bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloablative Intravenous Busulfan/Fludarabine Conditioning Does Not Facilitate Reliable Engraftment of Dual Umbilical Cord Blood Grafts in Adult Recipients.
Authors: Horwitz Mitchell , Chute John , Gasparetto Cristina , Long Gwynn , Rizzieri David , Morris Ashley , McPherson Jackie , Sullivan Keith , Chao Nelson
Published: April 2020

Bone marrow conditioning with the combination of myeloablative doses of intravenous busulfan (IVBu) with fludarabine (Flu) has gained wide acceptance as a viable alternative to the conventional busulfan and cyclophosphamide combination. When followed by transplantation with peripheral blood stem cells from matched related or unrelated donors, IVBu/Flu facilitates reliable engraftment with reduced toxicity (Blood. 2004;104:857). Whether this combination provides sufficient immunosuppression to facilitate engraftment of partially matched umbilical cord blood (UCB) grafts has yet to be reported. Ten patients with median age of 41 years (range 21–54 years) were enrolled in an IRB approved clinical trial with a primary endpoint to assess donor engraftment. Stopping rules were constructed to prevent graft failure from exceeding 20%. Disease characteristics include AML; CR1-1pt, CR2-3pts, MDS-4pts, CML-2pts. Seven of 10 patients had high-risk cytogenetic abnormalities. Conditioning consisted of fludarabine 40mg/m2 daily × 4 days and intravenous busulfan 130mg/m2 once daily × 4 days. Six patients received the drugs sequentially, and 4 concurrently. GvHD prophylaxis was provided by tacrolimus and mycophenolate mofetil. The cord blood grafts were at least 4 of 6 HLA-matched with the recipient and 3 of 6 HLA matched between grafts (low resolution class I, high resolution class II). The median combined cryopreserved total nucleated cell dose was 3.5 × 107/kg recipient body weight (range 2.1 to 4.4 × 107/kg). Engraftment of one or both UCB grafts was detected in only 4 of 10 recipients resulting in premature closure of the study as per graft failure stopping rules. None of the patients with donor engraftment achieved full donor chimerism and all ultimately died of relapsed disease. Of the 6 patients that experienced primary graft failure, 5 underwent second transplants (1 autologous, 4 haploidentical). One haploidentical and one autologous graft recipient remain alive and disease free 18 months and 13 months post transplant, respectively. In summary, 5 of 10 patients died of relapsed disease and 3 patients from treatment-related toxicity. Busulfan pharmacokinetic measurements were performed on the first and fourth dose. Interdose PK variability between day 1 and 4 was less than 15%. The mean and median AUCs were 4181 and 4195 μmol-min, respectively (range, 2634–5279 μmol-min). These levels are comparable to what has been previously reported for once daily IVBu dosing. We conclude that the myeloablative conditioning regimen of once daily IVBu with Flu provides insufficient immunosuppression to allow for engraftment of a partially matched umbilical cord blood graft.
CD34+ Viability Is a Critical Determinant of the Engraftment Potential of Umbilical Cord Blood (UCB) in Double Unit Transplantation.
Authors: Scaradavou Andromachi , Smith Katherine , Hawke Rebecca , Lee Sinda , Schaible Allison , Abboud Michelle , Collins Nancy , Kernan Nancy A. , Van den Brink Marcel , Barker Juliet N.
Published: April 2020

Compared to single unit historical controls double unit UCB transplantation (UCBT) has been associated with improved engraftment and survival in adult recipients, despite sustained donor engraftment of only one of the units. However, the mechanism of this advantage and unit predominance is not understood. Our hypothesis was that superior CD34+ and CD3+ viability (measured by 7-AAD staining) would be associated with unit predominance. Therefore, we evaluated 26 double unit UCBT recipients [median age 36 yrs (9–64); median weight 73 kg (40–111)] with high-risk hematologic malignancies who received myeloablative (n=15) or non-myeloablative (n=11) conditioning according to age and diagnosis. Units were 6/6 (n=2), 5/6 (n=25) and 4/6 (n=25) HLA-A, B antigen, DRB1 allele matched to the patient and ≥3/6 to each other. Flow cytometric evaluation was performed within 2 hours of thaw using CD45-FITC/CD34-PE/7-AAD/CD3-APC (dual-platform, modified ISHAGE protocol). 25/26 patients (96%) demonstrated donor engraftment by STR evaluation of day+21 bone marrow with one unit predominating (the “winner”). The % post-thaw CD34+ and CD3+ viability for the 26 double unit pairs is shown. Patients could be divided into 3 groups: 1) 10 in whom one of the 2 units had CD34+ viability ≥75% (always the winner) and the other <75% (always the loser); 2) 15 in whom both units had CD34+ viability ≥75% (either one was the winner); and 3) 1 in whom both units had CD34+ viability <75% and neither unit engrafted. CD3+ viability strongly correlated with CD34+ viability and was not an independent predictor of unit predominance. In univariate analysis infused viable CD3+ dose/kg was associated with unit predominance, while infused TNC/kg, viable CD34+/kg, CFU-total/kg, CFU-GM/kg, HLA-match and order of infusion were not. In summary, all engrafting units had CD34+ viability ≥75% and none of the units with CD34+ viability <75% engrafted (p: 0.007). Thus, in our analysis of unit predominance we have demonstrated that the % post-thaw CD34+ viability (but not the infused viable CD34+ dose) dictated the “engraftment potential” of a UCB unit, and, using the threshold of ≥75% viability, 12/52 units (23%) were unacceptable. When both CB units had CD34+ viability ≥75%, other factors (possibly related to CD3+ dose) determine unit predominance. Importantly, the finding that CD34+ viability is a marker of unit quality on the day of the transplant, if confirmed, has major implications for the clinical practice of UCBT. It suggests that double unit UCBT is superior because this approach significantly increases the chance that patients will receive at least one unit with engraftment potential.

