bluuebunny/crossref_metadata_2025 huggingface dataset:
Encouragement of Transplacental Stem Cell Transplantation.
Authors: Heldermon Coy , Sands Mark
Published: October 2019

Human fetomaternal cell trafficking has been demonstrated to result in persistent microchimerism of the mother and offspring from 20–100% of pregnancies. The level of microchimerism is small but in disease states may compose up to 0.96% of some cell types. This microchimersim could conceivably have therapeutic affects by serving as a reservoir of normally functioning stem cells. In order to determine if the level of microchimerism could be increased we have begun to explore methods to increase the number of stem cells and permeability of the placental barrier. To assess the affect of increasing stem cell number, granulocyte colony-stimulating factor (GCSF) was delivered subcutaneously during mid-gestation in pregnant heterozygous mucopolysaccharidosis VII (MPS VII, a deficiency of beta-glucuronidase) mice. We observed an increase in the proportion of sections from mutant offspring with demonstrable beta-glucuronidase producing cells from ∼2% in PBS treated mice to ∼25% in GCSF treated mice. To assess the affect of increasing placental permeability, pregnant MPS VII heterozygous mice were injected IV with vascular endothelial growth factor (VEGF) and a transfusion of 1 × 107 GFP positive bone marrow cells. We observed an increase in circulating GFP positive cells to 0.15% of circulating cells in 25% of the offspring of high dose VEGF-treated animals. Analysis of mutant offspring from the VEGF-treated animals reveals a dose effect of VEGF with 61% of sections from the group receiving the highest dose having demonstrable beta-glucuronidase producing cells. These studies demonstrate a proof-of-principle that microchimerism can be increased substantially and subsequent studies will focus on optimizing this transfer and analyzing therapeutic effect.
Novel Direct Evidence That Adult Bone Marrow-Derived Very Small Embryonic Like (VSEL) Stem Cells Are Mobilized into Peripheral Blood - Leukopheresis as a Potential Tool To Isolate Pluripotent Stem Cells for Therapeutic Pruposes.
Authors: Kucia Magda , Wysoczynski Marcin , Wan Wu , Ratajczak Mariusz Z.
Published: October 2019

Recently, we identified in adult bone marrow (BM) a population of very small embryonic like (VSEL) stem cells (Leukemia2006:20;857) that are

very small in size (∼3.5 μm), CXCR4+Oct-4+ SSEA-1+Sca-1+CD45−lin−, respond to SDF-1 gradient and contain large nuclei with open type of euchromatin.

We asked if VSEL are mobilized into peripheral blood (PB) similarly as BM-derived hematopoietic stem cells. To address this question, we evaluated VSEL level in PB after

pharmacological (G-CSF) and stress/organ damage-induced mobilization (toxic liver and skeletal muscle damage after exposure to CCl4 and cardiotoxin respectively).

The number of circulating VSEL in PB was measured by

FACS, direct histochemical staining of PB-cells for SSEA-1 and Oct-4 expression, and RQ-PCR analysis of pluripotent genes (Oct-4, Nanog).

Finally, VSEL sorted from the PB were tested for their pluripotency in our established assay based on formation of embryoid-like spheres by VSEL in co-cultures with C2C12 cells. We report that in steady state conditions, VSEL circulate at very low levels in PB (∼100–200 cells/ml) and that the number of these cells/ml of PB is five times higher in two month old mice as compared to one year old animals. Furthermore, we found that VSEL are mobilized into PB both by G-CSF- and tissue injury and the number of circulating VSEL increases 5–6 and 2–3 times after G-CSF and after liver or skeletal muscle injury, respectively. Furthermore, the mobilization of VSEL could be additionally enhanced by blocking CXCR4 receptor by T140 antagonist. Based on this, we provide the evidence that VSEL

is a mobile pool of primitive CXCR4+SSEA-1+Oct-4+ stem cells, is released into circulation during tissue/organ injury and their trafficking is regulated by SDF-1-CXCR4 axis.

Further studies will asses if the level of mobilized VSEL

correlates with overall regenerative potential of an organism and could become a helpful prognostic indicator for the clinical outcome from organ damage/injury (e.g., stroke, heart infarct).

Finally, mobilized VSEL could also be employed in regenerative medicine and we are testing this possibility in appropriate animal models.
Differential Transactivation Activities of Sumoylated CCAAT Enhancer Binding Protein alpha (C/EBPα) p42 Versus p30 May Contribute in Part, to Aberrant C/EBPα Activity in Acute Leukemias.
Authors: Khanna-Gupta Arati , Silver Matthew , Hankey William , Sun Hong , Berliner Nancy
Published: October 2019

