bluuebunny/crossref_metadata_2025 huggingface dataset:
Physical and Functional Association of the MRN Complex with Human Telomerase in Multiple Myeloma.
Authors: Driscoll James J. , Bertheau Robert C. , Shammas Masood A. , Prabhala Rao H. , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

Telomerase is a specialized RNA-directed DNA polymerase that extends telomeres of eukaryotic chromosomes, is repressed in normal human somatic cells but is activated during development and upon malignant transformation. There is abundant evidence that the regulation of telomerase is multifactorial in mammalian cells, involving telomerase gene expression, post-translational protein-protein interactions, and protein phosphorylation. Initial experiments indicated that the catalytic subunit(s) of human telomerase (hTERT) is highly elevated in Multiple Myeloma (MM) cell lines and MM patient samples relative to normal donor plasma cells. We then exploited this highly detectable level of hTERT to address the potential role of proteins that may physically associate with the telomerase complex in MM. To this end, we investigated whether the MRN complex, a trimeric structure that consists of Mre11, RAD50 and Nbs that is evolutionarily conserved and functions in DNA repair and homologous recombination physically associates with the human telomerase complex. The MRN complex is required in vivo for a 5′ to 3′ exonuclease activity that mediates DNA recombination at double-strand breaks (DSBs). We have observed that each of the three MRN components was associated with the catalytic subunit of human telomerase (hTERT) in MM cell lines as demonstrated by in vivo co-immunoprecipitation with an hTERT monoclonal antibody under non-denaturing conditions. In addition, polyclonal antibodies to each MRN component individually immunoprecipitated the hTERT catalytic subunit. We also detected that the telomerase complex accessory proteins TRF-1 and TRF-2 are also associated with hTERT and the MRN components. TRF-2 may function as bridge coupling hTERT to the MRN complex. The association of the MRN components with hTERT was increased following treatment of myeloma cells with DNA-damaging agents indicating a functional relationship between telomere maintenance and the DNA repair pathway. The functional role of this complex is being addressed by measuring telomerase activity, telomere length and homologous recombination activity. Based upon the data that abolition of exonuclease activity in MRN mutants resulted in shortened telomeric DNA tracts, we hypothesize that this elevated MRN complex expression and its interaction with hTERT in myeloma, provides the ability to maintain telomeres and may be an important therapeutic target in MM.
Osteonecrosis of the Jaw in Multiple Myeloma Patients. Experience of Two Hospitals.
Authors: Garcia-Garay Carmen , Gonzalez-Garcia Consuelo , Majado Maria Juliana , Santos Tomasa , Borrego Domingo
Published: April 2020

Introduction of bisphsophonates (BPs) treatment has changed the history of bone symptoms in multiple myeloma (MM) patients, as well as in metastasic cancers (MC). However, osteonecrosis of the jaw (ONJ) has been recently described in these patients. No clear mechanism is known, although length of BPs treatment, as well as dental procedures, have been argued as possible causes. We reviewed MM patients treated with BPs from two hospitals, in the last 7 years, with the aim of studying the incidence on ONJ and their correlation with: kind of BPs administered, time of treatment and dental procedures. MC patients treated with BPs were also reviewed in order to establish the influence of the disease in ONJ development. Non-parametric Mann-Whitney U-test was used in statistical analysis. From January 1999 to June 2006 a total of 225 MM patients were diagnosed in our two hospitals, 143 of them received BPs: 49 pamidronate (P) only, 64 zoledronic acid (Z) only, and 30 received P and Z (PZ) sequentially. P was administered at a doses of 90 mg every 4 weeks, and Z at a doses of 4 mgr every 4 weeks. 14 MM patients treated with BPs suffered ONJ: one patient (2%) treated with P, six (9,3%) with Z, and seven (23.3%) in the group receiving PZ. In the same period of time 353 MC patients were treated with BPs, two of them (0.6%) developed ONJ. Length of treatment, expressed in months, is shown in the table (in MC patients, only Z group was analyzed).

Months of treatment Diagnosis P Z PZ Total Expressed as median and range. MM without ONJ 22.5 (4–52) 9.5 (1–28) 38 (12–72) 14 (1–52) MM with ONJ 28 12 (7–28) 43.5 (24–59) 28 (7–59) MC 8 (1–41)

The longest administration of treatment was in PZ patients compared with P and Z(p<0.02), followed by P group that was longer than Z group (p<0.01). No difference was found between MC and MM groups in Z treatment length, neither between patients with and without ONJ inside each Z and ZP groups. Five ONJ patients suffered from dental procedures along BPs administration. In our series the incidence of ONJ is higher than that described in literature (4–10%), and it is very surprising the elevated incidence reached in patients treated with both BPs (23%). In our opinion, coincidental with other authors, the lasting of treatment might be one, but not the only, important factor, because in patients receiving P treatment duration was longer than Z but, ONJ incidence was lesser. For this reason we think that more potent BPs might have a role in ONJ development. Since MC patients receiving the same schedule of treatment with Z than MM do not develop ONJ so frequently, so it seems that the disease itself might play a role in ONJ incidence.
Velcade as Retreatment of Multiple Myeloma Patients Previously Responsive to Velcade.
Authors: Petrucci Maria Teresa , Gallucci Cristiano , Federico Vincenzo , Del Bianco Patrizia , Foa Robert
Published: April 2020

