bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting of mTOR in AML Is Associated with Decreased Growth of Leukemic Cells and Downregulation of VEGF.
Authors: Boehm Alexandra , Aichberger Karl J. , Mayerhofer Matthias , Krauth Maria-Theresa , Derdak Sophia , Pickl Winfried F. , Samorapoompichit Puchit , Sillaber Christian , Florian Stefan , Sonneck Karoline , Sperr Wolfgang R. , Valent Peter
Published: April 2020

The mammalian target of rapamycin (mTOR), a key-regulator of cell cycle progression, has recently been implicated in growth of neoplastic cells and tumor- and leukemia-associated angiogenesis. In addition, mTOR has been described as a regulator of expression of vascular endothelial growth factor (VEGF), a cytokine involved in tumor-related angiogenesis, in various neoplasms. We asked whether mTOR can be employed as a new therapeutic target in acute myeloid leukemia (AML) using the mTOR-targeting drug rapamycin and its derivatives RAD001 (everolimus) and CCI-779. As assessed by 3H-thymidine incorporation, rapamycin was found to counteract growth of AML cells in vitro in 9/11 patients examined as well as in the AML-derived cell lines U937, HL60, and KG1a. The effects of rapamycin on growth of AML cells were dose-dependent (primary AML cells: IC50: 10 pM - 1 nM; inhibition of 3H-thymidine uptake at 1 nM of rapamycin: 43±15 % of control; p<0.05) and associated with G1 cell cycle arrest as well as apoptosis. The same results were obtained with RAD001 and CCI-779. In all responsive cells analyzed, rapamycin and its derivatives decreased expression of VEGF in AML cells at the mRNA- and protein level. Exogenously added VEGF was found to revert the rapamycin-induced inhibition of growth in HL60 cells, but did not revert rapamycin effects in KG1a cells, U937 cells, or primary AML cells. In summary, our data suggest that rapamycin and its derivatives act antileukemic in AML cells by two distinct mechanisms, namely direct inhibition of cell growth and suppression of proangiogenic VEGF. mTOR may be a new interesting target in AML.
Nucleophosmin Protects Hematopoietic Cells from Stress-Induced Apoptosis through Inhibition of P53.
Authors: Pang Qishen , Li June , Zhang Xiaoling , Sejas Daniel P. , Bagby Grover C.
Published: April 2020

Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells including tumor and hematopoietic stem cells. Here we report that NPM protects hematopoietic cells from stress-induced apoptosis through inhibition of the tumor suppressor p53. Specifically, we forced expression of wild-type (WT) NPM or a mutant variant with a deletion of the C-terminal 120 aa of NPM (NPMΔC) by retroviral gene transfer in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid cell line MO7e (expresses low level of NPM) and the lymphoblast HSC536 cells derived from a Fanconi anemia (FA) patient in the C complementation group (expresses essentially undetectable NPM). Using a flow cytometric method for caspase 3 activation (early apoptosis), we demonstrated that overexpression of NPM but not the mutant NPMΔC confers MO7e and HSC536 cells resistance to apoptosis induced by growth factor deprivation and treatment with the chemotherapeutic drug etoposide. In addition, suppression of NPM expression by small interfering RNA targeting NPM in chronic myelogenous leukemia line K562 and FA-associated acute myelogenous leukemia cell line UoC-M1 increases etoposide-induced apoptosis, thus providing proof of concept evidence that the pathological elevations of NPM found in cancers and leukemias are important for maintaining cell survival and resistance to apoptosis. Because overexpression of the mutant NPMΔC, which lacks the p53-interacting domain, fails to confer cellular resistance to stress-induced apoptosis, we determined whether NPM protects cells from apoptotic cell death through a mechanism involving p53. We used the genetically matched p53 WT and null mouse bone marrow (BM) cells to show that overexpression of WT NPM protects against ionizing irradiation (IR)-induced apoptosis of wild-type but not p53-null BM cells. Moreover, NPM inhibits IR-induced p53 phosphorylation at Ser18 and transactivation, and interacts with p53 in bone marrow hematopoietic cells. Thus, this study not only demonstrates anti-apoptotic function of a proliferation-promoting protein but also suggests that cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival.
Forced Overexpression of Ahi-1 Confers a Proliferative Advantage on Primitive Murine Hematopoietic Cells and Enhances the Effects of BCR-ABL.
Authors: Zhou Leon , Ringrose Ashley , Jiang Xiaoyan
Published: April 2020

