bluuebunny/crossref_metadata_2025 huggingface dataset:
Putative Substrates of AKT in Multiple Myeloma Cells.
Authors: Yew Lee Ping , Chen Chih-Hao , Chu Bo-Feng , Ho Yen-Peng , Hsu Jung-Hsin
Published: October 2019

The serine/threonine protein kinase AKT (also known as protein kinase B) is thought to be a key mediator of signal transduction processes. The AKT kinase is activated in myeloma tumor cells. Frequent AKT activation in situ in myeloma cells of patient bone marrow has been demonstrated. IL-6 is a known growth factor of myeloma cells. Recent evidence indicates that AKT plays a critical role in IL-6-dependent expansion of multiple myeloma clones. A method to identify serine kinase substrates has been described. In that method, an antibody specific for the phosphomotif generated by the kinase is used to isolate phosphorylated substrates by immunoprecipitation, and the isolated proteins are identified by tandem mass spectrometry of peptides. This method has been adopted in identifying putative substrates of AKT in mutiple myeloma cells. AF-10 cells of myeloma cell line were treated with IL-6 to activate AKT, and the putative AKT substrates were isolated by immunoprecipitation with PAS (phosphospecific Ser/Thr AKT substrate) antibody. The isolated proteins were identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In Western blot probed with PAS antibody, intensities of putative AKT substrates increased with IL-6 treatment, but decreased when the cells were preincubated with wortmannin (a specific PI 3-kinase inhibitor) then treated with IL-6. Putative AKT substrates identified include: nonmuscle myosine-9, vimentin, and tubulin beta-2. All these proteins contain appropriate motifs of AKT substrates (R/KXRXXS/T, XXRXXS/T). Conformation studies and studies of the physiological significances of these putative substrates are undergoing. Recently, it has been reported that a novel isocoumarin derivative induces mitotic phase arrest and apoptosis of human mutiple myeloma cells by affecting tubulin assembly.
Effect of Bortezomib on the Autonomic Nervous System.
Authors: Crocchiolo Roberto , Ferrari Stefania , Calbi Valeria , Marcatti Magda , Guggiari Elena , Servida Paolo , Bernardi Massimo , Carro Ubaldo Del , Bonini Chiara , Tresoldi Moreno , Peccatori Jacopo , Ciceri Fabio , Sora Maria Grazia Natali
Published: October 2019

Background

Peripheral neuropathy is one of the most frequent adverse events associated to bortezomib therapy; among neuropathic syndromes, postural hypotension is described as well. However, information on disautonomic toxicity are limited and there are no studies addressed to this particular issue so far.

Before starting this study we had observed a case of dramatic acute autonomic failure during bortezomib treatment in a patient with advanced multiple myeloma, who developed severe orthostatic hypotension, tachycardia and impairment to tolerate the sitting and orthostatic posture for several weeks.

Methods

We prospectively studied 12 consecutive patients, treated with bortezomib at our Institution between February 2005 and July 2006. Assessment included neurological examination, INCAT Disability scale, INCAT sensory sum score, MRC sum score, nerve conduction studies, and standard cardiovascular autonomic tests including Lying to standing, Deep breathing and Postural hypotension. These tests were conducted baseline and after each bortezomib 21-days cycle up to three consecutive cycles.

Results

Two pts showed a clinically evident autonomic involvement with orthostatic hypotension; a change to a pathological score in 2 out of 3 tests anticipated clinical disautonomic signs in 1 and 11 weeks respectively. Clinical impairment showed to be reversible in both cases. Four pts presented only 1 pathological test (without any clinical sign or symptom of postural hypotension) and 6 patient had normal results. There was no apparent correlation between autonomic impairment and disease response to therapy or patients baseline characteristics (disease stage, Ig class, previous anti-myeloma therapies, concomitant thalidomide or anti-hypertensive treatment); furthermore, none had evidence of amyloidosis.

Conclusions

Our prospective study confirms that autonomic dysfunction is a potential adverse effect of bortezomib. Autonomic, non invasive, cardiovascular testing may reveal subclinical involvement and precede clinical evidence of disautonomic neuropathy. Further studies in larger cohorts of patients are needed to clarify the interactions between proteasome inhibitor activity and autonomic nervous system, in order to assess the frequency, the ability of available test in early pre-clinical diagnosis and the potential of the reversibility of neuropathic signs after drug discontinuation.
Conditioning with Treosulfan and Cyclophosphamide without Application of Antibodies in MHC Mismatch Transplantations in Mice.
Authors: Nogai Axel , Thiele Marc , Heimesaat Markus M. , Thiel Eckhard , Goebel Ulf B. , Uharek Lutz
Published: October 2019

BACKGROUND: Treosulfan is increasingly used in clinical conditioning regimens because of its myeloablative and immunosuppressive effects and the low hepatotoxicity compared to busulfane. The myeloablative and immunosuppressive characteristics of treosulfane was investigated in a murine MHC mismatch transplantation model.

METHODS: C57BL/10 (H-2Db) female mice were treated with treosulfan (2000 mg/kg) on day -3 to -1 and increasing doses of cyclophosphamide (without, 100 or 200mg/kg) at day -1, or 3000 mg/kg treosulfan with and without 200 mg/kg cyclophosphamide at day -1. Some mice were not transplanted to examine the myeloablative effect of the regimens, the rest of mice were transplanted with 10 × 10^6 marrow cells from Balb/c mice (H-2Dd) donors and monitored for donor-type chimerism determined by flow cytometry.

RESULTS: Mice treated with treosulfan and cyclophosphamid without transplantation died, indicating the myeloablative effect of the regimens. Ca. 50% of mice treated with 3000 mg/kg treosulfan and 200 mg/kg cyclophosphamide at day -1 died. Analysis of bone marrow cells in the remaining mice showed a complete rejection at day 55. Approximately 90% of the recipients of three daily doses of treosulfan and increasing doses of cyclophosphamide survived until the end of experiment (day +100). All hosts engrafted when 200 mg/kg cyclophosphamide was applied. 85% of mice engrafted after 100mg/kg cyclophosphamide. 100% of grafts were rejected in the group with treosulfan alone (2000 mg/kg).