Figure Figure
The Combination of 2-CDA and Rituximab in Patients with Chronic Lymphocytic Leukemia (CLL): A Prospective Multicenter Phase II Trial (SAKK 34/02).
Authors: Leupin Nicolas , Schuller Jan C. , Solenthaler Max , Tichelli Andre , Gratwohl Alois , Beretta Kurt , Bargetzi Mario J. , Himmelmann Andreas , Hanselmann Susanne , Zenhausern Reinhard
Published: April 2020

Introduction: This trial aimed to determine the efficacy and toxicity of an induction immunochemotherapy consisting of rituximab and cladribine (2-chlorodeoxyadenosine, 2-CDA) in patients (pts) suffering from chronic lymphocytic leukemia (CLL).

Methods: Inclusion criteria were CLL at first diagnosis or after one treatment with alkylating agents. The regimen consisted of four remission induction cycles. In cycle 1, 2-CDA (0.1 mg/kg/day) was administered for 5 days. In cycles 2-4, Rituximab (375 mg/m^2) was given on day 1 followed by 2-CDA (0.1 mg/kg body weight), in intervals of 28 days. Responding pts (complete remission (CR), very good partial remission (VGPR) or nodular partial remission (NPR)) underwent stem cell mobilization chemotherapy with Cyclophosphamide (4g/m^2 on day 2) G-CSF (10 microgram/kg s.c. daily, from day 4 on), and Rituximab (375 mg/m^2) on day 1 and 8 as in vivo purging. If no CR, VGPR or NPR was achieved, up to 4 cycles CHOP were administered. Primary endpoint was CR, secondary endpoints were VGPR, NPR and toxicity after induction and feasibility of stem cell mobilization. For response evaluation, staging procedures included clinical examination as well as bone marrow biopsies and CT-scans. A total of 41 pts was planned using Simon’s two-stage design with 5% significance and 80% power for the null hypothesis of CR rate < 25% and the alternative hypothesis of CR rate > 45%.

Results: 42 pts were included, median age 53.8 y (range 38 – 65), WHO performance status 0 in 33 pts and 1 in 9 pts, stage Binet B in 20 pts, Binet C in 8 pts and progressive A in 14 pts. 2 pts were not evaluable for response. 9 pts reached CR (22.5%, 95% CI: 11–38%). Overall response rate including 15 VGPR and 2 NPR was 65% (CI: 48–79%). Of the 14 non-responders, 8 underwent CHOP treatment of which 2 achieved VGPR. 20 patients underwent mobilization and 8 pts refused further protocol treatment. 14 pts had leucapheresis. Stem cell harvest was feasible in 7 pts, all with ≥ 2×10^6 cells/kg. Fever and infection were reported respectively in 13 and 9 pts. Infusion related adverse events of Rituximab were moderate and occurred mainly after the first infusion. 42 of 158 cycles (27%) were associated with grade 3 or 4 neutropenia and 6 of 158 cycles (4%) with grade 3 thrombocytopenia.

Conclusions: Although the expected CR rate was not achieved, a combination of Rituximab and 2-CDA is an effective and well tolerated treatment.
Identification of Driver and Passenger Mutations of FLT3 by High-Throughput DNA Sequence Analysis and Functional Assessment of Candidate Alleles.
Authors: Frohling Stefan , Scholl Claudia , Levine Ross L. , Loriaux Marc , Boggon Titus J. , Bernard Olivier A. , Berger Roland , Dohner Hartmut , Dohner Konstanze , Ebert Benjamin L. , Teckie Sewit , Golub Todd R. , Schittenhelm Marcus M. , Lee Benjamin H. , Griffin James D. , Stone Richard M. , Heinrich Michael C. , Deininger Michael W.N. , Druker Brian J. , Gilliland D. Gary
Published: April 2020

Activating mutations in the juxtamembrane domain or the activation loop of the receptor tyrosine kinase FLT3 occur in many cases of acute myeloid leukemia (AML), but it is not known whether genomic alterations outside these regions contribute to leukemogenesis. High-throughput DNA sequence analysis has provided insights into the mutational profiles of various cancers and represents a promising strategy for the identification of novel therapeutic targets. However, recognizing the subset of genomic alterations that are functionally relevant has proven difficult. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in a cohort of 222 adult AML patients without known FLT3 mutations and experimentally tested the functional consequences of each candidate leukemogenic allele by exogenous expression in BaF3 cells. DNA sequencing detected nine non-synonymous sequence variants in six exons that were not known single-nucleotide polymorphisms. Functional assessment of these alleles identified four novel activating mutations in the extracellular domain, the juxtamembrane domain, and the activation loop that induced constitutive kinase activity, differentially activated downstream signaling pathways, and conferred varying sensitivity to pharmacologic FLT3 inhibition. In contrast, the remaining five alleles, including mutations in highly conserved, key functional domains of FLT3, were not associated with increased kinase activity and aberrant signal transduction. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation, and underscore the importance of functional validation of candidate alleles discovered using high-throughput genomic screens, to distinguish between ‘driver’ mutations underlying cancer development, and biologically neutral ‘passenger’ alterations.
Dual Epigenetic Control of CCAAT/Enhancer Binding Protein α (C/EBPα) Expression in Acute Myeloid Leukemia.
Authors: Hackanson Bjoern , Bennett Kristi L. , Brena Romulo M. , Jiang Jinmai , Maharry Katie , Whitman Susan P. , Schmittgen Thomas D. , Luebbert Michael , Marcucci Guido , Bloomfield Clara D. , Plass Christoph
Published: April 2020

