey Clayton L. , Ryan Thomas M.
Published: October 2019

The generation of transgenic and gene targeted mouse models of human hemoglobinopathies provides valuable opportunities to test mechanisms of human globin gene regulation and experimental therapies. Yet mice do not naturally have a fetal hemoglobin, challenging our ability to adequately model the developmental onset of disease. Transgenic model systems that contain the entire human β-globin locus present obstacles to the study of human globin gene switching, including a fetal to adult globin gene switch that occurs too early in development. The generation of genetically engineered mice with a delayed human γ to β hemoglobin switch has been a major topic of interest for our laboratory. Delayed γ globin gene expression improves the clinical progression in patients as well as animal models with hemoglobinopathies. However, molecular mechanisms involved in globin gene switching are not well understood. In this study the transcriptional and epigenetic regulation of human γ to β hemoglobin switching are analyzed in novel human knock-in (KI) mouse models that complete the switch from fetal to adult hemoglobin after birth. These KI mice were generated by replacement of the adult mouse β-globin genes by homologous recombination in embryonic stem cells with a delayed switching human γ to β globin gene construct. Quantitative real-time PCR and HPLC were used to measure mouse and human embryonic, fetal, and adult globin genes through development and show that we have given the mouse a true fetal hemoglobin. Heterozygous mice express human β-like globin genes at a high level comparable to the adult mouse β globin genes. Mutations responsible for hereditary persistence of fetal hemoglobin (HPFH) in the γ globin promoter recapitulate the human phenotype in KI mice, with over 50 fold γ globin gene upregulation in adults. These HPFH KI mice also display higher γ globin levels at birth and markedly delayed γ globin gene downregulation in the weeks following birth. These studies in KI mice demonstrate that human β-like globin genes interacting with the mouse LCR are regulated in a manner similar to what is seen in humans and may be used to study the mechanisms of globin gene switching. Greater understanding of γ-globin gene regulation will be required for achieving the therapeutic goal of reactivating silenced γ-globin genes to ameliorate severe human hemoglobinopathies.
A Humanized Cooley’s Anemia Mouse Model.
Authors: Huo Yongliang , McConnell Sean C. , Ulrey Clayton L. , Zhang Ting-Ting , Yang Rui , Liu Shan-Run , Ryan Thomas M.
Published: October 2019

Cooley’s Anemia (CA) is a heterogeneous group of inherited disorders all marked by the complete absence of adult β-globin chains in red blood cells (RBCs). Newborns with CA are healthy because of the high level of fetal hemoglobin (HbF) present at birth. As HbF levels decline during the first year of life, the absence of functional adult β-globin genes results in a severe anemia that necessitates the initiation of regular blood transfusions for the remainder of life. CA has been difficult to study in murine models due to the lack of a human fetal hemoglobin equivalent in the mouse. This study reports a novel preclinical animal model of CA that survives solely on human fetal hemoglobin at birth and is blood transfusion dependent for life upon completion of the hemoglobin switch after birth. These humanized CA mice were generated by targeted gene replacement in embryonic stem cells of the adult mouse α-globin genes with human α-globin and the adult mouse β-globin genes with a delayed switching γδβ0-globin gene cassette. The nonfunctional human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1. Both wild-type and hereditary persistence of fetal hemoglobin (HPFH) promoter mutations were tested in the human γ-globin knock-in allele. Heterozygous knock-in mice exhibit β thalassemia intermedia. Newborn homozygous knock-in mice express 100% human hemoglobin in their RBCs, suffer from ineffective erythropoiesis, survive from one to ten days after birth, and are blood transfusion dependent for adult life. This is the first CA mouse model that recapitulates the temporal onset of the disease in human patients. This CA disease model is useful for the study of the regulation of globin gene expression, synthesis, and switching; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.
Increased Prothrombotic State in Patients with Advancing HIV Disease.
Authors: Lijfering Willem M. , Sprenger Herman G. , Georg Rita R. , van der Meulen Piet A. , van der Meer Jan
Published: October 2019

HIV-infected patients are at high risk for venous and arterial thrombosis. We hypothesized that advancing stages of HIV are associated with thrombophilic abnormalities that could predispose to thrombosis. A total of 109 consecutive HIV infected patients were included in the study and twice screened for currently known thrombophilic defects with a time interval of at least 3 months. Ten percent of HIV infected patients had confirmed decreased protein C levels (< 65 IU/dL), 41% had confirmed elevated factor VIII levels (> 150 IU/dL), 22% had confirmed high fibrinogen levels (> 3.5 g/L) and 60% had a confirmed free protein S deficiency (< 65 IU/dL). Median factor VIII levels were higher in patients with AIDS defining illness compared to non AIDS defining illness (226 IU/dL versus 149 IU/dL; P< 0.001), while median free protein S levels were lower in these subgroups (45 IU/dL, versus 58 IU/dL; P< 0.001). Advancing HIV, stratified by CD4 levels of > 500 cells/μL, 200–500 cells/μL or < 500 cells/μL, respectively, was associated with increasing factor VIII levels, fibrinogen levels and decreasing free protein S levels (Figure 1). When factor VIII levels were < 100 IU/dL, median fibrinogen levels were 2.4 g/L, which was 0.3 g/L lower than when factor VIII levels were 100–150 IU/dL (P=0.033) and 0.9 g/L lower than when factor VIII levels were > 150 IU/dL (P< 0.001). Free protein S levels were 70 IU/dL when factor VIII levels were < 100 IU/dL, which decreased to 60 IU/dL when factor VIII levels were 100–150 IU/dL (P=0.010) and to 52 IU/dL when factor VIII levels were > 150 IU/dL (P< 0.001). We conclude that HIV infected patients have multiple prolonged thrombophilic abnormalities compared to the normal population, which increase with advancing HIV disease, thus providing a biologic mechanism for the increased prevalence of venous and arterial thrombosis in HIV.

