bluuebunny/crossref_metadata_2025 huggingface dataset:
Epstein-Barr Virus Status Correlates with Poor Outcome and Is an Independent Prognostic Variable in De Novo Nodal Diffuse Large B Cell Lymphoma in Peru.
Authors: Beltran Brady , Morales Domingo , Riva Luis , de Mendoza Fernando Hurtado , Quinones Pilar , Yabar Alejandro , Carrasco-Yalan Antonio A.
Published: October 2019

BACKGROUND: Epstein Barr Virus (EBV) positive status is associated with poor survival in Hodgkin lymphoma and T cell NHL.

AIM: In order to define the prognostic impact of Epstein Barr Virus (EBV) infection in newly diagnostic nodal diffuse large B cell lymphoma (DLBCL), we investigated the EBV status in Peruvian DLBCL patients.

PATIENTS AND METHODS: Between January 2002 and December 2004, seventy-four patients, diagnosed with nodal DLBCL, were included in the analysis. Tissues from patients were analyzed for the presence of EBV encoded RNA (EBER) using the in situ hybridization (ISH)

RESULTS: Of the 74 cases, 53 cases (71.6%) were of the non Germinal Center DLBCL type (non GC). This group had a higher IPI than the Germinal Center DLBCL type (GC). Also the non GC DLBCL type had an inferior overall survival compared with GC DLBCL type, but the difference was not significant (p=0.171). Eleven cases (14.9%) were identified as EBER-positive. EBER positive was associated with an advanced age (> 60 years), poorer performance status, more advanced stage, higher IPI and poorer outcome to initial treatment. The EBER+ DLBCL patients demonstrated substantially poorer overall survival (EBER+ vs. EBER−): 1.1 months (95% CI, 0.0–2.6 months) vs. 15.5 months (1.0–30.0 months) respectively, (p=0.001). Most of the cases EBER (+) were of the non GC group. At the multivariable level, EBER status is an independent variable compared with the International Prognostic Index (IPI) (p=0.001) with a 3.2-fold (95% CI, 1.5–7.2) risk for death for positive cases.

CONCLUSION: EBV positive status in de novo, nodal DLBCL was present in 11/74 cases and is a powerful prognostic factor in this Peruvian population. EBV positive status correlated with advanced age, poor performance status, more advanced stage, non-GC group and short survival. New therapeutic strategies should be investigated in this poor prognostic subgroup.

Figure Figure
HAEMACARE Project: Reclassifing Hematologic Neoplasias According Morphological Data.
Authors: Giraldo Pilar , Latre Paz , Franco-García Esther , Alvarez Ramiro , Pascual Mar , Castilla Tomas , Sants Milena , Martos Carmen
Published: October 2019

Aims: The European Project HAEMACARE (VI Frame Research Program), is working in the reclassification of hematological neoplasias included in population based Cancer Registries of 13 European countries with the objective to improve and standardize morphological data validation.

Patients and methods: a multidisciplinary research group of the I+CS integrated by epidemiologists, hematologists, pathologists is working in the reclassification of Hodgkin disease (HD) included in the population based Cancer Registry of Zaragoza (CRZ) in order to reclassify according to WHO and ICD-O classifications and identify the non other specification (NOS) cases. From January 1995 to December 2000, a total of 141 patients diagnosed as Hodgkin disease are included. The sources of data were: Zaragoza population based Cancer Registry, Aragon hematological malignancies Registry (FEHHA) and from the records of public Hospitals and the National Deaths Index. All cases were identified from the CRZ and 35 cases (24.8%) NOS were re-studied by the hematologists and pathologists and reclassified according histological and morphological subtypes of WHO classification. Diagnostic criteria were set for LRCHD: scattered Hodgkin-Reed-Sternberg cells with a classical immunophenotype in a background of small lymphocytes without admixture of eosinophils and neutrophils and without sclerosis nodular lymphocyte predominant HD (NLPHD), mixed cellular (MCD) and lymphocyte-rich classical HD (LRCHD). In 24.8% of cases were necessary to process the samples again or review smears and applied immunochemistry techniques in order to establish accurate diagnosis according the new sub-classification in NLPHD or CHD and distinguish some cases of T-cell rich large B-cell lymphoma. In addition we have calculated the estimated survival according specific morphological subtypes.

Results:

Hodgkin Disease. Histological distribution ICD-0-3 No % males females mean age(y) mean surv(y) No deaths (%) *non other specification NOS* 35 24.8 15 20 43.7 5.8 13 (34.3) CHD 64 45.4 39 25 39.3 7.6 19 (29.7) MCD 21 14.9 15 6 46.3 7.7 4 (19.0) LRCHD 11 7.8 9 2 51.1 6.7 5 (45.5) NLPHD 10 7.0 6 4 51.2 8.3 2 (20.0) Total 141 84 57 43.2 7.1 43 (30.5)

The estimated survival analysis showed a mean of 7.6 years (95% CI: 6.7–8.4) for all cases, median not reached.

Comments: In our study has been necessary to reclassify the 24.8% of cases according WHO criteria. The incidence peaks are classical. The overall survival is similar to describe previously.
The Effects of Zoledronic Acid on Serum Lipids in Multiple Myeloma Patients.
Authors: Gozzetti Alessandro , Martini Giuseppe , Avanzati Annalisa , Franci Beatrice , Marchini Elena , Nuti Ranuccio , Lauria Francesco
Published: October 2019