Ahi-1 (Abelson helper integration site-1) is a novel gene that is commonly activated by proviral insertional mutagenesis in v-abl or myc-induced murine leukemias and lymphomas. Ahi-1 encodes a unique protein with known signaling features including SH3 and WD40-repeat domains but its normal function is unknown. Involvement of Ahi-1 in leukemogenesis is suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias and by the gross perturbations seen in the expression of human AHI-1 and its isoforms in several human leukemic cell lines, as well as in the primary lin−CD34+CD38− leukemic stem cell-enriched population in patients with chronic phase CML. To further investigate the role of Ahi-1 as a potential co-operating oncogene relevant to BCR-ABL-mediated leukemogenesis, we compared the biological behavior of primitive murine hematopoietic cells from the bone marrow of 5-FU-treated adult C57BL/6 mice after their transduction with MSCV-Ahi-1-IRES-YFP, MSCV-BCR-ABL-IRES-GFP retroviruses, either alone or in combination. Quantitative real-time RT-PCR analysis of RNA from FACS-purified lin−YFP+ (Ahi-1+), lin−GFP+ (BCR-ABL+) and lin−YFP+GFP+ (Ahi-1+ + BCR-ABL+)-transduced bone marrow cells showed that Ahi-1 transcripts were present at 40-fold higher levels in the Ahi-1-transduced cells by comparison to the control cells transduced with the empty MIY vector. Immediately post-transduction, the Ahi-1-transduced cells produced a similar number of colonies as the MIY-transduced control cells in semi-solid cultures containing Steel factor (SF) + IL-3 + IL-6 + EPO, although a small proportion of the Ahi-1+ CFCs (~10%) were already growth factor-independent. In addition, the proliferative activity of the FACS-purified lin−YFP+ (Ahi-1+) cells (as indicated by the rate of expansion of viable cells in a week in liquid cultures containing SF, IL-3, and IL-6) was ~3-fold higher than in cultures initiated with control (lin−YFP+) cells. Moreover, after 4 weeks in longterm culture-initiating cell (LTC-IC) assays, the Ahi-1+ cells produced 2x more CFCs than the control cells. All of these endpoints (proliferative activity, growth factor-dependence and CFC output in LTC-IC assays) are also perturbed by BCR-ABL transduction. Interestingly, in cells that were co-transduced with Ahi-1 and BCR-ABL, all of these effects were further enhanced as compared to cells transduced with either BCR-ABL alone (2–4-fold) or Ahi-1 alone (3–6-fold). Thus, overexpression of Ahi-1 alone deregulates the proliferation control of primitive murine hematopoietic cells and this is additive with the effects of BCR-ABL, suggesting that Ahi-1 and BCR-ABL can cooperate to promote the progression of BCR-ABL-associated diseases like CML.
A Multicentre Study on 100 Cases with Thrombotic Thrombocytopenic Purpura: The Role of Adamts-13 Activity and Anti-Adamts-13 Antibodies.
Authors: Peyvandi Flora , Ferrari Silvia , Lavoretano Silvia , Canciani Maria T. , Mannucci Pier M.
Published: April 2020

Background: The metalloprotease ADAMTS-13 disposes physiologically of the unusually large molecular forms of the von Willebrand factor (VWF), that have the property to aggregate platelets in the microcirculation in the presence of high shear forces. The congenital or acquired deficiency of ADAMTS-13 has been specifically associated with a diagnosis of thrombotic thrombocytopenic purpura (TTP), a rare microangiopathy characterised by the massive formation of occlusive platelet thrombi in the microcirculation.

Aim of the study: To investigate the pathogenic mechanism of TTP in 100 patients referred from 6 different countries and diagnosed on the basis of the presence of at least 3 of the followings laboratory abnormalities or clinical signs: thrombocytopenia, hemolytic anemia, elevated serum level of lactadehydrogenase and neurological symptoms compatible with focal ischemia.

Materials and methods: ADAMTS-13 levels were measured in all patients’ plasma by Collagen Binding assay as previously described (Gerritsen et al., Thromb Haemost 1999); anti-ADAMTS-13 neutralizing antibodies was measured only in patients with low ADAMTS-13 levels, whereas candidate gene mutations were searched only in patients who had low ADAMTS-13 levels (less than 20%) not explained by detectable anti-ADAMTS-13 antibodies, whether or not there was a family history of TTP.