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The Stanford V Regimen Is an Effective Treatment for Good Prognosis Patients with Hodgkin’s Disease.
Authors: Abuzetun Jamil Y. , Loberiza Fausto , Bast Martin , Vose Julie M. , Bierman Philip J. , Bociek R. Gregory , Armitage James O.
Published: October 2019

Despite recent therapeutic advances, the treatment of Hodgkin’s disease remains controversial. Among the most commonly used regimens are ABVD, Stanford V and BEACOPP. Studies of Stanford V have shown conflicting outcomes with some excellent and some much less good results. To address this discrepancy we reviewed 113 patients with previously untreated Hodgkin’s disease, all of whom were HIV negative, who received the Stanford V regimen from physicians in the Nebraska Lymphoma Study Group between January 1997 and January 2006. The median age was 36 years (range 15–88) with only 10 patients over 60 years of age. Fifty-two (46%) of the total were males. Seventy-eight (68%) had nodular sclerosis, subtype and 69 (61%) were stage I and II. Constitutional symptoms were present in 36 (32%) patients. Tumor bulk > 5cm was evident in 68 (60%). Four or more nodes were involved in 57 (50%) and 84 (78%) received radiotherapy. Median follow up was 63 months (range 8–114). Univariate analysis of the data identified 4 adverse risk factors which included age > 60 years, stages III or IV, constitutional symptoms, and mixed cellularity subtype. Complete remission or unconfirmed complete remission was achieved in 82(72%), 21 (18%) had partial response, 2 had progression of disease (PD) and 3 were unable to be evaluated. There were 5 early deaths; 3 of which were definitely related to the treatment. For the entire group, the 5 year overall survival (OS) was 84% and the 5-year event free survival (EFS) was 74%. However, the results were dependent on the presence or absence of the adverse risk factors of age >60, stage III or IV, non-nodular sclerosis histological subtype, and B symptoms as shown below:

Risk Factors 0 (N=53) 1–2(N=50) 3–4(N=10) OS 100% 90% 40% EFS 87% 82% 20%

The Stanford V regimen represented an excellent treatment for patients with stage IA and IIA nodular sclerosis Hodgkin’s disease under the age of 60 and patients with 1–2 adverse risk factors did only slightly less well. The regimen yielded a much poorer outcome in patients with multiple adverse risk factors.
Relationship between the Hematopoietic Phenotype and the Subtype of Fanconi Anemia Mice.
Authors: Navarro Susana , Rio Paula , Meza Nestor W. , Chinchon Raquel , Segovia Jose C. , Bueren Juan A.
Published: October 2019

Fanconi anemia (FA) is a rare inherited disease associated with aplastic anemia and cancer predisposition. Great progress has occurred at a molecular level that allowed the identification of 13 genes involved in the disease. Additionally, several FA mouse models have been developed with the purpose of investigating the biological basis of the disease, and of facilitating the development of new therapies for FA. Because of the genetic heterogeneity of FA, here we have investigated the relevance of different FA genes to preserve the function of the hematopoietic stem cells (HSCs) of the mouse. To this aim, we have comparatively investigated the hematopoietic phenotype of different FA mice: Fanca−/−, Fancc−/−, Fancd1/Brca2Δ27/Δ27and Fancd2−/−. Normal counts of peripheral blood (PB) cells were observed in the different FA mice. Flow cytometry analyses were also incapable of demonstrating significant changes in the hematopoietic subpopulations present in PB, bone marrow (BM) or spleen of the different FA mice. In contrast to these observations, a reduction in the content of the hematopoietic progenitors was observed both in the BM and the spleen of Fancd1/Brca2Δ27/Δ27 and Fancd2−/− mice, either in young or adult. Significantly, a similar reduction in hematopoietic progenitors was only observed in old Fanca−/− and Fancc−/− mice. Moreover, hematopoietic colonies from mice defective in FA proteins downstream the FA core complex were smaller, compared to colonies from wild type, Fanca−/− or Fancc−/− mice. As expected, hematopoietic progenitors from all studied FA mouse models were highly sensitive to the DNA cross-linking agent, mitomycin C (MMC). Concerning the function of the HSCs of these FA mice, reduced competitive repopulation ability was always observed, compared to WT HSCs. However, in animals defective in FA proteins downstream the FA core, CRA values progressively dropped along the transplantation period. Additionally, a spontaneous chromosomal instability was observed in bone marrow cells from Fancd1/Brca2Δ27/Δ27. These animals also showed a marked repopulation defect in their HSCs assessed in their natural physiological environment, when wild type cells were transplanted in unconditioned Brca2Δ27/Δ27 recipients. In summary, our results show a more severe defect in the functionality of the HSC compartment in mice with mutant FA proteins downstream the FA core complex.
Updated Survival Analyses after Prolonged Follow-Up of the Phase 2, Multicenter CREST Study of Bortezomib in Relapsed or Refractory Multiple Myeloma.
Authors: Jagannath Sundar , Barlogie Bart , Berenson James R. , Siegel David , Irwin D. , Richardson Paul , Niesvizky Ruben , Alexanian Raymond , Limentani Steven , Alsina Melissa , Esseltine Dixie-Lee , Anderson Kenneth C.
Published: October 2019

Background: The initial report of the CREST phase 2 study (Br J Haematol2004;127:165–72) demonstrated the substantial activity of bortezomib (VELCADE®, Vc) at two different dose levels in patients with relapsed or refractory multiple myeloma (MM). Here, we provide an updated analysis of overall survival (OS) after prolonged follow-up (median >5 years).