Nitrogen-containing bisphosphonates (N-BPs) inhibit osteoclast-mediated bone resorption and they are widely used for the treatment of postmenopausal osteoporosis, Paget’s disease and tumor-associated osteolysis. The mechanism of action of these drugs has not been completely clarified but it has been observed that N-BPs may inhibit squalene synthase, as ibandronate, or farnesyl pyrophosphate synthase, as alendronate or pamidronate, inducing also a reduction of cholesterol biosynthesis. Zoledronic acid (ZA) represents a novel N-BPs which has also antitumor activity. To explore the effects of ZA on serum lipids, twenty-six untreated consecutive patients (14 males/12 females; median age 71.4 ± 7.8, range 49–82 years) with SMM (14 IgG/k, 2 IgG/λ, 8 IgA/k 2 IgA/λ) were recruited between October 2004 and March 2005 from the Department of Hematology of the University of Siena. In particular, enrollment criteria were marrow plasmacytosis between 10–30%, no lytic bone lesions, no myeloma related symptoms. In order to enter the study, patients had not to be on treatment for hypercholesterolemia. Patients gave an informed consent before the enrollment. After baseline evaluation, SMM patients were sequentially assigned to receive no treatment (10 patients) or ZA (16 patients), 4 mg administered as a 15 min i.v. infusion at baseline and after 1, 2, 4 and 6 months. Data distribution was expressed as mean ± SD. Two-Sample T-test for independent data were used to evaluate the differences between groups. To test the effect of treatment, we used the Repeated Measures ANOVA Procedure. p value of less than 0.05 were considered significant. Analysis were performed using SPSS software for Windows, version 10.0 (SPSS ltd., Chicago, IL, USA). In all subjects, total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and C-terminal telopeptide of Type I collagen (CTX) were measured at baseline and after 1, 3 and 6 months. In the control group no significant changes were observed throughout the study period for any of the biochemical variables. As expected, CTX decreased significantly by 40–50% after ZA administration. In treated patients, we observed a progressive and significant reduction of TC with a maximum decrease of 13% at 6 months; moreover LDL-C decreased by 21% at 6 months while no significant differences were appreciated in HDL-C and TG. Also the indexes of cardiovascular risk improved after ZA administration: TC/HDL-C ratio progressively decreased by 17% and HDL-C/LDL-C ratio increased by 36% showing an effect that appears to be cumulative. In conclusion ZA is able to alter lipid profile more than older N-BPs as pamidronate or neridronate. This effect can be due to the potency of ZA to inhibit FPP synthase that is about 70-fold than pamidronate.
The Assessment of IL-6R Gene Using FISH in MM.
Authors: Min Hyun Jung , Kim Tae Young , Oh Bora , Lee Jae Hoon , Lee Dong Soon
Published: October 2019

Multiple myeloma (MM) is characterized by proliferation of plasma cell, mostly in the bone marrow. It is established that cytokines including IL-6 play a key role as growth and survival factor for plasma cells through the specific receptors on the cell surface. Abnormalities of chromosome 1q have frequently been reported in various cancers as well as multiple myeloma (MM). Recent studies suggest that amplification of chromosome 1q is a negative prognostic factor. To determine whether increase of the IL-6 receptors located at chromosome 1q21 stimulate secretion of cytokine IL-6 we performed a gene expression evaluation on 136 newly-diagnosed MM, analyzed by interphase FISH for IL-6R(1q21) amplification, Rb1(13q14) deletion, and IGH translocation and additional copies of 9,17,20. We found that the correlation of IL-6R gene and other major genetic risk factor in MM. IL-6R gene amplification (3 to 12 orange signal) was detected in 46 of 136 (34%). These results indicate that IL-6R gene occur the gain of chromosome 1q. It is suggest that the identification of interleukin-6 receptor associated with 1q amplification is useful in providing diagnostic information on multiple myeloma.
Bone Marrow Stem Cell Transplantation Restores Heme Oxygenase and Antioxidants Genes and Pancreatic Function in Type 1 Diabetes.
Authors: Wang Jishi , Peterson Stephen , Kruger Adam , McClung J. , Abraham N.G.
Published: October 2019

Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta(β)cells has been linked to a decrease in circulating stem cells and increased superoxide (O2)secondary to a decrease in heme oxygenase (HO) activity. Decrease HO-1 increases oxidative stress, resulting in β-cell destruction. O2 and β-cell destruction. Nonobese diabetic (NOD) mice (2 months old), which undergo pancreatic T-cell infiltration resulting in elevated were irradiated and reconstituted with bone marrow stem cells from BALB/c mice(<2 months old) using a perfusion technique, exhibited neither insulitis nor overt diabetes. The newly developed T-cells in BMT recipients were tolerant to both donor and host major histocompatability complex determinants. After six months, BMT-treated mice showed the same glucose tolerance as age-matched controls, HO activity was restored to the level of age-matched controls, and EC-SOD protein levels returned to normal. The increased HO activity after BMT was accompanied by a significant elevation of HO-1 protein and a significant decrease in O2-levels(μmol/mg protein 1.16±0.37 μmol/mg protein versus 2.81±0.37 in nontransplant mice). These results demonstrate that bone marrow-derived stem cell transplant restored the antioxidant genes HO-1 and EC-SOD as well as pancreatic function. In addition, we demonstrate that combining BMT and islet cell transplant can restore insulin production in severely diabetic animals. We have thus demonstrated in this animal model of type 1 diabetes that BMT is effective as both a preventive and therapeutic treatment.
Reversal of New-Onset Type 1 Diabetes in Mice and Induction of FoxP3+ Regulatory T Cells by Syngeneic Bone Marrow Transplantation.
Authors: Ouyang Jian , Wen Yanting , Chen Junhao , Liu Yong , Wu De Pei
Published: October 2019

Objective: Autologous bone marrow transplantation (ABMT) was introduced as a novel strategy to treat new-onset type 1 diabetes (T1D). Induction of CD4+CD25+ FoxP3+T regulatory lymphocytes (Tregs) helps maintain self-tolerance in autoimmune diabetes. Here, we were tying to investigate the effects of syngeneic bone marrow transplantation (syn-BMT) in T1D mice models and to investigate the role of Tregs in this context.