Velcade (bortezomib, PS 341) represents a novel class of anti-cancer drugs that, according to the randomized phase 3 trial (APEX) including 669 patients with relapsed multiple myeloma (MM), resulted more effective than high-dose dexamethasone as demonstrated by a significant improvement in response rate (43% vs 18%), median time to progression (6.2 vs 3.4 months) and 1-year survival rate (80% vs 67%, respectively). Its use in addition to dexamethasone in patients who had suboptimal responses to velcade alone has been associated with an improvement in responses without prohibitive toxicity, suggesting that the combination of these two drugs can overcome drug resistance. This agent is highly effective on extramedullary plasmacytomas and usually induces a very rapid response (after 1 or 2 cycles), independently of the previous therapies used. To evaluate the possibility of re-inducing a response in relapsed patients previously treated with velcade, we re-treated 3 patients with the same drug at 1.3 mg/m2 administered on days 1, 4, 8 and 11 of a 21-day treatment cycle for 8 cycles and dexamethasone 20 mg on the day of and on the day after each velcade dose. The patients (2 females and 1 man) previously treated with velcade as single agent after 47, 54 and 93 months, respectively, from diagnosis and after more than two lines of previous therapy, obtained a very good response without a complete disappearance of the monoclonal component. They were thereafter observed and re-treated with the same drug plus dexamethasone for a relapse documented after 7, 13 and 23 months, respectively. Velcade was well tolerated and the toxicity acceptable, without additional adverse events compared to the first administration. No patient experienced a herpes zoster reactivation and all side effects were typically reversible and disappeared upon drug suspension. Response was very rapid with a >50% decrease of the monoclonal component after 3, 4 and 5 cycles, respectively. It can be hypothesized that velcade, which offers great promise to overcome resistance to conventional chemotherapy, may not be susceptible to the most common drug resistance mechanisms. Based on the chronic nature of MM, drugs with documented efficacy, absence of cumulative toxicities and which do not develop resistance following repeated treatment are required. The responses observed in our relapsed MM patients who received velcade for the second time are encouraging. A longer follow-up and a greater number of patients will conclusively confirm the true efficacy of velcade as re-treatment drug. Updated results on a larger number of treated patients will be available at the time of presentation.
High Incidence of Varicella Herpes Zoster with Use of Bortezomib in Chinese Patients.
Authors: Tong Yin , Qian Jie Jing , Li Ying , Meng Hai Tao , Jin Jie
Published: April 2020

Bortezomib has been used for patients with refractory and relapsed multiple myeloma, non-Hodgkin lymphoma and leukemia in recent years. It has several complications. Here we report the complication of varicella herpes zoster after using bortezomib, which has higher incidence in Chinese patients. Ten patients were treated with bortezomib in our hematology centre. Among them, seven patients had refractory and relapsed multiple myeloma, two patients had refractory lymphoma (one of T cell lymphoma and one of mantle cell lymphoma) and one patient had refractory acute myeloblastic leukemia. All the patients received bortezomib (1.3 mg/m2) on days 1, 4, 8, and 11 of a 3-week cycle. Patients of multiple myeloma received dexamethasone simultaneously. The combination with liposomal doxorubicin was used on the patients of lymphoma. The patient of acute myeloblastic leukemia received bortezomib in combination with Amsacrine. After one cycle, 6 patients with multiple myeloma responded to treatment (four near complete remission, two partly remission). Two patients with lymphoma reached PR. The patient with leukemia had no response. A remarkable observation in our treatment was the high incidence of varicella herpes zoster. Six out of ten patients developed varicella herpes zoster. Among the six patients, two patients had previous infection of the zoster virus before. Most of the patients had the varicella herpes zoster infection after the complete of one cycle of bortezomib and were disappeared within one month by the use of antiviral treatment. The incidence of varicella hepers zoster after bortezomib was around 13% as reported. But the higher incidence of varicella herpes zoster were observed in our patients. Six of ten patients (60%) developed varicella zoster virus infection during treatment. We considered that the Chinese patients may be more liable to varicella zoster virus infection. And further observations should be made in the future. Prophylactic antiviral medication might be used in predisposed patients who receive bortezomib.
Zoledronic Acid for the Prevention of Bone Lesions in Patients with Monoclonal Gammopathy of Undetermined Significance (MGUS).
Authors: Berenson James R.
Published: April 2020

Recent studies have shown that approximately 5% of individuals over the age of 70 years have a monoclonal gammopathy and are at high risk for developing myeloma or a related lymphoproliferative disorder. Patients with monoclonal gammopathy of undetermined significance (MGUS) are at high risk for developing new fractures, particularly vertebral compression fractures. Currently, there is no treatment for this patient population. Among patients with advanced multiple myeloma, zoledronic acid has demonstrated efficacy in reducing skeletal complications. Therefore, a multicenter phase II trial is ongoing to evaluate zoledronic acid for patients with MGUS and a high risk for fractures. A total of 80 patients with MGUS and a T-score of less than −1.0 who have not received intravenous bisphosphonates within the past 2 years or oral bisphosphonates for more than 3 months within the past 2 years are eligible for enrollment. Eligible patients may not have had a change in hormonal therapy within 6 months of study entry. Patients will receive 4 mg zoledronic acid infused over 15 minutes at 0, 6, and 12 months. The primary endpoint is the change in bone mineral density (BMD) in the lumbar spine 1 month following the last infusion of zoledronic acid. Secondary endpoints include the mean change from baseline hip BMD, number of patients with new fractures, changes in serum M protein levels, number of patients who develop myeloma or other related malignancies, and safety of zoledronic acid in this patient population. The study commenced recruitment in 2004 and has enrolled 52 patients to date. Enrollment is continuing and expected to be complete by the end of 2006. This study may provide further support for the use of zoledronic acid in patients with MGUS who are at high risk for fracture development and currently do not receive any treatment.
Cord Blood Nucleated Cells Induce Delayed Proliferative and Cytotoxic T Cell Alloreactivity.
Authors: Mahmud Dolores , Chunduri Sandeep , Abbasian Javaneh , Maciejewski John , Iqbal Ronak , Rondelli Damiano
Published: April 2020