Methods: 54 patients were enrolled to receive Vc 1.0 mg/m2 (n=28) or 1.3 mg/m2 (n=26) on days 1, 4, 8, and 11 of a 21-day cycle, for up to 8 cycles. Dexamethasone (dex) 20 mg on the day of and day after each Vc dose was added for 16 (57%) patients in the 1.0 mg/m2 group and 12 (46%) patients in the 1.3 mg/m2 group, upon suboptimal response to Vc alone. A total of 17 (31%) patients continued to receive Vc ± dex in an extension study, 12 (43%) from the 1.0 mg/m2 and 5 (19%) from the 1.3 mg/m2 groups. Patients received a median of 3 (range, 1–7) prior regimens. Median time from diagnosis to first Vc dose was 2 years. In the 1.0 and 1.3 mg/m2 groups, respectively, mean age was 64 vs 60 years, 50% vs 35% of patients were male, 54% vs 65% had IgG MM, 58% vs 48% had β2-microglobulin ≥4 mg/L, and 29% vs 48% had abnormal cytogenetics. Response rate (CR+PR, EBMT criteria) to Vc alone was 30% vs 38%, and to Vc ± dex was 37% vs 50% in the 1.0 and 1.3 mg/m2 groups. OS from first dose of Vc in each dose group was analyzed using the Kaplan-Meier method.

Results: Median OS in the 1.0 and 1.3 mg/m2 groups was 26.8 months and 60.0 months, after median follow-up of 61 and 65 months, respectively (Figure). In the 1.0 mg/m2 group, 21 (75%) patients have died; the 1- and 2-year OS rates were 82% and 54%, respectively. In the 1.3 mg/m2 group, only 14/26 (54%) patients have died; 1- and 2-year OS rates were 81% and 69%, respectively.

Conclusions: Vc ± dex was active in relapsed or refractory MM at both the 1.0 mg/m2 and 1.3 mg/m2 dose levels and was associated with notable OS, particularly in patients treated with the approved Vc dose of 1.3 mg/m2. The difference in OS between dose groups suggests that the higher dose of Vc is more active. Figure. Kaplan-Meier analyses of OS in patients who received Vc 1.0 mg/m2 (n=28) or 1.3 mg/m2 (n=26).

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T-Cells Redirected Against CD70 for the Immunotherapy of Hematological Malignancies.
Authors: Shaffer Donald , Savoldo Barbara , Dotti Gianpietro , Spencer David , Kenney Shannon , Rooney Cliona M. , Gottschalk Stephen
Published: October 2019

BACKGROUND: CD70 is a tumor necrosis family member that is expressed on a broad spectrum of hematological malignancies including multiple myeloma, non-Hodgkin’s lymphomas and Hodgkin’s disease. In contrast to other already established immunotherapy targets such as CD19, CD20, or CD52, which are widely expressed in the hematopoietic system, CD70 expression is restricted to a subset of activated B and T cells, reducing potential ‘collateral damage’ when targeted by immunotherapy. Preclinical studies in animal models using monoclonal antibodies have validated CD70 as an immunotherapeutic target for malignancies and the aim of this study was to generate CD70-specific T cells for adoptive immunotherapy approaches.

METHODS and RESULTS: To create CD70-specific T cells we constructed a CD70-specific chimeric antigen receptor (CD70-CAR) consisting of domains derived from the CD70 receptor (CD27) and the T-cell receptor CD3-ζ chain. CD70-specific T cells were generated by transducing CD3/CD28-activated T cells with a SFG retroviral vector encoding the CD70-CAR. Cell surface expression of CD70-CARs was confirmed by FACS analysis on CD4- as well as CD8-positive T cells. CD70-specific T cells from healthy donors proliferated and produced IFN-γ as well as IL-2 in contrast to mock transduced T cells after coculture with CD70-positive myeloma cells (U266 and ARH-77) and lymphoblastoid cell lines. In cytotoxicity assays, CD70-specific T cells killed CD70-positive myeloma cell lines and lymphoblastoid cell lines where as activated lymphocytes that express CD70 at low levels and CD70-negatvie targets were not killed.

CONCLUSION: We have successfully constructed a CD70-CAR and demonstrate that CD70-specific T cells selectively recognize and kill malignant cells that express CD70 at high levels. Murine xenograft studies are in progress to confirm these findings in vivo. Adoptive immunotherapy with CD70-specific T cells may represent a promising immunotherapeutic approach for CD70-positive hematological malignancies.
CD4+ CD25high Regulatory T Cells in Newly Diagnosed Patients with Acute Myeloid Leukemia.
Authors: Szczepanski Miroslaw J. , Strauss Laura , Czystowska Malgorzata , Szajnik Marta , Welsh Ann , Foon Kenneth A. , Whiteside Theresa L. , Boyiadzis Michael
Published: October 2019

Regulatory CD4+CD25+ T cells (T reg) are critical regulators of immune tolerance. Increasing evidence supports the existence of elevated numbers of regulatory T cells in cancer patients. Although the increase of T cells seems to be a characteristic feature in most tumors the functional role of Treg, especially in patients with hematologic malignancies, has been less well defined. Mutlicolor flow cytomerty was used to investigated the frequency and phenotype of CD4+ CD25high T cells in the peripheral blood of thirty seven newly diagnosed acute myeloid leukemia (AML) patients (prior to any treatment) and thirty one healthy donors. The suppressive function of Treg was evaluated using CFSE-labeled fresh autologous CD4+CD25− T cells activated with an anti-CD3 antibody as responders. The percentage of circulating CD4+ CD25high Treg was higher (p < 0.0001) in the AML patients (2.9 ± 0.3%, range 0.1–7%) compared to healthy donors (0.4 ± 0.02%, range 0.1–1%). Percentages of T reg expressing Foxp3, CTLA-4, CD45 RO, Fas ligand (CD95) and GITR were significantly elevated in the circulation of AML patients compared to healthy donors: Foxp3 60vs32% p<0.001, CTLA-4 47vs21% p<0.01, CD45 RO 81vs53% p<0.002, Fas ligand 23vs4% p<0.001, GITR 65vs25% p<0.001. Suppression mediated by T reg co-incubated with proliferating autologous responders was also significantly higher (p<0.001) in AML than that mediated by control T reg (75 ± 6% vs 12% ± 4%). These results indicate that T reg accumulate in the periphery of patients with AML and mediate vigorous suppression. Modulation of the Treg suppressive activity, especially during the post-induction period, in AML patients may have therapeutic implications.
The Clinical Observation on Arsenic Trioxide with ATRA in Treating Acute Promyelocytic Leukemia for Six Years.
Authors: Zhong Zhou , Liu Zhen-cai , Li Jian-wei
Published: October 2019