Research design and methods: Syn-BMT was used to mimic the ABMT to T1D patients in human. Fraternal inbred multiple low doses of streptozotocin (mld-SZ) induced diabetic mice were used as both donors (n = 7) and recipients (n = 20). And normal fraternal inbred mice were also used as donors (n=13). After total body irritation to recipient mice, syn-BMT was given either on day 10 or on day 40 of T1D. Blood and urine glucose was observed. On day120 after syn-BMT, glucose tolerance was tested and then mice were killed. Insulin levels in sera were tested by ELISA and pancreata histological and morphometry were analyzed by HE staining. Tregs in spleens were tested by Flow cytometry analysis, FoxP3 protein was tested by western blot, and FoxP3 mRNA was tested by real time PCR.

Results: Syn-BMT, if applied when T1D is new-onset (10 d), can reverse blood glucose to close to normoglycemia without farther relapse, maintain blood insulin levels, improve glucose tolerance and ameliorate pancreata destruction. Syn-BMT leading to recovery of T1D results in the induction of Tregs, increased Foxp3 protein and mRNA expression in both the groups of diabetic mice receiving syn-BMT with either diabetic or normal donors. However, if given on a later disease stage (40 d), although syn-BMT helped reduce blood glucose for about 66 d and improved pancreata histological and morphometry, it showed relapse of diabetes further and decrement of insulin levels. Interestingly, this group did not show the increment of Tregs, Foxp3 protein or mRNA expression.

Conclusions: This study provided a rationale to treat human new-onset T1D with autologous HSC. Syn-BMT, if given when T1D is new-onset, is safe and is able to reverse the diabetic status. The induction of Tregs and Foxp3 in both mRNA and protein levels as the results of syn-BMT, may contribute to the immune tolerance after syn-BMT, and the observed improvement of new-onset T1D.
Optimizing Culture Conditions for Ex Vivo Expansion of Virus Specific T Cells.
Authors: Borelli Gabriel , Aarvak Tanja , Brunsvig Anne , Dyrhaug Marianne , Lundby Marianne , Hammerstad Hege , Rasmussen Anne Marie , Kvalheim Gunnar
Published: October 2019

Adoptive immunotherapy with virus specific CD8+ cytotoxic T lymphocytes (CTL) is currently an option for treatment of viral infections after allogeneic stem cell transplantation. A major obstacle for clinical grade production of virus specific CTL is to retain the antigen specific clones, which can be easily deleted during ex vivo expansion. Manipulation of CD3/CD28 engagement, depletion of T regulatory (Treg) cells before expansion and prestimulation with antigen presenting cells were explored in this study. Mononuclear cells were obtained from HLA-A2+ donors by leucapheresis. Lymphocytes were enriched by elutriation, stimulated with anti-CD3/anti-CD28high Dynabeads or Dynabeads® ClinExVivo™ CD3/CD28 and cultured for 10 days in CellGro media supplemented with human AB serum and IL-2. Virus specific CD8+ CTL were quantified by flow using pentamer staining. By using anti-CD3/ anti-CD28high Dynabeads a more balanced expansion of CD4+ and CD8+ subsets was obtained, while Dynabeads ClinExVivo CD3/CD28 allowed a higher expansion of CD8+ subset and therefore a higher expansion of the virus specific CTL. A high Dynabead: T cell ratio (3:1) deleted completely the virus specific CTL. By reducing this ratio we could retain the virus specific CTL after expansion. No benefits were observed by adding extra Dynabeads during expansion. It is known that Treg cells can inhibit expansion of all T cell subsets. By depleting Treg cells with CD25 Dynabeads prior to T cell expansion, we observed a significantly increased expansion of all T cells, including virus specific CTL. In some patients low numbers of virus specific CTL can be detected. To increase the numbers of antigen specific CTL we have included a prestimulation step using peptide-loaded mononuclear cells prior to expansion with Dynabeads, which gave more than 3000- fold expansion of virus specific CTL. We are currently exploring the phenotype profile of expanded CTL (CD62L, CCR7, CD57, CD27, CD28 and CTLA-4), functionality (cytokine secretion and proliferative capacity) and cytotoxicity. This protocol can be upgraded to clinical grade production of virus specific CTL for treatment of viral infections in immunocompromised patients.
InVitro Culture of Human Cord Blood Hematopoietic Stem/Progenitor Cells Interfered with Their Distribution to Other Bone Marrow Compartments after Intra-Bone Marrow Transplantation.
Authors: Ohishi Kohshi , Yamamura Kentaro , Masuya Masahiro , Katayama Naoyuki
Published: October 2019

Intra-bone marrow transplantation (IBMT) is a novel strategy for transplantation of hematopoietic stem cells because it can transfer various types of cells to bone marrow regardless of their homing capacity. However, reconstitution process of these cells after IBMT remains to be fully elucidated. Here, we investigated whether in vitro culture of cord blood hematopoietic stem/progenitor cells affects their reconstitution in bone marrow after IBMT. Freshly isolated AC133+ cells (5x104 cells/mouse) or all cells derived from AC133+ cells cultured with growth factors (stem cell factor, flt-3 ligand, and thrombopoietin) for 5 days were injected into the bone marrow of the left tibia in irradiated NOD/SCID mice. In the bone marrow of the injected left tibia, the engraftment levels of human CD45+ cells at 6 weeks after transplantation was not considerably different between transplantation of noncultured and cytokine-cultured cells (54±28% vs. 69±13%). However, the migration of transplanted cells to the bone marrow of other noninjected bones was extremely lower for cytokine-treated cells compared with noncultured cells (2±2% vs. 36±10%). Similar findings were observed for engraftment of CD34+ cells. To enhance the migration of cytokine-cultured cells after IBMT, we similarly transplanted cultured AC133+ cells into the bone marrow of the left tibia, assessed the engraftment in the injected and noninjected tibiae at 7 days after transplantation, and then subcutaneously administered G-CSF (250 μg/kg/d) for 5 days. Administration of G-CSF stimulated the migration of cytokine-cultured cells to the bone marrow of previously-aspirated right tibia but failed to induce their migration to intact bone marrow of femur. These data indicate that ex vivo manipulation of hematopoietic progenitor/stem cells adversely influences their migration properties to other bone marrow compartments after IBMT. Our data raise caution for future clinical applications of the IBMT method using ex vivo-manipulated hematopoietic stem cells.
Allogeneic Committed Hematopoietic Progenitors Are Protective Against Radiation.
Authors: Chen Benny J. , Deoliveira Divino , Chao Nelson J.
Published: October 2019