CCAAT enhancer binding protein alpha (C/EBPα) is the founding member of a family of basic region/leucine zipper (bzip) transcription factors and has been shown to be a master regulator of granulopoiesis It is expressed at high levels throughout myeloid differentiation and has been shown to bind to the promoters of multiple myeloid- specific genes at different stages of myeloid maturation. Profound hematopoietic abnormalities have been reported for mice nullizygous for C/EBPα, including a selective early block in the differentiation of granulocytes. Recently mutations in C/EBPα have been demonstrated in a subset of patients with AML presenting normal karyotypes. These mutations can result in the expression of a 30kD dominant negative C/EBPα isoform which contributes to loss of C/EBPα function. Since the molecular basis for this observation remains unknown, a complete understanding of the regulation of this key transcription factor during myelopoiesis is critical. C/EBPα was recently shown to be post-translationally modified by small ubiquitin-related modifier (SUMO) at a lysine residue (K159) which lies within a region of the C/EBPα protein that can negatively affect transcriptional activity. Sumoylation at K159 in the C/EBPα protein is thought to prevent association of the SWI/SNF chromatin remodeling complex with C/EBPα, thereby hampering transactivation. In order to demonstrate the role of sumoylation in mediating C/EBPα activity, we examined the effect of both the full length (p42) and the dominant negative (p30) isoforms of the C/EBPα protein on myeloid gene expression. We demonstrate that the levels of sumoylated p42C/EBPα decrease upon normal neutrophil maturation, and that transactivation of the myeloid-specific lactoferrin promoter reporter is significantly enhanced by a p42 sumoylation mutant of C/EBPα (K159A). Additionally, in oligonucleotide pull down assays, we show that sumoylated p42C/EBPα binds to the C/EBP site in the LF promoter in immature myeloid cells while loss of sumoylation correlates with loss of p42C/EBPα binding and LF expression in more mature cells. Based on these observations we conclude that sumoylated p42C/EBPα is associated with the negative regulation of LF in early myeloid cells. We show in addition, that p30 C/EBPα can also be sumoylated. In transactivation assays, however, sumoylated p42C/EBPα suppresses LF promoter activity more efficiently than p30C/EBPα in 293 cells. We are now in the process of confirming this finding in leukemic cell lines. This observation leads us to hypothesize that the differential transactivation potential of sumoylated p30 versus the p42 C/EBPα proteins may contribute, in part, to the aberrant activity of C/EBPα in acute leukemias.
The First Zinc Finger Domain of EVI1 Enhances Self-Renewal of Hematopoietic Progenitors.
Authors: Laricchia-Robbio Leopoldo , Nucifora Giuseppina
Published: October 2019

EVI1 was first identified as a preferential integration site of ecotropic retroviruses in the MDS1/EVI1 genomic locus leading to myeloid tumors in susceptible mice. Later studies showed that retroviral integration in the MDS1/EVI1 locus results in the emergence of a non-malignant dominant hematopoietic stem cell clone in non-susceptible mice strains, in non-human primates, and in patients, suggesting that a gene encoded by the locus could be deregulated by the retrovirus and affect the self-renewal potential of the cell. The locus encodes two genes. One of them, EVI1, has long been associated with myeloid leukemia and myelodysplastic syndrome. To understand whether EVI1 has a role in self-renewal control, we forcibly expressed EVI1 in the bone marrow progenitors of two mouse strains that differ in their proliferation and self-renewal potential. By comparing the response of the hematopoietic cells to EVI1, we show that EVI1 has a role in prolonging the self-renewal potential of the cells and that this ability of EVI1 is however limited and modulated by inherent strain-specific characteristics. To identify the region of EVI1 mediating this effect, we infected the bone marrow progenitors of the two murine strains with EVI1 wild type or with specific alternative EVI1 point mutants and compared the self-renewal potential of the cells. This approach allowed us to show that the first zinc finger domain of EVI1 is required to enhance the self-renewal of the hematopoietic progenitors. This function is mediated by two specific zinc finger motifs and their disruption by point mutations abolished this effect. These results suggest that the motifs interact with factors that regulate self-renewal. The cooperation between EVI1 and these unknown proteins in deregulation of self-renewal occurs inappropriately when EVI1 is deregulated, but based on EVI1 gene knock out studies it is likely that this function of EVI1 is required during embryonic development.
Mesodermal Patterning Activity of the Transcription Factor SCL.
Authors: Ismailoglu Ismail , Yeamans Gabriel , Daley George Q. , Perlingeiro Rita , Kyba Michael
Published: October 2019

The transcription factor SCL is critically required for the establishment of the hematopoietic lineage as well as for proper endothelial development, however it is not required for maintenance of HSCs or endothelial cells in the adult. Conflicting data exists regarding the developmental function of SCL, namely whether it acts as a master regulator, actively patterning mesoderm toward hematopoietic/endothelial development at the expense of other lineages, or is merely necessary to maintain the earliest committed hematopoietic and endothelial precursors. To answer this question in a mammalian model, we have engineered murine ES cells with a conditional doxycycline-inducible SCL transgene, and evaluated the effects of pulses of SCL expression at defined time points during in vitro development. During embryoid body differentiation, a pulse of SCL expression during mesodermal patterning results in enhanced hematopoiesis at later time points, as assayed by CFC activity and FACS analysis, as well significantly increasing the frequency of bipotent hematopoietic/endothelial hemangioblast CFCs. Concomitantly, SCL represses cardiac development, assayed by beating cardiomyocyte frequency, and molecular markers of the cardiac lineage. SCL also represses paraxial mesodermal developmental potential, severely reducing both the earliest PDGFαR+/Flk-1neg paraxial progenitors, as well as repressing levels of later markers including Pax3. Through chimeric embryoid bodies in which SCL cells are mixed with GFP-labelled ES cells, we show formally that this effect is cell-autonomous. Finally, we tested whether expression of SCL would enable hematopoiesis during ES cell monolayer differentiation. Hematopoiesis requires inter-germ layer inductive signaling which occurs in 3 dimensions but not in monolayer culture, thus ES cells do not spontaneously differentiate into blood in this 2 dimensional culture. We show that when mesoderm is produced during monolayer culture of wild-type ES cells, or control cells not expressing SCL, it is biased towards paraxial or cardiac differentiation. However when given a pulse of SCL expression, these pathways are repressed, PDGFαRneg/Flk-1+ presumptive lateral plate progenitors are produced, and abundant hematopoietic differentiation occurs. This data in the mammalian system thus strongly supports the former hypothesis, of an active role for SCL in mesodermal patterning.
Loss of Ku70 Results in a HSC Repopulating Defect.
Authors: Qing Yulan , Gerson Stanton L.
Published: October 2019