Objective: To observe the efficacy of arsenic trioxide(AS2O3) with ATRT for the treatment of patients with acute promyelocytic leukemia(APL).

Methods: Seventy-four patients with APL, from 2000 to 2006, were randomly divided into two groups. Thirty-eight patients as treating group received AS2O3+ATRA, another thirty-six patients as control group were treated only AS2O3+ARTA+chemotherapy(DA or HA).

Results: The first induced complete remission (CR) was 100%, the three-year relapse rate was 2.6%, the six-year continuous complete remission(CCR) rate was 94.6% for treating group. The rate of CR, the three-year relapse and CCR were 81.3%, 30.7% and 61.5% respectively for control group. The side effects such as myelosuppression, severe infection or septicemia, hepatic, renal and myocardial damage, and the digestive tract side effect were mild compared with that in control group(p<0.01). In treating group, the hyperleukocytosis was not observed, Incidence of the three-year treatment-related mortality (TRM) was 0%, the control group was 16.7%.

Conclusion: The combination of AS2O3 and ATRA can obtain a higher CCR, low relapse rate and mild treatment side effects.
Clinical Trials Underestimate Age of Chronic Myeloid Leukemia (CML) Patients: Epidemiological Study in a Representative Area in Germany.
Authors: Rohrbacher M. , Berger U. , Hochhaus A. , Metzgeroth G. , Adam K. , Lahaye T. , Saussele S. , Hasford J. , Heimpel H. , Hehlmann Rüdiger
Published: October 2019

Epidemiological information on chronic myeloproliferative disorders (CMPD), notably Philadelphia (Ph) and/or bcr/abl positive chronic myeloid leukemia (CML), is rare. National cancer registries and clinical trials differ with regard to median age of CML patients by 10 to 20 years (Table). Therefore, an evaluation was conducted in a defined area in Germany between 1998 and 2000 to determine incidences and compare clinical characteristics of Ph and/or bcr/abl positive CML patients participating and not participating in trials. 68 (37.4%) hospitals and 241 specialty practices (16.4%) reported 893 newly diagnosed CMPD patients. CML patients represented 24.9% of all cases with CMPD. The crude incidence of CML cases (n=218) was 0.79, that of the 172 Ph and/or bcr/abl positive CML cases 0.62 and that of CMML (n=61) 0.22. The incidence of CML and CMML cases combined was 1.01. 110 (64.0%) of the 172 Ph and/or bcr/abl positive CML patients participated in clinical studies, mainly CML Studies III and IIIA of the German CML Study Group. Median age was significantly different between patients participating and not participating in clinical trials: (54.1 vs. 64.8 years, p=0.0001). The chance for a Ph and/or bcr/abl positive CML patient < 65 years to be enrolled in a clinical study was 3.8 times higher than for a CML patient ≥ 65 years (OR=3.8, CI: 1.9–7.3). Male patients had a slightly higher probability to be enrolled in a study than females (OR=1.5 (CI: 0.8–2.8)). Our data indicate that 36% of the Ph and/or bcr/abl positive CML patients registered in a defined area of Germany are not treated in clinical trials, that elderly patients have a lower probability to be included in trials than younger patients and that patients participating in trials are 10.7 years younger than those who do not.

Age of CML Patients in Population based Registries and in Clinical Trials A) Registries Age mean (years ± S.D.) / Thames Cancer Registry, U.K. 65 (20–98) SEER Cancer Statistics Review, 1975–2004 68 (range not available) SEER cancer statistics review, 1973–1998 64 (range not available) B) Trials The Italian Cooperative Study Group on Chronic Myeloid Leukemia. N Engl J Med 1994 48 ± 14 Hehlmann et al., Blood 1994 48 (17–85) Guilhot et al., N Engl J Med 1997 50 (7–70) Hasford et al., JNCI 1998 49 (10–85) The Benelux CML Study Group. Blood 1998 56 (20–83) Baccarani et al., Blood 2002 45 ± 13 Hehlmann et al., Leukemia 2003 48 (10–83) O’Brien et al., N Engl J Med 2003 50 (18–70) Hehlmann et al., Blood 2007 49 (11–90)
Genes and Proteins Associated with the Tumor Microenvironment Are Differentially Expressed in Cured Versus Primary Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma.
Authors: Linderoth Johan , Edén Patrik , Ehinger Mats , Valcich Jeanette , Jerkeman Mats , Bendahl Pär-Ola , Berglund Mattias , Enblad Gunilla , Erlanson Martin , Roos Göran , Cavallin-Ståhl Eva
Published: October 2019

Background: The aim of this study was to identify possible differences in gene expression profiles (GEP) between two clinically very different patient populations, cured versus primary chemotherapy-refractory diffuse large B-cell lymphoma (DLBCL).