CCAAT/enhancer binding protein α (C/EBPα) is a master regulator for myeloid differentiation. As a potential tumor suppressor, its loss of function through mutations and posttranslational mechanisms has been thoroughly investigated in acute myeloid leukemia (AML). Recently, we have demonstrated the importance of epigenetic alterations in deregulating C/EBPα expression patterns in lung cancer and head and neck squamous cell carcinoma (HNSCC). In this study we investigated the role of DNA methylation and other epigenetic factors in the regulation of C/EBPα in AML using 94 patient samples and 7 leukemia cell lines. A comprehensive and quantitative DNA methylation analysis of C/EBPα’s large CpG island using MassARRAY (Sequenom©) technology and BioCOBRA identified a distinct and densely methylated upstream promoter region (−1423 bp to −1121 bp in relation to the transcription start site) in 20% (19 of 94) of AML patient samples and in five of seven leukemia cell lines, while the core promoter remained unmethylated. This aberrant DNA methylation pattern was associated with two generally prognostically favorable cytogenetic subgroups: inv(16)(p13q22) and t(15;17)(q22;q21). While DNA methylation levels in normal bone marrow samples (NBM) were very low (median: 0%, range: 0%–5%) when compared to NBM, we observed significantly higher DNA methylation levels in the inv(16) and t(15;17) cytogenetic subgroups, with median DNA methylation levels of 29% (range: 1% to 81%) and 5% (range: 1% to 94%), respectively (p<0.05). However, using semi-quantitative RT-PCR, we could not detect a significant correlation between DNA methylation of C/EBPα and its expression in the inv(16) subgroup. Interestingly, while epigenetic-targeting compounds induced up-regulation of C/EBPα mRNA in vitro, C/EBPα protein was diminished. This unexpected scenario was postulated to be compatible with the presence of a microRNA targeting C/EBPα mRNA. Using a computational microRNA prediction approach and functional studies, we show that C/EBPα mRNA is a target for microRNA-124a. We additionally demonstrate that microRNA-124a is frequently silenced by epigenetic mechanisms in AML, becomes upregulated following epigenetic treatment and targets the C/EBPα 3′-untranslated region (3′UTR). In this way, C/EBPα protein expression is reduced in a posttranscriptional manner. Our results indicate that epigenetic alterations of C/EBPα are a frequent event in AML and that epigenetic treatment can result in down-regulation of a key hematopoietic transcription factor.
Genome-Wide Identification of Aberrant Promoter Associated CpG Island Methylation in Acute Lymphoblastic Leukemia.
Authors: Kuang Shao-qing , Tong Weigang , Yang Hui , Lee Mathew K. , Fang Zhi-Hong , Jelinek Jeroslav , Issa Jean-Pierre , Garcia-Manero Guillermo
Published: April 2020

Aberrant DNA methylation is a common molecular feature of both pediatric and adult ALL. Specific methylation patterns predict for poor prognosis (Shen et al Blood 2004), and reactivation of epigenetically inactivated molecular pathways results in induction of leukemia cell death (Kuang et al. Oncogene 2007). Until now most studies of methylation in ALL have been based on arbitrary gene selection methods. To overcome this limitation and to study hundreds of promoter CpG islands simultaneously, we have developed a method that combines MCA (Methylated CpG Island Amplification) with either RDA (Representational Difference Analysis) or the Agilent Promoter Microarray platform. With these methods differentially methylated DNA treated with bisulfite is generated after mixing tester DNA (in our case DNA from de novo refractory Ph negative and MLL negative ALL patients) with driver DNA (normal B cell controls) and using specific restriction enzymes and several rounds of PCR. DNA fragments thus generated are either cloned (RDA) or labeled and spotted on the Agilent Array. Using this technology, that can potentially interrogate up to 17K promoters, we have identified 932 promoters targets of aberrant DNA methylation in poor risk ALL from patients that cannot be currently identified by standard molecular methods (Ph and MLL negative). The genes associated with these promoters are distributed through the human genome but an overrepresentation of methylated promoters located in chromosomes 3, 9, 11 and 19 was detected. Using molecular pathway clustering analysis, 404 of these genes are grouped together in 29 specific functional pathways. We have validated the methylation of 31 of these 923 genes by bisulfite pyrosequencing. Of these, 27 (87%) were confirmed to be hypermethylated in 23 human leukemia cell lines but not in normal controls (N=15). Methylation status analysis of these 27 genes allowed for the segregation of T cell versus B cell leukemia cell lines. Fifteen of these genes (GIPC2, RSPO1, MAGI1, CAST1, ADCY5, HSPA4L, OCLN, EFNA5, MSX2, GFPT2, GNA14, SALL1, MYO5B, ZNF382 and MN1) were also frequently hypermethylated in primary ALL samples. Expression analysis of 6 of these genes (GIPC2, MAGI1, ADCY5, HSPA4L, OCLN and GNA14) in leukemia cell lines further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with 5′-aza-2′-deoxycytidine and trichostatin A resulted in gene re-expression, further confirming the role of DNA methylation in their silencing. In summary, we have identified in excess of 900 targets of aberrant DNA methylation in ALL. The study of the epigenetically suppressed pathways represented by these genes should allow us to further understand the molecular pathogenesis of ALL and develop new prognostic biomarkers for patients with Ph and MLL negative disease.
Epigenetic Inactivation of the Secreted Frizzled-Related Protein Gene Family in Multiple Myeloma.
Authors: Jost Edgar , Gezer Deniz , Wilop Stefan , Herman James G. , Osieka Rainhardt , Galm Oliver
Published: April 2020

Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. Previous molecular studies have largely focused on acquired genetic aberrations in MM. There is increasing evidence that in addition to genetic aberrations epigenetic processes play a major role in carcinogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene function. This epigenetic event acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function. The Wnt pathway has been recognized to be essential for normal organ development, and a role for Wnt signal transduction at several stages of lymphocyte differentiation and in the self-renewal of hematopoietic stem cells could be demonstrated. Alterations in the Wnt pathway have been shown to contribute to the pathogenesis of various human malignancies. Epigenetic silencing of the family of secreted frizzled-related proteins (SFRPs), which act as Wnt antagonists, was recently reported in several solid tumors and in acute lymphoblastic leukemia. In order to investigate the potential role of abnormal Wnt signaling in MM, we determined the methylation status of the promoter-associated CpG islands of SFRP-1, 2, 4 and 5 in the MM cell lines U266, LP-1, RPMI-8226 and OPM-2. Methylation-specific polymerase chain reaction (MSP) analysis revealed that promoter hypermethylation of the SFRP genes was a frequent event in MM cell lines (SFRP-1: 2/4, SFRP-2: 2/4, SFRP-4: 1/4 and SFRP-5: 3/4). Aberrant methylation of SFRP-1 and SFRP-2 in MM cell lines was associated with transcriptional silencing, as determined by real-time reverse transcriptase polymerase chain reaction. Treatment of cell lines that carry a hypermethylated SFRP-1 and SFRP-2 gene, respectively, with the demethylating agent 5-aza-2′-deoxycytidine resulted in gene reexpression. We then analyzed by MSP the methylation status of SFRP-1, 2, 4 and 5 in 76 specimens obtained from MM patients. The frequency of aberrant methylation among the primary patient samples was 38.2% (29/76) for SFRP-1, 59.2% (45/76) for SFRP-2, 2.6% (2/76) for SFRP-4 and 7.6% (6/76) for SFRP-5. There was a high incidence of concomitant hypermethylation of SFRP-1 and SRFP-2. We conclude that promoter hypermethylation of the SFRP genes is a novel epigenetic event in MM that may contribute to aberrant activation of the Wnt pathway. Further studies are warranted to elucidate the functional consequences of aberrant Wnt signaling by downregulation of the SFRP genes in the pathogenesis of MM. Additionally, the increasing evidence for the important role of DNA methylation changes in malignant plasma cell disorders may serve as a basis for the use of epigenetically targeted therapeutic approaches in MM in the future.
Host Plasmacytoid or Conventional Dendritic Cells Alone Are Sufficient To Initiate Graft-Versus-Host Disease.
Authors: Koyama Motoko , Hashimoto Daigo , Aoyama Kazutoshi , Matsuoka Ken-ichi , Karube Kennosuke , Takeda Kiyoshi , Ohshima Koichi , Akashi Koichi , Tanimoto Mitsune , Harada Mine , Teshima Takanori
Published: April 2020

Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation. Alloantigen expression on host dendritic cells (DCs) is critical to initiate GVHD. DCs can be divided into two main subpopulations; conventional DCs (cDCs) and plasmacytoid DCs (pDCs), however, the contribution of each DC subset to elicit GVHD remains unclear. We examined the ability of cDCs and pDCs to initiate GVHD. pDCs, cDCs and B cells were isolated from C57BL/6 (B6: H–2b) mice treated with Flt3 ligand in order to expand DCs. pDCs were enriched from bone marrow by depleting CD3+, CD19+, CD11b+, and CD49b+ cells, followed by a FACS sorting of CD11cint B220+ cells. cDCs and B cells were sorted from splenocytes as CD11chi B220− cells and CD11c− B220+ cells, respectively. Isolated pDCs showed plasmacytoid morphology, produced IFN-α in response to CpG oligonucleotide. Although pDCs stimulated allogeneic T cells far less potently than cDCs, stimulation with CpG enhanced their allostimulatory capacity as potent as cDCs. We compared the ability of each DC subset to initiate GVHD by an add-back study of MHC class II-expressing DCs into MHC class II-deficient (II−/−) mice that were resistant to CD4-dependent GVHD. Lethally irradiated II−/− B6 mice were injected with 2 × 106 pDCs, cDCs or B cells from wild-type (II+/+) B6 mice on day -1 and injected with 2 × 106 CD4+ T cell from BALB/c (H–2d) mice on day 0. A flow cytometric analysis of the mesenteric lymph nodes on day +5 demonstrated significantly greater expansion of donor CD4+ T cells in recipients of pDCs or cDCs than those of B cells (Table). While injection of B cells did not cause any sign of GVHD, injection of pDCs or cDCs alone was sufficient to produce clinical and pathological GVHD (Table), thus breaking GVHD resistance of II−/− mice. We next examined the ability of pDCs to induce CD8-dependent GVHD in MHC-matched transplant using mice deficient in functional MHC class I expression (β2m−/−). Again, injection of pDCs or cDCs alone was sufficient to cause expansion of donor CD8+ T cells (p<0.05). We next asked whether signaling through Toll-like receptors (TLRs) could be required for pDCs to initiate GVHD. However, injection of pDCs isolated from MyD88/TRIF-double deficient mice was able to initiate GVHD as potent as wild-type pDCs, thus demonstrating that pDCs initiate GVHD in a TLR signaling independent manner. These results provide important information for developing strategies aimed at inactivating host DCs to prevent GVHD.

Impact of each APC subpopulation on GVHD APC Donor CD4 expansion (×103±SE) Clinical GVHD score (mean±SE) Pathological GVHD score (mean±SE) *p<0.05 compared with B cells B cell 0.1 ± 0.0 2.1 ± 0.2 2.1 ± 0.2 pDC 5.3 ± 2.4* 4.3 ± 0.3* 7.4 ± 0.5* cDC 9.7 ± 3.8 * 3.8 ± 0.5 * 7.2 ± 0.7*
A Shift in the Intestinal Microflora towards Pro-Inflammatory Bacteria and Signaling Via Toll-Like-Receptor 2 Triggers Early Onset of Graft-Versus-Host Disease.
Authors: Nogai Axel , Heimesaat Markus M. , Bereswill Stefan , Thiel Eckhard , Goebel Ulf B. , Uharek Lutz
Published: April 2020