Transplantation of HLA-mismatched nucleated cells from cord blood (CB) has reduced risks of graft rejection and severe acute graft-versus-host disease. In this study we analyzed the in-vitro alloantigen presenting capacity of cord blood nucleated cells. CB mononuclear cells (MNCs) or immunomagnetically-selected CD34+ cells, or CD14+ monocytes, were irradiated and tested as stimulators of allogeneic blood T cells in primary (stimulator:responder ratio = 1:1) or secondary (stimulator:responder ratio = 1:2) mixed leukocyte culture (MLC), or in cytotoxic T-lymphocytes (CTL) assays. CB-MNCs failed to induce allogeneic T cell proliferation in 6-days primary MLC, whereas CD34+ or CD14+ cells stimulated brisk T cell responses. A suppressive effect of CB-MNCs was ruled out since CD3+ cell-depletion of CB-MNCs did not restore CB immunogenicity and the addition of increasing doses of CB-MNCs did not inhibit T cell alloreactivity to CD34+ cells. Despite allogeneic T cells were unresponsive to CB-MNCs after primary and secondary MLC, T cell anergy was ruled out since T cells that were unresponsive after primary MLC proliferated potently in secondary MLC stimulated with CB CD34+ cells, and even more with CB monocyte-derived dendritic cells (Mo-DC) generated in-vitro with GM-CSF and IL-4. Interestingly, after co-culture with irradiated allogeneic T cells for 6 days, CB-MNCs showed a greater proportion of CD86+ cells and elicited allo- T cell proliferation. In addition, allo-CTL activity was induced by CB-MNCs only after restimulating effector cells for 3–4 weeks (26±7% lysis of antigen-specific PHA-blast at 50:1 E:T ratio), and was comparable to CTL activity induced after 1 week by Mo-DC generated from the same CB. When T cell effectors were stimulated by combining two incompatible cord blood MNCs mixed together, CTL activity was then detected after 4 weeks against both of them regardless of the CB:CB cell ratio. These results show an impaired allo-APC activity of CB-MNCs, without suppressive or tolerogenic activity. These findings might partially explain the initial engraftment of combined HLA mismatched CB grafts in vivo, however they also suggest that a delayed T cell response may occur due to CB-derived APCs activating CTLs.
FGFR3 Activates RSK2 To Mediate Hematopoietic Transformation through Both Tyrosine Phosphorylation of RSK2 and Activation of the MEK/ERK Pathway.
Authors: Kang Sumin , Dong Shaozhong , Gu Ting-lei , Cohen Michael S. , Lonial Sagar , Khoury H. Jean , Gilliland D. Gary , Taunton Jack , Polakiewicz Roberto D. , Chen Jing
Published: April 2020

Reccurent chromosomal translocation t(4;14)(p16.3;q32.3) that frequently occurs in patients with multiple myeloma is associated with ectopic overexpression of fibroblast growth factor receptor 3 (FGFR3), which sometimes contains the activation mutations such as K650E (TDII). To better understand the signaling properties of oncogenic FGFR3, we performed a mass spectrometry-based phospho-proteomics study to identify potential downstream signaling effectors that are tyrosine-phosphorylated due to expression of FGFR3. RSK2 (p90 ribosomal S6 kinase 2) was found to be specifically tyrosine-phosphorylated at Y488 and Y529 in hematopoietic Ba/F3 cells transformed by TEL-FGFR3 fusion, which is associated with t(4;12)(p16;p13) peripheral T-cell lymphoma. In addition, RSK2 was highly tyrosine-phosphorylated in t(4;14)-positive human myeloma cell lines expressing constitutively activated FGFR3 mutants. We found that expression of leukemogenic FGFR3 variants resulted in activation of RSK2 through the MEK/ERK pathway. The current model suggests that inactive ERK binds to the C-terminus of RSK2 in quiescent cells, prior to ERK-dependent phosphorylation and activation of RSK2 when ERK is activated. Interestingly, we found that FGFR3-dependent tyrosine phosphorylation at Y529 regulates the ERK-dependent activation of the serine/threonine kinase RSK2. Co-immunoprecipitation experiments revealed that substitution of Y529 significantly decreased inactive ERK binding to RSK2. This disrupted ERK-RSK2 association consequently attenuated phosphorylation and activation of RSK2 by ERK activated by FGFR3, in both in vitro kinase assays and an RSK2-specific CREB mediated transcription assay. Moreover, inhibition of RSK2 by specific siRNA or an RSK inhibitor fmk effectively induced apoptosis in FGFR3 transformed murine Ba/F3 and t(4;14) human myeloma cells, suggesting RSK2 is a critical signaling effector in FGFR3-induced hematopoietic transformation. Together, these data support a novel two-step model that leukemogenic FGFR3 activates RSK2 to mediate transformation signals by promoting both the ERK-RSK2 interaction and subsequent phosphorylation of RSK2 by ERK. Thus, RSK2 may represent an alternative therapeutic target in the treatment of diverse human malignancies associated with dysregulated FGFR3.
The Molecular Characteristics of T-Cell Receptor Repertoire of Clonal Expansion of T Lymphocytes in Leukemia Patients after Haploidentical Bone Marrow Transplantation.
Authors: Fu YueWen , Wu De Pei , Feng YuFeng , Chang WeiRong , Zhu ZiLin , Zhu Ping
Published: April 2020