Genomic integrity is essential for organism development and longevity and in large part is mediated by DNA repair proficiency. Non-homologous end-joining (NHEJ) is a specific pathway essential for DNA double-strand break repair in all cells and VDJ processing in B and T cells; and it is crucial for maintenance of mammalian genome. We hypothesize that NHEJ is also critical for hematopoietic stem cells (HSC) maintenance and function. Ku70 is a key component of the NHEJ pathway; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. Ku70-deficient mice were used to assess the importance of NHEJ pathway in HSC function. Ku70-deficient mice have decreased bone marrow cells (BMC), and significant reductions in the frequency of colony-forming units in culture (CFU-C) in the presence of cytokines including IL-3, IL-6, SCF and Epo. SKL (Sca1+, c-Kit+, Lin−) cells from Ku70-deficeint mice showed a similar proliferation rate compared with SKL cells from WT mice when cultured in vitro in the presence of cytokines; single cell culture experiment showed that SKL cells from Ku70-deficient mice and WT mice have similar clonogenic ability. Thus, Ku70-deficient SKL cells retain proliferation capacity although there is a deficiency in overall hematopoietic homeostasis. To address Ku70-deficient HSC function in vivo, Ku70-deficient BMCs were serially transplanted into lethally irradiated congenic wild-type mice. These Ku70-deficient marrow cells were able to reconstitute the myeloid lineages in both primary and secondary recipients, while over 95% of the T cells in the recipients were derived from the host. However, the tertiary whole marrow recipients died within 2 weeks of transplantation, while the WT BMCs recipients survived tertiary transplantation. Competitive repopulation assays were also performed between WT and Ku70-deficient BMCs. The results showed that when Ku70-deficient BMCs were mixed with WT competitor BMCs at a 1:1 ratio, Ku70-deficient BMCs were completely outcompeted by the WT BMCs. These data demonstrate that Ku70, a key component of NHEJ, is required for HSC function. Ku70 deficiency in the hematopoietic compartment results in a long-term repopulating defect and loss of competitive repopulation ability.
The Expression of Polycomb Group Genes Products, Bmi1 and Mel-18, Regulate the Function of Murine Primitive Hematopoietic Cells.
Authors: Kajiume Teruyuki , Sato Takashi , Kobayashi Masao
Published: October 2019

The Polycomb group (PcG) genes (bmi1 and mel-18) known as negative control factors of the Hox gene is thought to regulate the differentiation and self-renewal of hematopoietic stem cells (HSCs). The loss of mel-18 results in the promotion of HSC self-renewal, and the increase of mel-18 expression inversely leads to the differentiation of HSCs. On the other hand, the loss of bmi1 does not lead to self-renewal activity of HSCs. In this study we examined the effect of expression of bmi1 and mel-18 on the role of function in murine HSCs. Lineage-negative, Sca1-positive, and cKit-positive primitive hematopoietic cells were purified and the expression of PcG protein was evaluated from the intra-nuclear distribution of PcG proteins. The Bmi1-positive hematopoietic cells barely contained Mel-18, and the Mel-18-positive cells barely contained Bmi1. the frequency of positive cells for both Bmi1 and Mel-18 was less than 0.5% of purified primitive hematopoietic cells. The expression levels of the PcG genes, bmi1 and mel-18, in HSCs were knocked down by siRNA and then gene expression was assessed by quantitative real-time PCR. The introduction of siRNA against bmi1 or mell-18 resulted in approximate 50 to 60% decrease of each gene expression without affecting another gene expression. Primary colony-forming activity of knocked down cells in response to stem cell factor, thrombopoietin and the ligand for flt3 was not affected by the induction of siRNA. However, secondary colony-forming activity from primary colony-forming cells in bmi1-knockdown cells was significantly decreased when compared with that of control cells. Conversely, the mel-18-knockdown cells significantly increased, suggesting that mel-18-knockdown cells are capable of proliferating activity. Finally, bone marrow reconstitutive activity was examined by using Ly5.1 and Ly5.2 system. While the bmi1-knockdown marrow cells decreased the reconstitutive activity, the mel-18-knockdown marrow cells showed the increase of engraftment activity after 6 months of transplantation. From these results, we consider that mel-18 and bmi1 have reciprocal functions in HSCs. Mammalian PcG protein complexes can be classified into two distinct types, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). The Mel-18 protein is a constituent of mammalian PRC1 together with M33, Bmi1 or rae28, and Scmh1. The Mel-18 protein is composed of 342 amino acids and the N-terminal region of the 102 amino acid, which includes the RING finger motif, shares 93% homology with Bmi1 protein. In addition, its secondary structure shows high homology with the Mel-18 and Bmi1 proteins. We hypothesized that the opposite function is expressed in HSCs because Mel-18 and Bmi1 share the same structure and compete when in the complex form. These results suggest that mel-18 and bmi1 have inverse function in HSCs and that the balance of Bmi1 and Mel-18 may regulate the fate of self-renewal and differentiation in HSCs.
Erythroid Development in the Absence of Hemoglobin.
Authors: Liu Shan-Run , McConnell Sean C. , Huo Yongliang , Zhang Ting-Ting , Yang Rui , Ulrey Clayton L. , Ryan Thomas M.
Published: October 2019

The mammalian erythrocyte is a highly specialized blood cell that differentiates via an orderly series of committed progenitors in the bone marrow in a process termed erythropoiesis. Homeostasis of the erythron is carefully maintained by balancing the proliferation and destruction of early and late erythroid progenitors. In mature red blood cells over ninety-five percent of the protein is hemoglobin (Hb). What happens to committed erythroid cells in the absence of hemoglobin? To answer this question we have derived a novel line of embryonic stem (ES) cells from mouse embryos that have all eight adult alpha and beta globin genes knocked out. These “Null” Hb ES cells were injected into wild-type blastocysts to examine their in vivo potential to contribute to the tissues of developing chimeric mice. Examination of the peripheral blood and bone marrow of these chimeras by flow cytometry revealed that the “Null” Hb ES cells were able to produce normal levels of each type of white blood cell analyzed. However, “Null” erythrocytes were absent from the circulation and only early committed progenitors were found in the bone marrow. Very few “Null” erythroid cells matured beyond the proerythoblast to the basophilic erythroblast stage (Ter119low, CD71hi). To study this maturational block in more detail, an erythroid culture system was established by in vitro differentiation of the “Null” Hb ES cells. These pure erythroid progenitor (EP) cultures support and amplify the proerythroblast stage of development. Interestingly, EP cells could be derived from “Null” Hb ES cells demonstrating that Hb is not required for the development of proerythroblasts. “Null” derived EP cells express erythroid lineage markers (EKLF, GATA1, GypA, EpoR, Tal1), but express no adult globins or markers of other hematopoietic lineages (Mpl, GATA3, IL7R, PAX5, CEBPα, CD41b). Upon terminal differentiation most “Null” derived EP cells undergo apoptosis by 48 hours (7AAD−, Annexin V+) and are dead (7AAD+) by 72 hours. These “Null” Hb ES cells provide a novel experimental system to elucidate the role of hemoglobin during erythroid differentiation, maturation, and homeostasis.
The IPI Predicts Outcome in Patients with Diffuse Large B-Cell Lymphoma (DLBCL) Treated with Immunochemotherapy: A Report of the University of Iowa/Mayo Clinic SPORE
Authors: Habermann Thomas M. , Link B.K. , Maurer M.J. , Wooldridge J.E. , Geyer S.M. , Macon W.R. , Allmer C. , Ansell S.M. , Witzig T.E. , Weiner G.J. , Cerhan J.R.
Published: October 2019

