bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of LCR 5′HS4 and 5′HS1 Deletions on β-Like Globin Gene Expression in β-Yac Transgenic Mice.
Authors: Harju Susanna , Fedosyuk Halyna , Peterson Kenneth R. 
Published: April 2020

Abstract
A 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) was modified by homologous recombination to delete 2.9 Kb of cross-species conserved sequence similarity encompassing the LCR 5′HS4 (Δ5′HS4 β-YAC). Three transgenic mouse lines were established; each contained two intact copies of the β-globin locus as determined by long range restriction enzyme mapping (LRRM) and Southern blot hybridization analyses. Human ε-, γ- and β-globin, and mouse α- and ζ-globin mRNAs were measured by RNAse protection in hematopoietic tissues derived from staged embryos, fetuses and adult mice. No difference in the temporal pattern of globin transgene expression was observed between Δ5′HS4 β-YAC mice and wild-type β-YAC mice. In addition, quantitative per-copy human β-like globin mRNA levels were similar between Δ5′HS4 and wild-type β-YAC transgenic lines, although γ-globin gene expression was slightly increased in the fetal liver, while β-globin gene expression was slightly decreased in Δ5′HS4 β-YAC mice. These data are in contrast to data obtained from β-YAC mice containing a deletion of the 280 bp 5′HS4 core. In these mice, γ- and β-globin gene expression was significantly decreased during fetal definitive erythropoiesis and β-globin gene expression was decreased during adult definitive erythropoiesis. However, these data are consistent with the observation that deletion of the 5′HS core elements is more deleterious than large deletions of the 5′HSs. Together, the compiled deletion data supports the hypothesis that the LCR exists as a holocomplex in which the 5′HS cores form an active site and the flanking 5′HS regions constrain the holocomplex conformation. In this model, 5′HS core mutations are dominant negative, whereas larger deletions allow the LCR to fold into alternate holocomplex structures that function normally, albeit less efficiently.
To complete the study on the contribution of the individual 5′HSs to LCR function, a 0.8 Kb 5′HS1 fragment was deleted in the 213 Kb β-YAC by homologous recombination. Two ΔHS1 β-YAC transgenic lines have been established; four additional founders were recently identified. Of the two lines, one contains two intact copies of the globin locus; the other contains four deleted copies, one of which extends from the LCR through just 5′ to the β-globin gene. For both lines, ε-globin gene expression was markedly reduced, approximately 5–10 fold, during primitive erythropoiesis. Developmental expression profiles and levels of the γ- and β-globin genes (in the line that contains loci including the β-globin gene) were unaffected by deletion of 5′HS1. Breeding of the remaining four founders to obtain F1 and F2 progeny for similar structure/function studies is in progress. Decreased expression of the β-globin gene is the first phenotype ascribed to a 5′HS1 mutation, suggesting that this HS does indeed have a role in LCR function.
In Vivo and In Vitro Analysis of an Aγ-Globin Gene Silencer in Human β-Yac Transgenic Mice.
Authors: Harju Susanna , Peterson Kenneth R. 
Published: April 2020