Material and methods: Tumor samples from 37 patients with de novo DLBCL, stage II-IV, either in continuous primary complete remission (n=24) or with progressive disease during primary treatment (n=13) were selected from a larger study cohort and examined with respect to GEP using spotted 55K oligonucleotide arrays. Formalin fixed paraffin-embedded tissue for immunohistochemical analysis was available in 33 of 37 cases, and was used for confirmation on protein level.

Results: The top 86 genes with the greatest statistical ability of discriminating the two patient groups were chosen for further analysis. Seventy-nine were over-expressed in the cured cohort, many of them coding for proteins expressed in the tumor microenvironment: CD68, several proteolytic enzymes and proteins involved in remodelling of extra cellular matrix and inflammation. Furthermore, MHC class I molecules, CD3δ, NK transcript 4, and ICAM-1 were overexpressed indicating an enhanced immunological reaction. Immunohistochemistry showed that the frequency of cells expressing CD68, defining the macrophage population, was higher in the cured cohort and that expression of lysozyme, cathepsin D, UPAR, and ICAM-1, was mainly seen within the reactive cells. Tumor infiltrating CD8+ T-cells were more frequent within the cured cohort, corresponding to the increased MHC class I expression seen within this group.

Conclusions: In DLBCL, genes coding for antigens present in the tumor microenvironment are differentially expressed in patients with cured vs. chemotherapy-refractory disease. Our findings suggest that a reactive microenvironment, including tumor infiltrating T-cells and macrophages, may have impact on outcome of chemotherapy in DLBCL.
Molecular Analysis of WASP Gene - First Report from India.
Authors: Jacob Rintu Rebecca , Rajasekar Reena , Edison Eunice S. , George Biju , Mathews Vikram , Srivastava Alok , Chandy Mammen
Published: October 2019

Wiskott Aldrich Syndrome (WAS) is an X-linked recessive disorder associated with severe thrombocytopenia, eczema, bloody diarrhea, profound immunodeficiency and an increased risk of malignancies. More than 200 mutations have been described in the gene encoding the WAS protein (WASP). We report here the molecular basis of WAS in six patients from India. Diagnosis of WAS/X- linked thrombocytopenia (XLT) was based on low platelet count (9000– 1, 25,000/mm3), presence of small platelets (mean: 6.2fl) and detection of WASP by flowcytometry. Patients evaluated in the study could be classified as classical WAS (n=3) and XLT (n=3) depending on the clinical severity of their disease (Zhu et.al 1995). All 12 exons and flanking splice-sites of WAS gene were amplified by polymerase chain reaction (PCR) using primers and PCR conditions adopted with modifications from Kwan et al (PNAS1995;92:4706). Mutation analysis was accomplished by conformation sensitive gel electrophoresis (CSGE) and direct sequencing. Abnormal banding pattern was observed only in 4 patients after CSGE analysis. c.995 C>T could not be detected by CSGE but was detected by direct sequencing. The types of molecular defects identified in five patients were as follows: nonsense - 2, deletion - 1 and duplication - 1. Three of these alterations (see table) have been reported previously and are associated with classical WAS. In patients detected to have nonsense mutations, flowcytometric analysis of WASP exhibited high values (table) not consistent with reported western blotting data in these mutations. c.307+10-11dupC was observed in two patients (brothers) with thrombocytopenia, small platelets and mild bleeding. This duplication is not a reported polymorphism. Though this duplication changes the acceptor splice site prediction score from 0.83 to 0.91, its functional significance has not been determined and requires further evaluation. The sixth patient in our study had thrombocytopenia (15,000–45,000/mm3), small platelets (4.8–7.2 fl), mild bleeding, similar family history and normal expression of WASP (86.2%). No mutation has been identified in the coding region of WAS gene in this patient either by CSGE or direct sequencing. Our data shows that the molecular basis for WAS is heterogenous in India. This is the first study evaluating mutations in WAS gene from India. Further mutation screening of patients with WAS will allow the genetic definition, phenotype-genotype corelation, carrier detection and prenatal diagnosis of this condition.

Molecular defects observed in WAS patients Patient ID Flow cytometry (%) Mutation *-Reported mutation, ♦-Functional Significance not known PD-22 WASP D1-98.5, WASPB9 - 94.8 c.665C>T, p.Arg211X* PD-16 WASP B9- 6.7 c. 1035-1036 delG, p.Gly314ValfsX120* PD-21 WASP D1-97.2, WASP B9-91.2 c.995 C>T, p. Arg321X* PD-34, PD-35 (siblings) WASP D1-3.6, WASP B9-2; D1-1.8, B9-1 c.307+10-11 dupC♦ PD-28 WASP D1 - 86.2 Mutation not detected
Hyaluronidase and IFN-γ from Activated T-Cells Are Key Mediators To Induce Immunosuppressive Activity of Mesenchymal Stem Cells.
Authors: Sato Kazuya , Katsutoshi Ozaki , Oh Iekuni , Matsu Haruko , Tatara Raine , Meguro Akiko , Hatanaka Keiko , Nagai Tadashi , Muroi Kazuo , Ozawa Keiya
Published: October 2019

Mesenchymal stem cells (MSC) have been shown to suppress T-cell proliferation in vitro and are being clinically applied to treatment for steroid-refractory graft-versus-host disease (GVHD). However, the molecular mechanisms of immunosuppressive activity of MSC have not been fully understood. We have recently reported that nitric oxide (NO) is one of the key molecules for T-cell suppression by MSC (Blood, 2007;228–34). Inducible NO synthase (iNOS) expression in MSC and NO production were exclusively detected upon interaction with activated T-cells. An inhibitor of NOS restored the proliferation of T-cells. We have also shown that both IFN-γ and NF-κB mediate NO production by MSC (BBRC, 2007;80–90), however the factor(s) from T-cells that induce immunosuppressive activity of MSC have not been elucidated. In the present study, we focused on hyaluronic acid (HA), a major extracellular matrix component, because

low molecular weight (LMW) HA is known to play an essential role in regulation of inflammation and infection, HA induces NOS in macrophages through a NF-κB-dependent mechanism, and T-cells are reported to express hyaluronidase-2 mRNA after activation.

