bluuebunny/crossref_metadata_2025 huggingface dataset:
Therapeutic Angiogenesis in Critical Limb Ischemia by Implantation of Autologous Hematopoietic Cells.
Authors: Besalduch Juan , Lara Raul , Sampol Antonia , Gutierrez Antonio , Lozano Pacual , Galmes Antonio
Published: April 2020

Patients with critical limb ischemia in whom surgical repair is not possible because bad distal vasculature, have an ominous prognosis. This study describes a technique for treatment of patients with critical limb ischemia with implantation of autologous peripheral mobilized hematopoietic cells, with the aim to stimulate angiogenesis.

Twelve patients (9M, 3F), with a median age of 58 years (22–78) with critical limb ischemia were treated by injections of peripheral mobilized blood cells in the gastrocnemius muscle of the affected limb. The inclusion criteria were patients with chronic limb ischemia grade III-IV by Doppler examination, plethismography and angiography, with failure to respond to non-surgical treatment and some with previous surgery, and that were no candidates for surgical revascularization. The patients were treated by G-CSF (Neupogen, Amgen) 5μ /kg weight/d during 5 days. At day fifth and sixth we obtained peripheral mononuclear blood cells by means of a CS-3000 Plus (Baxter) blood cell separator. The unmanipulated cells were injected in the affected limb in 2 ml aliquots into the gastrocnemius muscle. Each product of a single apheresis (volume 50 ml) were injected in a sole limb. Six patients received injections only in one limb. Each patient were evaluated regularly for rest pain, amount of required analgesia, healing of the ulcers, peak walking time and, Doppler, plethismographic and angiographic findings. The mean number of injected CD34+ cells in each limb was 0.77 x 106/kg. There were no secondary effects for the mobilization and injection of cells in the 12 studied patients. Moreover, in 7 patients studied with a median follow-up of 1 year, 5 showed an improvement of all parameters, specially pain at rest, peak walking time and skin throphic lesions. Two patients suffered early amputation of the affected extremity because obstruction of an old by-pass.

Despite the short follow-up and small patient series, this technique is expected to achieve relief of pain and decrease in major amputations in this critical population. However, comparative studies with longer follow-up should be done to confirm the benefits of this technique.
Total Therapy 2 (TT2) for Multiple Myeloma (MM): Thalidomide (T) Effects Superior Complete Response (CR) and Event-Free Survival (EFS); Similar Overall Survival (OS) Linked to Shorter Post-Relapse Survival.
Authors: Tricot Guido , Rasmussen Erik , Anaissie Elias , van Rhee Frits , Zangari Maurizio , Hollmig Klaus , Jackson Lisa , Epstein Joshua , Shaughnessy John , Crowley John , Barlogie Bart
Published: April 2020

Background: Autotransplants have become the standard of care for patients with MM. TT2, a prospective randomized phase III trial, evaluated whether adding T, effective in end-stage MM, could improve the outcome of patients receiving melphalan (MEL200)-based tandem autotransplants.

Methods: Between October 1998 and February 2004, 668 patients were randomized to receive T (n=323) or no T (n=345), with the primary objective to increase the 5-yr EFS rate from 40% to 50%. TT2 consisted of 4 phases: induction with VAD-DCEP-CAD with PBSC collection, DCEP; tandem autotransplants with MEL 200; D-PACE consolidation; interferon maintenance with added DEX pulsing during the 1st year. The median follow-up is 3 years.

Results: Patient baseline characteristics in the 2 arms were similar. T increased CR from 41% to 59% (p<0.001) (Figure 1a); 5-yr EFS was superior for the T arm (54% vs. 42%, p=0.017), whereas 5-yr OS was not different at 67% and 62% (p=0.9), due to shorter post-relapse survival of patients initially randomized to T (13 vs 32mos, p<0.001) (Figure 1b). Of 437 patients with interphase FISH data, amp1q21 and chromosome 13q14 deletion (del13q14) both conferred short OS (p<0.001, p=0.003) and EFS (p<0.001, p<0.001), independent of metaphase cytogenetic abnormalities (CA) (p<0.001); these 3 genetic features alone accounted for 35% of OS and 28% of EFS variability. T improved EFS both in patients without and with one or more of these 3 genetic abnormalities. High amp1q21 indices accounted for shorter post-relapse survival (p<.001), independent of initial T randomization (p=.5). The post-relapse therapies were similar among the 2 arms and included T in all previously not on T and most previously on T, often together with bortezomib = DEX (VTD regimen; Barlogie, Blood, 2004).

Conclusions: Intensive chemotherapy before and following tandem autotransplants in TT2 effected unprecedented high 5-yr EFS (48%) and OS rates (65%). T significantly increased CR frequency and extended EFS but not OS. A shorter post-relapse survival could be traced to high amp1q21 index at relapse (more often present on T arm).

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Bacterial Endotoxin LPS but Not LTA Can Enhance Osteogenic Differentiation of Human Mesenchymal Stem Cells.
Authors: Chan Godfrey ChiFung , Mo F.Y. , Yip K.H.K. , Li J. , Law H. , Lau Y.L.
Published: April 2020

Background & Objective: Dental implant requires osseointegration for anchoring and human’s oral cavity has plenty of bacterial oral flora. Whether these bacteria have any effects on the human mesenchymal Stem Cells (MSCs) that can differentiate into osteoblasts remains unknown. We therefore investigated the effect of bacterial endotoxins commonly found in the oral cavity and gastrointestinal tract, namely lipopolysaccharides (LPS, Escherichia coli) and lipoteichoic acid (LTA, Streptococcus pyogenes), on the proliferation and osteogenic differentiation of MSCs.

Methods: Human MSCs are derived from bone marrow (BM) of normal healthy donors. The culture condition, immunophenotyping determination and tests of differentiating functions of the human MSCs were similar to what we reported previously (Li J, Br J Haematol 2004). The proliferation of MSCs under either a 3-day or a prolonged 7-day endotoxins challenge was evaluated by XTT assay. The extent of osteogenic differentiation was examined under microscopy and measured by the increase in alkaline phosphatase (ALP) activity at day 10 and the calcium mineralization/deposition per unit volume of protein at day 14.