Results: Plasma levels of ADAMTS-13 were severely reduced (<10% of normal) in 48% of the cases, moderately reduced (between 10 and 46%) in 24% and normal (>46%) in 28%. An antibody was identified as the cause of the deficiency in 38% the cases: the great majority of the patients with detectable inhibitor (87%) had severe ADAMTS-13 deficiency but 5 (13%) had moderately reduced ADAMTS-13 activity (ranging from 11 to 25%). Mutation analysis of the ADAMTS13 gene, carried on 15 patients, revealed a genetic variant in only two patients born from consanguineous marriages. The first case was a double heterozygosity for a 29 base pair deletion in exon 3 (291–319del) and a single nucleotide insertion in exon 29 (4143insA). The 290–319del and 4143insA mutations lead to premature stops at codons 368 and 1387 respectively. The second one consisted in a homozygosity for a 6 base pair deletion in exon 23 (2930–2935del). This deletion leads to a substitution of Cys 977 by Trp, and a deletion of 2 aminoacids, Ala and Arg, at residues 978 and 979 respectively.

Conclusion: The study of this large series of patients with TTP indicates that in nearly one third of patients ADAMTS-13 levels were normal. In more than half of the patients with low plasma level of the protease, ADAMTS-13 deficiency was not due to the presence of inactivating antibodies or gene abnormalities. An unexpected observation was that an inhibitor was also found in 5 patients with moderately reduced levels of ADAMTS-13.
A Meta-Analysis to Determine the Risk of Heparin Induced Thrombocytopenia (HIT) and Isolated Thrombocytopenia in Prophylaxis Studies Comparing Unfractioneted Heparin (UFH) and Low Molecular Weight Heparin (LMWH).
Authors: Martel Nadine , Wells Philip S.
Published: April 2020

HIT is an uncommon but potentially devastating complication of anticoagulation with UFH or LMWH. The absolute risk of HIT and thrombocytopenia are not clearly defined and no summary data to provide odds ratio is available. We conducted a meta-analysis to determine and compare the incidences of HIT in surgical or medical patients receiving thromboprophylaxis with either UFH or LMWH. We searched MEDLINE-OVID and MEDLINE-PubMed using and combining the following terms: heparin induced thrombocytopenia, low molecular weight heparin, prophylaxis, randomized controlled trials, prospective studies. The function Explode was used. Search was limited to humans from 1984 to 2004. Over 400 abstracts were reviewed and then 91 articles were independently reviewed by two authors, without any restriction of article language. Included studies were those comparing prophylactic UFH and LMWH and measuring HIT (defined as platelets drop > 50% or < 100 X 109/L AND positive laboratory HIT assay) or thrombocytopenia (defined as platelets drop > 50% or < 100 X 109/L) as outcomes. Studies defining thrombocytopenia with lower thresholds were excluded because cases could have been missed. Extracted data included patient characteristics, drug regimens, HIT, thrombocytopenia and venous thromboembolism rates. Disagreements were resolved by consensus. Eligible studies were included into the meta-analysis using a random-effects model to determine the odds ratio for the incidences of HIT and thrombocytopenia between UFH and LMWH. Funnel plots were made to assess possible publication bias. 17 articles were eligible with a total of 8500 patients: 2 RCTs measuring HIT; 10 RCTs measuring thrombocytopenia, and 5 prospective non-randomized studies with comparison groups measuring HIT. Three analysis were performed and all favoured the use of LMWH: 1) 2 RCTs measuring HIT showed an OR of 0.10 (95% confidence interval [CI] 0.01–0.77; p=0.03); 2) all 7 studies measuring HIT showed an OR of 0.11 (95%CI= 0.05–0.26; p< 0.00001); 3) 12 RCTs measuring thrombocytopenia showed an OR of 0.45 (95% CI= 0.26–0.80; p=0.006). Comparing the rates in the 7 studies measuring HIT UFH resulted in HIT in 3.4% (95%CI=2.6% to 4.3%) of cases and LMWH resulted in HIT in 0.2% (95% CI=0.1% to 0.6%), a statistically significant difference (p<0.0001). This meta-analysis confirms the lower incidences of HIT and thrombocytopenia with LMWH prophylaxis compared to UFH. Absolute rates of HIT with LMWH are very low. The HIT rates should be considered when determining the drug of choice for thromboprophylaxis in surgical and medical patients.
The Association between Tissue Factor-Exposing Microparticles and the Activation of the Coagulation System in Cancer Patients.
Authors: Weltermann Ansgar , Hron Gregor , Kollars Marietta , Kornek Gabriela , Quehenberger Peter , Eichinger Sabine , Kyrle Paul Alexander
Published: April 2020

Background: Malignancy is a major risk factor of venous thromboembolism. In addition to circumstantial factors such as chemotherapy, the pathogenesis of thrombosis in cancer patients is directly influenced by tumor related factors. In this respect, the expression of tissue factor (TF) on tumor cells is considered to play an important role. Besides its cellular expression, more recent studies focused on TF exposure on circulating microparticles. The role of these TF exposing microparticles with regard to the pathogenesis of VTE in cancer patients, however, has not yet been investigated. We therefore conducted a prospective case control study in order to determine (a) the number and cellular origin of TF exposing microparticles in cancer patients and (b) the association between these microparticles and the activation of the coagulation system.