Abstract
Autonomous silencing of gene expression is one mechanism operative in the control of human β-like globin gene switching and is best exemplified by the ε-globin gene. Experiments using variously truncated Aγ-globin genes linked to LCR sequences suggested that a region of the Aγ-globin gene between −730 to −378 relative to the mRNA CAP site may function as an adult stage-specific silencer element. A 5.4 Kb marked Aγ-globin gene (Aγm) inserted between LCR 5′HS1 and the ε-globin gene in a β-YAC (Aγm 5′ ε β-YAC) was silenced in transgenic mice during adult definitive erythropoiesis, even in the absence of an adult β-globin gene. In contrast, when a marked β-globin gene (βm) was inserted in this same location in another β-YAC (βm 5′ ε β-YAC), the βm-globin gene was expressed throughout ontogeny. From these data we concluded that: 1) any gene located near the LCR will be strongly expressed throughout ontogeny, unless some gene-specific silencing mechanism exists, 2) competition between the γ- and β-globin genes for interaction with the LCR is not the exclusive mechanism controlling γ- to β-globin gene switching, and 3) that the Aγm-globin gene was autonomously silenced. A -730 to -378 deletion of the Aγm-globin gene was introduced into the Aγm 5′ ε β-YAC via homologous recombination to produce a Δ1s Aγm 5′ ε β-YAC. This YAC was microinjected and six founders were obtained. Four transgenic lines were established carrying at least one full-length β-globin locus and two were established that lacked the adult β-globin gene. All founders containing an intact β-globin gene expressed the Δ1s Aγm-globin during adult erythropoiesis (45% – 122% relative to human β-globin expression). In one line examined in detail, the Δ1s Aγm-globin gene was expressed in the embryonic yolk sac, fetal liver, and adult blood. ε-globin gene expression was not detected in the embryonic yolk sac and expression of the normally located γ-globin genes was not observed at any developmental stage. β-globin gene expression was observed in the fetal liver and adult blood, although its expression was decreased. To further delineate the function of the Δ1s fragment, transient transfection assays to test silencer function and protein-DNA interaction assays were performed. Silencer activity of the352 bp Δ1s fragment was examined using a series of pGL2 luciferase reporter plasmids that were synthesized to include the Δ1s fragment; these were electroporated into various cells. Electrophoretic mobility shift assays (EMSAs) and DNAse I footprinting were employed to begin assessment of protein binding within the Δ1s fragment. A 50 bp DNA fragment spanning −713 to −664 of the Δ1s element was used in EMSAs; DNA binding activity was observed in K562 nuclear extracts. These preliminary data suggest that the −730 to −378 γ-globin gene silencer binds a repressor protein complex.
B Locus YAC Mice Carrying G Gene Promoter Mutations: Insights into the Mechanism of Action of the -175 HPFH Mutation.
Authors: Yu Man , Ye Xin , Han Hemei , Stafford Mary , Navas Patrick A. , Li Qiliang , Stamatoyannopoulos George 
Published: April 2020

Abstract
Non-deletional HPFHs are g gene promoter mutations, which are associated with HbF elevation in heterozygous adult individuals. To investigate the mechanism whereby these mutations result in g gene activation, the −198 (T→C) and −175 (T→C) mutations were introduced into the promoter of the Ag gene in the context of a 213 kb b-globin locus YAC. The mutated YAC DNA was then used to produce transgenic mice. Expression of the human g and b globin gene was examined by RNase protection assay in blood of the adult transgenic mice carrying either −198 or −175 mutations. The level of g gene expression ranged from 4.0 to 8.5% of the human b mRNA in the −198 mutant transgenic mice, and from 25 to 41% in the −175 mutant mice. In contrast, there was no detectable level of g gene expression in the blood of wild type transgenic mice. The ratios of g/b mRNA in the mutant transgenic mice correspond to the levels of Hb F in adult individuals carrying the corresponding mutations: Hb F in heterozygotes for HPFH −198 and −175 mutations ranges from 3.5 to 10% and from 36 to 41%, respectively. To delineate the mechanism of g gene reactivation by the HPFH mutations, the CACCC box in the g gene promoter was deleted in the context of the mutant YAC. In contrast to the −175 HPFH bYAC mice, in the doubly mutated −175 HPFH DAgCACCC bYAC mice, expression of the g gene in the adult blood of the transgenic mice was undetectable. These results provide strong evidence that an intact CACCC box is required for g gene activation in the −175 HPFH mutation. We conclude that YAC mice can faithfully reproduce the phenotypes of nondeletional HPFH mutations and be used for the investigation of the mechanisms by which these mutations activate the g gene.
Poor Outcome of Allogeneic Bone Marrow Transplantation Recipients Requiring Mechanical Ventilation: Should We Restrict Intensive Care Support?.
Authors: Aubron Cécile , Azoulay Elie , Blot François , Mira Jean-Paul , Thiery Guillaume , Raynard Bruno , Buzyn Agnes , Socié Gérard , Arnaud Philippe , Pene Frédéric 
Published: April 2020