Results: There was no significant effect of LPS and LTA on the growth and proliferation of MSCs, even under a relatively high dose. However, continued LPS challenge on MSCs under osteogenic culture condition was shown to increase the ALP activity and calcium deposition in a dose-dependent manner (100ng/ml, 1 ug/ml, 10ug/ml). No such phenomenon can be identified when LTA challenge was used.

Conclusions: LPS and LTA did not show any significant effect on the proliferation and growth of human MSCs. However, LPS enhanced the osteogenic differentiation of MSCs in a dose-dependent manner. Our finding suggests that the endotoxin from bacteria commonly found in the oral cavity and gut does not have any negative impact on MSCs induced osteogenesis.
Mobilization of Stem Cells from the Bone Marrow Is Required for Neovascularization of Ischemic Limbs in Mice.
Authors: Kim Jeong-A , Lee Chang -Hoon , Yoon Jin-A. , Min Woo-Sung , Kim Chun-Choo
Published: April 2020

We examined whether the injection of bone marrow mononuclear cells (BM-MNCs) or mesenchymal stem cells (MSCs) might augment angiogenesis and collateral vessel formation in a mouse model of hind limb ischemia. C57BL/6 BM-MNCs were isolated by centrifugation through a Histopaque density gradient and MSCs were obtained from C57BL/6 bone marrow and cultured in low-glucose DMEM media. Unilateral hind limb ischemia was surgically induced in C57BL/6 mice (control; n=4), and autologous BM-MNCs (Group 1; n=4, 1.8±0.2 x107/animal) or MSCs (Group 2; n=4, 1.0±0.14 x106/animal) or BM-MNCs and MSCs (Group 3; n=4, 2.3±0.1 x107 and 1.1±0.21 x106/animal) were transplanted into the ischemic tissue. Six weeks after transplantation, the group 1, group 2 and group 3 had a higher capillary/muscle ratio (0.82±0.12 vs 0.85±0.08 vs 0.97 ±0.03) than control (0.46±0.12, p<0.05) (Fig. 1). This result suggested that direct local transplantation of autologous BM-MNCs or MSCs seems to be a useful strategy for therapeutic neovascularization in ischemic tissues.

Next, we evaluated whether bone marrow derived stem cells were participated in the process of local injected stem cells forming new vessels. In general, mobilizing stem cells from bone marrow to local site, MMP-9 has been known as an important molecule. So we used the MMP-9 deficient KO mice and wild type, 129SvEv mice were used in the experiments. Autologous BM-MNCs and MSCs were transplanted into the ischemic limb in MMP-9 (−/−) (n=4) after unilateral hind limb ischemia was surgically induced and then the same experiments was done in MMP-9 (+/+) mice (n=4). The number of the injected BM-MNCs and MSCs was 2.2±0.05 x107 and 0.87±0.17 x106/animal in MMP-9 (−/−). And the number of the injected BM-MNCs and MSCs was 2.1±0.17 x107 and 0.98±0.09 x106/animal in MMP-9 (+/+). No difference was seen in the BM-MNCs and MSCs were injected or not (0.52±0.07 vs 0.49±0.03,) in MMP-9 (−/−). But, in the case that BM-MNCs and MSCs were injected, the higher capillary/muscle ratio was seen in MMP-9 (+/+) compared to control (0.86 ±0.09 vs 0.49±0.03, P<0.05) (Fig 2). This data indicated that the mobilization of bone marrow derived stem cells would have an important role in the neovasculrization although the stem cells were injected directly into the muscle of ischemic limb.

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Aberrant DNA Methylation of the C/EBPalpha Promoter Region in Hematopoietic Malignancies.
Authors: Jost Edgar , do Nicole , Herman James G. , Osieka Rainhardt , Galm Oliver
Published: April 2020

The CCAAT/enhancer binding protein alpha (C/EBPalpha) is a transcription factor implicated in myeloid differentiation. Loss of function of C/EBPalpha may impair granulocytic differentiation and thus contribute to leukemogenesis. Inactivation mutations of C/EBPalpha have been reported in hematopoietic malignancies, especially in acute myeloid leukemia (AML). Hypermethylation of CpG islands within gene promoter region is associated with transcriptional inactivation and represents an important mechanism of gene silencing in hematological malignancies. This epigenetic phenomenon acts as an alternative to mutations and deletions to disrupt gene function. In this study, we have analyzed the methylation status of the promoter-associated CpG island of the C/EBPalpha gene in hematopoietic cell lines and primary blood and bone marrow samples from patients with AML, multiple myeloma (MM) and non-Hodgkin’s lymphoma (NHL) by methylation-specific polymerase chain reaction (MSP). C/EBPalpha promoter methylation in L540 cells was associated with transcriptional repression, and treatment with the demethylating 5-aza-2′-deoxycytidine resulted in C/EBPalpha upregulation. MSP analysis of primary patient samples revealed C/EBPalpha hypermethylation in 19.3 % (11/57) of AML, 27.1 % (13/48) of MM and 46.7 % (7/15) of NHL. Aberrant C/EBPalpha methylation was found in AML of all FAB subtypes and throughout all cytogenetic risk groups. In MM, C/EBPalpha hypermethylation occurred in all Salmon and Durie stages as well as in plasma cell leukemia. Our data indicate that hypermethylation of the transcription factor C/EBPalpha is a common finding in various hematopoietic malignancies and may contribute, in addition to genetic aberrations, to the malignant phenotype. Our growing knowledge about epigenetic aberrations not only gives further insight in the pathogenesis of hematopoietic malignancies but also provides a rationale and molecular basis for therapeutic approaches with demethylating agents.
A Single Nucleotide Polymorphism and Its Haplotype of ZNF42 Transcriptional Modulator Gene Predisposes Individuals to High-Risk Acute Myelogenous Leukemia (AML).
Authors: Lee Il-Kwon , Choi Jeong-Hwa , Lee Yu Ra , Kim Yeo-Kyeoung , Kim Hee Nam , Park Kyeong-Soo , Lee Je-Jung , Shin Myung Geun , Choi Chan , Kook Hoon , Hwang Tai Ju , Park Moo Rim , Jo Deog Yeon , Won Jong Ho , Kim Hyeoung-Joon
Published: April 2020