Whole-body irradiation may lead to bone marrow failure and death. It was previously reported that congenic myeloerythroid-restricted progenitors are able to radioprotect lethally irradiated animals. However, this approach will not be practical because syngeneic/congenic donors are rarely available in humans. To solve this problem, we investigated whether allogeneic committed progenitors are also radioprotective. Hematopoietic committed progenitors were isolated by FACS based on the presence of early progenitor marker CD244 and the absence of stem cell marker CD150 (CD244+CD150−). BALB/c mice (H2d) were lethally irradiated with 8.5 Gy. Within 4 hours of irradiation, the irradiated mice were infused with 5x105 sorted hematopoietic progenitors from major histocompatibility complex mistmatched C57BL/6 donors (H2b). As shown in the Figure B, all the mice in the radiation control group died within 15 days post irradiation (median survival time: 13 days). Infusion of hematopoietic committed progenitors significantly prolonged the survival of the lethally irradiated mice (P=0.0018, median survival time: 28 days). These results are similar to the results obtained from congenic hematopoietic progenitors using 1x105 cells (Figure A, P<0.0001, median survival time: 10 days vs. 28 days). These data suggest that allogeneic hematopoietic committed progenitor cells are also able to mediate radioprotective effects. Similar to the congenic hematopoietic committed progenitors, allogeneic progenitors may also exert radioprotective effects by jumpstarting hematologic recovery post irradiation. These cells may be stockpiled and used as “off-the-shelf” products for radiation injury and other applications.

Figure Figure
Immunosuppressant Sinomenine Promotes Differentiation of Bone Marrow Progenitors to IL-10-Producing Ialow Regulatory Dendritic Cells Leading to the Prolongation of Allograft Survival.
Authors: Wang Quanxing , Wu Jianchun , Wang Jianli , Liu Yushan , Chen Guoyou , Cao Xuetao
Published: October 2019

Sinomenine (SN), an immunosuppressive compound derived from the Chinese medicinal plant Sinomenium acutum, effectively inhibits both proinflammatory factor production and lymphocyte proliferation. However, the effects of SN on dendritic cells (DC), the professional antigen-presenting cells, have not been elucidated. In this study, we examined the effects of SN on DC generation, maturation and function. We found that SN affected DC in a dose dependent manner; significantly inhibiting surface expression of MHC class II, CD86, and CD40, production of IL-12, TNF-a, IL-1b, and T cell-allostimulatory activity. SN targets antigen-presenting capacity of DC not only via the MHC class II pathway, but also impairs DC migration by inhibiting LPS-induced up-regulation of CCR7 and CXCR4 expression. These inhibitory effects on DC may be partially due to SN-mediated suppression of NF-kB and p38 MAPK pathways. Further study showed that SN-treated DC (DC-SN) induce donor-specific T cell hyporesponsiveness and trigger generation of IL-10-producing T regulatory-like cells in vitro. In vivo transplant studies revealed that, compared to pretreatment with immature DC, pretreatment of recipients with DC-SN could significantly prolong allograft survival, although long-term allograft acceptance was not achieved. Importantly, pretreatment of recipients with DC-SN in combination with SN administration profoundly prolonged allograft survival, resulting in long-term survival of 30% of allografts. Increased generation of CD4+CD25+ Treg cells and enhanced microchimerism in recipients brought on by combination treatment may contribute to this extension of allograft lifespan. Our results demonstrate that SN can promote bone marrow progenitors differentiation to IL-10-producing (Ialow) regulatory DC which contribute to induction of tolerance by increase generation of Treg cells.
Development of Tools for T Cell Repertoire Analysis (TCRB Spectratyping) for the Canine Model of Hematopoietic Cell Transplantation.
Authors: Kiianitsa Konstantin , Lesnikov Vladimir , Jordan Robert , Georges George E.
Published: October 2019

Analysis of the recombinatorial diversity of rearranged T cell receptor genes in mature T cells is an essential tool in the evaluation of immune status and immune reconstitution in hematopoietic cell transplantation studies. While the spectratyping technique is available in human clinical research and mouse models, the canine genome has not been sufficiently annotated to simply implement the assay on the dog TCRB locus. To this end, we have annotated by bioinformatics and experimentally all canine TCRBV segments, as well as TCRBD, TCRBC and most of TCRBJ segments. Of all 31 canine TCRBV found, 23 were functional and 8 were pseudogenes. A multiplex PCR-based assay was further designed to analyze the entire TCRBV spectratype in a set of 4 reactions each containing 4 to 5 V-segment specific forward primers and a common C-segment specific reverse primer. Direct sequencing of RT-PCR products confirmed that all amplified genes originated from predicted V-segments and that the designed V-specific PCR primers did not cross-react with other TCRBV family members. The usefulness of the spectratyping technique for canine model of transplantation was further demonstrated in analysis of T-cell repertoire reconstitution of irradiated dogs at different time points of recovery. Moreover, our simple and rapid V (J) annotation strategy relies on internet resources open to the general public and does not require specialized training in bioinformatics. It can be readily applied for de novo identification and mapping of TCRB gene families in other animal species where genome sequence drafts become available.