Pattern recognition receptors such as Toll-like-receptors (TLR) and NOD proteins bind specifically to microbial components and activate innate immune responses. Reducing the bacterial load by antibiotic treatment has been shown to ameliorate the severity of graft-versus-host-disease (GvHD) after allogenic stem cell transplantation. The impact of bacteria and TLRs on the induction of T-cell-alloreactivity and GvHD was investigated in a murine transplantation model. C57B/10 wild-type (wt) mice served as recipients of MHC-mismatched Balb/c grafts after conditioning with treosulfan and cyclophosphamide. Analyses of the gut microbiota in fecal samples showed a pronounced increase of luminal load of both gram-positive and gram-negative bacteria within 11 days after stem cell transplantation. The increase in the Escherichia coli load was significantly correlated with the severity of graft-versus-host-disease and with decreased survival rates. Analysis of the kinetics of fecal E. coli load and the histologial and clinical GvHD scores revealed that the increase of the fecal E. coli load is detected simultanously to the onset of clinical overt GvHD. Because TLR 2 and 4 represent the major receptors for gram-positive and gram-negative bacteria, respectively, the incidence and severity of GvHD was investigated in TLR2, TLR4 and TLR2/4 deficient mice on a C57B/10 background. The onset of GvHD was delayed in recipient mice lacking TLR2 and both TLR2 and 4 (p<0.05) whereas a single TLR4 deficiency had no influence. Interestingly, there was no correlation of E. coli load and GvHD in TLR2-deficient mice.Taken together, these results support potential roles of TLR2-dependent signaling and gut bacterial components in the induction and early onset of intestinal GvHD. In expansion of these findings, our data elucidate the role of the innate immune system and its receptors for the induction of alloreactive T-cell responses, providing evidence that modulation of the intestinal gut flora might prevent or reduce GvHD in humans.
Lymphopenia Promotes the Development of Graft-Versus-Host Disease upon Donor Leukocyte Infusion.
Authors: Sachs Jessica , Sykes Megan
Published: April 2020

In murine models of hematopoeitic cell transplantation (HCT), a graft-versus-leukemia (GVL) effect can be effectively separated from graft-versus-host disease (GVHD) by giving delayed donor-leukocyte infusions (DLI) to established mixed hematopoietic chimeras (MC) (Mapara, Transplantation 2003). In these models, the absence of conditioning-induced inflammation at the time of DLI prevents trafficking to GVHD target tissues, limiting the GVH response to the lymphohematopoeitic system (Chakraverty, J Exp Med 2006). While clinical trials have shown that the same outcome is possible in humans, some patients develop severe GVHD following delayed DLI (Dey, Blood Biol Marrow Transplant 2003). In contrast to mice, who recover T cell numbers quickly after HCT through de novo thymopoiesis, humans remain lymphopenic for many months. The current study investigates the role of recipient lymphopenia in the development of GVHD following delayed DLI to MC. Allogeneic (B10.A) or syngeneic (B6) lymphocytes (30 × 106 whole splenocytes) and bone marrow cells (10 × 106) were administered to unconditioned RAG–1 knockout (KO) B6 recipients or to established MC, and animals were followed for development of GVHD. By day 7 after injection, the RAG KO recipients of allogeneic lymphocytes (allogeneic group) had lost significantly more weight (p<0.001) and had significantly higher clinical GVHD scores (p<0.0001) than RAG KO recipients of syngeneic lymphocytes (syngeneic group) or MC recipients of allogeneic lymphocytes (MC group). Histologic evaluation on day 7 showed significantly increased GVHD in liver and colon of the allogeneic group. Chimerism analysis on day 7 showed the presence of an ongoing lymphohematopoietic GVH response (LGVHR) in both the allogeneic and MC groups, though the response was slower in the MC group. When followed over time, clinical GVHD in the allogeneic group resolved by day 56, weight differences disappeared by day 65, but mild histologic GVHD persisted as late as day 239. Importantly, the same dose of allogeneic T cells given to freshly conditioned animals results in rapid, lethal GVHD, whereas these unconditioned, lymphopenic animals developed only a relatively mild, largely self-limited GVHD. These results demonstrate that in the absence of conditioning-induced inflammation, lymphopenic recipients of allogeneic T cells do develop mild GVHD, consistent with the possibility that recipient lymphopenia contributes to the GVHD seen in human MC recipients of DLI. The mechanisms by which lymphopenia promotes GVHD in non-conditioned MC recipients of DLI require further investigation, but may include the absence of regulatory T cells, the potential for lymphopenia-induced proliferation of T cells, and the presence of infection-induced inflammation.
Adenoviral Vector-Mediated Transfer of the Indian Hedgehog Gene Modulates Lymphomyelopoiesis In Vivo.
Authors: Kobune Masayoshi , Kawano Yutaka , Sasaki Katsunori , Takimoto Rishu , Kato Junji , Hamada Hirofumi , Niitsu Yoshiro
Published: April 2020

Indian hedgehog (Ihh) plays an essential role in angiogenesis, hematogenesis and epiphysis formation during embryogenesis. In the present study, we injected an adenoviral vector (Adv) carrying the mock-control (adv-control) or Ihh gene (Adv-Ihh) into SCID or BALB/c mice to evaluate the effects of lhh on the regulation of postnatal hematopoiesis in vivo. After the intravenous injection of Adv-Ihh, the expression of vector-derived Ihh mRNA was detected in the liver. Four weeks after administration of Adv-Ihh to SCID mice, we observed an increase in the number of c-Kit+ cells and clonogenic cells/105 mononuclear cells in the bone marrow as compared with adv-control administrated mice. Moreover, after administration of Adv-Ihh to BALB/c mice, the number of splenic B220+IgMlowCD23intCD21int B lymphocytes (Figure 1) and CD4+ T lymphocytes (Figure 2) was strongly increased. Furthermore, the number of thymic double negative (DN)2, DN3, CD8+ immature single positive and CD4+/CD8− cells was significantly elevated relative to the number in mice that received the control Adv vector. Our results suggest that enhanced signaling by Ihh can modulate the proliferation and differentiation of splenic B lymphocytes and thymic T lymphocytes during BM hematopoiesis in vivo. Thus, modulation of the Hh signaling pathway may provide a therapeutic strategy to stimulate lymphomyelopoiesis in vivo.