CONCLUSION: A chemotherapeutic regimen for achieving a long term survival in a MHC-mismatch situation without additional need of antibodies was established in mice. While investigated for the practicability in an animal model as alternative to TBI, the high rate of engraftments in a MHC mismatch situation makes the chemotherapeutic regimen suitable for clinical situations.
Low-Dose Cyclosporin A Does Not Abrogate the Suppressive Function of In Vitro Expanded Human CD4+CD25+ Regulatory T Cells.
Authors: Piseshka Biserka , Eder Ruediger , Boeld Tina J. , Andreesen Reinhard , Holler Ernst , Hoffmann Petra , Edinger Matthias
Published: October 2019

Graft-versus-host disease (GVHD) is a major complication after allogeneic stem cell transplantation caused by donor T cells in the stem cell graft. Prophylactic treatment of GVHD usually comprises immunosuppressive medication including cyclosporin A (CsA) that blocks calcineurin phosphatase activity and thereby inhibits nuclear factor of activated T cells (NFAT)-mediated cytokine production. CD4+CD25+ natural regulatory T (Treg) cells have been shown to suppress the proliferation and cytokine secretion of conventional CD4+ and CD8+ T cells in vitro and to protect from GVHD lethality in murine models of allogeneic BMT. As the adoptive transfer of human CD4+CD25+ Treg cells as a means of GVHD prophylaxis is currently under investigation, we aimed to explore the influence of CsA on survival, cytokine production and suppressive activity of this T cell subpopulation. To this end, in vitro expanded human CD4+CD25high and CD4+CD25− T cells were stimulated with anti-CD3 and anti-CD28 antibodies and simultaneously exposed to various concentrations of CsA. While T cell death occurred in both in vitro expanded subpopulations at high CsA concentrations of 1000 ng/ml, with only 10 to 20% viable cells after 72h exposure, CD4+CD25high T cells showed significantly enhanced resistance to lower CsA concentrations of 100 ng/ml with still approx. 60% viable cells, as compared to only 20% viability in expanded CD4+CD25− T cells. At this lower concentration, CsA also blocked the proliferation of the two subpopulations in response to anti-CD3/CD28 stimulation, as determined by CFSE-dilution, and inhibited the IL-2 and IFN-γ production of CD4+CD25− T cells as well as the IL-10 production by expanded CD4+CD25high Treg cells, as shown by intracellular staining after stimulation with PMA/ionomycin. Addition of exogenous IL-2 protected both subpopulations from CsA-induced cell death and restored their proliferative capacity but was unable to abrogate the block in cytokine production. Surprisingly, the suppressive activity of in vitro expanded CD4+CD25high Treg cells was neither affected by a pre-incubation of the cells with 100 ng/ml CsA, nor by its presence during the suppression assay. These results indicate that in vitro expanded CD4+CD25+ Treg cells are less sensitive to CsA-induced apoptosis and that their yet unknown mechanism of suppression does not involve the calcineurin pathway.
Human CD4+CD25+CD127− T Cells Show Potent Dose-Dependent Inhibition of Allogeneic DC-Driven MLRs.
Authors: Protheroe Rachel E. , Steward Colin G. , Mazza Graziella , Wraith David C.
Published: October 2019

Graft versus host disease (GVHD) is the primary cause of transplant related morbidity and mortality, limiting the widespread application of haemopoietic stem cell transplantation (HSCT). Evidence from murine models supports the role of CD4+CD25+ regulatory T cells in the suppression of GVHD. Human evidence regarding the role of regulatory T cells in alloresponses is conflicting and may reflect the difficulty in defining and isolating the regulatory T cell population in humans. We have investigated the use of peripheral blood monocyte-derived dendritic cells (DCs) as stimulator cells in allogeneic mixed lymphocyte reactions (MLRs), as a means of assessing the in vitro suppressive function of regulatory T cells in human alloresponses. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adult volunteers. Magnetically isolated CD4+CD25+ T cells were combined with 50×103 autologous PBMCs or 20×103 autologous CD4+ T cells as responders, and 5×103 allogeneic irradiated DCs. Proliferation was assessed by tritiated thymidine incorporation. The CD4+CD25+ cells were anergic and demonstrated dose-dependent suppression of responder cell proliferation in the DC-driven allogeneic MLR. Greater than 50% suppression was seen with CD4+CD25+ T cells co-cultured with responder PBMCs at ratios of 1:4 to 1:32. Furthermore, depletion of CD4+CD25+ T cells from whole CD4+ responder cells resulted in enhanced proliferation and an increase in the amplitude of the MLR. Flow cytometry indicated that the majority of the magnetically isolated CD4+CD25+ T cells were FoxP3+ on intracellular staining and demonstrated down-regulation of cell surface expression of the IL-7 receptor (CD127). The potent suppression demonstrated here by CD4+CD25+CD127− T cells at ratios of 1:32 responder cells, suggests that these cells have a potential role for suppressing alloresponses at physiological levels. Moreover, this assay provides the basis for future investigation into regulatory T cell function in patients post-HSCT.
Decitabine Induces Responses in Patients with Myelodysplastic Syndrome (MDS) after Failure of Azacitidine Therapy.
Authors: Borthakur Gautam , Ravandi-Kashani Farhad , Cortes Jorge , Jabbour Elias , Faderl Stefan , O’Brien Susan , Verstovsek Srdan , Garcia-Manero Guillermo , Newman Beth Ann , Giles Francis , Issa Jean-Pierre , Kantarjian Hagop M.
Published: October 2019

Background

The hypomethylating agents azacitidine and decitabine are active in MDS and approved in the U.S.A. for this indication. The comparative efficacy of these two agents is not established. Responses to one drug after treatment failure with the other may offer evidence for superior efficacy.