ZNF42 (MZF-1 or Human Zinc finger protein 42) is a hematopoietic transcription factor regulating the differentiation and proliferation of myeloid cells. ZNF42 belongs to the Krupple-class zinc finger protein, which is expressed in myeloid progenitor cells. As a transcriptional regulator it has been found to be pivotal in hematopoietic development, especially in granulopoiesis. ZNF42 is a bi-functional transcriptional regulator, by repressing transcription in non-hematopoietic cells, but trans-activating hematopoiesis-related genes. Despite the important role of ZNF42 in differentiation and proliferation in the myeloid lineage cells, molecular epidemiologic studies of ZNF42 have not been conducted. In this case-control study, we conducted a comprehensive analysis of the genomic region of ZNF42, including SNP discovery by re-sequencing of the promoter and exon-encompassing regions, haplotype construction and construction of linkage disequilibrium (LD) map and comparison LD structure among four different populations data from the International HapMap project. In total, 275 de novo AML patients plus age- and sex-matched controls were recruited and four coding non-synonymous SNPs were genotyped by Pyrosequencing for this study. All genotypes frequencies were in Hardy-Weinberg equilibrium. A non-synonymous SNP (G/A altering amino acid R51H) revealed strong association with increased susceptibility to AML. Relative to individuals with the GG genotype, those with the A allele (AA + AG) had a 4.8-fold (95% CI, 3.3–7.0) (p=4.111e-17) risk of development of AML. Also four common (> 5% frequency, cumulative frequency of over 96%) haplotypes were identified and the frequencies of the common haplotypes predicted were similar between cases and controls. When we studied if there is haplotype-based association to examine the contribution of common genetic variation at the ZNF42 locus to AML risk among Korean, two haplotypes (G-G and A-G from R51H-R130Q) showed statistically significant association.. These results suggest that ZNF42 variants and its haplotypes are significantly associated with increased susceptibility to AML.
Lineage-Specific Expression of Mouse Abcg2 during Hematopoiesis Is Regulated by Alternative Use of Multiple Leader Exons and Promoters.
Authors: Zong Yang , Zhou Sheng , Sorrentino Brian P.
Published: April 2020

ABCG2 encodes a transmembrane transporter associated with multi-drug resistance in various cancer cells. ABCG2 is also highly expressed in hematopoietic stem cells (HSCs) and is down-regulated in most committed progenitors, while expression is sharply up-regulated during erythroid differentiation and significant levels of protein expression are detected on mature red blood cells. The expression of ABCG2 in these hematopoietic cells likely reflects a protective role against both endogenous and exogenous toxins. The mechanisms for regulation of ABCG2 expression in hematopoietic cells are poorly understood. We have recently identified three novel leader exons (termed E1a, E1b and E1c) located in the 5′ untranslated region (5′-UTR) of mouse Abcg2 mRNA by EST database searches and reverse-transcription PCR (RT-PCR). In genomic DNA, the distance of these noncoding exons from exon 2, which contains the translation start codon, is 58.5 kb for E1a, 15.0 kb for E1b and 5.1 kb for E1c. In a mouse erythroid cell line (MEL), RT-PCR analysis showed that the transcript containing E1b exon was the only isoform detected. Furthermore, in primary Ter119+ erythroid cells from mouse bone marrow (BM), real time PCR showed that expression levels of the E1b-containing transcript were at least 100 fold greater than those of the E1a transcript. For comparison, cDNA was isolated from c-Kit+, Sca-1+, Lin- (KSL) BM cells, a population devoid of erythroid cells but highly enriched for repopulating HSCs. Real-time PCR showed that the E1a-containing transcript was the major expressed isoform in KSL cells, while the E1b transcript was present at significantly lower levels. The differential expression pattern of Abcg2 mRNA isoforms in HSCs and erythroid cells indicates that at least 2 different promoters control Abcg2 transcription during hematopoiesis. We also analyzed the human EST database and found 3 different ESTs all containing exon 2, similar to mouse Abcg2 gene. RT-PCR analysis of cDNA derived from human placenta and BM mononuclear cells showed expression of all 3 isoforms, which was confirmed by plasmid cloning and sequencing. These results indicate that the human ABCG2 locus also has three leader exons which are alternatively used and give rise to three isoforms of ABCG2 mRNA that differ in their 5′-UTR. We are currently defining the expression pattern of these isoforms in different human hematopoietic cell subpopulations. In summary, our data show the expression of Abcg2 during hematopoiesis is regulated at the level of transcription by alternative use of multiple noncoding leader exons in lineage-specific manner. Functional analysis of different putative promoter regions is underway and should provide a better understanding of the developmental regulation of Abcg2 expression in hematopoiesis.
A Role for Twist in Thymocyte Development.
Authors: Sugiyama Daisuke , Sugiyama Hiroko , Speck Nancy A.
Published: April 2020