Abstract
In the recent years, a better selection of patients and early management of organ dysfunction allowed an improved survival of critically ill hematological patients. However allogeneic bone marrow transplantation (BMT) recipients admitted to the ICU with severe organ dysfunction still have poor survival rates. We performed a large retrospective multicenter study to analyse the features and short-term and long-term outcomes of allogeneic BMT recipients admitted to the ICU. Between 1997 and 2003, 220 allogeneic BMT recipients were referred to 3 ICUs. Patients were classified in three groups, on the basis of time since BMT : group 1 (&lt; Day 30) n= 73, group 2 (Day 31 to day 100) n= 59, group 3 (&gt; day 100) n=88. The ICU overall mortality rate was 51% (112/220), 56% (41/73) in the group 1, 54% (32/59) in the group 2 and 44% (39/88) in the group 3. Twenty percent of patients (45/220) were alive at 6 months after ICU admission and 14% (31/220) were alive one year after. None of the pre-admission characteristics (age, underlying disease and disease status at the time of transplantation, type of transplant) was associated with prognosis in the ICU. Mortality rate was highly correlated with the number of organ dysfunctions as measured by the Logistic Organ Dysfunction (LOD) score at admission (LOD score &lt; 5 : 30%, LOD score = 6 to 10 : 56%, LOD score ≥ 11 : 96%). Of the 119 patients who required invasive mechanical ventilation, 83% died (98/118). Among the ventilated survivors, 11% (13/118) and 6% (7 /118) remained alive at 6 months and one year respectively. In a multivariate logistic regression analysis, the requirement of invasive mechanical ventilation (OR 19.5, CI 95% 8.8–45.3, p&lt;0.0001) and hepatic dysfunction with elevation of serum bilirubin &gt;68 μmol/L (OR 4.7, CI 95% 1.9–11.4, p=0.0006) were independently associated with mortality. Although the requirement of invasive mechanical ventilation remains desperately associated with a high mortality rate, some allogeneic BMT recipients may have a good long-term outcome. Assessment of other organ failures, and particularly hepatic dysfunction, may lead to early identification of patients with very high probability of death.
Differential Requirements for Survivin in Hematopoietic Cell Development.
Authors: Xu Yanfei , Gurbuxani Sandeep , Keerthivasan Ganesan , Wickrema Amittha , Crispino John D. 
Published: April 2020

Abstract
The development of the complete repertoire of blood cells from a common progenitor, the hematopoietic stem cell, is a tightly controlled process that is regulated, in part, by the activity of lineage specific transcription factors. Despite our knowledge of these factors, the mechanisms that regulate the formation and growth of distinct, but closely related lineages, such as erythroid cells and megakaryocytes, remain largely uncharacterized. Here we show that Survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express Survivin throughout their maturation, up to the terminal stage of differentiation (orthochromatic), even after the cells exit the cell cycle. This is surprising because Survivin is generally expressed in a cell cycle dependent manner and not thought to be expressed in terminally differentiated cells. In contrast, purified murine megakaryocytes express nearly 5-fold lower levels of Survivin mRNA compared to erythroid cells. To investigate whether Survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring either the human Survivin cDNA or a mouse Survivin shRNA, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of Survivin promoted the terminal differentiation of red blood cells, while its reduction, by RNA interference, inhibited their differentiation. In contrast, downregulation of Survivin facilitated the expansion of megakaryocytes, and its overexpression antagonized megakaryocyte formation. In addition, consistent with a role for survivin in erythropoiesis, downregulation of Survivin expression in MEL cells led to a block in terminal differentiation. Finally, since caspase activity is known to be required for erythroid maturation, we investigated whether survivin associated with cleaved caspase-3 in erythroid cells. Immunofluorescence revealed that Survivin and cleaved caspase-3 co-localized to discrete foci within the cytoplasm of erythroid cells at the orthochromatic stage of development. Based on these findings, we hypothesize that Survivin cooperates with cleaved caspase-3 in terminal maturation of red blood cells. Together, our findings demonstrate that Survivin plays multiple, distinct roles in hematopoiesis.
Rituximab Plus Hypercvad (R-HCVAD) Alternating with Rituximab Plus High-Dose Methotrexate-Cytarabine (R-M/A) in Untreated Mantle Cell Lymphoma (MCL): Prolonged Follow-Up Confirms High Rates of Failure-Free Survival (FFS) and Overall Survival (OS).
Authors: Romaguera Jorge Enrique , Fayad Luis , Rodriguez Maria A. , Hagemeister Fredrick B. , Pro Barbara , McLaughlin Peter , Younes Anas , Samaniego Felipe , Goy Andre , Sarris Andreas H. , Dang Nam H. , Medeiros L. Jeffrey , Katz Ruth , Gagneja Harish , Samuels Barry I. , Cabanillas Fernando 
Published: April 2020

Abstract
Introduction: Aggressive MCL has a poor prognosis with a 21-40% complete remission (CR) after CHOP and a duration of response of only 10-16 months. More intense therapy could improve these statistics. Rituximab is effective in MCL and has minimal toxicity.
Methods: A prospective phase II trial of R-HCVAD (considered to be one cycle) alternating every 21 days with R- M/A (considered to be another cycle) as described earlier (Ann Oncol. 13, suppl 2, 2002 #24). Prophylaxis with mesna, calcium leucovorin, prednisone eyedrops, G-CSF, antibacterial, antifungal, and antiviral therapy. CBC with differential and platelet counts X 2-3/week. Re-staging every 2 cycles including upper and lower endoscopies. Patients in complete remission (CR) after 6 courses of a planned 6-8 cycles were not offered consolidation with stem cell transplant. Post-treatment evaluation was performed every 3 months for 1 yr, every 4 months for 2 yrs, every 6 months for 2 years, then annually.
Results: Of 100 patients registered, one was ineligible and two decided to not receive the treatment after registration, leaving 97 evaluable for analysis of response, survival and toxicity. An analysis of response after the first 6 cycles shows an 87% CR/CRu rate. With a median follow up of 40 months, the 3-year FFS and overall survival (OS) were 67% and 81%, respectively. Adverse factors for FFS were:

Grade 4 hematologic toxicity was significant. Five patients died during treatment of sepsis (3), pulmonary hemorrhage (1), and unknown cause (1). Four patients developed myelodysplasia/acute myelogenous leukemia after treatment and while in CR and three have died, for a total of 8 deaths in the study (8%).
Conclusion: R-HCVAD alternating with R-M/A without stem cell transplant is an effective regimen for treatment of aggressive untreated MCL, specially for patients ≤ 65 years old. Toxicity is as expected for an intense regimen. This encouraging data warrants continued follow-up and comparison with existing/new therapies in future trials.
Anti-Tumor Activity of Single-Agent CCI-779 for Relapsed Mantle Cell Lymphoma: A Phase II Trial in the North Central Cancer Treatment Group.
Authors: Witzig Thomas , Geyer Susan , Ghobrial Irene , Inwards David , Fonseca Rafael , Kaufmann Scott , Kurtin Paul , Colgan Joseph , Morice William , Alberts Steven , Salim Muhammed , Rowland Kendrith , Dakhil Shaker , Morton Roscoe , Stella Philip 
Published: April 2020

Abstract
Purpose: Mantle cell lymphoma (MCL) is characterized by a t(11;14) resulting in overexpression of cyclin D1, a member of the phosphatidylinosital 3 kinase (PI3K) pathway. This study tested whether CCI-779, which inhibits the PI3K pathway at the level of the mammalian target of rapamycin (mTOR) could produce tumor responses in patients (pts) with MCL.
Patients and Methods: Eligible pts had biopsy-proven, cyclin D1 positive MCL and had relapsed or were refractory to therapy. Pts received CCI-779 250 mg IV every week as a single agent. Pts were re-staged after 1 cycle (4 doses) and every 3 cycles thereafter. Pts with a tumor response after 6 cycles were eligible to continue drug for a total of 12 cycles or 2 cycles after complete remission (CR) and then were observed.
Results: Thirty-five pts were enrolled and evaluable for toxicity; 1 patient had MCL by histology but was cyclin D1 negative and ineligible for efficacy evaluation. The median age was 70 years (range, 38–89), 91% were stage 4, and 69% had ≥ 2 extranodal sites. Pts had received a median of 3 prior therapies (range, 1–11) and 54% were refractory to their last treatment. The overall response rate was 38% (13/34) with 1 CR (3%) and 12 PRs (35%), surpassing the pre-defined criteria for a promising agent. Responses tended to occur rapidly with median time to response of 1 month (range, 1–8). To date, 26 patients have progressed, with a median time-to-progression of 6.8 months (95% CI: 3.8 – 9.7). Median duration of response for the 13 responders was 5.7 months (95% CI: 5.2 – 13.2). Overall, 32 out of 35 patients who received treatment had grade 3 or 4 toxicity. The most common toxicities were hematologic with grade 3 (n=24) or grade 4 (n=4). Thrombocytopenia was the most frequent grade 3/4 toxicity (n=25) and the largest cause of dose-reductions, although counts typically recovered within one week. Only 4 patients could tolerate sustained 250 mg per week throughout their treatment (including one who went on to alternate treatment after 1 cycle) and the median dose/month was 175 mg.
Conclusions: Single-agent CCI-779 has substantial anti-tumor activity in relapsed MCL. This study demonstrates that agents, which selectively target cellular pathways dysregulated in MCL cells can produce therapeutic benefit. The high response rate warrants further studies of this agent in MCL, but the high incidence of hematologic toxicity suggests that a lower dose should be explored. CCI-779 at 25mg is currently being evaluated in MCL through an NCCTG trial
Secondary Amenorrhoea after Hodgkin’s Lymphoma Is Influenced by Age at Treatment, Stage of Disease, Chemotherapy Regimen, and the Use of Oral Contraceptives during Therapy: A Report from the German Hodgkin Lymphoma Study Group (GHSG).
Authors: Behringer Karolin , Breuer Kai , Reineke Thomas , May Michael , Nogova Lucia , Klimm Beate , Diehl Volker , Engert Andreas 
Published: April 2020