A series of such information suggests that activated T-cells regulate immunosuppressive activity of MSC through HA signaling. We investigated the molecular events in the interaction between MSC and activated T-cells, and demonstrated that HA may play an important role in the scenario. First, ELISA showed that MSC produce a large amount of HA. RT-PCR revealed that among three hyaluronic acid synthase (HAS), mRNA for HAS-1 but neither HAS-2 nor HAS-3 is constitutively expressed by MSC. Flow cytometric analysis also showed that MSC express CD44, one of the receptors for HA. In macrophages, either IFN-γ or LMW HA is known to induce NO production. Therefore, we investigated whether IFN-γ or LMW HA can induce NO production via NF-κB signaling in MSC. As a result, only in the presence of IFN-γ, LMW HA but not high molecular weight (HMW) HA induced expression of iNOS in MSC, followed by NO production from MSC. We found that prostaglandin E2 (PGE2), which was previously reported as another immunosuppressive factor of MSC, is also induced by the combination of IFN-γ and LMW HA. Since hyaluronidase activity in activated T-cells has already been reported, these data support a novel hypothesis that HMW HA produced by MSC is digested by hyaluronidase from activated T-cells, and that resultant LMW HA cooperates with IFN-γ from activated T-cells in inducing iNOS expression in MSC. Locally produced NO through the above mechanisms may suppress activated T-cell proliferation. These findings would be important to further potentiate and regulate the immunomodulatory activity of MSC in a variety of applications.
Human B Cell Development from Adult Bone Marrow Is Profoundly Dependent on IL-7 Due to Decreased B Lymphoid Generative Capacity.
Authors: Parrish Yasmin K. , Baez Ineavely , Milford Terry-Ann M. , Rogerio Jaqueline W. , Sahakian Eva , Kagoda Mercy , Benitez Abigail , Huang Grace , Payne Kimberly J.
Published: October 2019

IL-7 is critical for normal murine B cell development, however its role in human B lymphopoiesis is controversial. Here we examine the role of IL-7 in human B cell development using a novel in vitro model based on co-culturing cord blood (CB) and adult bone marrow (BM) hematopoietic stem cells (HSCs) on primary stroma from adult human BM. Addition of IL-7 to this co-culture model increased B cell production by ∼40-fold. IL-7-induced increases were dose-dependent and specific to CD19+ cells. Flow cytometry analysis of STAT5 phosphorylation, and expression of the IL-7Rα and the proliferation antigen, ki-67, indicate that IL-7 acts directly on B cell precursors and that this effect is at least partially mediated through increased proliferation. Ongoing studies are assessing the effects of IL-7 on apoptosis among B cell precursors. IL-7 effects appeared to be most profound in cultures initiated with adult human BM HSCs where few, if any, human B lineage cells are generated in the absence of either mouse or human IL-7 activity. However, when co-cultures initiated with CB and BM HSCs were compared, IL-7-induced increases were similar in magnitude, and B cell precursors responded similarly to IL-7. Therefore we compared CB and BM HSCs with respect to B lymphoid generative capacity. The ability of CB and BM HSCs to generate CD19- progeny in our cultures was not significantly different, however the in vitro B lymphoid generative capacity of adult BM HSCs was 40 times less than that of CB HSCs. Our results provide evidence that IL-7 is critical for B cell production from HSCs in adult BM due to their low B lymphoid generative capacity as compared to HSCs in CB. These studies have implications for B cell recovery following transplantation with stem cells from CB versus BM and suggest that B cell recovery could be impacted by availability of IL-7.
In Patients with Aplastic Anaemia, Conditioning with Reduced Intensity Followed by Infusion of T-Cell Depleted Grafts Is Associated with Limited Transplant Related Morbidity and Universal Survival.
Authors: Novitzky Nicolas , Thomas Valda , Du Toit Cecile E. , McDonald Andrew B.
Published: October 2019

Introduction: While the survival of patients receiving allogeneic stem cell transplantation for aplastic anaemia has improved significantly, a substantial number of patients develop extensive graft vs. host disease (GvHD), with the associated morbidity and mortality. T-cell depletion of grafts decreases the incidence and severity of GvHD but may result in greater risk of graft failure.

Patients and methods: Between 2004 and 2007, seventeen patients with marrow aplasia were conditioned with fludarabine 30 mg/m2 for 5 consecutive days (150 mg/m2) followed by cyclophosphamide 120 mg/kg. In addition 2 (one with Fanconi’s anaemia) received busulfan 8 mg/kg. All had received more than 30 units of blood products and 2 had lymphocytotoxic antibodies that required HLA matched platelet transfusions. HLA identical sibling donors were mobilised with filgrastim (5–10 ug/kg) for 5 days and underwent large volume apheresis. Donor cells were quantitated and the CD34+ cell number determined. PBPC grafts were then incubated “in the bag” with alumtuzumab (1 mg/1010 mononuclear cells) at 20°C for 30 minutes and infused without further processing. Patients commenced cyclosporin from day −1 and continued until day 90 post transplantation. End points were engraftment, OS, DFS, incidence and severity of GvHD.