The International Prognostic Factor Index (IPI) predicts survival in DLBCL in patients treated with chemotherapy. The Revised IPI (R-IPI) has been reported to be a simpler and more accurate predictor of outcome in patients treated with immunochemotherapy (rituximab and anthracycline-based chemotherapy). We evaluated the predictive value of the IPI and the R-IPI in an observational cohort of unselected patients treated with R-CHOP. Consecutive, newly diagnosed patients age 18 years and older with DLBCL were prospectively offered enrollment into our Lymphoma SPORE Registry. Pathology was centrally reviewed, and composite lymphomas and history of concurrent or prior cancers were excluded. All patients were actively followed for progression free progression (PFS) and overall survival (OS). Here we report on patients enrolled from 9/2002 – 6/2006. 229 patients with a median age of 62 years (range 20–93) were evaluated. 56% were >60 years of age, 16% had a performance score ≥2, 54% had an elevated LDH, 19% had >1 extranodal site, and 51% were stage III/IV. During follow-up, there were 63 progressions (28%) and 45 deaths (20%), and the median follow-up time for living patients was 34 months (range 6–61 months). As shown in the table and figure, the IPI and R-IPI were predictive for both PFS and OS (all p<0.001). The predictive ability of the IPI as measured by 3-year concordance index was stronger for the IPI (0.71) compared to the R-IPI (0.67) and the bootstrap 95% confidence interval for the difference (0.01, 0.08) indicates that this difference was statistically significant. While all factors in the IPI were statistically significant (p<0.05) predictors of OS individually, when included in a multivariate model, an elevated LDH (HR=1.5; p = 0.32) and >1 extranodal sites (HR=1.0; p = 0.96) were no longer significant; similar results were obtained for PFS. The IPI remains a strong predictor of PFS and OS in the immunochemotherapy era.

Figure Figure Group % Pats 3 Y PFS HR 95% CI 3 Y OS HR 95% CI Standard IPI Low (0,1) 41% 87% 1.0 ref 93% 1.0 ref Low-Int (2) 22% 62% 3.1 (1.4, 6.8) 74% 4.2 (1.6, 11.1) High-Int (3) 21% 60% 3.1 (1.4, 6.8) 77% 3.4 (1.2, 9.7) High (4,5) 15% 39% 7.2 (3.4, 15.2) 47% 10.2 (4.0, 26.5) R-IPI Very good (0) 11% 96% 1.0 ref 100% 1.0 ref Good (1,2) 53% 74% 6.2 (0.8, 46.0) 84% - - Poor (3–5) 36% 52% 13.5 (1.8, 98.9) 65% - -
R-HCVAD/R-MTX-ARAC Is an Effective Regimen for Untreated Diffuse Large B-Cell Lymphoma (DLBCL) with Aggressive Features in Patients Younger Than 60 Years
Authors: Fayad Luis , Pro Barbara , Hagemeister Fredrick , Cabanillas Fernando , Samaniego Felipe , McLaughlin Peter , Wang Michael , Neelapu Sattva , Kwak Larry , Romaguera Jorge , Rodriguez Maria A.
Published: October 2019

Background: R-HCVAD (rituximab-cyclphosphamide, vincristine, dexamethasone, doxorubicin) alternating with R-MTX-ARAC (rituximab, methotrexate-cytarabine) is a chemoimmunotherapy regimen with activity in ALL, mantle cell, and Burkitt’s lymphomas. Histological differentiation between BL. atypical BL, and DLBCL with high-grade features can be difficult. We treated patients with DLBCL with high-grade histological features, and/or poor-risk IPI. Patients who received at least one dose of R-HCVAD were included in this cohort.

Methods: Twenty-seven consecutive patients younger than 60 years, with information collected prospectively in the NCCN database, treated between 7/2002 and 10/05 at M.D. Anderson Cancer Center, 17 (63%) male, 15 (55%) HI and High IPI, 17 (63%) ↑LDH, 14 (52%) with Stage III/IV, 2 (5%) with PS>1, 60% had a Ki-67 higher than 80%.

Results: ORR was of 100% with (95%) CR/CRu. With a median follow-up of 26 months, 2 patients failed: One died of disease, and another received a SCT when the patient was in PR. Four patients have died, three of disease, one of pulmonary embolism. The 3-year OS is 96% (95% CI 77%–100%); 3-year FFS is 86% (95% CI 64%–100%). The 3-year FFS for patients with L/LI risk (2-failures of 12 pts) was 69% and for patients with HI/H age-adjusted IPI (0 failures of 15 pts) 100%.