Study Aims

Evaluate the efficacy of decitabine in MDS after failure of azacitidine therapy.

Study Group and Treatment

Adults with MDS who have progressed or failed to respond after at least three courses of azacitidine were eligible. Other entry criteria were standard including performance status 0–2, normal liver, kidney, and cardiac functions, and informed consent. Patients received decitabine 20 mg/m2 IV/D x 5 every four weeks. Response was evaluated by the modified IWG criteria (Cheson, Blood 2006).

Results

Fourteen patients have been treated to date. Their median age was 74 years (range 58–85 years). Patients had received prior azacitidine for a median of 4 courses (range 1 to 9); number of azacitidine courses 3 or more in 12 patients (86%); hemoglobin less than 10 g/dL in 7 (50%); thrombocytopenia < 100 x 109/L in 11 (78%); chromosomal abnormalities in 6 (43%); marrow or peripheral blast 5% or more in 13 (93%). Overall, 5 patients (36%) achieved IWG response: CR in 3 (21%), PR in 1 (7%), marrow CR +/− other hematologic improvements (HI) in 1 (7%). Improvement of thrombocytopenia was noted in 2/5 patients (40%) with pretreatment platelets < 50 x 109/L. Median remission duration was 5.3 months; median survival was 6.0 months. Extramedullary toxicities were minimal.

Conclusions

Decitabine is active in MDS following failure of azacitidine therapy. Comparative randomized studies of decitabine versus azacitidine in MDS may be indicated.
Enhancement of Dendritic Cell Therapy for Malignancy by Co-Activation of CD1d Restricted NKT Cells, NK Cells, and Antigen CTL.
Authors: Hagi Tomomi , Truong Vy , So Hang-Fai , Wang Qiao , Morley Jacqueline S. , Nieda Mie , Nicol Andrew
Published: October 2019

Human Vα24+ Natural Killer T (NKT) cells are cytotoxic against a range of malignancies, including human lymphoma and myeloid leukaemia in vitro and in murine models in vivo. Human NKT cells are activated by α-galactosylceramide (KRN7000) when presented by the MHC Class I-like molecule, CD1d, expressed by antigen presenting cells, particularly dendritic cells (DCs). Successful activation of human NKT cell antitumor activity in a clinical setting, particularly if this also activates natural killer (NK) cells and enhances tumor-specific cytotoxic T lymphocyte (CTL) activity, has the potential to be more potent and effective than current strategies focusing entirely on activation of peptide antigen specific CTL. In order to evaluate this we initiated a clinical study aiming to determine whether all three cell types could be activated with a single therapy. Melanoma was evaluated because of the known responsiveness to DC based immune therapy and the availability of well-defined peptide tumor antigens amenable to evaluation in a large number of subjects. Subjects with stage IV metastatic melanoma who were HLA-A*0201 were randomized to receive multiple intradermal or intravenous treatments with autologous mature monocyte derived DC pulsed with KRN7000, 4 peptide antigens (Mart-1, gp100, tyrosinase and Mage 3) or both. Peripheral blood NKT cells, NK cells and CD8+ CTL specific for the four melanoma antigens were monitored before, during, and after the series of treatments using 5 color flow cytometry. Antigen specific CTL were monitored using tetramers obtained from Beckman Coulter. Our results were that DC pulsed with KRN7000 + peptide were able to activate NKT cells and NK cells as well as increase the number of tumour antigen specific CTL. Activation of NKT and NK cells was most marked after intravenous treatment. However increased tumor antigen specific CTL were observed after treatment by both routes. Minor disease responses and stabilization were noted in subjects with non-bulky disease. We conclude that activation of NKT, NK and antigen specific CTL is possible with a single modality of immune therapy and that this is associated with disease responses.
Prospective Evaluation of Procalcitonin in Adults after Prolonged Neutropenia.
Authors: Cornillon Jerome J. , Bouteloup Marie M. , Lambert Claude C. , Devanlay Camille C. , Guyotat Denis D.
Published: October 2019

Infectious disease are persisting problem in patients with prolonged aplasia and a main cause to death. Determination of localization and germ remains difficult. The clinical presentation is poor and C Reactive Protein (CRP) is not specific. Treatment is empirical and uses broad spectrum antibiotic and antifungal therapy with sometimes moderate efficacy. Monotherapy or bitherapy in intial fewer are frequently discussed and no consensus exists. However, in sepsis, it is recommanded to use bitherapy in initial phase. Procalcitonin (PCT) is a pro-peptide of calcitonin produced mainly but not only in the C-cells of the thyroid glands. Several studies show PCT levels in plasma increase during infectious diseases in immunocompetent patients. In febrile neutropenia, data of PCT is scare and controversal. To determine the diagnostic and prognostic values of PCT, we assessed daily PCT level before and during the first febrile episode in 58 consecutive adults. Between November 2004 - April 2006, 32 patients received induction, or consolidation treatment for acute leukemia and 26 patients allogeneic or autologous hematopoietic stem cells transplantation. The mean duration of neutropenia was 17 days (range 5–43). Patients were treated with imipen only in first line except when clinical criteria of severity were presents (14 patients). Twenty-six percent had a clinically localized infectious (LI) which was documented in 14/15 patients, 45% had non documented fever and 29% bacteriemia. Twenty-one percent had a gram - bacillus and 29% gram+. No death was noted in this period. Mean duration of fever is 3.3 days (1–11). The means of CRP and PCT at day 0 and day 1 are presented in table 1. No difference exists in PCT before the first day of fewer (day -1). Not correlation was noted between different etiologies of fever and PCT or CRP. However, a trend of PCT values in bacteriemia was noted at day 1 (p=.07). The predictive value of PCT level at day 1 was calculated for bacteriemia. With a cut-off at 0.03, the negative predictive value was 78% (positive predictive value: 55%). In conclusions, neither PCT nor CRP levels can anticipate infections. Nevertheless, at day +1, PCT level < 0.03 could exclude the possibility of bacteriemia and therefore avoid the use of bitherapy before bacteriologic results. More patients are necesseray to confirm these preliminary results.