Figure Figure Figure Figure
Expression of miR-188 and 362 Induced by Erythropoietin Stimulation in a Human Erythrocytic Leukemia Cell Line.
Authors: Sugiura Keiichi , Kosaka Nobuyoshi , Yamamoto Yusuke , Yoshioka Yusuke , Miyazaki Hiroshi , Komatsu Norio , Ochiya Takahiro , Kato Takashi
Published: April 2020

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level and participate in a lot of biological processes. Several studies indicated that miRNAs play an important role in hematopoietic system. It has not been elucidated well about miRNAs involved in cytokine-regulated molecular mechanisms in cellular proliferation and differentiation. We screened specific miRNAs expressed in human leukemia cell lines, UT-7 and its sublines UT-7/GM, UT-7/EPO and UT-7/TPO, which exhibit hematopoietic lineage properties depending on cytokine stimuli by GM-CSF, erythropoietin (EPO), or thrombopoietin (TPO), respectively. The expression profiles of 470 miRNAs in all were analyzed using LNA-based microarray between UT-7/GM, UT-7/EPO and UT-7/TPO cells. The initial microarray data were validated, and specific expressions of several miRNAs were confirmed using quantitative real-time PCR. Especially, the expression levels of miR-188 and 362 clustered on X chromosome were significantly higher in UT-7/EPO cells as compared with those in UT-7, UT-7/GM and UT-7/TPO cells. To assess the effect of EPO on the expression of miR-188 and 362, UT-7/EPO cells were cultured under various concentration of EPO (0 to 10 U/ml) after cytokine withdrawal for 24 hours. Quantification of miRNAs expression by real-time PCR showed that miR-188 and 362 were increased 2- to 20-fold by EPO stimuli, but both expressions were not in dose-dependent manner. Furthermore, the addition of EPO (1 ng/ml) to growth factor-derived UT-7 cells which were cultured for 1 month caused a 4- to 50-fold increase of miR-362 expression; while miR-188 expression was not increased. Similarly, EPO induced the expression of miR-362 in erythroleukemic cell line TF-1. The expression levels of miR-188 and miR-362 were significantly higher in UT-7/EPO with erythrocytic features than UT-7/GM and UT-7/TPO cells, in addition, expressions of both miRNAs were induced by EPO stimuli in UT-7/EPO cells, suggesting that miR-188 and miR-362 are involved in lineage specific molecular mechanisms of erythropoiesis.
Impaired Repopulating Activity of Fus-Deficient Hematopoietic Stem Cells.
Authors: Sugawara Takeaki , Iwama Atsushi
Published: April 2020

RNA-binding protein FUS (also known as TLS) was originally identified in chromosomal translocation in human myxoid liposarcoma. The FUS gene is also translocated with the transcription factor gene ERG in human myeloid leukemia with recurrent chromosomal translocation t(16;21). Multiple data suggest that wild-type FUS is also involved in the development of leukemia as one of the downstream targets for oncoproteins including BCR-ABL. However, little is known about the role of FUS in the normal hematopoiesis. The previous report demonstrated that Fus-deficient (Fus−/−) newborn mice, which die shortly after birth because they cannot suckle, have a non-cell-autonomous defect in B lymphocyte development. No cell-autonomous defect of Fus−/− hematopoietic cells has been documented. Here we report the detailed analyses of the Fus−/− fetal liver hematopoietic stem cells (HSCs). Fus−/− fetal livers at embryonic day 14.5 were smaller in size and exhibited a significant reduction in hematopoietic cell numbers by 60% compared with the wild type (WT). Nonetheless, no significant difference was observed in the proportion of stem/progenitor cell fraction (lineage-marker-c-Kit+Sca-1+; KSL) as well as colony-forming cells between WT and Fus−/− fetal livers. Fus−/− KSL cells proliferated and differentiated almost normally in vitro. To examine in vivo repopulating activity, we transplanted fetal liver cells to lethally irradiated CD45.1 recipients with competitor bone marrow cells. Fus−/− fetal liver donor cells reconstituted recipients’ hematopoiesis for the long term and contributed to all cell lineages including B lymphocytes. In contrast to the in vitro results, however, the chimerism of donor-derived cells was significantly lower in recipients receiving Fus−/− fetal liver cells compared with WT controls (approximately 2-fold reduction). This trend was reproducible with both unfractionated and purified KSL fetal liver test cells. Our data demonstrated that the proto-oncogene Fus is involved in the maintenance of normal HSC functions. Detailed analyses on the underlying mechanisms are in progress.
Functional Interaction of c-Kit Receptor and Interleukin-3 Receptor.
Authors: Ye Zhijia , Weissman Sherman M.
Published: April 2020