Twist-1 and Twist-2 are basic helix-loop-helix (bHLH)- containing transcription factors important for mesoderm and muscle formation, and for osteoblast differentiation. The Twist proteins were previously shown to interact both physically and genetically with Runx2, a member of the core binding factor family of transcription factors (Bialek et al. Dev. Cell6: 423, 2004). Twist-1 and Twist-2 transiently inhibit Runx2 function during bone formation by inhibiting DNA binding by the Runx2 Runt domain. Since the Runt domains of the core binding factors are highly conserved, this raised the possibility that the Twist proteins may also regulate Runx1 function in hematopoiesis. To address this hypothesis we used Charlie Chaplin (CC) mice, which contain a hypomorphic Twist-1 allele generated by N-ethyl-N-nitrosourea (ENU) mutagenesis (Justice, Nat. Rev. Genet.1: 109, 2000). The CC allele encodes a single amino acid substitution (Ser192Pro) in the Twist box of Twist-1, which normally interacts with the Runt domain. CC/CC mice die from an unknown cause shortly after birth, and have small thymuses. No other hematopoietic organs show significant differences in cellularity. The number of bone marrow hematopoietic progenitors is almost identical among CC/CC, CC/+, and +/+ littermates as assessed by flowcytometry and colony forming assays. The ratios of CD4/CD8 single positive cells, and DN1-4 thymocytes are unperturbed, suggesting that T cell maturation is normal in CC/CC fetuses. However, CC/CC thymuses are reconstituted by wild type bone marrow at a lower efficiency than are wild type thymuses in fetal thymus organ culture assays, suggesting that Twist-1 may have an important non-cell autonomous role in thymocyte development.
Complex Regulation of the Homeobox Transcription Factor TGIF: Differential Expression of Multiple 5′ Isoforms.
Authors: Hamid Rizwan , Patterson Johnequia , Martincic Danko , Brandt Stephen J.
Published: April 2020

TG-interacting factor (TGIF) is a transcriptional repressor belonging to the TALE (three amino acid loop extension) class of homeobox proteins. In addition to its involvement by mutation or deletion in the inherited craniofacial disorder holoprosencephaly, we have shown that expression of TGIF is highly predictive of relapse and survival in acute myelogenous leukemia (AML). To better understand how TGIF expression is regulated, we characterized its genomic structure using expressed sequence tag analysis, reverse transcriptase-coupled PCR, and rapid amplification of cDNA ends. These studies revealed a complex pattern of gene expression, with 15 splice isoforms and 11 alternative 5′ exons spread over 40 kb of DNA, suggesting the presence of multiple promoters. Real-time and semi-quantitative PCR analysis showed these isoforms were differentially expressed in various adult tissues, leukemic cell lines, and AML blasts. Further, isoform C was found to be the major RNA product in hematopoietic cells, contributing significantly to total TGIF expression in leukemic cell lines TF1a, U937, AML-193, KG-1a, Kasumi-1, K562, GDM-1, HL-60 and AML blasts. This analysis suggests that altered splicing and/or expression of specific isoforms could be responsible for the reduced levels of TGIF message observed in AML blasts and cell lines. The unusually complex structure of the TGIF gene may enable its precise regulation during normal hematopoiesis and may be relevant to its reduced expression in myeloid leukemias.
A Novel Switch Culture System for Proliferation and Differentiation of Monocytic Cells: ERK Activation Is Not Sufficient for Macrophage Differentiation by M-CSF.
Authors: Suzu Shinya , Harada Hideki , Kimura Fumihiko , Motoyoshi Kazuo , Okada Seiji
Published: April 2020

M-CSF, a ligand for Fms, is an essential cytokine for proliferation and differentiation of monocytes/macrophages. However, molecular events responsible for M-CSF/Fms-mediated differentiation and their differences to proliferation-inducing signals are poorly understood. To clarify the difference, it is required to establish cell culture systems, in which distinct responses (proliferation versus differentiation) can be assessed independently in same cells. Here, we report the establishment of a novel culture system useful for analyzing the differences. TF-1-fms leukemia cells proliferate in response to M-CSF, but tend to differentiate into macrophages in the presence of a phorbol ester TPA. Interestingly, in the presence of TPA, M-CSF was likely to hardly yield the initiation of proliferation signal but markedly accelerate the TPA-triggered macrophage differentiation process. The combination of both TPA and M-CSF caused more drastic morphologic change than the treatment with TPA alone. The cells cultured with TPA and M-CSF showed increased granularity and higher phagocytic activity than those cultured with TPA alone. However, there was no significant difference in the numbers of viable cells and in the percentage of PI-positive dead cells between the two treatments, suggesting that the differentiation-accelerating effect did not reflect the survival-enhancing activity of M-CSF but the factor actively transmitted differentiation signals to TF-1-fms in the presence of TPA. The differentiation by TPA/M-CSF apparently correlated with elevated phosphorylation of ERK, a possible regulator of differentiation in a number of cell systems. However, and of importance, a specific MEK inhibitor, PD98059, inhibited the ERK phosphorylation but not macrophage differentiation. The result raised the possibility that elevated ERK activation was not necessarily sufficient for macrophage differentiation of TF-1-fms cells mediated by M-CSF. The experiments with TF-1-fms cells expressing HIV-1 Nef protein further supported the hypothesis: TF-1-fms cells over-expressing Nef showed impaired macrophage differentiation response but increased ERK phosphorylation (Suzu S et al., Blood 2005). Therefore, our switch culture system provides a useful tool for identifying molecules regulating the M-CSF/Fms-mediated macrophage differentiation.
Leptin-Induce Proliferation Mediated by JAK/STAT and MAPK Activation in Acute Myelogenous Leukemia Cells.
Authors: Park Hyun Ki , Kim Ju Young , Yoon Jin Sun , Kim Eun Shil , Ahn Kwang Sung , Yoon Sung Soo , Kim Byoung Kook , Lee Young Yiul
Published: April 2020