Table 1 PCT D−1 PCT H0 PCT D+1 CRP H0 CRP D+1 localized infections .11 .13 .17 49.33 94.14 SIRS .20 .23 .25 49.56 94.73 sepsis .15 .19 .95 52.69 104.54 Total .16 .19 .44 50.57 97.92
Split-Liver Transplantation in a Child with Liver Failure Based on Chronic Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation from the Same Donor.
Authors: Englert Cornelia Nicole , Ganschow Rainer , Beckmann Matthias , Janka-Schaub Gritta , Burdelski Martin
Published: October 2019

We report a case of a 6-year-old boy who developed chronic graft-versus-host-disease (GVHD) of the liver, the intestines and the skin following allogeneic hematopoietic stem cell transplantation.

The boy received allogeneic hematopoietic stem cell transplantation by the age of two years because of early recurrence of acute lypmphoblastic leukemia. The chimerism analysis showed complete chimerism. In the following year he developed GVHD despite adequate immunosuppressive therapy. Liver biopsy showed liver-GVHD resulting in liver cirrhosis by the age of five years.

Liver transplantation was performed with a split liver segment from the same unrelated donor as of the stem cells. Immunosuppressive therapy consisted of low dose steroids and low dose cyclosporine. The postoperative course was uneventful. Graft function was excellent and we performed protocol biopsies seven days and three weeks after transplantation which did not show any signs of rejection or GVHD.

To our knowledge this is the first report on a liver transplantation following allogeneic hematopoietic stem cell transplantation from the same unrelated donor.

In the latest liver biopsy three months after liver transplantation slight signs of GVHD could not be ruled out completely so that the low dose cyclosporine therapy is still continued.

Further biopsies will show if the immunosuppressive therapy can be discontinued.
Does Polyoma (BK) Virus Contribute to Development of Hemorrhagic Cystitis (HC) in Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation (UD HSCT) Recipients? A Prospective Evaluation.
Authors: Patah Poliana A. , Szewczyk Nicholas A. , Gilman Lisa , Gulbis Alison , Neumann Joyce , Walker Julia A. , Petropoulos Demetrios , El-Zimaity Maha , Anderlini Paolo , Tarrand Jeffrey , Champlin Richard E. , de Lima Marcos
Published: October 2019

BK virus infection is highly prevalent in humans, and has been associated with development of HC after HSCT. Previously we determined that UD HSCT is independently associated with higher prevalence of HC (El-Zimaity et al. Blood 2004). In order to further investigate the association of BK with HC, we hypothesized that patients who have positive urine PCR for BK virus before UD transplant have a higher incidence of HC.

Methods: we studied 62 consecutive patients transplanted from 09/05 to 05/06. Preparative regimens were ablative (n=26) or reduced intensity (n=36); 15 patients (23%) received cyclophosphamide-containing regimens. GVHD prophylaxis was tacrolimus and mini-methotrexate. Stem cell source was bone marrow (n=28), peripheral blood (n=25) or umbilical cord (n=9). BK virus quantitative PCR was performed on urine samples collected upon admission.

Results: Median age was 53 years (range, 19–67). Diagnoses were leukemias (n=36), multiple myeloma (n=2), Hodgkin’s disease (n=4), non-Hodgkin’s lymphomas (n=16), and other (n=4). Median time to platelet engraftment was 16 days (range, 0–62; n=51). Median follow-up is 97 days. BK PCR was positive in 28 patients (45%). Number of viral copies ranged from 300 to > 200 million copies. Eleven patients (18%) developed HC, at a median of 25 days after HSCT. HC was of grade 1 (n=1), grade 2 (n=4), grade 3 (n=5), grade 4 (n=1; required bilateral nephrostomies). In the PCR positive group, 7 patients (25%) had HC, versus 4 (12%) in the PCR-negative group (hazard ratio = 3.4 for a positive PCR; log-rank p = 0.057). 100-day cumulative incidence of HC is shown in the figure. 45% of CB recipients developed HC.

Incidence of HC was not statistically significantly increased among recipients of ablative or cyclophosphamide-containing regimens. Likewise, development of grade II-IV acute GVHD (n=18, 29%) was not associated with higher rates of HC. Six patients developed HC before platelet engraftment. Viral load did not correlate with development of HC. Four patients had viruria of 50–200million/mL; only one developed HC.

Conclusion: BK viruria pre-HSCT may be a risk factor for development of HC; further study is needed in a large cohort of patients.

Figure Figure
Adjustment of Immonosuppressants Based on T Lymphocyte Chimerism Following Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Wang Chun , Wan Liping , Yan Shike , Jiang Jieling , Yang Jun , Qin Youwen
Published: October 2019