Hematopoiesis is regulated by a number of growth factors and cytokines, among which stem cell factor (SCF) plays a critical role for normal hematopoiesis. An important feature of SCF is its strong capacity to synergize with the cytokines including interleukin-3(IL-3), granulocyte-marcrophage colony-stimulation factor (GM-CSF) and erythropoietin to regulate proliferation and differentiations of hematopoietic progenitor cells. The mouse hematopoietic progenitor cell line EML contains two populations of lineage negative cells, distinguished by the expression or absence of CD34. The two sub-populations express similar levels of the SCF receptor c-kit, but only the CD34+ population replicates in the presence of SCF alone, while the CD34 negative population replicates in the presence of IL3 but this replication is synergistically stimulated by the addition of SCF. Our previous studies indicated that synergistic proliferation of the mouse hematopoietic progenitor cell line EML induced by SCF in combination with IL-3 was probably mediated by the synergistic increases in tyrosine phosphorylation of c-Kit and β-chain of IL-3 receptor, which is shared with GM-CSF receptor and interleukin-5 receptor. Conversely, the decreased response of c-kit to SCF alone in the CD34 negative population may be a result of the increased expression of IL3 receptor in these cells. Trans-phosphorylation between c-Kit and the β-chain of IL-3 receptor was also observed in EML cells. In studies of the molecular mechanism behind the functional interactions between SCF and IL-3 we found that c-Kit and IL-3 receptor β-chain form a complex in EML cells. Antibody Ab-1 specific against the Ig-like domain 4 in the extracellular region of c-Kit blocked not only cell proliferation induced by SCF but also synergistic proliferation induced by SCF plus IL-3. Consistent with this finding, Ab-1 inhibited phosphorylation of c-Kit induced by SCF or IL-3, and synergistic phosphorylation of c-kit induced by SCF plus IL-3 respectively. SCF and IL-3 in combination synergistically activated the protein kinases Akt and Erks, the downstream mediators of c-Kit and IL-3R. Phosphorylation of Erks is independent on the phosphatidylinositol 3-kinase (PI3-kinase) pathway in EML cells. These data suggested the possibility that the synergistic growth of EML cells induced by SCF in combination with IL-3 was mediated by multiple mechanisms that could include trans- and synergistic phosphorylation of c-Kit and the β-chain of IL-3 receptor, PI3-kinase dependent synergistic phosphorylation of Akt and PI3-kinase independent synergistic phosphorylation of Erks. The trans- and synergistic phosphorylation of c-Kit and β-chain of IL-3 receptor was mediated by physical interaction of two receptors.
MicroRNA Analysis by In Situ Hibridization in Hodgkin Lymphoma.
Authors: Navarro Alfons , Martinez Antonio , Balagué Olga , Gaya Anna , Pons Aina , Urbano-Ispizua Alvaro , Montserrat Emili , Monzo Mariano
Published: April 2020

Hodgkin lymphoma (HL) is a neoplasm characterized by the presence of relatively few tumoral cells, Hodgkin and Reed-Sternberg cells in an important non-neoplastic environment. The mature microRNAs (miRNAs) are small RNA molecules (20–25 nt), that act inhibiting the mRNA translation to protein by binding to 3′ UTR region of mRNA. In a previous work we detect a 25 miRNA signature of HL. Some of them inhibit the expression of key genes related to B lymphomagenesis. The aim of the study was to determine if the differential expression of miRNAs in tumoral tissue versus reactive lymph nodes, is due to genetic alterations in the Hodgkin/Reed Sternberg cells or alterations in the non-tumoral microenvironment. Fluorescein (FITC) 5′ labelled locked-nuclei-acid-incorporated (LNA) miRNA ribo probes for miR-21, 134, 138, 155 (miRCURY™ LNA detection, Exiqon) were used in 20 cases of HL formalin-fixed paraffin embedded tissue sections on silane coated slides (Vision BioSystem). Chromogenic in situ hybridization was done in an automated platform Bond Max (Vision Biosystems) with minor modifications. Pre-treatment of the slides was performed with Protease 1 for 10 min at 37°C. A total amount of 300 microliter of 25nM probe was hybridized in 1x sodium chloride-sodium citrate hybridization buffer (SSC) (Innogenetics) up to 50° C for 2 hours. We used a pre-diluted mouse anti-FITC antibody (Vision BioSystems) for 20–60 minutes followed by a goat anti-mouse linked to thousands of horse radish peroxidase (HRP) sites (Refine Detection System, Vision BioSystems). DAB was used as a chromogen reacting for 10 minutes and hematoxilyn was used as a counterstain. By functional and target analysis, we selected three miRNAs, miR-21 (PTEN), miR-134 (J-Chain) and miR-138 (PU.1), from the cHL signature that seemed to have a role in tumorigenesis process and analyzed them by CISH. In all cases a cytoplasmic signal was demonstrated in Hodgkin and Reed-Sternberg cells. Moreover, a nuclear signal was identified in reactive tumor infiltrating lymphocytes for miR-21 and miR-138. This nuclear signal may be due to crossreactivity with primary miRNA in these cells. We analyzed miR-155 as positive control of in situ hibridization and we demonstrated a cytoplasmic signal in Hodgkin and Reed Sternberg cells, as well as in scattered reactive lymphocytes and activated histiocytes as previously reported. In conclusion, miR-21, miR-134 and miR-138 play a role in HL lymphomagenesis since their expression is preferentially localized in Hodgkin/Reed Sternberg cells. The study of the other miRNAs of our signature can help explain the changes that appear in the atypical cells as well in the reactive cellular microenvironment in HL.
The Association between Hepatitis C Infection and Lymphoma in AIDS.
Authors: Ghanem Zuhair , Singh Amandeep , Zahran Ali , Maroules Michael
Published: April 2020

Background: Many studies have demonstrated a possible association between hepatitis C and a wide spectrum of lymphoproliferative malignancies. Increased hepatitis C virus (HCV) prevalence among patients with non-Hodgkin lymphoma (NHL), suggesting that HCV infection increases NHL risk through chronic immune stimulation. Most of the previous studies excluded patients with immunodeficiency. No study looked at the association between established chronic HCV infection and lymphoma in AIDS patients. However, previous studies found no relation between HCV infection and lymphoma in HIV patients. The aim of our study is to determine the correlation between chronic HCV infection and lymphoma in AIDS patients.

Methods: A hospital based retrospective case control study was performed in a tertiary care center. The medical records of 603 AIDS patients were reviewed between January 2001 and December 2005. 284 patients with both AIDS and hepatitis C in the first group and 319 control patients with AIDS only in the second group. The diagnosis of hepatitis C and AIDS was determined retrospectively from the patients medical records. Only patients with AIDS were included in the study. AIDS was defined as CD4 < 200 or any AIDS defining illness.

Results: Among the 284 patients with both AIDS and HCV infection 7 (2.4%) patients were found to have lymphoma at any stage of the disease. On the other hand, 8 (2.4%) patients were found in the control group. By using the Fisher Exact Probability Test there was no statistically significant increase in the incidence of lymphoma among AIDS patients with HCV infection compared to the control group (p=0.823063, and Odds Ratio =0.9824). The type of lymphoma was not determined giving the small number of cases and that the majority of lymphoma in AIDS are NHL cases.