Figure 1. TCRB spectratype of fetal canine thymus. (A) RT-PCR using TCRBV family-specific forward primers (V1 through V26) and a common C region-specific reverse primer. (B) Amplification products specific for family V1 (high abundance) and V26 (low abundance) were copied in run-off reactions with the fluorescent C-specific primer and resolved on capillary gel. Figure 1. TCRB spectratype of fetal canine thymus. (A) RT-PCR using TCRBV family-specific forward primers (V1 through V26) and a common C region-specific reverse primer. (B) Amplification products specific for family V1 (high abundance) and V26 (low abundance) were copied in run-off reactions with the fluorescent C-specific primer and resolved on capillary gel.
A Vector Expressing PML-RARα Fused to GM-CSF Is an Effective DNA Vaccine for Inducing Specific Immune Response to APL Cells.
Authors: Li Yangqiu , Cen Dongzhi , Hu Gang , Zhou Yubing , Chen Shaohua , Yang Lijian , Przybylski Grzegorz K. , Schmidt Christian A.
Published: October 2019

Despite combinations of different therapeutic strategies have significantly increased survival, acute leukemia is still not curable. To further improve outcome, specific immunotherapy might be one of the best choice. DNA vaccines have been showed leading to strong and persistent cell-mediated and humoral immune response to the antigen encoded by the plasmid. However, little data exist regarding the DNA vaccines in acute leukemia, there are few studies reported that PML-RARα DNA vaccines were developed, but the host immune response were weakly, due to the weak immunogenicity of tumor antigens. In order to improve the effect of DNA vaccine for acute promyelocytic leukemia (APL) therapy, we have used a full-length human GM-CSF (hGM-CSF) sequence fused to PML-RARα breakpoint-drived sequence and develop a vector coexpressing PML-RARα gene and hGM-CSF gene, which was expected to to promote T cells response in host. PML-RARα fusion gene segment and the hGM-CSF gene were amplified from NB4 cells or pORF-hGM-CSF plasmid. Both PCR products were cloned into PIRES plasmid respectively to construct a recombinant plasmid PML-RARα-IRES-hGM-CSF. The recombinant plasmids were then transfected into K562 or A549 cells respectively. The expression of the PML-RARα/GM-CSF mRNA and protein in transfected cells were identified by RT-PCR, dot blotting, ELISA and Western-Blot respectively. By in vivo assays, BALB/c mice were vaccinated at 6–8 week of age with a total of 200 μg DNA in normal saline, injected into two sites in the quadriceps muscles on day 0, 7 and 21. The plasmid containing the same PML-RARα segment and blank plasmid served as controls. Two weeks after the final DNA boost, both PML-RARα/GM-CSF mRNA and protein, serum INF-γ and anti-NB4 cells specific cytotoxicity of splenocytes following 7 days of stimulation in vitro with freeze thawing NB4 cells and recombinant human IL-2 were assessed by ELISA and LDH assays. The results showed that the sequence of the fragments inserted in multi-clone site (MCS) A and MCS B of PIRES plasmid were absolutely correct by double restriction enzyme cutting analysis (Xba I/Sal I) and sequence analysis, the PML-RARα/GM-CSF mRNA and protein could be identified in transfected K562 or A549 cells and in mice quadriceps muscles. The level of serum INF-γ and cytotoxicity of splenocytes against NB4 cells from immunized mice was significant increased than that from control groups. In conclusions, the vector expressing PML-RARα and hGM-CSF was successfully constructed, which can more effective immune response and anti-APL cells effect in animal models than that from plasmid containing single PML-RARα segment. It could be farther used in the research as PML-RARα DNA vaccine for APL.
IFN-g-Stimulated Mesenchymal Stromal Cells Acquire the Ability To Cross-Present Exogenous Soluble Antigens and Lead to an Effective Immune Response.
Authors: François Moïra , Romieu-Mourez Raphaëlle , Stock-Martineau Sophie , Galipeau Jacques
Published: October 2019

Antigen cross-presentation is the mechanism by which exogenous antigens can be presented by major histocompatibility complex (MHC) class I molecules to CD8+ T cells. This process is efficiently performed by professional antigen presenting cells (APC) such as dendritic cells and macrophages. Recently, we and others have shown that IFN-g enables the upregulation of the expression of MHC class I & II molecules by marrow-derived Mesenchymal Stromal Cells (MSCs) and that MHC II-mediated antigen presentation can lead to cell-mediated protective immunity to xenoantigens [Stagg et al., Blood March 2006]. These findings led us to investigate whether MSCs also possess the ability to cross-present antigens via MHC class I. Using antigen presentation assays performed on murine MSC in the presence of MHC class I-restricted ovalbumin (OVA)-specific CD8+ T hybridoma cells or purified primary CD8+ T lymphocytes from OT1 transgenic mice, we demonstrated that MSC can robustly and effectively cross-present exogenous antigens via MHC class I molecules upon IFN-g pre-treatment in a manner comparable to professional APCs like macrophages. Use of transporter associated with antigen processing (TAP-1)-deficient mice and proteasome inhibitors suggested a MHC class I machinery-dependent pathway. Cross-presentation by IFN-g-activated MSC was also observed to be suppressed by TGF-b and regulated by cell density. In vivo, IFN-g-treated, OVA-pulsed MSCs administered to normal C57Bl/6 mice led to an effective OVA-specific, T cell cytotoxic immune response, leading to the rejection of OVA-expressing EG7 lymphoma cells. In conclusion, our findings suggest that cross-presentation properties of MSC could play a role in their effectiveness as conditional APCs in vivo and this property may be exploited as a therapeutic cell-based immune biopharmaceutical for treatment of cancer or infectious disease.
Aberrant Subcellular Localization of Azurophil Granule Proteins in Myeloid Leukemia Favors Peptide Antigen Presentation on MHC-I and Susceptibility to Killing by Cytotoxic T Lymphocytes.
Authors: Alatrash Gheath , Ono Yoko , Lu Sijie , Sergeeva Anna , Wang Changqing , Wieder Eric , Molldrem Jeffrey J.
Published: October 2019