Conclusions: R-HCVAD/R-MTC-ARAC is a very active regimen for DLBCL. Toxicity was mostly hematological and infectious. We are currently doing a prospective randomized phase II study comparing this regimen to standard R-CHOP in poor risk younger patients.
The Addition of Rituximab to CHOP Chemotherapy Improves Response Rate, Failure-Free Survival and Overall Survival for Follicular Grade 3 Lymphoma Compared to CHOP Alone
Authors: Overman Michael J. , Feng Lei , Pro Barbara , McLaughlin Peter , Hess Mark , Samaniego Felipe , Younes Anas , Romaguera Jorge E. , Hagemeister Frederick B. , Kwak Larry , Cabanillas Fernando , Rodriguez Maria A. , Fayad Luis E.
Published: October 2019

Background: FL3 is a subcategory of follicular lymphomas that is challenging in that it behaves aggressively like large cell lymphomas. If treated with CHOP, however it has a clinical course of relapse and treatment failure similar to grade 1–2 follicular lymphoma. We looked at the outcome of FL3 patients treated with RCHOP, combining rituximab with CHOP. There are no large study reports of this regimen’s results in FL3 to our knowledge.

Patients and Methods: We retrospectively reviewed the records of 45 patients with follicular grade 3 lymphoma who were treated with rituximab and the combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) at The University of Texas MD Anderson Cancer Center (UTMDACC). Response rate (RR), failure-free survival (FFS), and overall survival (OS) were estimated and a historical comparison to 111 CHOP only treated patients was made.

Results: The International Prognostic Index (IPI) distribution was: 47% Low, 36% Low-Intermediate, 13% High-intermediate, and 4% High-risk. The complete response rate was 96%. Forty-four out of 45 patients are still alive. Median follow-up is 3.5 years. The 3-year FFS rate according to IPI was 80% (95% CI: 64% to 100%) in low risk, 81% in low-intermediate (95% CI 64% to 100%), and was 50% (95% CI: 25% to 100%) in high-intermediate/high-risk patient group. The addition of rituximab to CHOP improved both 5-year FFS, 71% (95% CI: 58% to 87%) compared to 44% of CHOP alone (95% CI: 36% to 55%) with p-value of 0.019 and 5-year OS, 98% (95% CI: 93% to 100%) compared to 75% (95% CI: 67% to 84) with p-valule of 0.0034. The addition of rituximab to CHOP improve the FFS compared to CHOP alone when subgroups of IPI were analyzed and compared (p=.002)

Conclusion: The addition of rituximab to CHOP provided a high response rate and excellent early survival. Poor risk patients continue to demonstrate a high rate of failure despite the use of rituximab.
The Contact Activation Coagulation Pathway Is Involved in (Recurrent) Coronary Disease.
Authors: Dielis Arne W.J.H. , Govers-Riemslag Jose W.P. , Winkens Mark , Wijers-Holys Patricia , Hamulyak Karly , Spronk Henri M.H. , Waltenberger Johannes , Cate Hugo ten
Published: October 2019

The classic contact activation coagulation system emerges as a potentially important mechanism in arterial thrombosis. Recent studies not only indicate that coagulation factor (F) XII is associated with atherothrombotic coronary disease, but also that it contributes to clot formation via activation of downstream FXI. The time course of contact activation after an acute myocardial infarction (AMI), has not been studied. In addition, its relevance for arterial thrombosis has not been addressed in prospective studies. We measured the plasma concentrations of activated FXII (FXIIa), FXIa and Kallikrein in complex with their natural inhibitors in 135 consecutive patients with a documented first AMI (chest pain between 30 minutes and 24 hours accompanied by ST-segment elevation on ECG and/or elevated plasma levels of creatine kinase and troponin T). FXIIa-C1 inhibitor, kallikrein-C1 inhibitor, FXIa-C1 inhibitor and FXIa-antitrypsin complexes were measured in established homemade ELISAs (Govers-Riemslag et al. J Thromb Haemost2007; 5: 1896–1903). Mean FXIIa-C1 inhibitor complexes were highest on admission (0.081 U/mL) as compared to day 4 (0.065 U/ml), when most patients were still hospitalized and treated with low molecular weight heparin (LMWH) and dual platelet inhibition (aspirin and clopidogrel). At 90 and 180 days after admission mean FXIIa-C1 inhibitor values were 0.064 U/mL, (P<0.05 vs. admission) and 0.067 U/mL, respectively. Except for 1 patient, kallikrein-C1 inhibitor complexes were undetectable at all time points. Mean FXIa-complexes were highest on day 4, in both the FXIa-C1 inhibitor assay (0.64 U/mL) and the FXIa-antitrypsin assay (0.33 U/mL, P<0.01 vs. admission). Mean FXIa-C1 inhibitor complexes decreased to 0.46 and 0.44 U/mL after 90 and 180 days (both P<0.05 vs. day 4), while FXIa-antitrypsin complexes decreased to 0.29 U/mL after 90 and 180 days (both P<0.0001 vs. day 4). Surprisingly, patients with one or more recurrent events (n=15; combined endpoint of death, recurrent MI, stroke, angiographic or percutaneous intervention) during follow-up of 180 days had lower FXIa-C1 inhibitor complexes on admission than those without such events (mean values 0.24 U/mL versus 0.67 U/mL in patients without events; P<0.05). These results indicate that the contact activation coagulation system is activated in atherothrombotic coronary disease. The degree of activation (reflected in FXIa-C1 inhibitor complexes) is inversely related to outcome, which may prognostically indicate that patients with evidence of intrinsic hypercoagulability on admission respond better to subsequent treatment (dual platelet inhibitors plus LMWH) than patients with less pronounced coagulation activity.
Expression of Complement Regulatory Protein CD55 on Tumor Cells Has an Adverse Effect on the Outcome of Follicular Lymphoma Patients Treated with Immunochemotherapy
Authors: Sommarhem Antti , Leppä Sirpa , Karjalainen-Lindsberg Marja-Liisa , Meri Seppo
Published: October 2019