Materials and Methods: A total of 20 patients with advanced colorectal cancer (Dukes D) and 20 healthy, age- and sex-matched controls were included. Microparticles were isolated from plasma, stained with annexin V, cell-specific antibodies and analysed by flow cytometry. The activation of the coagulation system was measured by D-dimer (ELISA).

Results: Compared to the controls, the total number of TF exposing microparticles was significantly higher in the patient group (32.5 ± 23.9 vs. 18.1 ± 10.0 k/ml plasma, p = 0.007). These finding was due to a higher number of TF exposing microparticles originating from thrombocytes (12.9 ± 7.9 vs. 6.5 ± 2.9 k/ml plasma, p = 0.017) and from monocytes (9.1 ± 6.5 vs. 5.5 ± 1.4 k/ml plasma, p = 0.11) in cancer patients. Likewise, D-Dimer levels were higher among cancer patients as compared to the controls (1.61 ± 1.58 vs. 0.40 ± 0.36 μg/ml, p = 0.001). The number of TF exposing microparticles correlated with D-dimer (r = 0.67, p < 0.001).

Conclusion: Both the significant elevation of TF exposing microparticles in the circulation of cancer patients and the association between these microparticles and the activation of the coagulation system suggest a potential role of TF exposing microparticles in the pathogenesis of venous thromboembolism in cancer patients.
Role of Congenital and Acquired Thrombophilic Factors in the Failure of Thrombolysis in Patients with Acute Myocardial Infarction.
Authors: Fossati Carolina , Patti Giuseppe , Pasceri Vincenzo , Giannetti Barbara , Nusca Annunziata , Mega Simona , Annibali Ombretta , Di Sciascio Germano , Avvisati Giuseppe
Published: April 2020

In patients with acute myocardial infarction, a persistently occluded infarct related coronary artery, despite a correct thrombolysis, is associated with an unfavourable prognosis. Therefore, identification of variables predictive of ineffective thrombolysis is crucial to identify patients at higher risk of thombolysis failure. To investigate whether or not acquired or congenital thrombophilic factors had a role in the ineffective thrombolysis we designed this study in which patients treated with intravenous thrombolysis for a ST segment elevation myocardial infarction were blind tested for the thrombophilic factors on the occasion of the coronary angiography performed within 30 days from the thrombolytic treatment. Patients with known factors influencing metabolism and circulating levels of homocysteine were excluded from this study. From October 2003 to May 2004, 104 consecutive patients treated with intravenous thrombolysis for a ST segment elevation myocardial infarction were available for this study, 3of these refused to participate in the study All patients underwent,within 30 days from thrombolysis, a coronary angiography and of the 101 participating in the study, 40 resulted occluded while 61 had a patent artery. In these 101 patients we blind tested the levels of ATIII,PC,PS; moreover, we determined also the levels of homocysteine, the presence of Lupus Anticoagulant (by mean of DRVVT and Silica Clotting Time) and ACA of IgG type as well as the Plasminogen levels. Furthermore, blood samples were also analysed by PCR technique for the presence of Factor V Leiden, the G20210A Factor II mutation and the C677T mutation in the MTHFR gene. Surprisingly, patients with MTHFR 677TT homozygosis had a significantly higher prevalence of occluded infarct artery (73%) vs those with MTHFR 677CT/CC genotype (30%, P=0.0008); frequency of MTHFR 677TT homozygosis was 4-fold higher in patients with occluded vs those with a patent vessel (40% vs 10%, P=0.0008). MTHFR 677TT genotype predicted the risk of failed thrombolysis with a specificity of 90% and multivariate analysis showed that MTHFR 677TT homozygosis was independently associated with an occluded artery (odds ratio 3.8, 95% confidence interval 1.1–9.1; P=0.03). None of the other studied factors at multivariate analysis influenced the thrombolysis failure. Moreover, patients with occluded infarct vessel and MTHFR 677TT genotype had the highest homocysteine levels (P=0.011). Our findings indicate that in patients with acute myocardial infarction, MTHFR 677TT homozygous is independently associated with a persistently occluded infarct-related artery after thrombolysis.
Efficacy and Safety of Yttrium 90 (90Y) Ibritumomab Tiuxetan (Zevalin®) Therapy with Rituximab Maintenance in Patients with Untreated Low-Grade Follicular Lymphoma.
Authors: Sweetenham John William , Dicke Karel , Arcaroli John , Kogel Karen , Rana Tahir M. , Rice Laura L.
Published: April 2020

Background: Recent trials have demonstrated the efficacy of rituximab induction therapy, when followed by maintenance rituximab, in patients with untreated low-grade follicular NHL. Earlier studies, employing low-dose total-body irradiation as an initial treatment for follicular lymphoma (FL), have previously shown the inherent radiosensitivity of the disease. Based on these findings, we evaluated the efficacy and safety profile of frontline 90Y ibritumomab tiuxetan (Zevalin®) delivered prior to maintenance rituximab in patients with low-grade FL.