Leptin, secreted as a product of the ob gene that is mainly produced by adipose tissue, has been involved in the regulation of energy metabolism. Recently, leptin has been suggested to stimulate normal cells as well as various cancer cells including acute myelogenous leukemia (AML). However, the molecular mechanism of leptin as a proliferative effector is not poorly understood in AML. In the present study, we show that leptin-induced cellular proliferation is mediated by JAK/STAT and MAPK activation in AML. Expression of the long form (Ob-Rb) and the short form (Ob-Ra) of leptin receptor (Ob-R) was observed in 10 AML cell lines examined, and up-regulated by treatment of leptin. Leptin induced the proliferation of HEL, which was shown to express the highest Ob-R among AML cell lines, in a dose-dependent manner. Treatment with 100ng/ml of leptin enhanced the expression of Janus kinase 2 (JAK2) phosphorylation, signal transducer and activator of transcription (STAT)-3 phosphorylation and mitogen-activated protein kinases (MAPKs) in HEL. Blocking of STAT3 phosphorylation with a specific inhibitor, AG490 (50 μM), significantly reduced leptin-induced ERK1/2 phosphorylation and cellular proliferation of HEL, whereas blocking of ERK1/2 activation by a specific ERK1/2 kinase inhibitor, PD98059 (25 μM), did not affect the STAT3 phosphorylation and leptin-induced proliferation in HEL. Furthermore, knockdown of Ob-R expression with small interfering RNA (siRNA) reduced leptin-induced proliferation of HEL, and also significantly attenuated leptin-induced STAT3 and ERK1/2 activation. This results provides that leptin promotes AML cell growth by activating JAK/STAT3 and MAPK, although not directly dependent on ERK. Blocking as direct receptor level could be a rational therapeutic strategy of AML.
Pegfilgrastim Compared to Granulocyte Colony Stimulating Factor (G-CSF) with Hyper-CVAD Chemotherapy Regimen for Aggressive Lymphoid Malignancy.
Authors: Lane Steven W. , Crawford Julie , Kenealy Melita , Cull Gavin , Seymour John F. , Prince Miles , Marlton Paula , Gill Devinder , Mollee Peter N.
Published: April 2020

Aim

Pegfilgrastim (Neulasta®) has proven efficacy as supportive therapy in a variety of chemotherapy regimens, but has not been studied in dose intensive, rapidly cycling regimens such as hyper-CVAD. We examined whether pegfilgrastim leads to similar kinetics of neutrophil recovery as daily G-CSF.

Methods

Using retrospective analysis, patients receiving pegfilgrastim were matched with controls (G-CSF) for cycle of chemotherapy, prior chemotherapy, dose of cytarabine received, age (<60 or >60), diagnosis (ALL or NHL) and bone marrow involvement. The primary endpoint was duration of grade IV neutropenia (ANC <500/ul). Secondary endpoints included time to neutrophil recovery, incidence of febrile neutropenia, positive blood cultures and delay in subsequent chemotherapy.

Results

Between 01/1999 – 06/2005 we identified 124 pegfilgrastim supported cycles in 43 patients. Cases were successfully matched to 124 G-CSF supported cycles from 38 controls. There were no significant differences between cases and controls with regards to prior chemotherapy, age, sex, cytarabine dose, diagnosis or bone marrow involvement. The median duration of grade IV neutropenia was 4 days in both groups (p =0.55).

Pegfilgrastim (95% CI) G-CSF controls (95% CI) P value Grade IV neutropenia - All cycles 4 days (0–11) 4 days (0–10) 0.55 - A cycles 2 days (0–7) 2 days (0–6) 0.65 - B cycles 6 days (2–12) 6 days (0–10) 0.70 Time to ANC >500/ul 13 days (10–19) 14 days (8–18) 0.75 Time to ANC >2000/ul 14 days (11–29) 14.5 days (9–22) 0.27 Febrile Neutropenia 29% 38% 0.16 Positive Blood Cultures 11% 12% 0.85 Delay in next cycle 44% 47% 0.75

Conclusions

A single dose of pegfilgrastim is as effective as daily G-CSF for supporting the hyper-CVAD chemotherapy regimen.
Motexafin Gadolinium Upregulates pAkt Levels and Synergizes with Inhibitors of Akt Phosphorylation.
Authors: Ramos Jason , Sirisawad Mint , Miller Richard A. , Naumovski Louie
Published: April 2020

Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective expanded porphyrin that targets oxidative stress related proteins. MGd treatment of the follicular lymphoma-derived cell line HF-1 resulted in growth suppression and apoptosis, whereas MGd treatment of the Burkitt’s lymphoma-derived cell line Ramos resulted in growth suppression but not apoptosis. Since phosphorylation status of Akt/PKB kinase is regulated by oxidative stress, we monitored total and phosphorylated Akt (pAkt) in MGd treated HF-1 and Ramos cells. Levels of total and pAkt increased within 30 min after MGd treatment of HF-1 but within 4 hrs began to show a progressive decline to below baseline levels prior to cells undergoing apoptosis. Levels of total Akt did not increase in MGd treated Ramos cells but pAkt increased about 2 fold within 8 h and remained persistently elevated. Since pAkt activates survival pathways, we determined if MGd-induced cell death could be enhanced by inhibiting phosphorylation of Akt. The addition of specific inhibitors of Akt phosphorylation (SH-5 or Akt inhibitor 1) reduced pAkt levels in MGd treated HF-1 and Ramos cells and synergistically enhanced MGd-induced cell death. MGd was also evaluated in combination with celecoxib, an inhibitor of Akt phosphorylation, or docetaxel, a microtubule inhibitor which can decrease Akt phosphorylation. The combination of MGd/celecoxib or MGd/docetaxel resulted in decreased Akt phosphorylation and in synergistic cytotoxicity compared to either agent alone. These data point to a potential protective role for pAkt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by combining it with agents that inhibit Akt phosphorylation.
Efficacy and Safety of Epoetin beta 30 000 IU Once Weekly in Anemic Patients with Non-Myeloid Malignancies Receiving Chemotherapy.
Authors: Damaj Gandhi , Garidi Reda , Rodon Philippe , Bordessoule Dominique , Spaeth Dominique , Fiorentini Paola , Bergougnoux Loïc
Published: April 2020

Background: Once weekly (QW) epoetin beta (NeoRecormon®) 30 000 IU rapidly increases hemoglobin (Hb) levels and prevents transfusions in patients with lymphoid malignancies (Cazzola et al 2003). The aim of this study was to evaluate the efficacy and safety of epoetin beta 30 000 IU QW in patients with non-myeloid malignancies.