The addition of rituximab to chemotherapy has considerably improved the outcome of follicular lymphoma (FL) patients, but the lengths of remissions are still variable. Activation of the C system has been shown to be an important effector mechanism of rituximab. The aim of this study was to evaluate whether differences in the expression of CD20 and C regulatory molecules (C-REG) in lymphoma cells correlate to the clinical course of FL. We used oligonucleotide microarray analysis to study mRNA levels of CD20, CD46 (MCP), CD55 (DAF) and CD59 (protectin) in the FL tissue of 23 patients treated with R-CHOP. The median follow-up time was 55 months. The patients were divided into long-term (progression free survival (PFS) >35 mo) and short-term (PFS <21 mo) responders, and mRNA levels were compared between the groups. High CD55 expression was observed more often in FL patients who relapsed early resulting in a shorter progression free survival (p=0.027). Median PFS time for patients with a low CD55 level was significantly longer than for those with high CD55 expression (not reached vs. 20 mo, p=0.003). The results were validated prospectively by analyzing the protein levels of CD20 and C-REGs with flow cytometry. The results of a pilot study of 12 FL patients showed that relative expression of CD20 to CD55 in CD20 positive cell population was higher in patients in remission in comparison to ones who relapsed. According to Kaplan-Meier estimates, the patients with low CD20/CD55 and CD20/C-REGave (average C regulator expression) ratios relapsed more often than the other patients. The results suggest that the expression of C regulators, especially of CD55, affects the efficacy of immunochemotherapy, and that C regulatory molecules represent an effective escape mechanism of immunochemotherapy in FL.
The Role of Serial Testing for Heparin/PF4 Antibodies in Evolving HITT: A Unique Phenomenon of Delayed Seroconversion with Thrombosis.
Authors: Sabnani Indu , Cohen Marc , Baran David A. , Tsang Patricia , Arroyo Luis H. , Camacho Margarita , Zucker Mark J.
Published: October 2019

In the appropriate clinical setting, a negative ELISA test for Hep/PF4 antibodies (Ab) has traditionally been considered sufficient to rule out HIT/HITT. We describe a series of 20 cases of HIT/HITT in which the ELISA at the onset of thrombocytopenia was negative despite high (6/8) “4T’s” scores (Thrombocytopenia, Timing of thrombocytopenia in relation to heparin exposure, Thrombosis, and no oTher explanation of thrombocytopenia) but showed strong seroconversion subsequently. This suggests that the negative predictive value of the ELISA test may be too low to definitively exclude HITT. We retrospectively reviewed 494 consecutive Hep/PF4 ELISA assays (sensitivity >90%) performed on 395 patients (pts). Of the 292 pts who tested negative, 73 pts underwent repeat ELISA testing due to the development of a new thrombotic event or the persistence of strong clinical suspicion of HIT/HITT. Twenty of the 73 pts (27%) were observed to have initial negative Hep/PF4 titers (mean = 0.13) at the time of thrombocytopenia but a positive assay (mean = 1.5) on follow-up testing (figure 1 right panel). All 20 pts presented with high “4T’s” scores (6 of 8) at the time of the negative ELISA and had a history of recent heparin exposure. The M:F ratio was 11:9. Eleven pts had prior cardiopulmonary bypass while six had left ventricular assist devices. Fifteen pts (75%) had >50% drop in platelet count (figure 1 left panel). Mean platelet count at the time of negative ELISA was 71x109/l (vs. baseline mean platelet count of 171x109/l) (figure 2). Fourteen of 15 pts (for which data was available) had thrombotic events (9 arterial, 5 venous). Strong seroconversion was seen in all pts (mean = 8 days after initial test) and surprisingly, it accompanied platelet recovery in 15 of 20 pts (figure 1 left panel), with mean platelet count of 136x109/l at the time of the positive ELISA (figure 2). Argatroban was used as a direct thrombin inhibitor (DTI) in 16/20 pts. The prognosis was poor: 10 pts (50%) succumbed with multi-system organ failure.

Conclusion: We have described 20 cases of HIT/HITT with initial negative Hep/PF4 Ab titers at the time of thrombocytopenia, followed by delayed seroconversion. This observation suggests that in the proper clinical setting one isolated negative ELISA result cannot exclude evolving HIT/HITT. Follow-up testing may be informative even as the platelet count begins to recover. As HIT/HITT may have potentially fatal outcome, clinicians should maintain a high degree of suspicion despite a negative serology by ELISA, especially in pts with “4T’s” scores ≥ six. Empiric treatment with DTI’s may be warranted until two consecutive ELISA tests are negative.

Figure Figure Figure Figure
Absolute Lymphocyte Count Is Independent of the Anaplastic Lymphoma Kinase and Predicts Survival in Primary Anaplastic Large Cell Lymphoma.
Authors: Porrata Luis F. , Ristow Kay , Witzig Thomas E. , Haberman Thomas M. , Inwards David J. , Ansell Stephen M. , Micallef Ivana N. , Johnston Patrick B. , Colgan Joseph , White William L. , Markovic Svetomir N.
Published: October 2019

Peripheral blood absolute lymphocyte count (ALC) at diagnosis is a predictor of survival in B-cell lymphomas. The role of ALC at diagnosis on survival in T-cell lymphoma has not been studied. Thus, we studied the role of ALC at diagnosis on clinical outcome in adult patients with primary anaplastic large cell lymphoma (PALCL) that were diagnosed, treated, and followed at the Mayo Clinic, Rochester. Between 1985 and 2006, 50 patients with PALCL qualified for the study. ALC was identified to be a strong predictor for complete response (CR), area under the curve (AUC = 0.83, p < 0.002). The median follow-up was 31.8 months (range: 1–212.6 months). ALC, as a continuous variable was a predictor for overall survival (OS) (HR = 0.143; 95%CI = 0.042–0.416; p < 0.0001) and progression-free survival (PFS) (HR = 0.150; 95%CI = 0.047–0.415; p < 0.0001) .Superior OS and PFS (Figure 1) were observed with an ALC ≥ 1.0 x 109/L (N = 31) versus an ALC < 1.0 x 109/L (N=19) (median OS: not reached vs 7.5 months, OS rates at 5 years, 81% vs 36%, p < 0.0006, respectively; and median PFS: not reached vs 6.3 months; PFS rates at 5 years, 77% vs 37%, p < 0.0007, respectively). Multivariate analysis demonstrated ALC to be an independent prognostic indicator for OS (HR = 0.196; 95%CI = 0.125–0.693; p < 0.002) and PFS (HR = 0.191; 95%CI = 0.053–0.559; p < 0.0008) when compared to anaplastic lymphoma kinase, international prognostic index, and cutaneous versus systemic presentation.