Twenty-eight patients with a median age of 38 (15~54) years received allogeneic peripheral blood stem cell transplantation (allo-HSCT). Diagnoses included AML (n=7), ALL (n=2), CML (n=13), MM (n=4), MDS (n=1) and advanced ovarian cancer (n=1). Thirteen patients of them had progressive diseases. Myeloablative conditioning was given to 12 patients and non-myeloablative conditioning to 16 patients. FACS sorted granulocytes, T lymphocytes, B lymphocytes and NK cells from peripheral blood of all patients were analyzed for PCR amplification of short tandem repeats. The purity of sorted cells was above 96%. On day 7 posttransplant, the chimerism of NK cells was the highest in 88.5% patients, indicating that NK cells were the earliest engrafting cells. The subpopulations of granulocytes, B lymphocytes and NK cells reached complete chimerism (CC) on a median of day 14 posttransplant, while T lymphocytes were the latest subpopulation to reach CC in both myeloablative and non-myeloablative transplant on a median of day 21 and day 28 posttransplant, respectively. Seven patients with myeloid malignancy had molecular omen of graft rejection or disease relapse. Three patients of them had chimerism of donor T cells shifting from CC to MC only, while other subpopulation of cells remained CC. T cell chimerism in 2 patients of them remained lowest level among all subpopulation of cells. Recipient T cells had decreasing MC in 5 cases after discontinuance of immunosuppressant and donor lymphocyte infusion, 3 of them achieved CC again. Eight cases developed acute GVHD in 19 stable CC patients. Seven of them had GVHD after CC was achieved by all cell subsets. None of the patients in unstable CC group developed GVHD before they had graft rejection or relapse. The rate of acute GVHD of CC group was higher than MC group(χ2=4.25, P<0.05). We conclude that donor T lymphocytes are the earliest subpopulation of cells to subside during the course of graft rejection and disease relapse in myeloid malignancy. Patients responding to adjustment of immunosuppressants and/or DLI could achieve CC of T lymphocyte fraction again. CC of T lymphocyte could be a marker of stable engraftment of donor cells. Progressive MC of T cell is the omen of graft rejection and disease relapse. Immunosuppressant dosage should be adjusted according to T lymphocyte chimerism for different patients. GVHD prevention should be preferred in patients who reach CC in T cell fraction in one month after transplant. Therefore we suggest the patients might be given higher dose and longer period of immunosuppressants in 3 months posttransplant. In order to promote the engraftment of donor cell and avoid disease recurrence, the immunosuppressants might be tapered in the patients who do not reach CC in T cell fraction in one month after transplant and the patients with progressive MC in T cell.
Polymorphisms of MBL2, Encoding Mannan-Binding Lectin, Influences the Risk of Sepsis in Multiple Myeloma Patients during Autologous Stem Cell Transplantation (ASCT).
Authors: Molle Ingolf , Peterslund Niels A. , Steffensen Rudi , Thiel Steffen
Published: October 2019

Polymorphisms of various immune defence genes interfere with the risk of severe infections in critically ill patients. This includes studies on autologous and allogeneic stem cell transplantation (ASCT).

Mannan-binding lectin (MBL) is a plasma protein which is able to recognize carbohydrate structures forming pathogen associated molecular patterns on the surface of bacteria and viruses. When binding to pathogens occur the complement system is activated leading killing of the microorganisms. Lack of MBL may lower the ability to resist infections.

The gene MBL2, encoding mannan-binding lectin (MBL), contains three well-characterized single nucleotide polymorphisms B (G54D), C (G57E), and D (R52C) (collectively termed O in contrast to the wild-type A). The presence of O variants greatly reduces the effector functions of MBL.

We examined the impact of the polymorphisms on the occurrence of severe infections related to ASCT in patients with multiple myeloma. Patients were genotyped with PCR techniques using aliquots of peripheral stem cells from apheresis. Infectious complications were recorded retrospectively from clinical records and database extractions from the Department of Microbiology. One hundred and thirteen consecutive patients were studied. During ASCT 71 patients (63%) had fever above 38.5°C despite prophylactic antibiotics. Eleven (10%) patients had proven sepsis. MBL2 analyses: 4/71 patients with AA genotype had sepsis versus 7/41 with AO/OO genotype (p=0.09). Two lethal cases of sepsis were seen in the AO/OO patients, none in the AA patients. In multivariate analyses the risk of sepsis was significantly lower in AA patients: OR 0.15 (95% CI: 0.03–0.74), p=0.02. Wild-type MBL2, associated with a high function of mannose-binding lectin, probably reduces the risk of sepsis in myeloma patients during ASCT. Myeloma patients with variant MBL2 may be candidates for future MBL replacement trials.
Single Center Analysis of Risk Factors for Puberty Disorders in Children after Allogeneic Haematopoietic Stem Cell Transplantation (HSCT).
Authors: Wojcik Dorota M. , Barg Ewa , Niedzielska Ewa , Kalwak Krzysztof , Ussowicz Marek , Doroszko Adrian , Chybicka Alicja
Published: October 2019

Background

Preparative regimen consisting of high dose chemotherapy HDC/T and/or cranial irradiation (CI) in combination with prolonged steroid therapy may cause abnormal gonadal function.

Objective

Evaluate puberty disorders in children occurring after HSCT as single center experience.

Material and methods

The study group consisting of 21 patients (13 girls and 8 boys) age 10–20 years at grafting (median 14 years) was prospectively evaluated post grafting. The indications for HSCT were: ALL (5), AML (4), CML (5), MDS (2) and NHL, JMML, SAA, Blackfan Diamond anemia, RMS. According to donor availability 11 children had mached sib donors (MSD), 7 matched unrelated donors (MUD) and 3 patients HLA-mismatched related donors. The preparative regimens consisted of HDC/T usually BU/MEL (2pts); BU/CY/VP (5pts); BU/CY/ATG (4pts) and total body irradiation (TBI) received 3 patients with 12 Gy. CI prior to grafting was delivered to 5 children: 18 Gy (4) and 24 Gy (1). Prolonged high doses steroids (beyond day +28) received 7 children prior to- and 10 children after grafting. Endocrine function after allo-HSCT was evaluated from 10 to 42 (median 26 months). Analysis of LH, FSH, PRL, oestradiol and testosterone levels, fT3, fT4, aTPO, TRH test and LHRH test was performed in all study patients. Bone age according to Pyle-Grulich method was estimated.