The recognition of leukemia-associated antigens (LAA) by immune cells is essential for generating anti-leukemia responses. Whether elicited by vaccine or hematopoietic stem cell transplantation (HSCT), anti-leukemia immunity requires that cytotoxic T lymphocytes (CTL) recognize and mount an effective response against leukemia cells. PR1 is a nonomeric HLA-A2-restricted peptide (VLQELNVTV) derived from the myeloid-restricted serine proteases neutrophil elastase (ELA2) and proteinase-3 (PRTN3), recognized azurophil granule proteins. PR1-specific cytotoxic T lymphocytes (PR1-CTL) have been shown to preferentially recognize and kill myeloid leukemia cells. We hypothesized that aberrant protein expression in target leukemia cells predisposes to CTL killing. To determine the subcellular localization of ELA2 and PRTN3 in leukemia and in healthy donor (HD) neutrophils, high-speed ultracentrifugation of leukapheresed cells from patients with AML and CML in blast crises yielded 4 fractions: cytoplasmic, nuclear, membrane and golgi/endoplasmic reticulum (ER). Western blot analyses identified higher levels of ELA2 and PRTN3 in cytoplasmic, nuclear, and golgi/ER fractions in leukemia, compared to HD granulocytes. Confocal microscopy of leukapheresed myeloid blasts and HD granulocytes confirmed these findings. We further hypothesized that cross-presentation of PR1 is necessary for priming anti-leukemia immunity. Confocal microscopy of the human B-cell line HMy.CIR-A2, co-incubated with either purified or recombinant ELA2 and PRTN3 demonstrated co-localization of both proteins with LAMP-2, a marker of lysosomes and early endosomes, and small distribution in cytoplasm after 2 hours. Uptake of ELA2 and PRTN3 by HMy.CIR-A2 cells was additionally confirmed by Western blots. Priming of naive PR1-CTL by co-culture of HD lymphocytes with PRTN3 or ELA2-transfected HMy.CIR-A2 cells was confirmed by cell proliferation assays using BrDU. This could be abrogated by addition of lactacystin and cyclohexamide to cell culture. Together, these results demonstrate that ELA2 and PRTN3 are aberrantly localized preferentially in gogli/ER and cytoplasmic fractions in leukemia, rather than azurophil granules, which are often absent or deficient in myeloid leukemia. Similar forced subcellular localization of these proteins in B cells leads to their susceptibility to PR1-CTL killing. Therefore, we conclude that the natural mistrafficking of PRTN3 and ELA2 in myeloid leukemia is necessary to render cells susceptible to CTL attack and that cross-presentation of ELA2 and PRTN3 contributes to priming of a PR1-CTL response.
Targeting Late Relapsed Acute Myeloid Leukemia Cells by Cytotoxic T Cells Generated from Late but Not Early Leukemic Antigen-Presenting Dendritic Cells.
Authors: Liang Yin , Lien Lily , Han Shuhong , Tong Chun-Rong , Gao Zhi-Yong , Wu Tong , Tang Jih-Luh , Lu Dao-Pei , Chang Lung-Ji
Published: October 2019

Cancer-specific cytotoxic T lymphocytes (CTL) have great potential in cancer immunotherapy. We performed a longitudinal study of CTL primed with dendritic cells (DC) exposed to leukemic antigens or DC directly differentiated from leukemic cells in an acute myeloid leukemia (AML) patient. The patient received three HLA locus-mismatched allogeneic bone marrow transplantation, relapsed around day 43, and after a brief remission, suffered an ongoing multi-focal extramedullary relapse. CTL generated against AML cells early during the course of disease (early-AML) effectively targeted the early-AML cells as demonstrated by in vitro CTL assays. The patient received multiple infusions of immune cells specific to the early-AML cells in a pilot trial. While the patient established stable remission in the peripheral blood, extensive extramedullary relapse had occurred. The CTL prepared from DC exposed to the early-AML cells failed to target the late relapsed AML (late-AML) cells isolated from pleural effusion and peripheral blood. Further characterization of the late-AML cells showed complete and partial loss of class II and class I HLA expression, respectively. Interestingly, CTL prepared from the late-AML-derived DC were able to kill the late relapsed cancer cells. This result suggests that the late-AML cells, although escaped from the early-AML-derived CTL, expressed novel immunogenic leukemic antigens. It appeared that selective growth of AML cells resistant to the early-AML-specific immune cells had occurred in vivo. However, CTL primed with the late-AML-derived DC were able to kill the late relapsed cancer cells in vitro. Thus, AML could evolve in vivo under active immunotherapy, but this may be coped with preparation of cancer-specific immune cells using the late-AML cells.
Epidemiology of S pneumoniae Infection in Hematology Patients in Ile de France: Serotypes Covered by the Polysaccharide 23-Valent (PS23) and the Heltavalent Conjugate Vaccine.
Authors: Debbache Karima , Varon Emmanuelle , Hicheri Yosr , Legrand Patrick , Donay Jean-Luc , Ribaud Patricia , Cordonnier Catherine
Published: October 2019

Patients suffering from hematologic malignancies, mainly from lymphoproliferative diseases, and stem cell transplant recipients are at high risk of S pneumoniae invasive infections. The epidemiology of S pneumoniae in the community may vary from one country to an other. Few data are available for hematology patients on the clinical relevance of the two available, PS23 (Pneumo 23®), and heptavalent conjugate vaccine Prevnar® (PC7) according to the serotypes encountered in this population.

Objective: The aim of this retrospective study was to look at the serotype distribution of S pneumoniae infecting hematology patients in Ile de France, and their proportion covered by PS23 and/or PC7.

Methods: All the patients with S pneumoniae infections occurring between Jan 2004–Dec 2006 in 2 hematology departments of Ile de France (Henri Mondor and Saint-Louis hospitals) were identified from the laboratory registries. Only patient with invasive infections (blood cultures, CSF infection, pneumonia, pleural effusion) were included. Serotyping was performed at the CNRP in Paris. Among 40 consecutive cases, serotype was performed in 25 cases occurring in 22 patients. The remaining 15 strains were either not available or did not regrow.