Methods: Patients were eligible for treatment if they presented with histologically confirmed, previously untreated low-grade FL. Additional eligibility criteria included stage III or IV disease and a need for therapeutic intervention because of constitutional symptoms, evidence of disease progression, or organ dysfunction directly attributable to NHL. Patients were excluded if there was evidence of histologic transformation to aggressive disease. On day 1, patients received an initial infusion of rituximab (250 mg/m2) followed by an imaging dose of Indium 111 (111In) ibritumomab tiuxetan (5 mCi). One week later, a second infusion of rituximab (250 mg/m2) was given, proceeded by an injection of 90Y ibritumomab tiuxetan (0.3 or 0.4 mCi/kg, depending upon platelet count). Rituximab maintenance therapy (375 mg/m2 x 4) was scheduled at 6-month intervals over 2 years. Endpoints included clinical response rates using standard International Workshop Response Criteria (IWRC) and toxicity.

Results: Ten patients, presenting with grade I or II FL, have been treated with 90Y ibritumomab tiuxetan, and 8 are evaluable for treatment response. The median age of patients was 58 years (range, 40–82) and 50% (5/10) of patients had stage IV disease. Delivered doses of 90Y ibritumomab tiuxetan ranged from 21–32 mCi. Toxicities were primarily hematologic with grade 3 cytopenia occurring in 38% (3/8) of patients. Of the 8 patients qualifying for evaluation, 5 (62%) had complete responses and 3 (38%) had partial responses to 90Y ibritumomab tiuxetan induction.

Conclusions: These data illustrate that frontline 90Y ibritumomab tiuxetan therapy produces high rates of response (100% in this small population) in patients with low-grade FL. Toxicities following administration of radiolabeled antibody were manageable. Further follow-up is needed to determine the long-term efficacy of combining frontline radioimmunotherapy with antibody-based maintenance.
SGN-30 (Anti-CD30 Monoclonal Antibody) Is Active and Well Tolerated in Patients with Refractory or Recurrent Systemic Anaplastic Large Cell Lymphoma.
Authors: Forero-Torres Andres , Bernstein Steven , Gopal Ajay , Foss Francine , Leonard John , Rosenblatt Joseph , Bartlett Nancy , Younes Anas , Fisher David , Cheson Bruce , Carabasi Matthew , Bociek Gregory , Hart Bruce , McDonald Michael
Published: April 2020

SGN-30 is a chimeric mAb which recognizes the CD30 antigen found on tumor cells from patients (pts) with Hodgkin’s disease (HD) and anaplastic large cell lymphoma (ALCL). In preclinical experiments, SGN-30 was shown to have antitumor activity in both in vitro and in vivo models of HD and ALCL. The results of a multi-dose phase I study showed minimal toxicity associated with doses of 2–12 mg/kg as six weekly IV infusions. One complete response (CR) was seen in the three non-Hodgkin’s lymphoma patients (2 ALCL, 1 diffuse large B-cell lymphoma) accrued to the study and two patients demonstrated stable disease (SD) over time. A phase II multi-dose study was initiated to further evaluate the safety, antitumor activity and pharmacokinetics of six weekly IV infusions of 6 mg/kg of SGN-30 in pts with relapsed or refractory HD or systemic ALCL (sALCL). Five patients (2M, 3F) with ALCL have been enrolled, with a median age of 52 (range 33–75) and 3 median prior therapies (range 2–5). Multiple doses of SGN-30 have been well tolerated in all of the pts. Drug-related adverse events have been typically mild and consistent with mAb administration. No drug-related grade 3/4 events have been observed. Two patients have had objective responses with one patient achieving a CR. The patient’s baseline CT scan showed a 5.1 x 2.0 cm chest wall mass in the lower left anterior lateral chest wall representing local lymphoma recurrence. After six doses of SGN-30 the mass disappeared completely with some edema in the chest wall but no obvious residual mass. The duration of response is pending at this time, and it will be presented in the meeting. Another patient experienced a partial response. The patient, who entered the study with constitutional symptoms and extensive cutaneous and nodal disease, had resolution of all skin lesions, significant reduction in adenopathy and improvement in constitutional symptoms after completion of her SGN-30 therapy. The patient progressed after discontinuation of therapy. Of the other three patients, one had SD and two progressed. These early results are promising and accrual to the trial continues.
Intermediate-Dose Interleukin-2 Demonstrates Activity in Patients with Relapsed or Refractory Cutaneous T-Cell Lymphoma.
Authors: Foss Francine M. , Higgins Beth
Published: April 2020