The clinical and experimental results indicated that the T cell immune reconstitution was slow after haploidentical hematopoietic stem cell transplantation and existed high rate of GVHD and infection. This study was to analyze the molecular characteristics of T cell clones during immune reconstitution in leukemia patients after haploidentical bone marrow transplantation (H-BMT). Reverse transcriptase-polymerase chain reaction (RT-PCR) amplified 24 subfamily genes of TCRVB from peripheral blood lymphocytes of nine leukemia patients underwent H-BMT, five normal donors as control. The PCR products were further analyzed by genescane to evaluate the clonality of VB subfamily and characteristics of CDR3 and usage rate in VB subfamily. The monoclonal bands which associated with GVHD obtained and sequenced. Compared the sequences of TCRVB CDR3 gene repertoire with other sequences associated with GVHD had been reported before. In +10~+19m, the usage of TCRVB subfamilies still restricted. It can detected that some VB subfamilies were missing, others expanded in monoclonal or oligoclonal. 4 patients in stable condition showed that 9~14 VB subfamilies expressed and more than 50 percent were polyclone. In other 5 patients which have GVHD or CMV-pp65(+), the usage of TCRVB decreased obviously(p<0.05), the express of CDR3 were monoclonal or oligoclonal, only 30 percent were polyclonal. No common monoclone VB subfamilies expressed. After treatment and GVHD controled, the usage of VB subfamilies was increased and CDR3 polymorphism increased in 2 patients. Analyzed the sequences associated with GVHD and found that none of the clones appeared to share any similarity in amino acid motif. In +10~+19m, the usage of TCRVB subfamilies still skewing. In stable condition, there were 9~14 VB subfamilies expressed and dominated by polyclones, in active condition, the express of VB subfamilies decreased and dominated by monoclones or oligoclones. A group of CDR3 molecules related to GVHD showed no common amino acid motif.
High Anti-Tumor Activity of Natural Killer Cells after Non-Myeloablative Conditioning and Allogeneic Stem Cell Transplantation.
Authors: Fischer Lars , Penack Olaf , Stroux Andrea , Gentilini Chiara , Thiel Eckhard , Uharek Lutz
Published: April 2020

Background: It is hypothesized that enhanced graft-versus-leukemia activity of natural killer (NK) cells contributes to the clinical efficacy of non-myeloablative allogeneic hemotopoietic stem cell transplantation (HSCT). We compared NK cell cytotoxic activity after transplantation with full and reduced dose regimens with normal values obtained from healthy volunteers.

Methods&Material: Using a flow cytometric assay that detects CD107a expression after coincubation with leukemia cell lines as a marker for NK cell degranulation we prospectively quantified and characterized NK cells mediating anti-tumor activity in patients after HSCT. Mononuclear cells (MNC) were isolated from peripheral blood of 17 healthy individuals and 31 patients transplanted with G-CSF mobilized peripheral blood stem cells at day +30. MNC were incubated with leukemia cell lines HL60 and K562 (effector/target ratio 1:1) and expression of CD107a was measured after 3 hours. The absolute number of degranulating (CD107a+) NK cells was calculated.

Results: Thirty one patients (five with ALL, four with NHL, one with Aplastic Anemia and 21 with AML) were enrolled, of whome 22 were transplanted from a matched related donor, seven from a matched unrelated donor, and two from a haploidentical donor. Nine patients had been transplanted after conditioning with 2 Gy TBI only, whereas 22 patients had received various conventional dose regimens. NK cell counts at day +30 after HSCT with conventional conditioning were comparable to normal values, but percentage and number of degranulating cells were significantly reduced (2.4% and 7/μl vs. 6.2% and 18/μl; p<0.0001 and p=0.0003). In contrast, in patients after 2 Gy TBI the percentage of degranulating NK cells was comparable to healthy donors (7.6%; p=0.9), and interestingly, absolute numbers of all and of degranulating NK cells (41/μl; p=0.004) were higher than normal values and consequently higher than in patients with conventional dose conditioning.

Conclusions: Using this new method we were able to exactly quantify tumor-reactive NK cells after HSCT in a feasible way. We found a high cytotoxic activity of NK cells towards leukemia cell lines in patients undergoing non-myeloablative conditioning with 2Gy TBI. Further studies to determine the clinical impact of these findings are needed.
Rapamycin Induces Balb/C Murine CD4+CD25+Foxp3+ T Cells Proliferations.
Authors: Yang Mingzhen , Wu De Pei , Yuan Yanhui , Cen Jiannong , Chang Weirong , Zhu Ziling
Published: April 2020

Rapamycin(RAPA) is an immunosuppressive agent, it inhibits T lymphocyte activation and proliferation by suppressed antigen and cytokine(interleukin-2, interleukin-4 and interleukin-15), it was always used to treat autoimmune disease and graft versus host disease. RAPA can selectively expand mice CD4+CD25+FoxP3+ regulatory T cells in vitro, in this study, we demonstrated RAPA induce mice CD4+ CD25+FoxP3+ regulatory T cells proliferation in vivo. Balb/C mice were used between 8–10 weeks of age, weight was 20±2g, RAPA was given to Balb/C murine 0.4mg/day intragastric administration according to man dose, the same mice of age and weight were given with steriled water as the control, all mice were kept under specific pathogen-free conditions. Drinking water and food were steriled. After three weeks, peripheral blood was collected and spleen cells were prepared, CD4+CD25+T cells were detected with FCM(CD4-pcy5, CD25-FITC), the relative levels of foxp3 mRNA were determined by real-time quantitative RT-PCR in total splenocytes. The CD4+CD25+T cell of peripheral blood of experimental group and control group was (9.24±4.16)% and (4.32±1.26)%, respectively (P<0.01), and CD4+CD25+T cell of experimental mice splenocytes was (22.99±10.59)%, while control group was (7.37± 2.91)% (P<0.01). real-time quantitative RT-PCR showed that the levels of foxp3 mRNA of experimental mice splenocytes was 6 folds than control group(P<0.01). CD4+CD25+T cells and CD+CD25−T cells were enriched with CD4+CD25+T regulate cells isolation kits from experimental Balb/C mice, we used mixed leukocyte reaction for CD4+CD25+T cells suppressor function. The CD4+CD25+T cells can inhibit the proliferation of CD4+CD25−T cells and inhibition ratio was about 50%. Our result demonstrated that RAPA can induce Balb/C murine CD4+CD25+Foxp3+T cells proliferation in vivo, which suggested RAPA could increase CD4+CD25+Foxp3+T cells on the autoimmune disease and graft versus host disease treatment.
Immune Mobilization of Autologous Peripheral Blood Progenitor Cells: IL-2 with GM-CSF and G-CSF Results in Effective Mobilization without Delay in Engraftment.
Authors: Hill John M. , Wu Jia-Yan , Webber Susan M. , Sharlin Ortal , Kendall Melinda , McGregor David , Ernstoff Marc J. , Szczepiorkowski Zbigniew M. , Meehan Kenneth R.
Published: April 2020