Methods: Adult anemic patients (Hb 8–12 g/dl) with non-myeloid malignancies, WHO performance status 0–2 and who were scheduled to receive chemotherapy (CT) entered into this multicenter, open-label, single-arm study. Patients received epoetin beta 30 000 IU QW for 16 weeks. Primary efficacy endpoint was the % of Hb responders during epoetin beta therapy. Hb response was defined to account for the Hb level at baseline: Hb level of 13 g/dl achieved in patients with baseline Hb 11–12 g/dl or Hb increase of 2 g/dl and/or Hb level of 12 g/dl achieved in those with baseline Hb < 11g/dl.

Results: This analysis reports data of the first 98 patients (mean age 63.4 ± 12.4 years; 61% solid; 39% hematological malignancies). Overall, 67% of patients responded to epoetin beta treatment, with a greater number of patients with hematological malignancies (78.9%) obtaining a Hb response. Median time to response was 43 days. Mean Hb level at baseline was 10.1 ±1.04 g/dl. Hb levels increased to a mean of 12.8 ±1.75 g/dl over a median treatment period of 14 weeks with epoetin beta. Median Hb increase of 1.5 g/dl and 2 g/dl at 7 and 10 weeks, respectively, were recorded. A greater number of patients receiving non-platinum (Pt)-based CT responded (73.5%) than those receiving Pt-based CT (51.7%). At least one thromboembolic event (TEE) was observed in 5% of the patients. Only one TEE was considered to be related to the study treatment.

Conclusions: Epoetin beta 30 000 IU QW is an effective and safe treatment of anemia in patients with non-myeloid malignancies, regardless of CT type and appears particularly effective in patients not receiving Pt-based CT or with hematological malignancies.
siRNA Knock down of MPL Receptor in Mouse Hematopoietic Progenitors.
Authors: Souyri Michele , Fleury Maud , Cocault Laurence
Published: April 2020

We recently showed that the expression of the thrombopoietin (TPO) receptor, MPL, could have different impacts on the generation and the expansion of hematopoietic stem cells (HSC) during establishment of definitive hematopoiesis in the mouse embryo. In order to investigate further MPL/TPO signaling pathways involved in the emergence or in the amplification and maturation of HSCs as well as their downstream targets, we set up RNA interference to knock down Mpl expression in hematopoietic progenitors and HSCs. We used the retrovirus pSUPERretro for stable and efficient delivery of siRNAs in primary cells. Three siRNAs were first tested in a model of rat fibroblast overexpressing Mpl (FREmpl) which get transformed in the presence of TPO. This transformation requires pathways involved in TPO-induced proliferation and differentiation of hematopoietic cell lines (Challiet et al., Oncogene19:2033, 2000). Transduced cells were selected and amplified. Northern blot analysis indicated that the 3 siRNAs were able to knock down Mpl expression (65 to 72% inhibition). However, decrease of the protein level and inhibition of TPO mediated transformation was observed only with 2 out of the 3 siRNAs. Inhibition of transformation was accompanied with an inhibition of Stat5 nuclear translocation.

We next introduced these siRNAs in bone marrow cells from 5-FU treated adult C57Bl6 mice, and showed that they were also able to inactivate Mpl expression in primary hematopoietic progenitors after culture of the cells for 7 days in the presence of SCF, Flt3 ligand, IL-3, IL-6 and TPO (1,1% of MPL+ cells instead of 23% in the control population). Further molecular analysis as well as in vivo an in vitro study of these cells is under current investigation and will be presented.
Cyclin D1 Mediates Resistance towards p53-Independent and -Dependent Apoptosis through Anti-Apoptotic Factors and Molecular Chaperones.
Authors: Roue Gael , Pichereau Vianney , Lincet Hubert , Troussard Xavier , Sola Brigitte
Published: April 2020

Cyclin D1 is a key regulator of the G1 to S transition of the cell cycle, physiologically absent in normal lymphocytes, but overexpressed in some lymphoproliferative disorders such as mantle cell lymphoma or multiple myeloma. While cyclin D1 has also been shown to control other aspects of the cellular fate, such as cellular senescence, apoptosis or tumorigenesis, its precise role in the pathogenesis of B-cell lymphomas and leukemias is still unclear. In this context, using various cell lines derived from the pre-B murine cell line BaF3 and conditionally expressing cyclin D1, we have previously reported that when expressed at a level comparable to the one detected in tumoral cells, cyclin D1 impairs cell proliferation mainly by inducing apoptosis. We now report that the BaF3-derived cell line BD1-A21, which constitutively expresses a low but notable level of cyclin D1, presents a recurrent resistance towards various apoptotic stimuli involving different signaling pathways, such as cytokine withdrawal, etoposide treatment and staurosporine-mediated kinases inhibition, when compared to the parental BaF3 cells. This lack of sensitivity towards apoptosis was correlated in cyclin D1-expressing cells with an increase in the basal levels of several antiapoptotic factors such as IAPs (c-IAP1, c-IAP2, XIAP and survivin) or the death receptors pathway inhibitor cFLIP, and the relative maintenance of these factors in IL-3-starved cells. 2D-PAGE analysis also pointed out in cyclin D1 expressing cells the accumulation of a series of molecular chaperones, which the most overexpressed is hsp70, concording with previous works describing the cyclin D1-dependent synthesis of hsp70 in lymphoid cells. Consistently with a protective role of hsp70 in cyclin D1-expressing cells, its pharmacological inhibition by the mean of the flavonoid quercetin allowed to re-sensitize BD1-A21 cells to the apoptotic stimulus evoked by IL-3 starvation. Hsp70 protective efect appeared to act upstream of mitochondrion, as determined by mesurement of mitochondrial membrane potential and western-blot or FACS analysis of the main Bcl-2 family members. The relationship between cyclin D1 constitutive expression, antiapoptotic factors and chaperones accumulation, and resistance to apoptosis is currently under investigation.
Growth Inhibition or Apoptosis after Treatment with Motexafin Gadolinium and Celecoxib Is Cell Line Dependent and Correlates with p27 Levels.
Authors: Ramos Jason , Sirisawad Mint , Chen Jun , Miller Richard A. , Naumovski Louie
Published: April 2020