Interleukin-2 (IL-2) has demonstrated activity as an antineoplastic and biomodulatory agent, but its activity in hematologic malignancies has not been fully defined. Cutaneous T-cell lymphoma (CTCL) has been characterized as a disorder of mature clonal CD4+CD7− cells with a predominantly Th2 cytokine profile and a blunted response to Th1 and Fas-associated apoptosis. Durable responses have been reported in several advanced CTCL patients in prior studies using high dose IL-2, but the capacity of IL-2 as an immune modulator to upregulate Th1 cytokine secretion and to facilitate a cytotoxic T-cell response in CTCL has not been studied. To further explore the activity of IL-2 to restore a Th1 phenotype and to induce an antitumor response in CTCL, we treated 11 patients with advanced or refractory CTCL with intermediate dose IL-2 (11 MIU subcutaneously daily x 4 days for 6 weeks with 2 weeks rest). The median age was 60 (range 55–72) among 6 females and 5 males. Disease was stage IB-IIA in 4, IIB in 2, III-IVB in 5, and 7 had circulating Sezary cells. Four had evidence of significant lymphadenopathy on CT scans. The mean prior therapies was 3, with 4 patients having received 3 or more systemic cytotoxic therapies. Five patients had prior electron beam irradiation, and 5 had prior biological therapies, including IFN in 3, bexarotene in 4, and interleukin-12 in one. The median cycles administered was 2 (range 1–8). Two patients were discontinued for toxicity, one with grade 3 constitutional symptoms and one who had severe underlying cardiovascular disease and developed bilateral lower extremity deep venous thromboses. Overall, therapy was well tolerated with grade 3 fatigue in 3 patients and grade 2 eosinophilia in one. Response data includes: 4 patients with partial response, including 3 with Sezary syndrome and one with diffuse plaque stage disease; 3 patients with stable disease; 2 patients with progressive disease; 2 patients discontinued before completing one cycle and were inevaluable for response. One patient with diffuse pruritis, erythroderma and circulating Sezary cells completed 8 cycles of therapy and had a complete response in the skin but persistence of a low number of Sezary cells in the blood. Immunophenotypic analysis of circulating lymphocytes revealed an overall increase in CD4+CD25+ cells during therapy, suggesting a direct immunomodulatory effect of IL-2. In summary, our experience with intermediate dose IL-2 demonstrates that the therapy was well tolerated and was associated with biological activity in patients with extensive and refractory CTCL, with responses noted in 3 of 7 patients with Sezary syndrome. Based on our results, we intent to explore the biomodulatory effects of IL-2 on both normal T-cells and Sezary cells to attempt to define the role of IL-2 as a single agent or in combination with other biological agents in patients with CTCL.
Results of a Phase II Trial of Oral Bexarotene Combined with Interferon Alfa-2b for Patients with Cutaneous T-Cell Lymphoma (CTCL).
Authors: Straus David J. , Duvic Madeleine , Kuzel Timothy , Horwitz Steven , Demierre Marie-France , Myskowski Patricia , Maignan Kathleen , Steckel Steven
Published: April 2020