We initiated an immune mobilization trial in an attempt to mobilize cytotoxic effector cells, along with CD34+ hematopoietic progenitor cells. A Prospective Phase I trial was initiated using dose escalation of IL-2, in combination with GM-CSF and G-CSF. IL-2 began on Day 0 and continued as a daily SQ injection for 11 days. On Day 7, GM-CSF (7.5 mcg/kg/d) and G-CSF (5 mcg/kg/d) were initiated for 5 days (Days 7–11). On Day 11, leukapheresis was started if the peripheral blood CD34 + cell count was > 5 cells/mcl. The endpoint of collection was ≥ 3 × 106 CD34+ cells/kg. After collection, patients received melphalan (200 mg/m2) followed by infusion of cryopreserved stem cells. Post-transplant GM-CSF began on Day +5 and terminated once the ANC reached 5000 cells/mcl. To date, 9 patients have been treated (myeloma, n = 8; NHL, n = 1) and 7 patients are evaluable. Six patients received IL-2 at Dose Level 1 (6 × 105 IU/m2/d). The remaining 3 patients received IL-2 at Dose Level 2 (1 × 106 IU/m2/d). The MTD of IL-2 has not been reached. One patient (NHL) was removed from the study due to progressive disease. The remaining 8 patients completed the regimen. Toxicities have been mild and have included Grade 2 fever (n=1) on Dose Level 2. All patients were successfully mobilized. The median number of CD34+ cells/kg and MNC/kg collected were 3.4 × 106 (range 2.8 – 4.4 × 106/kg) and 9.5 × 108 (range 0.4 – 1.7 × 109), respectively. Two large volume leukaphereses were required (median; range 1 – 3). Following transplant, the ANC recovered on Day 13 (median; range: 10 – 14 d) and platelets recovered on Day 12 (median; range 0 – 13 d). These preliminary results demonstrate that immune mobilization and collection of an adequate number of hematopoietic progenitor cells is feasible without suppression of hematopoiesis. Toxicities are minimal but the MTD of IL-2 has not yet been reached. Post-transplant engraftment is not delayed. As patient accrual continues, we are currently evaluating the qualitative and quantitative components of the collected cells, including Th1 vs. Th2 cells and the types of dendritic cells mobilized.
Engineering a Disulfide Bond To Stabilize the Calcium Binding Loop of Activated Protein C Eliminates Its Anticoagulant but Not Protective Signaling Properties.
Authors: Bae Jong-Sup , Yang Likui , Manithody Chandrashekhara , Rezaie Alireza R.
Published: April 2020

In addition to its potent anticoagulant activity, activated protein C (APC) also exhibits anti-inflammatory and cytoprotective properties. The latter properties of APC are thought to be responsible for its beneficial effect in treating severe sepsis patients. A higher incidence of bleeding due to its anticoagulant function has been found to be a major drawback of APC as an effective anti-inflammatory drug. Thus, there is intense interest in developing APC variants that have no anticoagulant activity, but have normal protective properties. In this study, we have prepared a protein C variant in which an engineered disulfide bond between two β-sheets has stabilized the functionally critical Ca2+-binding 70–80 loop of the molecule. The 70–80 loop of this mutant no longer binds Ca2+ and the activation of the mutant by thrombin has been enhanced 60–80-fold independent of thrombomodulin. The anticoagulant activity of the APC variant has been essentially eliminated as determined by both factor Va degradation and plasma-based clotting assays. However, the endothelial protein C receptor- and protease-activated receptor-1-dependent protective signaling properties of the APC variant have remained intact as determined by staurosporine-induced endothelial cell apoptosis, thrombin-induced endothelial cell permeability and TNF-α-mediated neutrophil adhesion and migration assays. These results suggest that modulation of the structure and/or activity of APC by Ca2+ is required for its anticoagulant, but not for its anti-inflammatory activity, thus the protein C variant may potentially be a safer drug for treating severe sepsis patients in both activated and zymogenic forms.
Predictive Markers for Acute Graft-Versus-Host Disease in the Peripheral Blood - Surface Receptor Profile Defines Alloreactive T Cells In Vivo.
Authors: Beilhack Andreas , Zeiser Robert , Schulz Stephan , Olson Janelle A. , Nishimura Ryosei , Leveson-Gower Dennis , Negrin Robert S.
Published: April 2020

Acute graft-versus-host disease (aGVHD) still results in high morbidity and mortality in patients undergoing allogeneic hematopoietic cell transplantation (aHCT). Early diagnosis of aGVHD remains difficult and is made based on clinical symptoms and histological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestations. Therefore, it is important to develop predictive assays to identify patients at risk of developing aGVHD for improved disease prevention.