Results:

Abnormal gonadal function was present in 12/21 children (9 girls, 3 boys) in average 28 months after HSCT Primary amenorrhea occurred in 3, secondary amenorrhea in 6 girls. Delayed bone age was documented in 2 children in average 30 mths post grafting. No significant correlation was found between children’s age during allo-HSCT and occurrence of puberty disorders. Gender of children had no effect on evalated variables. Time elapsing from HSCT did not influence the occurrence of abnormal gonadal function. No association was found between abnormal gonadal function and TBI, prolonged high doses steroids prior to- and after HSCT. Occurrence of puberty disorders was statistically significantly blunted by conditioning regimen containing Cyclophosphamide (p=0,019) and Busulfan (p=0,0179) while Melfalan did not turn out such a risk factor. PRL levels and thyroid function do not have any determination on occurrence of abnormal gonadal function after HSCT.

Conclusion:

Abnormal gonadal function as late effect frequently occure after HSCT. High-dose Cyclophoshamide and Busulphan tretaed children often had blunted puberty. Melfalan and TBI, however, did not cause such an effect reported by others. Endocrinological care for patients after HSCT is necessary in order to: - Discover the impaired endocrine function - Initiate correct hormonal substitution therapy - Significantly improve the quality of life after allo- HSCT and - Secondary prevent treatment related complications.
Campath-1H-Containing Reduced Intensity Regimen Followed by Unrelated Allogeneic Stem Cell Transplantation in Patients with Advanced Hematologic Malignancies.
Authors: Magalhaes-Silverma Margarida , Carter Thomas , Schlueter Annette , Gingrich Roger
Published: October 2019

BACKGROUND: Graft versus host disease (GVHD) remains a major cause of mortality after unrelated transplants. Reduced intensity conditioning regimens (RIC) followed by allogeneic stem cell transplantation (SCT) with in vivo T-cell depletion using Campath-1H have been shown to decrease the incidence of GVHD. We report our experience with such an approach in 19 patients (pts) with advanced hematologic malignacies who undergone unrelated SCT.

PATIENTS AND METHODS: Between October 2001 and June 2003, 19 pts with a median age of 56 years (range 18–70) not eligible for ablative SCT received Campath-1H 20 mg/m2, fludarabine 25 mg/m2 × 5 days and either cyclophosphamide 1 g/m2 × 2 days (n=16pts), melphalan 140mg/m2 (1 pt) or busulfan 0.8 mg/m2 × 8 doses (n=2pts) followed by G-CSF stimulated peripheral blood (n=13) or unmanipulated bone marrow (n=6). GVHD prophylaxis consisted of cyclosporin at least until T+60 and mycophenolate mofetil through T+30. DLI was allowed for residual disease/progressive disease or mixed chimerism after T+ 60, in the absence of acute GVHD. Seven pts had a previous autologous transplant. Ten pts had myeloid diseases (AML=6, CML Ph negative=1, CML accelerated phase =1, therapy related MDS=1, myelofibrosis=1); 9 pts lymphoid (HD=3, NHD=5, PLL=1); Only 5 pts were in CR at transplant. Six pts received bone marrow witha median CD34+ cells infused of 3.7 × 106/Kg, and 13 pts received peripheral blood with a median of 4.5 × 106/Kg CD34+ cells infused.

RESULTS:19 pts achieved ANC>0.5 × 106/L within a median of 12 days (range 8–16); 2 pts rejected the graft and 1 had autologous reconstitutuion; 10/19 pts achieved sustained platelet engraftment at a median of 16 days (range7–27). 2/17 pts with sustained engraftment developed acute GVHD (grade IV T+30 and grade III T+53); 7/14 at risk pts had CMV reactivation; TRM at T+100 was 16% (disease progression in 2 and GVHD in 1). Nine pts received DLI (5 for mixed chimerism and 4 pts for persistent/progressive disease). After DLI, 5 pts had GVHD limited to skin. None of the pts receiving DLI for persistent/progressive disease responded. Response to transplant was as follows: CR in 3/6 AML pts, CR in 1/1 pt with myelofibrosis, CR in 3/3 HD pts, CR in 3/5 NHL pts. Five pts (26%) remain alive in CR at T+ 36, 42, 50, 56 and 66 months. Causes of death include disease progression/relapse in 7 pts, CMV disease in 1 pt, sepsis in 2 pts, GVHD in 3 pts (2 underwent a second transplant), other malignancy in 1 pt.

CONCLUSION: These results confirm that unrelated SCT with in vivo T -cell depletion using Campath-1H is well tolerated. This approach is associated with low TRM even in pts with advanced disease, and some can achieve long-term disease control. Disease progression/relapse remains a major impediment to this approach.
Submyeloablative Cord Blood Transplant Corrects Clinical Defects Seen in IPEX Syndrome.
Authors: Lucas Kenneth G. , Comito Melanie A. , Ungar David R. , Groh Brandt P.
Published: October 2019