Abstract
Purpose: Long term survivors of successfully treated Hodgkin’s disease (HD) are at risk for late complications. Among these, infertility for female patients is of major importance affecting many young women. The subject of this analysis is to evaluate the menstrual status in young women after HD therapy.
Patients and methods: From 1994–1998, the German Hodgkin Lymphoma Study Group (GHSG) conducted clinical trials for early, intermediate and advanced-stage HD (HD7-HD9) involving a total of 3186 patients. A questionnaire was used to evaluate the menstrual status of young female patients after therapy. The following factors were assessed by multivariate analysis for their influence on amenorrhoea: age, treatment, stage, and the use of oral contraceptives during chemotherapy.
Results: The data presented were last updated in May 2004. A total of 405 women aged &lt;40 years answered the questionnaire referring the menstrual status after HD therapy. For most women (89.6%), menstruation before the beginning of therapy was regular. After a median follow-up of 3.2 years, 51.6% of the women receiving 8 cycles of dose-escalated BEACOPP had continuous amenorrhoea. Amenorrhoea was significantly more frequent in women receiving 8 cycles of dose-escalated BEACOPP compared to women treated with ABVD alone, COPP/ABVD or standard BEACOPP (p=0.0066). Moreover, amenorrhoea after therapy was most pronounced in women with advanced-stage HD (p&lt;0.0001), in those older than 30 years at treatment (p= 0.0065), and in women who did not take oral contraceptives during chemotherapy (p=0.0002).
Conclusion: Most women who are treated for advanced-stage HD experience amenorrhoea after therapy. Amenorrhoea is significantly more frequent in women with advanced-stage HD receiving 8 cycles of dose-escalated BEACOPP and in women older than 30 years at treatment. Furthermore, the present data suggest a possible protection of oral contraceptives against amenorrhoea.
Inhibition of Retinoic Acid Signaling by the cdx-hox Pathway Is Essential for Blood Cell Formation during Embryogenesis.
Authors: Davidson Alan J. , Wang Yuan , Daley George Q. , Zon Leonard I. 
Published: April 2020

Abstract
The zebrafish mutant kugelig (kgg) is caused by a defect in the caudal-related homeobox gene cdx4 and has a deficit in both ‘primitive’ and ‘definitive’ hematopoietic stem cells. The embryonic anemia in the mutants can be rescued by overexpressing hox genes such as hoxb7a and hoxa9a, but not hoxb8a. This suggests that specific hox genes are required to make mesoderm competent to form blood. To further explore this, we undertook a microarray analysis to identify differentially expressed genes in kgg mutants and wild-type embryos. We found that raldh2, an enzyme required for retinoic acid (RA) production, is overexpressed in kgg mutants during the early stages of blood formation. This data led us to hypothesize that RA may act to suppress blood formation and that the cdx-hox pathway functions to limit RA production, thereby permitting blood formation to occur. To test this, we treated wild-type zebrafish embryos with RA and found that they became severely anemic. Treating kgg embryos with DEAB, a chemical that blocks raldh2 activity, restored hematopoiesis in kgg mutants. Expression of hoxa9a was not rescued in these treated embryos, indicating that RA acts downstream of the hox genes. DEAB also induced an expansion of erythroid cells in wild-type embryos, thus supporting the notion that the levels of RA during development are a critical determinant for blood formation. By performing a time-course rescue experiment, we determined that DEAB is effective when scl + hematopoietic progenitors are first formed from mesoderm, suggesting that RA acts upstream of the blood-inducer scl. In support of this, SCL overexpression rescues GATA-1 expression in embryos treated with RA. We next looked at the effects of DEAB and RA on the formation of mouse hematopoietic progenitors arising from ES cell-derived embryoid bodies (EBs). Addition of DEAB to EBs between days 2 to 3 of development resulted in a 5–8 fold increase in ‘primitive’ erythroid colonies (CFU-Ep), analogous to our results in zebrafish embryos. In contrast, RA treatment caused a general inhibition in the growth of all colony types. Taken together, these results suggest a new model in which suppression of RA by the cdx-hox pathway is necessary for yolk sac hematopoiesis to occur. This model provides an explanation for how hox genes control the spatiotemporal formation of hematopoietic tissue during organogenesis and may shed new light on the pathogenesis of leukemias involving translocations of the cdx , hox, and retinoic acid receptors.