Motexafin gadolinium (MGd, Xcytrin®) is a tumor selective expanded porphyrin belonging to the class of compounds known as texaphyrins. MGd is a redox mediator that may be directly cytotoxic or growth inhibitory to various tumor cells and can potentiate the effects of chemotherapeutic agents and radiation therapy. Initial promising activity has been seen in an ongoing phase II study of MGd as a single agent in patients with lymphoma. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, has been shown to induce apoptosis and growth inhibition and potentiate the effects of chemotherapeutic agents. To determine potential synergistic effects of MGd and celecoxib, we treated lymphoma cell lines HF-1, Ramos, DHL-4 and Raji with MGd, celecoxib or the combination. Apoptosis was a prominent feature in two of the cell lines (HF-1 and Ramos) treated with MGd/celecoxib, whereas, growth inhibition was more prominent in the other two (DHL-4 and Raji). In Ramos cells, MGd/celecoxib treatment was synergistic in inducing apoptosis and resulted in activation of a caspase cascade as demonstrated by cleavage of caspases-9, -8, -3 and cleavage of substrates Bid and PARP. Two other specific COX-2 inhibitors, rofecoxib and valdecoxib did not synergize with MGd to induce apoptosis in Ramos cells, suggesting that celecoxib potentiation of MGd is a COX-2-independent activity of celecoxib. MGd/celecoxib-induced apoptosis of HF-1 and Ramos correlated with a decrease in p27Kip1 levels whereas MGd/celecoxib-induced growth inhibition of DHL-4 and Raji correlated with increased p27Kip1 levels. Cell cycle profiles of MGd/celecoxib treated Ramos and Raji cells revealed G1 accumulation only in Raji cells, consistent with elevated levels of active p27Kip1. Our pre-clinical data suggest that celecoxib enhances the efficacy of MGd and indicates a role for p27 in the response of cells to MGd and celecoxib.
Preliminary Study of Inducing Differentiation and Apoptosis in a Novel Human Acute Monocytic Leukemic Cell Line SHI-1.
Authors: Chen Suning , Xue Yongquan , Wu Yafang , Pan Jinlan , Wu De Pei
Published: April 2020

Objective

To explore the inducing differentiation and apoptosis in a novel human acute monocytic leukemic cell line SHI-1.

Methods

Cell morphological analysis, NBT reduction test, the expression of CD11b and CD14, cell cycle analysis and binding of annexinV tested by FCM, DNA electrophoresis were performed to evaluate whether tributyrin (TB), arsenic trioxide (As2O3), and 12-O-tetradecanoyl-phorbol-13 acetate (TPA) could induce the differentiation and apoptosis in the SHI-1 cell line.

Results

The results of morphological analysis, NBT reduction test, and FCM assay showed that TB could induce differentiation of SHI-1 cells. The SHI-1 cell line presented typical apoptotic evidences including morphology features, DNA ladders and the increased apoptotic rate determined by FCM. Moreover, the SHI-1 cell line presented typical apoptotic morphology after being treated with As2O3. The apoptotic rate of the SHI-1 cell line treated with As2O3 was increased determined by FCM. The expression level of CD14 was upregulated while that of CD11b was unchanged in the SHI-1 cell line. Most of the SHI-1 cells adhered to the flask after being treated with TPA for 24 hours.

Conclusions

TB may produce dual effects of differentiation and apoptosis. The cell line could be induced to differentiation partially or apoptosis after being treated with 0.5μM, 1.0μM, and 2.0μM As2O3. TPA could induce differentiation and adhesion of the SHI-1 cell line.
Cell Penetrating Mechanism of Bax Inhibiting Peptides.
Authors: Gomez Jose A. , Yoshida Tomoyuki , Lam Minh , Distelhorst Clark W. , Matsuyama Shigemi
Published: April 2020

Plasma membrane is known to have a high degree of selectivity for molecular trafficking, and it does not allow the penetration of peptides larger than 3 amino acids. Previously known exceptions of large peptides that penetrate the plasma membrane are the Arginine rich peptides such as human immunodeficiency virus (HIV)-tat peptides. However, the mechanism of cell penetration of these peptides is largely unknown. Bax Inhibiting Peptides (BIP) are penta-peptides derived from the Bax binding domain of Ku70. At present, three types of BIP have been developed. Those are: VPMLK, VPTLK, and VPALR. All of these three BIPs directly bind Bax and inhibit Bax-mediated cell death in cultured cells as well as in animal study. Surprisingly, BIPs are cell permeable and autonomously enter the cytoplasm of the cells within 1 hr. Therefore BIPs are recognized as new members of cell penetration peptides. In this study, we investigated the mechanism of cell penetration of BIPs. DAMI cells (a human megakaryocyte cell line) and HeLa cells were used to investigate the detailed mechanism of cell penetration of BIPs. To detect the cell entry of BIPs, fluorescent dyes (fluorescein or tetramethylrodhamine) were conjugated to the N-terminus of BIPs and the cytoplasmic localization of BIPs was confirmed by confocal microscopy. Cell Penetration activities of BIPs were detected at 1 uM concentration in the culture medium. The significant accumulation of BIPs in the cytoplasm were detected within 1 hour of incubation both at 4 °C and 37 °C, suggesting that ATP-independent mechanism of cell penetration of BIP exists. However, cellular uptake of BIPs reaches plateau at 100 uM at 4 °C, whereas it increases in a dose dependent manner up to 1 mM at 37 °C without any sign of cytotoxicity. These results suggest that there are at least two mechanisms contributing to the cell penetration of BIPs that are, “ATP-independent (4 °C)” and “ATP-dependent (37 °C)” mechanisms. In addition to BIPs, we generated a series of mutated BIPs that do not bind Bax but retain cell-penetrating activities. We performed competition assay using fluorescence dye-labeled and non-labeled BIP (and the mutant BIPs), and the preliminary results suggest that there is a specific receptor for each peptide for its delivery into the cells. Our data also indicates that BIPs can deliver a cargo molecule (e.g. fluorescent dye) with at least the same molecular weight. Unlike other cell penetrating peptides, BIP has minimum toxicity due to its nature to inhibit Bax-mediated cell death. Along with the new data showing that BIP protects cells from pathological damages in cell culture and animal model, we will discuss the potential application of BIPs as a new type of drug delivery tool.
Expression of the Smad3 Transgene Restores Radiosensitivity and Migratory Capacity to a Smad3−/− Clonal Bone Marrow Stromal Cell Line.
Authors: Epperly Michael W. , Cao Shaonan , Zhang Xichen , Greenberger Emily E. , Goff Julie , Wang Hong , Bahnson Al , Shields Donna , Franicola Darcy , Greenberger Joel S.
Published: April 2020