IPEX syndrome (immune dysfunction, polyendocrinopathy, enteropathy, X-linked) is a disorder of regulatory T cell function caused by mutations in the FOXP3 gene. This disorder is generally associated with a fatal outcome early in life from complications related to bleeding, infection, or enteritis. Allogeneic stem cell transplant (SCT) can be curative for this disorder, but pre-transplant disease-related complications may preclude a full myeloablative conditioning regimen. We report a seven year old boy with IPEX whose pre-transplant course was complicated by recurrent laryngeal papillomas and severe airway obstruction, frequent pulmonary infections, and recurrent gastrointestinal hemorrhage. Due to these problems he underwent a submyeloablative conditioning regimen consisting of fludarabine 30 mg/m2/day for 6 days, Busulfan 0.8 mg/kg/dose every 6 hours for 2 days, then anti-thymocyte globulin 2.5 mg/kg/dose for 4 days. Mycophenylate and cyclosporine were used for graft versus host disease (GVHD) prophylaxis. This patient received an HLA 5/6 matched (A,B,DR) unrelated donor cord blood transplant with 1.1 ×108 total nucleated cells/kg and 3 × 105 CD34+ cells/kg. His diarrhea and airway issues resolved following the conditioning regimen. The patient achieved myeloid engraftment on day 14, and was platelet and red cell transfusion independent by day 29 and 56, respectively. He had 81% and 98% donor chimerism at 2 and 9 months post-transplant, respectively. While tapering cylcosporine he developed graft versus host disease of the lower GI tract, which was sucessfully treated with a course of corticosteroids. He also experienced reactivation of EBV and was treated with 4 weekly doses (375 mg/m2) of anti-CD20 monoclonal antibody. This patient has had full resolution of his clinical symptoms and is currently 17 months post-transplant, with no signs of GVHD. For those children who do not respond or have only partial improvement following immunosuppressive therapy, allogeneic SCT using a submyeloablative approach can be well tolerated and result in sustained donor chimerism.
HSV ICP0 Inhibits TLR-Mediated NF-κB Response to TLR Signaling.
Authors: Tolba Khaled A. , Bowers William , Tan Yaohong , Daubeuf Sandrine , Federoff Howard J. , Rosenblatt Joseph D.
Published: October 2019

HSV infection activates a robust innate response through engagement of multiple pattern recognition receptors (PRR) including both TLR (TLR2 and TLR9) as well as non-TLR. Signaling events downstream of these receptors activate NF-κB and IRF3 responsive genes and initiate an innate inflammatory response aimed at controlling viral replication and spread. In this work, we studied immune suppressive activity of a replication-defective HSV virus and identified the immediate early protein ICP0 as a negative regulator of both NF-κB and IRF3 signaling. ICP0 possesses an ubiquitin E3 ligase function through its NH2 RING domain, as well as de-ubiquitinating activity through its association with the cellular de-ubiquitinating enzyme USP7 (HAUSP). We show that these two domains of ICP0 function independently to suppress IRF3 and NF-κB signaling, respectively and, in the process, effectively shut down host innate immunity to HSV infection. Although ICP0 inhibition of IRF3 has been reported, inhibition of TLR-mediated NF-κB response has not been previously described. We show that ICP0 globally inhibits NF-κB response to all TLR receptors as well as IL-1R. ICP0 exerts this activity by associating with USP7 and altering its cellular localization from a nuclear to cytoplasmic protein. In the cytosol, USP7 associates with and de-ubiquitinates TRAF6 and IKK-γ (NEMO), two signaling components of the TLR-mediated NF-κB pathway that are poly-ubiquitinated upon TLR activation. ICP-0 expression vectors harboring point mutations/deletions that target the RING domain E3 ligase function or compromise ICP-0 ability to bind USP7 would selectively inhibit its ability to interfere with either IRF3 or NF-kB signaling, respectively. In support of this, knockdown of endogenous USP7 by RNAi severely impaired ICP0-mediated inhibition of NF-κB response while leaving its capacity to inhibit IRF3 intact. In contrast, over-expression of USP-7 was sufficient to inhibit TLR-mediated NF-κB response. Ability of ICP-0 to inhibit both IRF3 and NF-κB signaling pathways, the former through its E3 ligase function and the latter through its association with USP-7, affords HSV comprehensive protection from host immunity during repeated cycles of lytic infection and reactivation from latency. The work also identifies a rare example of how two seemingly contradictory biologic functions resident within ICP0, namely ubiquitin E3 ligase activity at the NH2 terminus RING domain and de-ubiquitinating activity through association with USP-7, could cooperate to inhibit multiple signaling pathways necessary for efficient silencing of innate immunity. Finally, the work also identifies a previously unknown function for USP7 in regulating innate signaling beyond its known function as a regulator of p53.
The Prevalence of Anemia in Older Nursing Facility (NF) Residents with Chronic Kidney Disease (CKD).
Authors: Tangalos Eric G. , Erwin Gary , Stuart Bruce , Molotsky Sandra , Dybicz Sharon , Barlev Arie , Sciarra Angela , Audhya Paul
Published: October 2019

Anemia and CKD contribute to poor outcomes in the elderly. An understanding of chronic health conditions which may affect older NF residents, such as anemia and CKD, are important to providing optimal care to these residents. The objective of this study was to determine the proportion of NF residents who had CKD, anemia, and anemia associated with CKD, and to evaluate the use of anemia therapies in residents with CKD and Hb < 11 g/dL. ALTER (Anemia and associated outcomes in Long-TErm care Residents) was conducted in a representative sample of 24 NFs with Minimum Data Set (MDS) and prescription claims data available for the 1-year period before chart abstraction. Eligibility criteria included: ≥ 65 years of age, ≥ 1 hemoglobin (Hb) documentation, available records to estimate glomerular filtration rates (eGFR), not receiving dialysis, and not comatose. The first record to estimate GFR was used to analyze CKD status and the first subsequent Hb record was used to assess anemia status. CKD was conservatively defined as eGFR < 60mL/min/1.73m2 (MDRD equation). Anemia was defined according to the WHO criteria of < 13 g/dL for men and < 12 g/dL for women. Treatment was evaluated for residents with CKD and a Hb < 11g/dL (NKF/KFDOQI Guidelines, 2003). A total of 1501 residents were eligible: 80.5% were white, 75.1% were female and the mean (SD) age was 84.1 (8.1) years. Anemia was identified in 54.7% (784/1434) of the residents and CKD was identified in 45.7% (686/1501) of the residents. Both CKD and anemia were diagnosed in 31.8% (210/661) of the residents. Only 15.6% (26/210) and 7.2% (12/210) of residents with CKD and Hb < 11 g/dL received iron therapy and erythropoiesis-stimulating protein treatment, respectively. The prevalence of select co-morbid conditions, which may also be associated with anemia, in residents with CKD and Hb < 11 g/dL were: diabetes, 43.1%; heart failure, 43.1%; and chronic infection, 25.7%. In summary, anemia associated with CKD is highly prevalent in older NF residents. Conventional treatment to correct anemia in residents with CKD was uncommon.
Hyperglycemia in Patients with Acute Myeloid Leukemia Is Associated with Increased Hospital Mortality.
Authors: Ali Naeem A. , O’Brien James M. , Blum William , Klisovic Rebecca B. , Marcucci Guido , Phillips Gary , Marsh Clay B. , Lemeshow Stanley , Byrd John C. , Grever Michael R.
Published: October 2019