Using bioluminescence imaging (BLI) we have recently demonstrated that aGVHD pathogenesis is tightly spatially and temporally regulated in a murine model across major histocompatibility barriers. Therefore, we asked whether insights in the timing of aGVHD initiation and effector phase could allow for the development of a diagnostic test whereby specific cell surface profiles can predict the onset of aGVHD.

FVB/N (H-2q, Thy1.1+, 4x106) or C57Bl/6 (H-2b, Thy1.1+, 4x106) splenocytes plus bone marrow (BM) cells (5x106) were transplanted into conditioned (2x400rad) allogeneic Balb/c (H-2d, Thy1.2+) or syngeneic (2x450rad) C57Bl/6 (H-2b, Thy1.2+) recipients. Allogeneic recipients developed the first clinical signs of aGVHD starting at day+6 which progressed rapidly to animal death typically by day+14. However, BLI revealed that aGVHD target organ infiltration had already occurred days earlier. On day+3 after aHCT, dividing (BrdU+) donor derived allogeneic T cells (Thy1.1+) were still confined to the T cell areas in secondary lymphoid organs. Between day+3 and day+4 T cells appeared in the red pulp of the spleen, indicating likely entry into the blood circulation. Thereafter, increasing aGVHD target organ infiltration became apparent. These findings prompted us to analyze peripheral blood (PB) samples from allogeneic vs. syngeneic recipients (day+1 to +6). Until day+3 after aHCT, no significant PB T cell numbers were detectable. However, by day+4 we found a dramatic increase of PB T cells (mostly CD4+, by day+5 mostly CD8+) that were CD44hi and expressed the homing receptors α4β7 integrin, αEβ7 integrin, CCR9, E-selectin ligand, P-selectin ligand, CCR5, and CXCR3 in allogeneic recipients, but not in syngeneic or BM controls. These T cell populations could be verified as clonally expanded (CFSElo) donor-derived alloreactive T cell subsets (Thy1.1+).

In summary, alloreactive T cells could be identified by the timed up-regulation of a panel of distinct homing receptors. Furthermore, the transition between aGVHD initiation and effector phase emerged as an early diagnostic window for the detection of alloreactive T cells in the peripheral blood days before aGVHD onset. Taken together, this approach could predict aGVHD in order to tailor immunosuppressive therapy for individual patients.
Clinical Outcomes in Patients Receiving Venous Thromboembolism (VTE) Prophylaxis after Orthopedic Surgery (OS). A Systematic Review and Meta-Analysis of Proportions.
Authors: Lazo-Langner Alejandro , Coyle Douglas , Barrowman Nicholas J. , Ramsay Tim , Wells Philip S. , Scarvelis Dimitrios , Forgie Melissa A. , Rodger Marc
Published: April 2020

Major VTE is the most frequent complication of OS. Current recommendations are to administer prophylaxis with an anticoagulant agent for at least 7–10 days. Numerous studies have evaluated different agents for this purpose. Although the most recent studies are usually methodologically sound, several studies have been hampered by inappropriate designs and insufficient sample sizes. In order to help with the design of future trials we conducted a systematic review of randomized trials evaluating short-term (< 15 days) administration of anticoagulants for VTE prophylaxis in OS and performed a MA of simple proportions to estimate the overall incidence of major VTE (proximal VTE, pulmonary embolism (PE), or death from PE), total VTE (proximal and distal VTE, PE or death from PE), and major bleeding episodes (as defined by the authors and defined using a definition similar to the one proposed by the International Society on Thrombosis and Haemostasis-ISTH). We included randomized trials comparing different drugs for VTE prophylaxis in OS (hip and knee arthroplasty and hip fracture surgery) using systematic evaluation of VTE (ultrasound or venography, pulmonary angiography, tomographic angiography, or ventilation perfusion lung scan). Heterogeneity of proportions was evaluated using a chi ² test and pooled estimates of proportions were obtained using a fixed or a random effects model as appropriate. In the latter the weights were estimated as proposed by Laird and Mosteller. We retrieved 55 studies (135 research arms) which enrolled 42,131 patients. The percentage and variance of major and total VTE and major bleeding are shown in table 1. The total number of events and the number of evaluable patients are shown in table 2.

We found differences in the percentage of clinical outcomes associated with the use of different agents for VTE prophylaxis after OS, however, because of the analytical strategy used no estimation of odds or risk reduction can be derived from this data. We believe that these estimates will be of help for the design of future studies.
A Novel Cytokine, FPT025, Regulates Myeloid Growth and Differentiation Via the M-CSF Receptor.
Authors: Lin Haishan , Huang Min Mei , Leo Cindy , Ji May , Wu Ge , Zhou Aileen , Halenbeck Robert , Qin Minmin , Linnemann Thomas , Behrens Dirk , Giese Scott , Bosch Elizabeth , Justin Katrina , Pham Amber , Lee Ernestine , Hestir Kevin , Zhang Hongbing , Wong Justin , Wang Yan , Chu Keting , Doberstein Steve , Williams Lewis T. , Hollenbaugh Diane
Published: April 2020