Results: Median age was 22 (7–49) years and 5/17 were females. Median CD34+ cell infused was 3.93 x 106/kg, containing median of 22.2 x 14/kg CFU-GM. The median “ex vivo” alumtuzumab dose employed was 10 mg (range 5–10 mg). There was universal engraftment and leukocytes reached 0.5 x 109/L at a median of 14 days (8–17) post transplantation. Other than haematological suppression, the toxicity of the conditioning was limited to grade I–II intestinal side effects. Seven individuals did not develop pyrexia following infusion of the graft. No patient developed GvHD. Two subjects had CMV reactivation (pp65+) and responded to therapy with gancyclovir. Another two developed haemorrhagic cystitis due to BK virus. Donor-recipient sex mismatch was present in 8 instances and post transplant bone marrow karyotype analyisis showed that haematopoiesis was of donor’s origin in each case. One patient developed delayed graft failure 6 months post transplantation and had a satisfactory recovery following a second T- cell depleted transplant with similar conditioning. Another developed mixed chimerism at 8 months and received DLI. All patients survive at a median of 630 days (range 60–1433) with normal blood parameters.

Conclusion: We conclude that purine analogues in combination with alkylators are a sufficiently intense immunosuppressive conditioning to allow successful engraftment of T-cell depleted PBPC grafts. Alumtuzumab “in the bag” led to excellent GvHD prophylaxis, leading to minimal morbidity of the procedure.
Molecular Mechanisms of Spindle Checkpoint-Apoptosis Linkage and Karyotypic Stability in Stem Cells.
Authors: Mantel Charlie , Rhorabough Sara , Guo Ying , Lee Man-Ryul , Han Myung-Kwan , Kim Kye-Seong , Broxmeyer Hal E.
Published: October 2019

Ex-vivo expansion of human HSC prior to bone marrow transplantation is still an unrealized goal that could greatly extend the usefulness of this mainstay strategy for treating numerous human hematologic diseases. The safety of this process for potential use in humans depends in large part on the maintenance of karyotypic stability of HSC during expansion, a lack of which could contribute to serious, even fatal, complications such as cancer, and could also contribute to engraftment failure. The spindle checkpoint and its linkage to apoptosis initiation is one of the most important cellular processes that helps maintain chromosomal stability in rapidly proliferating cell populations by removing aneuploid and karyotypically abnormal cells via activation of cell death programs. Detailed understanding of the molecular regulation of this vital cell cycle checkpoint is important to maximize safety of in-vitro HSC expansion techniques. It is widely accepted that mammalian cells enter the next G1-phase with 4N DNA after slippage from prolonged drug-induced mitotic block caused by activation of the transient spindle checkpoint that it is from this state that polyploid/aneuploid cells initiate apoptosis. However, definitive biochemical evidence for G1 is scarce or unconvincing; in part because of methods of protein extraction required for immunoblot analysis that cannot take into account the cell cycle heterogeneity of cell cultures. We used single-cell-intracellular-flow-cytometric analysis to define important factors determining cell fate after mitotic slippage. Results from human and mouse embryonic stem cells that reenter polyploid cell cycles are compared to human somatic hematopoietic cells that die after MS. We now report for the first time that phosphorylation status of pRb, p53, CDK1, and cyclin B1 levels are important for cell fate/apoptosis decision in mitotic-slippage cells, which occurs in a unique, intervening, non-G1, tetraploid subphase. Hyperphosphorylated Rb was extremely abundant in mitotic-slippage cells, a cell signaling event usually associated with early G1-S phase transition. P53 was phosphorylated at sites known to be associated with apoptosis regulation. Cyclin A and B1 were undetectable in mitotic slippage cells; yet, CDK1 was phosphorylated at sites typically associated with its activation. Evidence is also presented raising the possibility of cyclin B1-independent CDK1 activity in mitotic-slippage cells. These findings challenge the current models of spindle checkpoint-apoptosis linkages. Our new model could have important implications for methods to maintain karyotypic stability during ex-vivo HSC expansion.
Expansion of BCR-ABL Positive Leukemic Stem Cells Is Dependent on Hedgehog Pathway Activation.
Authors: Dierks Christine , Beigi Ronak , Stegert Mario , Zirlik Katja , Schmitt-Graeff Annette , Veelken Hendrik , Warmuth Markus
Published: October 2019

Chronic Myeloid Leukemia is characterized by the expansion of a leukemic stem cell clone carrying a Philadelphia translocation, which outgrows the non-malignant haematopoietic stem cells. Here we show that BCR-ABL directly enhances self renewal of haematopoietic stem and progenitor cells by activating the hedgehog signaling pathway through upregulation of SMO. Induction of hedgehog signaling in regular bone marrow by overexpression of SMO or loss of PTCH induces expansion of short term repopulating cells, but also enhances the expansion of long term repopulating haematopoietic stem cells. Pharmacological inhibition of SMO activity in BCR-ABL positive bone marrow cultures induced apoptosis in BCR-ABL positive differentiated cells and inhibited colony forming capacity of BCR-ABL positive self renewing cells in vitro. While loss of SMO in regular haematopoiesis only impacts the reconstitution of CD8 positive T-cells and short term repopulating cells (transplantation of SMO−/− fetal liver cells), the development of BCR-ABL positive leukemias was impaired and retransplantability abolished in the absence of SMO expression. Combined treatment of leukemic mice with AMN107 (Abl inhibitor) and Cyclopamine (SMO inhibitor) lead to a reduction of Bcr-Abl positive self-renewing cells in vivo and enhanced the time to relapse more than 3-fold compared to mice treated with AMN107 alone. Thus we conclude that BCR-ABL enhances the self renewal of leukemic stem cells through intrinsic activation of hedgehog signaling by upregulation of SMO. Therefore Hh pathway inhibition alone or in combination with Abl inhibitors could serve as an effective therapeutic strategy to reduce the malignant stem cell pool in BCR-ABL positive leukemias.
Interstitial Lung Disease in Patients with Diffuse Large B-Cell Lymphoma Receiving CHOP-Based Chemotherapy Is Associated with the Use of Rituximab.
Authors: Go Ronald S. , Riggle Kevin M. , Beier-Hanratty Sue A. , Gundrum Jacob D. , Schroeder Jonean E. , Wright Linda J. , Manske Brian A.
Published: October 2019

Background: Several cases of chemotherapy-induced interstitial lung disease (ILD) or pneumonitis have been reported in recent years in patients with lymphoma. The potential roles of rituximab (R) and granulocyte colony stimulating factor (GCSF), agents more commonly used in recent years, are suggested.