Patients with acute myeloid leukemia (AML) who receive therapy for their disease are at significant risk for infections. One risk factor for infections is hyperglycemia which can often be observed in medically ill patients. In post-operative critically ill patients, intensive insulin therapy has been shown to reduce the incidence of nosocomial infections. (Van den Berghe, 2001) In one study of patients with acute lymphoblastic leukemia, hyperglycemia during induction therapy was associated with more infections and shorter disease free survival. (Weiser, 2004) We hypothesized that hyperglycemia is associated with worse outcomes in AML due to increased incidence and severity of infectious complications. We performed a retrospective cohort study to determine if there was an association between hyperglycemia and hospital mortality in hospitalized patients with AML. Four-hundred and sixty-four adult patients were identified through a search of our administrative databases as having the diagnosis of acute myeloid leukemia over a three year period. These individuals experienced 1,004 admissions to the hospital. The occurrence of increasing hyperglycemia, defined as the proportion of glucose assessments above normal (>110 mg/dL), during all hospital days was associated with increased odds of hospital mortality [OR 1.24, 95% CI 1.13–1.36, p<0.001) after adjusting for co-variates like age, gender, diabetes and other co-morbidities. In addition, the odds of developing sepsis [OR 1.08, 95% CI 1.02–1.14, p=0.007], severe sepsis [OR 1.16, 95% CI 1.08–1.26, p<0.001), respiratory [OR 1.23, 95% CI 1.07–1.40, p=0.003] or renal failure [OR 1.14, 95% CI 1.01–1.30, p=0.04] were increased with increasing hyperglycemia. Because duration of neutropenia is known to increase the risk of infectious complications, we included leukopenia (WBC<1000) as a possible mediator in the risk-adjusting models. Independently, leukopenia was associated with the development of severe sepsis, however inclusion of this co-variate did not appreciably change the relationship between hyperglycemia and sepsis [OR 1.07, 95% CI 1.01–1.16, p=0.01], severe sepsis [OR 1.18, 95% CI 1.09–1.28, p<0.001] or mortality [OR 1.24, 95% CI 1.12–1.35, p<0.001]. Finally, after adjusting for the diagnosis of severe sepsis, hyperglycemia remained significantly associated with mortality, but the point estimate was reduced (OR from 1.24 to 1.16) suggesting that the association between abnormal glucose regulation and hospital mortality may be partially mediated by the development of severe sepsis. Based on these findings we are performing a more formal analysis of the relationship between hyperglycemia and the morbidity and mortality associated with treatment of patients with AML in a prospective study.
Optimising Haemoglobin Response to Exogenous Erythropoietin in Anaemic Patients Treated in Routine Haemato-Oncology Practice.
Authors: Agrawal Samir G. , Greaves Paul , Lim Christina , Poole Chris D. , Walker Mel D.
Published: October 2019

Patients’ response to treatment with human recombinant erythropoietin (Epo) for chemotherapy-associated anaemia varies between 50% and 70%. The cost-effectiveness of Epo may be maximized by targeting treatment at patients most likely to respond. In this study, modified ASH/ASCO guidelines were combined with a systematic approach to iron supplementation in order to maximize haemoglobin (Hb) response in iron deficient patients. Patients with haematological malignancy and chemotherapy-associated anaemia received Epo alone (n = 25) or Epo plus intravenous iron sucrose (iv iron) if functional iron deficiency was present (n=24). The cohort had a male/female ratio of 48:52% and a mean age 62.8 years (SD 12.8; range 24 to 81). Baseline measurements were repeated at 4-weekly intervals for a period of 32 weeks. Overall, 80% of cases experienced a Hb response greater than 1g/dl, with 65% of patients achieving a major Hb response (≥ 2g/dl increase above baseline). There was no difference in Hb response between the Epo alone vs. Epo plus iv iron groups (∅ 2.72 vs. 2.84 g/dl; p=0.831), indicating equivalent outcomes for functionally iron deficient patients treated with effective iv iron supplementation and iron replete patients. General Linear Modelling was used to explore the independent contributions of demographics, baseline iron parameters, and disease progression in predicting Hb response to Epo. In the final model three independent variables emerged as significant, which together accounted for 40% of variance in Hb response. Progression of the underlying malignancy was associated with a mean reduction in Hb response of 1.9g/dl (p<0.001). Lower baseline Hb was associated with an increased response - each 1g/dl decrement was associated with a mean increased response of 0.87g/dl (p=0.012). Early change in reticulocyte count (between baseline and early follow up (< 4 weeks) was found to be positively associated with Hb response - each additional 1% change was associated with a 0.016g/dl increase in Hb response (p=0.006). These findings are in contrast to previous reports that the response to Epo was

independent of the outcome of the underlying malignancy and diminished at lower Hb levels.

Disease progression, baseline Hb and early increases in reticulocytes were all highly significantly correlated with the Hb response to Epo. In fact, in this cohort, all cases of non-response to Epo or loss of a response were associated with disease progression. To maximise response rates to Epo requires detection of functional iron deficiency and supplementation with iv iron. Furthermore, early reticulocyte response and disease progression should be closely followed and can help guide when to stop Epo therapy. This would allow rational use of resources and improve the health economics of Epo therapy by focusing on those most likely to benefit.