Results: The 22 patients had acute leukemia (n=6), CML (n=1), myelodysplastic syndrome (n=1) or lymphoproliferative disease (n=14) (M/F: 1.4; median age: 54 y). Seventeen had received a stem cell transplant (SCT) (allo: 9; auto: 8) a median of 12 months before. The most frequent serotypes were 9V (n=7) and 19F (n=6). 19/25 (76%) of the strains were covered by PC7 and PS23, and 4/25 (16%) were covered only by PS23. Only 2 were not covered by any vaccine.

Conclusion: We found that most strains of S pneumoniae responsible for invasive infections in hematology patients from Ile de France, including SCT recipients, were covered by PS23 (92%), and PC7 (76%). Only 2/25 strains were covered neither by PS23 nor by PC7. These data make relevant the development of prospective studies and vaccination programs in the high risk hematology patients.
Herpes Simplex Virus Encephalitis (HSE) after Allogeneic Hematopoietic Stem Cell Transplatation (alloHSCT): Impact of Early Diagnosis and Treatment on Outcome.
Authors: Cavattoni Irene M. , Morello Enrico , Casini Marco , Decaminada Norma , Pagani Leonardo , Fabris Pietro , Cortelazzo Sergio
Published: October 2019

Introduction: HSE is a rare but very severe central nervous system (CNS) infection caused by Herpes simplex virus type 1 (HSV-1) in almost all cases, and characterized by a high morbidity and mortality rate (97 and 70% without therapy, respectively). Immunosuppression, is a known risk factor and both early diagnosis and treatment are critical points for a good outcome in this setting. Hereby we reported two cases of HSE after alloHSCT.

Patients: A 35-year-old female with relapsed multiple myeloma, underwent alloHSCT from a matched unrelated donor (MUD). On day +120, she developed an acute onset of fever, drowsiness, and generalized seizures. After 48 hours, a rapid deterioration of neurological symtoms was observed and the brain MRI showed acute encephalitis with high signal intensity in the temporal lobe and insula cortex (fig. 1). The HSV-PCR from cerebrospinal fluid (CSF) was positive for HSV-1, and a treatment with acyclovir 30 mg/Kg/d was initiated, 72 hours after the hospital admission. A 66 year-old male with high risk AML underwent MUD alloHSCT. Thirteen months after transplant, an acute sialoadenitis and HSV-1 gengivostomatitis occurred, followed by fever and focal seizures. Since typical lesions of HSE were demonstrated by brain MRI, antiviral therapy with aciclovir was promptly administrated, within 24 hours from the symptoms onset. HSV-PCR on CSF was performed two days later, and proved negative, probably due to ongoing antiviral therapy and delay to CSF analysis. After 21 days of antiviral treatment, both patients recovered from the acute phase, but in the first case we observed serious neurological sequelae that required prolonged unsuccessful cognitive rehabilitation. The second patient didn’t experienced significant residual neurological impairment, and didn’t require any other supportive measure. Both pts have had chronic GVHD and received prolonged courses of immunosuppressive therapy; they experienced recurrent CMV infections thereafter, and were under high dose foscarnet therapy at the diagnosis of HSE, but without HSV prophylaxis.

Conclusion: the time from diagnosis-to-antiviral therapy, and the level of consciousness at the onset of symptoms are two major risk factors impairing morbidity and mortality of HSE. In heavily immunosuppressed alloHSCT pts with initial focal neurological signs, an early and aggressive diagnostic approach should be performed as emergency, even if they are under anti-CMV therapy; prompt acyclovir administration is therefore warranted in these settings, in order to prevent severe disability or mortality.

Figure Figure
Similar Background of Graft-Versus-Host Disease (GVHD) Following Allogeneic Blood and Marrow Transplantation (Tx) in Korean and Japanese Populations Allows a Uniform Foundation To Perform Large-Scale Clinical Studies in the Region.
Authors: Kim Sung-Won , Lee Jong-Wook , Kim Hyeoung-Joon , Fukuda Takahiro , Kanda Yoshinobu , Kim Hee-Je , Min Woo-Sung , Mori Shin-ichiro , Tanosaki Ryuji , Lee Je-Hwan , Lee Kyoo-Hyung , Yamashita Takuya , Sakamaki Hisashi , Sohn Sang-Kyun , Chae Yee-Soo , Ohshima Kumi , Ichinohe Tatsuo , Kanda Junya , Kasai Masaharu , Kobayashi Naoki , Ando Kiyoshi , Onizuka Masahito , Taniguchi Shuichi , Seo Sachiko , Yang Deok-Hwan , Kim Yeo-Kyeoung , Shin Ho-Jin , Takaue Yoichi , Kim Chun-Choo
Published: October 2019

Background: Although it has been reported that the incidence of acute GVHD (aGVHD) and early non-relapse mortality (NRM) after HLA-identical sibling BMT are different between Japanese and other ethnicities, including white American, African American and Irish populations (Oh, Blood.2005), there has been no previous comparison among Asian countries.

Patients and Methods: We retrospectively surveyed the data of 1933 patients (pts) (996 Korean and 937 Japanese) who underwent allo-Tx for leukemia or MDS between Jan, 2000 and Dec, 2005. The median age of the pts was 37 yrs (range, 16–70; 34 yrs vs 43 yrs, respectively) and the median follow-up of surviving pts was 1046 days (11–2435). Unexpectedly, the pts’ backgrounds, including age, underlying disease, disease status at Tx, stem cell source, donor type, No. of HLA-mismatched (MM) antigen/allele, conditioning regimen, in vivo T-cell depletion with ATG or Campath-1H and GVHD prophylaxis were significantly different between the two groups.