To identify novel protein therapeutics we have utilized an integrated screening system in which a collection of full-length human cDNA clones, encoding virtually all secreted proteins and receptors, was assembled. Soluble secreted proteins were expressed in a high-throughput format using human cells as the expression host and tested in high-throughput cell-based assays. Through this approach, a novel cytokine, FPT025, was identified in a human monocyte proliferation assay. FPT025 has no apparent sequence homology to known cytokines or any other genes and is expressed in human spleen, skin, brain, and other tissues. The purified recombinant FPT025 protein stimulated human primary monocyte proliferation and/or survival. FPT025 protein specifically bound to human primary monocytes and activated ERK1/2 phosphorylation in primary monocytes as well as in a human monocytic cell line, THP-1. FPT025 promoted formation of the myeloid lineage colonies, CFU-M, in a human bone marrow colony formation assay and enhanced proliferation of cells with myeloid cell surface markers from human monocytes. To identify the receptor of FPT025, a collection of extracellular domains (ECD) of transmembrane proteins was screened for their ability to block FPT025 activation of monocyte proliferation. In this screen, we found that FPT025 binds to the M-CSF receptor (M-CSFR) with high affinity. Furthermore, we showed that the binding of FPT025 to M-CSFR was specific and could be competed by M-CSF. The soluble ECD of M-CSFR inhibited both the binding of FPT025 to M-CSFR and the activity of FPT025 in monocyte proliferation. Therefore, FPT025 functions as a novel ligand of the M-CSF receptor and participates in the regulation of myeloid lineage differentiation, proliferation, and survival.
Morpholino Knockdown of Jak2a Inhibits Primitive and Definitive Hematopoiesis in Zebrafish.
Authors: Lin Hui-feng , Zhang Jing , Handin Robert I.
Published: April 2020

In zebrafish, the primitive wave of hematopoiesis is specified at 3 somites and is marked by two lateral stripes, which express early hematopoietic genes. The coalescence of these stripes forms the intermediate cell mass (ICM). This phase of hematopoiesis lasts about 4 days with erythrocytes entering the circulation 26 hpf. Between 36 and 42 hpf, the definitive wave of hematopoiesis commences. It is marked by the presence of hematopoietic progenitor cells in the dorsal aortic wall. Later in day 3 of development, hematopoietic stem cells from the dorsal aorta migrate into the interstitium of the mesonephros, which becomes the major hematopoietic organ in adult fish. Jak2 is a member of the Janus kinase family of non-receptor tyrosine kinases which plays an important role in hematopoiesis by transmitting cytokine-induced signals to associated STATs (signal transcription and activation transducers). The phosphorylated STATS migrate to the nucleus and induce gene transcription. The knockout of Jak2 in mice is embryonic lethal with a severe defect in erythropoiesis. We and others have observed that Jak2 is duplicated in zebrafish with one of the duplicated genes, jak2a, expressed exclusively in hematopoietic tissues. We have knocked down jak2a with anti-sense morpholinos to assess its role in hematopoiesis. O-dianisidine staining of embryos 2dpf shows a reduction in the primitive wave of erythropoiesis in the jak2a morphants. Using the CD41-GFP transgenic fish line we observed markedly reduced and, in some cases, the complete absence of circulating thrombocytes in jak2a morphants. To help understand how jak2a knockdown impairs hematopoiesis we examined the expression of a panel of early hematopoietic genes in the ICM of 20 to 24 hpf fish by whole mount in situ hybridization (WISH). There was an overall reduction in the expression of lmo2, scl, gata-1 and gata-2. Upon closer observation it was apparent that the reduction was due to the loss of certain cells from the ICM with normal staining intensity in the remaining cells. We also observed reduced cyclin D1 expression and elevated caspase 3 activity, which strongly suggested programmed cell death was activated in the jak2a morphant. We believe that there is a selective loss due to cell cycle arrest of the more mature hematopoietic cells, which require the expression of jak2a for their survival with retention of early hematopoietic progenitors and hematopoietic stem cells in the ICM. These observations in developing zebrafish embryos establish the important role of jak2a in hematopoiesis and provide new insights into the stage of hematopoiesis where jak2a is critically important.
Tumor Cell-Derived HLA-B-Associated Transkript 3 (BAT3) Is a Ligand for NKp30 and Activates NK Cells.
Authors: von Strandmann Elke Pogge , Simhadri Venkateswara R. , von Tresckow Bastian , Sasse Stefanie , Reiners Katrin S. , Hansen Hinrich P. , Rothe Achim , Borchmann Peter , McKinnon Peter J. , Hallek Michael , Engert Andreas
Published: April 2020

Natural-killer (NK)-cells are lymphocytes of the innate immune system, which directly attack tumor and virus-infected cells. They provide a link between innate and adaptive immunity through crosstalk with dendritic cells (DCs) and mediate T-cell activation. Among the activating and inhibitory receptors that regulate NK cell activity, the orphan NKp30 receptor (NCR3, CD337) plays a special role as NKp30 does not only induce target cell lysis but is also crucial for the interaction between NK cells and dendritic cells. However, so far the cellular ligands for NKp30 have remained elusive.

Using a yeast two hybrid approach with the extracellular NKp30 sequence as bait we were able to isolate a putative NKp30 ligand from a tumor cDNA library. Sequence analysis revealed that the cDNA encoded for BAT3 (HLA-B-associated transcript 3) which originally had been cloned from the human major histocompatibility complex by chromosome walking and mapped to chromosome 6p21.3 within the HLA complex. BAT3 was described as a nuclear protein characterized by an N-terminal ubiquitin-like region, a polyproline stretch and a highly conserved BAG-(Bcl-2-associated athanogene) domain. Until now, the function of BAT3 in mammals is not clearly defined. With BAT3-specific antibodies and by means of laser scanner microscopy and Western Blotting we demonstrate that tumor cell lines and immature as well as mature DCs express BAT3. Upon co-cultivation with NK cells or exposure to stress signals such as heat shock, we observed that BAT3 translocates from the nucleus to the cell-surface and the protein is released from tumor cells into the extracellular environment. BAT3 binds directly and specifically to NKp30 on NK cells and triggers NK cell-mediated cytokine release and NKp30-dependent cytotoxicity. The inhibition of endogenous BAT3 using polyclonal antibodies prevents tumor rejection in vivo, demonstrating that BAT3 is necessary for the tumor rejection in a xenograft tumor model.