Figure 1 Figure 1.
Generation of Regulatory Dendritic Cells by Administration of Donor Alloantigen in Mouse Transplantation Model.
Authors: Shirasugi Nozomu , Aramaki Osamu , Zhang Qi , Iwami Daiki , Ando Kiyoshi , Niimi Masanori
Published: October 2019

Backgrounds: Various lines of evidence indicate that regulatory cells control peripheral immune responses in self-tolerance and transplantation. Here we show that administration of donor alloantigen via the trachea generated not only CD4+ regulatory cells but also regulatory dendritic cells (DCs) and induced hyporesponsiveness to fully allogeneic grafts in mouse transplantation model.

Methods and Results: CBA mice (H-2k) received administration of C57BL/10 (B10: H-2b) splenocytes via the trachea (intratracheal delivery; ITD). Seven days later, the CBA mice underwent transplantation of B10 hearts. Untreated CBA recipients rejected B10 allografts acutely (median survival time [MST], 8 days). ITD of B10 splenocytes induced prolongation of B10 allograft survival in CBA recipients (MST, 67 days). To determine whether regulatory cells were generated by ITD treatment, adoptive transfer study was conducted. Seven days after CBA mice (primary recipients) were given B10 splenocytes by ITD, splenocytes from the primary recipients were adoptively transferred into naive secondary CBA recipients that underwent B10 cardiac grafting the same day. Secondary CBA recipients given not only whole but also CD4+ splenocytes from ITD-treated CBA mice (primary recipients) 7 days before had significantly prolonged survival of B10 allografts (MST, 78 and 68 days, respectively, compared to those in control secondary CBA recipients with adoptive transfer of naive whole or CD4+ splenocytes, MST, 11 and 13 days, respectively). Interestingly, adoptive transfer of CD11c+ cells from ITD-treated CBA mice also induced prolongation of B10 allograft survival in naïve secondary recipients (MST, 85 days, compared to that in control secondary recipients with adoptive transfer of naive CD11c+ cells, MST, 9 days). These CD11c+ cells produced interleukin (IL)-10 upon activation. Experiments using anti-IL-10 monoclonal antibody revealed that both induction and effector function of the regulatory cells required IL-10.

Conclusion: Our data indicate that ITD of donor alloantigen induced hyporesponsiveness to fully allogeneic grafts, and generated regulatory cells that contained both DCs and CD4+ cells in our model.

C57BL/10 allograft survival in secondary recipients (naive CBA recipients) with adoptive transfer Adoptively transferred cells Adoptively transferred from; Allograft survival (days) ITD: intratracheal delivery of C57BL/10 alloantigen Whole splenocytes (5x107) ITD-treated CBA mice 49, 63, 71, 85, 89, >100 naive CBA mice 8, 8, 9, 13, 14, 18 CD4+ splenocytes (2x107) ITD-treated CBA mice 60, 61, 68 ,69, >100 naive CBA mice 8, 11, 12, 14, 14, 24 CD11c+ cells (1x106) ITD-treated CBA mice 26, 54, 65, 85, 86, 91, >100 naive CBA mice 7, 7, 9, 13,14
Peptide-Major Histocompatibility Complex Class I Tetramers with Enhanced Coreceptor Binding Properties Enable Visualization of Low Avidity Leukemia-Associated Antigen-Specific CD8+ T Cells.
Authors: Melenhorst J. Joseph , Scheinberg Phillip , Gostick Emma , Chattopadhyay Pratip K. , Lissina Anna , Cole David K. , Roederer Mario , Bornstein Ethan , Hensel Nancy F. , Douek Daniel C. , Sewell Andrew K. , Barrett A. John , Price David A.
Published: October 2019

There is substantial evidence to indicate that CD8+ T cells specific for epitopes derived from overexpressed antigens form part of the immune response to leukemia in patients both prior to and after stem cell transplantation (SCT) and following adoptive immunotherapy with donor leukocyte infusion. However, the immunobiology of these CD8+ T cell populations has proven difficult to study directly ex vivo due to technical obstacles that preclude the reliable detection of low frequency CD8+ T cells that bind antigen poorly. We engineered a mutation (Q115E) in the heavy chain a2 domain of HLA-A*0201 that moderately enhances monomeric CD8 binding without impacting the TCR binding platform. Combined flow cytometric studies and biophysical analysis of recombinant TCR/peptide-major histocompatibility complex class I (pMHCI) interactions showed that tetrameric complexes of these coreceptor-enhanced HLA-A*0201 molecules were more sensitive reagents for the detection of specific CD8+ T cells at the clonal level compared to corresponding tetramers produced with wildtype HLA-A*0201 complexes; thus, the threshold of detection was enhanced approximately twofold relative to the monomeric TCR/pMHCI cut-off (KD=80mM) observed with the wildtype tetramer. These novel reagents were incorporated in a polychromatic flow cytometric panel to detect CD8+ T cell populations specific for HLA-A*0201-restricted epitopes derived from Wilm’s tumor-1 (WT1) and proteinase 3 (PR1) in the bone marrow and peripheral blood of patients with acute myeloid leukemia before SCT. The HLA-A*0201-restricted CMV pp65-derived epitope NLVPMVATV, which induces high avidity cognate CD8+ T cell populations, was used as a control. Both wildtype and coreceptor-enhanced tetramers consistently identified populations of NLVPMVATV-specific CD8+ T cells that were identical in magnitude; molecular analysis of TCR usage in these populations after high resolution flow cytometric sorting confirmed the equivalent cognate binding properties of these reagents. In contrast, coreceptor-enhanced tetramers enabled the visualization and simultaneous immunophenotyping of CD8+ T cell populations specific for PR1 and WT1 in patients with AML that were not detectable with the corresponding wildtype reagent. These results provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8+ T cell populations by flow cytometry; such coreceptor-enhanced tetrameric reagents are likely to be useful in a multitude of settings for the detection of tumor-specific and autoreactive CD8+ T cells.
Modulation of Tryptophan Catabolism by Acute Myeloid Leukemia Cells Acts as a General Mechanism of Immune Tolerance Via the Induction of T Regulatory Cells.
Authors: Curti Antonio , Salvestrini Valentina , Aluigi Michela , Trabanelli Sara , Ottaviani Emanuela , Durelli Ilaria , Horenstein Alberto L. , Fiore Francesca , Massaia Massimo , Piccioli Milena , Pileri Stefano , Martinelli Giovanni , Baccarani Michele , Lemoli Roberto M.
Published: October 2019

Indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the conversion of tryptophan into kynurenine, has been identified as a novel immunosuppressive agent by inhibiting T-cell proliferation and is involved in tolerance induction to tumors. We have recently shown that IDO protein is constitutively expressed in a significant subset of newly diagnosed acute myeloid leukemia (AML) patients, resulting in tryptophan catabolism along the kynurenine pathway and in the inhibition of allogeneic T-cell proliferation. Moreover, we demonstrated that IDO-expressing AML cells are capable to promote the differentiation of new CD4+CD25+Foxp3+ T regulatory cells (Treg cells). AML cells may be differentiated into leukemic dendritic cells (AML-DCs) which have increased immunogenicity and may be used as vaccine against leukemia. We in vitro generated DCs from 7 AML samples according to standard procedures and tested IDO expression and activity. At baseline, 5/7 AML samples expressed IDO, whereas 2/7 did not. After differentiation into DCs, IDO+ AML samples showed an up-regulation of IDO mRNA and protein, and IDO− AML cells turned positive. IDO-expressing AML-DCs were capable to catabolize tryptophan into kynurenine metabolite and, functionally, they inhibited allogeneic T-cell proliferation through an IDO-dependent mechanism. Similarly to undifferentiated IDO+ AML blasts, IDO-expressing AML-DCs induced a population of CD4+CD25+Foxp3+ Treg cells, which were capable to suppress in vitro allogeneic naïve T-cell proliferation. These data identify IDO-mediated catabolism as a general tolerogenic mechanism in AML cells, including AML-DCs and raise several concerns for the use of AML-DCs as cellular vaccine against leukemia.
Malignant B Cells Skew the Balance between Treg Cell and TH17 Cell Differentiation in B-Cell Non-Hodgkin Lymphoma (NHL).
Authors: Yang Zhi-Zhang , Novak Anne J. , Witzig Thomas E. , Ansell Stephen M.
Published: October 2019

Numerous clinical therapies have attempted to modulate tumor cell immunity, but for the most part, have proven unsuccessful. The inability to produce or augment an effective immune response is due in part to regulatory T (Treg) cells, which inhibit CD4 and CD8 T cell function. Our group has recently shown that Treg cell numbers are elevated in NHL tumors and that NHL B cells induce the development of Treg cells thereby inhibiting anti-tumor responses. The ability of NHL B cells to direct the cellular composition of their microenvironment is critical to our understanding of tumor immunity and we therefore wanted to determine if NHL B cells also directed the expansion or reduction of other T cell populations. IL-17-secreting CD4+ T cells (TH17), a newly characterized CD4+ T helper cell lineage, promote inflammation and play an important role in autoimmune disease. IL-17 has been shown to inhibit tumor cell growth suggesting a potential role for TH17 cells in anti-tumor immunity. We therefore set out to determine if TH17 cells were present in NHL tumors and whether or not their numbers were regulated by NHL B cells. Using unsorted mononuclear cells from malignant lymph nodes, we were unable to detect IL-17 expression in resting CD4+ T cells or CD4+ T cells activated with PMA/Ionomycin stimulation (less than 1%). However, IL-17-secreting CD4+ T cells could be detected in significant numbers in inflammatory tonsil and normal PBMCs. Interestingly, depletion of CD19+ NHL B cells from mononuclear cells obtained from patient biopsies resulted in detection of a clear population of IL-17-secreting CD4+ T cells (5%). These results suggest that NHL B cells suppress TH17 cell differentiation. The frequency of IL-17-secreting CD4+ T cells could not be further enhanced by the addition of exogenous TGF-b and IL-6, a cytokine combination favoring for TH17 differentiation, suggesting a further impairment of TH17 cell differentiation in the tumor microenvironment. In contrast, Foxp3 expression could be detected in resting CD4+ T cells (30%) and could be induced in CD4+CD25−Foxp3− T cells activated with TCR stimulation (28%). Contrary to the inhibition of TGF-b-mediated TH17 differentiation, Foxp3 expression could be dramatically upregulated by TGF-b in intratumoral CD4+ T cells (35%). In addition, lymphoma B cells strongly enhanced Foxp3 expression in intratumoral CD4+CD25−Foxp3−. Furthermore, when added together, the frequency of Foxp3+ T cells and Foxp3-inducible cells reached up to 60% of CD4+ T cells in tumor microenvironment of B-cell NHL. These findings suggest that the balance of effector TH17 cells and inhibitory Treg cells is disrupted in B-cell NHL and significantly favors the development of inhibitory Treg cells. Our data indicate that lymphoma B cells are key factor in regulating differentiation of intratumoral CD4+ T cells toward inhibitory CD4+ T cells.