An intact Smad3 gene product is critical for a functioning signal transduction pathway following TGF binding to the TGF-β receptor. We have previously established Smad3−/− and Smad3+/+ long term bone marrow cultures (LTBMCs) and isolated clonal bone marrow stromal cell lines from each. The Smad3−/− cells were smaller in size but had a faster cell doubling time (24 hours compared to 48 hours) and increased saturation density compared to +/+ cells (15.3 ± 1.0 x 105 cells/25 mm2 flask compared to 3.8 ± 0.1 x 105, p = 0.003). The plating efficiency of the lines was similar (18.3 ± 2.7 compared to 15.5 ± 1.7, p = 0.417). We transfected the Smad3−/− cell line with a retrovirus containing the Smad3 transgene, and selected a subclone expressing the transgene mRNA, designated Smad3−/−(3). Smad3−/−(3) cells were increased in size to that of Smad3+/+ cells, and showed decreased cell saturation density. Using the Cytoworks computer controlled cell tracking Bioreactor, we measured the migration of each clonal line. Tissue culture wells of 100 cells per well were followed for 5 days tracking each cell in quadruplicate wells per cell line. Smad3+/+ cells migrated significantly faster over 5 days in culture compared to Smad3−/− cells. (The average velocities were 0.62 μm/min for Smad3+/+ and 0.36 μm/min for Smad3−/−, p<0.0001). Over 5 days, the average velocities for Smad3+/+ cells were 0.51, 0.51, 0.52, 0.72, 0.91 μm/min, and for Smad3−/− cells were 0.28, 0.38, 0.41, 0.37, 0.35 μm/min. The 5 p-values comparing these cell lines were all <0.0001. The 7 day clonagenic irradiation survival curve showed that Smad3+/+ and Smad3−/−(3) cells were significantly more sensitive (D0 = 1.75 ± 0.03 and 1.51 ± 0.07 Gy, respectively) compared to the Smad3−/− cell line (D0 = 2.43 ± 0.06 Gy, p=0.0016 and 0.0103). These results demonstrate a concordance of radioresistance and decreased migratory capacity in bone marrow stromal cells devoid of a functioning Smad3 gene product, and restoration of both properties following overexpression of the transgene product. These data may help explain the decreased radiation fibrosis observed in Smad3−/− mice.
Expression of Cadherins in Bone Marrow Stromal Cells Affects Capacity Supporting Expansion of CD34+ Cord Blood Cells.
Authors: Liu Jin-Hwang , Liu Chin-Cheng , Yang Ming-Jie
Published: April 2020

Ex vivo expansion of hematopoietic progenitor cells (HPCs) has been used to circumvent the limitation of umbilical cord blood applied on adult HPC transplantation and may facilitate ex vivo gene manipulation. Nonetheless, the nature of these signals provided by the stromal microenvironment and mechanisms by which stromal cells regulate the growth of stem cells remain to be defined. With the help of retrovirally-expressing human telomerase reverse transcriptase (hTERT) in marrow stromal cells, we isolated 16 human bone marrow stromal cell lines. After a 7-day short stromal cells-dependent co-culture, the cord blood CD34+ cells were 1.0- to 14.5-fold expanded. Among the adhesion molecules in stromal cells examined, expression of N-cadherin and P-cadherin in stromal cells were found to correlate with capacities of stromal cells to support cord blood HPC expansion and be predictive of HPC-expanding capacities.
Immunosupressive Property of Mesenchymal Stem Cells (MSCs) in Mixed Lymphocyte Reactions In Vitro.
Authors: Li Jianyong , Meng Lijuan , Zhu Yu , Lu Hua , Ruan Changgeng
Published: April 2020

Meesnchymal stem cells (MSCs) were successfully used in the prevention and treatment of graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. To further explore the immunosuppressive property of human bone marrow (MSCs) in alloantigen-induced mixed lymphocyte reactions (MLRs) in vitro, human bone marrow MSCs and lymphocytes were prepared from healthy volunteers. MSCs were expanded in vitro in Mesencult serum free media. MSCs were cocultured with one-way MLRs and bidirectional MLRs, responder cells were labeled with carboxyfluorescein diacetate- succinimidyl ester (CFSE) in bidirectional MLRs. Cell Counting Kit-8(CCK-8)kit was used in cell proliferation detection, T-cell subsets were analyzed by flow cytometry (FCM). The results showed that MSCs were positive for CD105, CD73, CD13, CD90 and were negative for hematopoietic cell markers. In one-way MLRs, MSCs down-regulated alloantigen-induced lymphocyte expansion in a dose-dependent and MHC-independend manner. In two-way MLRs, MSCs suppressed proliferation of CFSE positive cells. T cell subsets were changed: Th2 and Tc2 were down-regulated. Th2 was reduced from 1.70% to 0.65%, and Tc2 reduced from 1.10% to 0.47%, while Th1 and Tc1 were unaffected. T cells that became CD69+, which was an early activation marker, were significantly up-regulated from 7.14% to 26.12% and CD4+CD25+T regulatory cells (CD4+CD25+Tr) were up-regulated from 4.04% to 6.19%, which indicating that suppression did not interfere with activation phase of T cells and might be mediated by CD4+CD25+Tr partly. We conclede that MSCs down-regulated alloantigen-induced lymphocyte expansion. The immunosupressive effect might involve in post-activation phase of T cells. CD4+CD25+Tr might contribute to the suppressory activity of MSCs.