Bexarotene, a synthetic retinoid ligand for the retinoid X receptor (RXR), is a highly active agent in the treatment of relapsed or refractory CTCL with a response rate of ~45% (Arch Dermatol137: 581–93, 2001; J Clin Oncol19: 2456–71, 2001). Interferon alfa has a similar response rate although the doses and responses in the literature have varied. Promising results have been reported with combinations of retinoids and interferon alfa, including bexarotene (J Am Acad Dermatol50: 375–9, 2004). To explore this combination, a phase II trial was designed in which patients with CTCL were treated with bexarotene 300 mg/m2/day for at least 8 weeks. If less than a complete response was seen at this time, interferon alfa-2b at a dose of 3 million units escalated to 5 million units subcutaneously 3 times weekly was added for another 8 weeks. Responding patients could continue treatment beyond 16 weeks. Twenty-two patients with CTCL were enrolled in the trial: median age 58 years (22–79); 9 males, 13 females, TNM stages: IB (3), II (7), III (4), IV (8). One patient had no prior treatment; the rest had relapsed or refractory disease with a mean of 49 months from initial diagnosis to enrollment. Four patients had previously received bexarotene and 2 interferon alfa. Seventeen patients completed 16 weeks of treatment and are assessable for response, 1 has not completed 16 weeks and 4 received less than 16 weeks (1 early death, 1 non-compliant, 1 withdrawal of consent because of side effects, and 1 myocarditis, probably not treatment-related). Using reproducible objective criteria for response utilized in the single agent bexarotene trials cited above (Primary Endpoint Classification), 6 of 17 patients achieved a partial response and 1 a complete response (objective response rate 41%, 95% confidence intervals 18%–67%). Three of the 6 partial responses occurred with bexarotene alone at 8 weeks. Median duration of response was 2.7 months, range 1.1–7.6 months. Reversible grade 3 or 4 toxicities seen in more than 1 patient were hypercholesterolemia (2 pts.), hypertriglyceridemia (4 pts.), neutropenia (3 pts.), lymphopenia (2 pts.) and elevated AST in 2 patients. Lipid lowering agents were used in all patients. Six patients died, all of progressive disease from 1.6–29.9 months following initiation of treatment. The major response rate for the combination of bexarotene and interferon alfa is similar to that for bexarotene alone. Despite the wide confidence intervals that indicate that a small advantage might be missed by not increasing accrual, the clinical usefulness of this combination is limited by the inconvenience of interferon injections. Further trials in CTCL are planned combining bexarotene with other agents, including active cytotoxics.
Cbl Regulates CXCR4-Mediated Chemotaxis and CXCR4 Receptor Internalization.
Authors: Lane Heather C. , Anand Appakkudal R. , Fernandis Aaron Z. , Ganju Ramesh K.
Published: April 2020

CXCL12-induced and CXCR4-mediated chemotaxis plays a critical role in the trafficking of lymphocytes, which modulates inflammatory and immune responses. The CXCR4 receptor is down-modulated by its ligand CXCL12, which has been shown to inhibit HIV entry into the cells. However, the molecular mechanisms that regulate CXCL12-induced chemotaxis and down-modulation of the CXCR4 receptor are not well known. We have previously shown that CXCL12 induces the tyrosine phosphorylation of the signaling molecule Cbl and its association with the tyrosine phosphatase SHP2. In the present investigation, we have further explored the role of Cbl in CXCR4-mediated chemotaxis and CXCR4 down-modulation. Cbl, a 120 kd protein that contains a tyrosine kinase binding domain (TKB), can function as an adaptor molecule by binding to various proteins. It also contains a RING finger domain and has been shown to negatively regulate signaling by directing the ubiquitination and degradation of signaling molecules. We found that CXCR4 L1.2 cells overexpressing wild-type Cbl or G306E-Cbl (deletion in the TKB domain) showed reduced migration to CXCL12, only 50% of the vector control. Cells overexpressing 70Z-Cbl (deletion in the RING finger domain) showed no difference in migration as compared to the vector control. We next examined CXCR4 expression in these cells by FACS analysis. We found that cells overexpressing wild-type Cbl and G306E-Cbl had approximately 60% CXCR4 receptor expression as compared to the vector control, while the cells overexpressing 70Z-Cbl had no difference in receptor expression. Cells were then internalized by stimulation with CXCL12 or HIV gp120, and after 2 hours approximately 80% of the vector control cells had internalized, as compared to approximately 90% of the cells overexpressing wild-type Cbl and G306E-Cbl, and 60% of the cells overexpressing 70Z-Cbl. Overexpression of wild-type Cbl or G306E-Cbl slightly increased internalization of the CXCR4 receptor, while 70Z-Cbl impeded this process. This indicates that wild-type Cbl enhances internalization, while the G306E mutation in the tyrosine kinase binding domain has no effect on this function of Cbl. Our results suggest that the RING finger domain of Cbl is important for internalization of the CXCR4 receptor. In addition, we observed that there was no difference in the phosphorylation of Erk 1/2 between the vector control cells and those overexpressing wild-type Cbl, 70Z-Cbl, or G306E-Cbl after stimulation with CXCL12, indicating that these changes in migration and receptor expression act in a MAPK-independent pathway. Taken together, these studies suggest that Cbl plays an important role in CXCR4-mediated chemotaxis and the CXCR4 internalization process. These results may lead to better understanding of HIV pathogenesis and inflammatory responses mediated by CXCR4.
Suppression of Negative Immune Regulators PD-1 and PD-L1 Expression on Lymphocytes by Inflammatory Cytokines.
Authors: Zhong Xuemei , Bai Chunyan , Rothstein Thomas L.
Published: April 2020