Results: (Table) In a multivariate analysis, Tx from a serologically HLA-1-antigen MM donor, Tx from an unrelated donor (URD), pts with high-risk disease at Tx, no in vivo T-cell depletion and a conditioning regimen that included TBI ≥ 10 Gy were associated with an increased risk of grade II–IV aGVHD. Overall, race was not a risk factor for grade II–IV (P=0.19) or III–IV (P=0.98) aGVHD. The incidence of chronic GVHD (cGVHD) in Japanese was significantly lower than that in Korean (P=0.016). In a multivariate analysis, Tx from a genetically HLA-allele MM donor, Tx from an URD, pts with performance status 2–4 at Tx, pts with high-risk disease at Tx, pts with ALL, female to male Tx and pts’ age ≥ 40 yrs were associated with poor OS. Race was not a risk factor for OS. The incidence of relapse adjusted for the underlying disease status was associated with grade II–IV aGVHD (RR 0.75, 95% CI 0.61–0.94, P=0.010), but not cGVHD (P=0.63).

Conclusions: Despite their different medical backgrounds, our data confirmed that the biology of GVHD is very similar between these two Asian populations, and this provides a uniform foundation for large-scale clinical studies in the region.

Korean (n=996) Japanese (n=937) Grade II–IV/III–IV aGVHD 37%/15% 44%/19% Median onset of aGVHD day 28 day 21 cGVHD 60% 47% 3-yr OS in standard/high risk 70%/42% 65%/41% NRM 25% 30% Relapse 25% 24%
Refractory Autoimmune Hemolytic Anemia Following Unrelated Cord Blood Transplantation for Acute Lymphoblastic Leukaemia: A Case Report and Review of the Literature.
Authors: Liu Huilan , Geng Liangquan , Tong Juan , Wang Zuyi , Sun Zimin , Yao Wen , Yuan Xiao , Chen Jie
Published: October 2019

Autoimmune haemolytic anaemia (AIHA) after hematopoietic stem cell transplantation(HSCT) is a rare severe complication particularly in the paediatric population who underwent umbilical cord blood transplantation(UCBT).The pathogenesis of the post-HSCT AIHA is poorly understood, which frequently difficult to treat and overall prognosis is often poor. We present a case of a 13-year-old boy who developed AIHA 6 months after UCBT with a minor ABO blood group incompatiblity, 5/6 HLA matching umbilical cord blood unit from an unrelated donor was used for high risk acute lymphocyte leukemia in third remmision. Following conditioning with busulfan, cyclophosphamide, and antithymocyte globulin (ATG), the patient underwent transplantation and attained sustained hemopoietic reconstitution without severe graft-versus-host disease. At 6 months following UCBT,he developed severe acute autoimmune hemolytic anemia associated with a “warm” IgG antibody. He was being treated with packed RBC transfusions, cyclosporine, steroids and immunoglobulins, His AIHA was partly resolved. Hemolysis recurred after steroid tapering and partially responded to rituximab (375 mg/m(2) once weekly ×4 and plasma exchange.Despite a range of complex treatment regimen, the unstable hemolytic anemia was present and repeated severe infectious episodes occurred.We reduced suppression,strengthened antibiotic therapy,Pulmonary infection and hemolysis could not be controlled, the patient died of respiratory failure and circulatory collapse finally. This case report highlights the difficulty in managing posttransplant AIHA,Further studies about the pathogenesis of AIHA associated in HSCT recipients are needed.
Low-Dose Azacitidine for Relapse of MDS/AML after Unrelated Donor Peripheral Blood Stem Cell Transplantation.
Authors: Rossetti James M. , Shadduck Richard K. , Thatikonda Chandana , Sahovic Entezam , Lister John
Published: October 2019

Background: Post-transplant relapse in patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) is difficult to manage. Cytogenetic relapse and decreasing donor chimerism often precedes morphological relapse. Weaning of immunosuppressive agents and donor lymphocyte infusion (DLI) may induce graft versus tumor effect (GVT), but is of limited value for patients with existing GVHD. In addition, the degree of GVT in high-risk myeloid disease is suboptimal. We present our experience using low-dose azacitidine (AZA) for cytogenetic relapse post-transplantation.

Methods: Six patients with high-risk myeloid malignancy with cytogenetic relapse after matched unrelated donor peripheral blood stem cell transplantation were treated with low-dose AZA at 25 mg/m2 SC or IV for 5 days. An average of 2 cycles of AZA were given (range = 1 to 3). There were 3 females and 3 males with a mean age of 48 years (range = 31 to 59 years). Four had high-grade MDS (including 2 with treatment related disease) and 2 had high-risk AML. Conditioning consisted of fludarabine and ablative doses of busulfan in all patients. Cytogenetic relapse was seen by FISH testing within 187 days post-transplant (range = 30 to 730 days). AZA was given after an initial attempt to wean immunosuppression, which was not possible in 3 patients due to existing GVHD. All patients tolerated AZA well, without major toxicity. DLI was possible in 3 patients following AZA. A reduction in cytogenetic abnormalities (by FISH) and increase in donor chimerism (by FISH or STR) was observed in 5 out of 6 patients (83%) within 21 days post-AZA (range = 7 to 71 days). Three of the 5 responders demonstrated improvement after 1 cycle. The other 2 responders improved after 2 cycles given 28 and 60 days apart, respectively. One of these patients responded to a second AZA cycle after failing DLI. AZA did not appear to induce or worsen GVHD in any patient. One patient remains in CR 4 months after 1 cycle of AZA. Another patient demonstrated ongoing improvement in chimerism until her death from previously existing GVHD 20 days after a third cycle of AZA. The remaining 3 responders relapsed within 30 days from time of first response (range = 17 to 43 days).

Conclusions: Low-dose AZA appears to have activity in post-transplant relapse of MDS and AML. This low-dose regimen appears to be well tolerated, however, response to AZA is short-lived in the majority of patients. Further investigation is planned to improve the durability of response by giving AZA at regular intervals from the time of early relapse. The utility of AZA as a preparatory regimen pre-DLI should also be explored.