Thus, BAT3 is the long sought cellular NKp30 ligand and exhibits a novel mechanism by which a nuclear protein activates NK cells via NKp30-engagement after its release to the cell-surface and the cell environment. The isolation of the first cellular NKp30-ligand provides the basis for a better molecular understanding of NK cell-regulation and allows the development of clinical applications targeting NKp30 for the immunotherapy.
The Unexpected Role of C3a-C3aR Axis in Maturation of Erythroid Cells - Implications for Pathogenesis of Hypoxia-Related Polycythemias.
Authors: Ratajczak Janina , Reca Ryan , Machalinski Bogdan , Maciejewski Jaroslaw , Laughlin Mary , Ratajczak Mariusz Z.
Published: April 2020

Hypoxia leads both to secondary polycythemia and activation of the complement (C) cascade in bone marrow (BM). We reported that C is activated in BM during hypoxia by the IgM-dependent classical pathway (Blood2006, 107:835–840). These observations prompted us to study the potential role of C cleavage fragments in erythropoiesis. We noticed that human and murine cells from the erythroid lineage express functional G-protein coupled C3aR and that C3 cleavage fragment - anaphylatoxin C3a enhances SDF-1-mediated chemotaxis of erythroid progenitors and induces in normal erythroblasts i) adhesion to fibronectin, ii) calcium flux and iii) phosphorylation of MAPKp42/44. To further explore a potential role of the C3a-C3aR axis in erythropoiesis we focused on C3aR−/ − mice. We noticed that these animals as compared to wild type (wt) littermates have i) lower hematocrits and ii) display delayed recovery of hematocrit values after sublethal irradiation. More importantly, we noticed that the number of clonogeneic BFU-E progenitors in BM of C3aR−/ − mice, but not CFU-GM and CFU-Meg, is reduced by ~65%. Thus, in order to learn more about the role of the C3a-C3aR axis in erythropoiesis we moved to an in vitro model and costimulated normal human CD34+ or murine SKL cells with C3a or desArgC3a in the presence of suboptimal doses of EpO and KL, but no effect on BFU-E colony formation was observed. We found, however, that C3a increases in early erythroid cells expression of mRNA for VEGF and MMP-2 - which are important for maturation/enucleation and egress of erythrocytes from the BM into peripheral blood. These data were subsequently confirmed by ELISA (VEGF) and zymography (MMP-2). Furthermore, we noticed that at the morphological level C3aR−/ − mice as compared to wt littermates had significantly reduced number of erytropoietic islands in BM. This suggests that in the absence of the C3aR, maturing erythroblasts do not traffic properly in the marrow microenvironment in BM and do not associate with C3 producing central macrophages in erythropoietic islands. In conclusion the C3a-C3aR axis modulates the interaction of erythroid progenitors with the hematopoietic environment and we conclude that it plays an important and underappreciated role in developmental trafficking of erythroid cells in BM. Thus C3a may enhance the erythropoietic effects of erythropoietin in polycythemias related to hypoxia. Furthermore, since hypoxia activates C, we postulate that C cleavage fragments could be involved in the pathogenesis of hypoxia-related polycythemias and C3aR antagonists could find a potential supportive role in controlling erythropoiesis in these patients.
Epo Receptor Circuits to Irs2, Foxo3a, Trb3 and Bim1 Expression, and Erythroblast Survival.
Authors: Bogacheva Olga , Bogachev Oleg , Sathyanarayana Pradeep , Menon Madhu P. , Kapelle William S. , Houde Estelle , Wojchowski Don M.
Published: April 2020

Epo cytoprotection of developing erythroblasts mechanistically is thought to involve 1] PI3K and Akt pathways (eg. mTOR, Foxo3A inhibition) as engaged via a distal EpoR PY479 site, and 2] anti-apoptotic effects as exerted by EpoR proximal box1 and PY343 signals (eg. Bcl-xl transcription). To globally assess Epo-regulation of survival- associated genes, a maximally Epo-responsive cohort of bone-marrow derived KitposCD71high erythroblasts was prepared, purified and used in Affymetrix 430–2.0 array based profiling of Epo responsive genes. Quadruplicate array analyses and RT-QPCR revealed selective modulation of PI3K pathway associated factors Irs2, p85-alpha, Trb3, and Foxo3a as well as the Bcl2-related factor Bim1. Irs2, p85-alpha and Trb3 were induced three- to five-fold, while Foxo3a was rapidly down-modulated. As studied using knocked-in minimal EpoR alleles, induction of Irs2 and Trb3 (a pseudo-kinase Akt inhibitor) depended sharply upon an EpoR PY343 Stat5 binding site. Epo down-modulation of Foxo3a (as well as Trb2 and Bim1), in contrast, occurred via EpoR PY-independent routes. Unexpectedly, the EpoR allele, EpoR-H-Y343 also protected erythroblasts from LY294002 and wortmanin induced apoptosis, while an EpoR-HM-F343 allele was hyper-sensitive. For EpoRH, this proved to correlate specifically with Y343 dependent phosphorylation of GAB2. PY343 couples specifically to Stat5, and phospho-Stat5 therefore is proposed to recruit GAB2 and enable its PY452 phosphorylation. GAB2 then provides a shunt to PI3K activation. Findings provide new insight into Epo-regulation of a select set of PI3K-associated genes, and core EpoR cytoprotective mechanisms.