Objective: We wanted to determine the prevalence of ILD and identify risk factors in patients with diffuse large B-cell lymphoma (DLCL) who received cyclophosphamide/doxorubicin/vincristine/prednisone (CHOP)-based chemotherapy.

Methods: Selection criteria included newly diagnosed DLCL patients treated at our institution from 2000–2006 who received CHOP with or without R and had at least 3 serial CT or PET/CT scans during chemotherapy. ILD was defined as new bilateral interstitial pulmonary infiltrates not likely representing fluid overload or fibrosis. One radiologist blinded to clinical data reviewed all imaging studies.

Results: Out of a total of 174 new cases of DLCL, 73 met our study criteria. Reasons for exclusion included no chemotherapy (45), <3 imaging studies (46), and non-CHOP-based chemotherapy (10). Among the 73 study patients, 52 (71%) received R in addition to CHOP. Eleven (15.1%) patients developed ILD, all in the subgroup that received RCHOP (P = 0.027). Most occurred between cycles 2 and 4 of RCHOP (81.8%) and persisted until after completion of chemotherapy (63.6%). Nine (81.8%) patients with ILD were asymptomatic and never required treatment or delay of RCHOP. The remaining 2 patients became symptomatic (1 hospitalized), were empirically treated for atypical pneumonia with clinical recovery, and had delay of RCHOP. All patients received the intended number of courses of RCHOP. Univariate analysis of potential ILD risk factors among those who received RCHOP showed a trend with the subgroup that either had GCSF or cardiopulmonary disease (P = 0.09). Multivariate analysis using a two-variable model suggests that the use of GCSF or presence of cardiopulmonary disease (P = 0.065) and a high (3–5) international prognostic index score (P = 0.13) need further investigation as risk factors.

Conclusions: In our cohort of DLCL patients receiving CHOP-based chemotherapy, ILD was common and significantly associated with the use of R. While most cases were asymptomatic, self-limited, and did not require delay of chemotherapy, more serious presentation could occur. The mechanism of ILD is unknown and requires further investigation.
Expression Profile Analysis of Genes Related to Resistance/Sensibility to Prednisolone, Daunorubicin, L-Asparaginase and Vincristine in Childhood Acute Lymphoblastic Leukemia.
Authors: Silveira Vanessa S. , Scrideli Carlos A. , Moreno Daniel A. , Yunes Jose A. , Brandalise Silvia R. , Toledo Silvia , Lee Maria L.M. , Petrili Antonio S. , Odoni-Filho Vicente , Bendit Israel , Tone Luiz G.
Published: October 2019

Resistance to anti-leukemic agents in childhood ALL has been strongly associated with prognosis. The aim of this study was to analyze the expression profile of genes related to resistance/sensibility of 4 anti-leukemic agents and their correlations with age, diagnosis WBC count, risk group classification, molecular cytogenetics, induction treatment response and event-free survival (EFS) in ALL children. Were analyzed the expression profile of 19 genes related to resistance/sensibility to vincristine (RPRP2, CD44, TCF5, KCNN1, TIF1), prednisolo ne (F8A, CDK2AP1, BLVRB, CD69), daunorubicin (MAP3K12, SHOCK2, PDCH9, EGF1, KCNN4) and L-asparaginase (GPR56, MAN1, SCGF, IGFBP7, GATA3) by real-time quantitative PCR, using SYBR Green approach and melting curve analysis, with GUSB as housekeeping gene. Relative quantification was done using the 2-DDCT method (efficiencies from 1.9 to 2.1 to all the analyzed genes). These genes were chosen according to those described on microarray analysis by Holleman et al. (NEJM, 351:533, 2004). From 154 consecutive children admitted to ALL treatment in the 3 participating institutions, 139 had the gene expression profile analyzed. Sixteen were excluded due to lack of good quality RNA/cDNA. The follow up ranges from 36–60 months. All patients were classified and treated according the Brazilian ALL treatment protocol (GBTLI-99). Association among the variables analyzed and the gene expression levels was evaluated by Mann-Whitney and chi-square tests. EFS analysis was performed using Kaplan-Meier and log-rank test and multivariate analysis by the Cox proportional model to define independent prognostic factors. Correlation between the genes was analyzed by Spearman test. The 4-years EFS was 75,5% to all patients analyzed (81,4% to standard risk and 68,7% to high risk). Statistical correlation was observed among the genes of resistance of daunorrubicin, L-asp and vincristine (P<0,001). There is a significant association (p<0,05) between the levels of expression of the genes TIF1A, BLVRB, CD44, SCFBP1, TCFL5 and risk group; CD44, MAP3K12, SHOCK2 and CD10 antigen and GPR56, SHOCK2 with favorable event. The overexpression of SHOCK2 and GPR56 genes were also associated to better 4y-EFS in univariate (86,3% x 68,7% (p:0,04) and 86,2% x 72,2% (p:0,03) respectively) and multivariate analysis (p:0,03 and 0,09 respectively). No significant differences were observed in the other variables analyzed. The present data suggest that the expression of some analyzed genes have influence in the resistence/sensibility to the treatment and outcome in childhood ALL; especially over-expression of the SHOCK2 and GPR15 genes were associated with higher sensibility to daunorrubicin and L-asp respectively.