Conclusion: Our data showed that among AIDS patients, co infection with Hepatitis C virus doesn’t increase the incidence of lymphoma. Giving the fact that AIDS is a low immunity status, this conclusion supports the theory that chronic immune stimulation plays a role in the pathogenesis of lymphoma in HCV infection.
Interleukin-15 Upregulates the Expression of NK-Cell Activating Receptors and Concomitantly Increases the NK Cytotoxicity in Patients with Acute Myeloid Leukemia.
Authors: Szczepanski Miroslaw J. , Czystowska Malgorzata , Szajnik Marta , Welsh Ann , Foon Kenneth A. , Whiteside Theresa L. , Boyiadzis Michael
Published: April 2020

Natural killer (NK) cells lyse malignant cells without prior antigen-specific priming and play a critical role in the innate immune response. A balance of signals from activating and inhibiting receptors expressed on each NK cell controls its activity. The growth, differentiation and survival of NK cells have been found to be dependent on interleukin-15 (IL-15). Using multicolor flow cytometry we investigated the receptor repertoire and also measured the NK cell activity in twenty three patients with newly diagnosed acute myeloid leukemia (AML) prior to any treatment. Further, we investigated the ex-vivo effect of IL-15 on the NK cell repertoire and NK cell cytotoxicity. The percentage of circulating NK cells was lower (p<0.0001) in the AML patients (6%± 0.7, range 1–17%) compared to the NK cells of healthy donors (12%± 1, range 9–17%). The expression of the activating natural cytotoxicity (NCR) receptors NKp30 and NKp46 and the C-type lectin receptors NKG2D and NKG2C was significantly decreased in the AML patients compared to the NK cells of healthy donors: NKp30 24 vs 51% p<0.0001, NKp46 32 vs 73% p<0.0001, NKG2D 43 vs 83% p<0.0001, NKG2C 17 vs 28% p<0.03. In addition, the receptor expression (mean fluorescence intensity, MFI) was also significantly lower in AML patients compared to healthy donors. No significant differences in the expression of the NCR NKp44 and the NK- cell inhibitory receptors were observed. Furthermore, the NK cytotoxicity in the AML patients at diagnosis was significantly lower (p<0.0003) compared to the NK cytotoxicity of healthy donors (4 vs 75 LU). When NK cells obtained from AML patients were cultured with IL-15, significant increases in the expression of the NK- cell activating receptors (Table 1) were observed. The upregulation of the activating receptors was associated with a concomitant significant increase (p<0.001) of the NK cell cytotoxicity (4 vs 70 LU). The data suggest that IL-15, a homeostatic NK cell cytokine, can upregulate the expression of activating receptors and concomitantly increase the NK lytic activity. The use of IL-15 as a platform for NK- based therapies for AML patients should be considered in the future.

Table 1. The effect of IL-15 on the receptor expression in AML patients NK cell Activating Receptors NK cells in AML pts at diagnosis NK cells in AML pts after IL-15 stimulation p value % positive, MFI % positive, MFI NKp30 24, 2.6 84, 6.0 0.001 NKp44 4, 2.4 71, 6.7 0.001 NKp46 32, 2.6 83, 7 0.002 NKG2C 17, 2.2 58, 6.9 0.005 NKG2D 43, 2.5 87, 7.8 0.01
hDectin-1 Mediates Cross-Presentation of Epitopes Via the Cytosolic Pathway.
Authors: Grünebach Frank , Weck Markus M. , Appel Silke , Werth Daniela , Sinzger Christian , Bringmann Anita , Kanz Lothar , Brossart Peter
Published: April 2020

Human (h)Dectin-1 is a member of the C-type-lectin-like receptor family that was shown to be the major receptor for fungal β-glucans and to play an important role in cellular responses mediated by these carbohydrates. It is mainly expressed on human DCs and macrophages. In our study, we observed that activation of monocyte-derived dendritic cells (MDCs) with TLR3 ligand (poly I:C) but not with TLR ligand 7/8 (R848) resulted in down-regulation of hDectin-1 expression and this down-regulation correlated with a reduced uptake of apoptotic cells in phagocytosis assays. In order to analyze the possible cross-presentation of engulfed antigens we used CMV infected human fibroblasts (HFF). We found that hDectin-1 is involved in the uptake of CMV-infected HFF leading to cross-presentation of CMV-derived peptides on MHC class I molecules and activation of CMV pp65-specific CD8+ T-lymphocytes. To further delineate the pathway leading to presentation, we pretreated MDCs with compounds that inhibit processing of antigens at defined steps during presentation. Cytosolic protein degradation is performed by the proteasome, a large multicatalytic protease complex. Lactacystin specifically inhibits the 20S and 26S proteasome activity by targeting the catalytic subunit. In standard 51Cr-release assays, addition of lactacystin completely inhibited the presentation of pp65-derived peptides indicating the involvement of the proteasome in these process. The fungal product brefeldin A blocks the MHC class I processing pathway by specifically inhibiting the vesicular egress from the ER and the Golgi complex. In line with previous findings, incubation with brefeldin A almost completely abolished the lysis of MDCs incubated with CMV+ HFF. To further analyze whether the cross-presentation of CMV-derived peptides on HLA class I molecules was dependent on lysosomal proteases, MDCs that were co-incubated with HFF as above were treated with the lysosomotropic agent chloroquine that prevents acidification of the lysosomal compartment involved in the exogenous pathway of antigen presentation. The addition of chloroquine had no effect on the cross-presentation of CMV-derived epitopes on HLA class I-molecules. Summarized, the data reported here show that hDectin-1 functions not only as a pattern recognition receptor in innate immunity but is also involved in the clearing of apoptotic cells and cross-presentation of cellular antigens on MHC class I molecules to specific CTLs.