The strength and duration of lymphocyte activation is determined by the net effect of positive and negative regulatory co-signaling molecules. The inhibitory receptor PD-1 and its ligand PD-L1 are both expressed on activated lymphocytes. Mice deficient of PD-1 or PD-L1 demonstrate lymphocyte hyperactivity and are prone to autoimmunity. We have shown that the expression and function of PD-1 on activated B lymphocytes can be down-regulated by certain inflammatory cytokines, rendering them resistant to PD-L1 mediated suppression. Here we further report that both PD-1 and PD-L1 expression on activated T lymphocytes can be down-regulated in a similar fashion. Thus, in addition to stimulating positive regulators, inflammatory cytokines may boost immune responses and break self-tolerance by inhibiting a negative regulator on T and B lymphocytes. Our findings may indicate a potential mechanism for the link between chronic inflammation and autoimmunity.
The Effect of Aging on Hematopoietic Stem Cell Homing and Engraftment.
Authors: Liang Ying , Van Zant Gary , Szilvassy Stephen J.
Published: April 2020

The efficiency with which hematopoietic stem (HSC) and progenitor cells (HPC) home to bone marrow (BM) critically impacts their engraftment after transplantation. Little is known about the effects of aging on this parameter. The present study was thus initiated to test the hypothesis that homing efficiency of HSCs and HPCs to the BM will vary with aging of the donor, recipient, or both. Young or old C57BL/6 BM cells were intravenously injected into lethally irradiated old or young Ly-5 congenic recipient mice. Three or 24 hours later, the numbers of HPCs or HSCs that could be recovered from the BM and spleen were assayed using an in vitro colony-forming cell assay or an in vivo limiting-dilution competitive repopulating unit (CRU) assay and compared to the number of such cells that were initially transplanted. The frequency of CRU in old BM was two-fold higher than that in young BM (~1 per 10,000 vs. 24,000 cells). However, old CRU homed less efficiently to young BM after 24 hours than did young HSCs (3% vs. 10%). The proliferative potential of individual HSCs (measured as the overall level of engraftment in mice transplanted with 1 CRU) did not change with donor age, but was reduced by advanced recipient age, and was also reduced by prior transplantation as observed previously. HSC differentiation potential (defined by the proportion of lymphocytes and myeloid cells in mice transplanted with 1 CRU) was also skewed toward myelopoiesis at the expense of lymphopoiesis with both donor and recipient age. Donor and recipient aging exerted similar effects on the 3 hour BM and spleen seeding efficiency of HPCs, both leading to a 40% decrease in BM homing, and a 5-fold decrease in spleen homing compared with young HPCs transplanted into young recipients. In terms of the rate of hematopoietic engraftment, advanced recipient age resulted in slower erythrocyte and platelet recovery, but moderately accelerated leukocyte regeneration compared with young cells transplanted into young recipients. Conversely, old donor cells exhibited faster leukocyte and platelet recovery, but erythrocyte recovery was essentially unchanged compared to young donor cells. In sum, these data suggest that primitive hematopoietic cells undergo intrinsic aging-related changes in homing efficiency, proliferation/differentiation potential, and engraftment capability that are also extrinsically affected by an older BM microenvironment. These findings have important implications for clinical stem cell transplantation where older individuals often serve as donors for elderly patients.
Repopulation Advantage of Blm−/− Cells in the Primary Recipients Can Be Reversed by Cisplatin Treatment.
Authors: Park Youngji , Luo Guangbin , Gerson Stanton
Published: April 2020

Deficiencies in DNA repair-related genes such RecQ DNA helicases lead to human genetic disorders such as Bloom’s syndrome, Werner’s syndrome, and Rothmund-Thomson syndrome characterized by premature aging and cancer predisposition. We hypothesized that deficiency in RecQ DNA helicases may lead to drug hypersensitivity and/or stem cell failure after serial transplantation. Several genotoxic agents were tested on the bone marrow cells isolated from Bloom (Blm)−/− and RecQ4−/− knockout mice and compared to wildtype bone marrow cells. RecQ4−/− bone marrow showed mild sensitivity to γ-ray irradiation and very mild sensitivity to cisplatin. RecQ4−/− bone marrow did not show sensitivity to etoposide. However, Blm−/− bone marrow cells did not show hypersensitivity to either γ-ray irradiation or etoposide treatment, implying each RecQ DNA helicase may have different roles for DNA repair and/or proliferation in bone marrow cells.

To assess how deficiency in RecQ DNA helicase affects hematopoietic stem cell function, serial transplantation capacity into lethally irradiated recipients was compared between bone marrow cells isolated from Blm−/− (Ly5.2) and wildtype (Ly5.1) mice. Competitive repopulating capacity was monitored by Ly5.1 / Ly5.2 marker analysis of peripheral blood every three weeks. There was no difference in early repopulating capacity between Blm−/− and wildtype in primary transplants at 3, 6 and 9 weeks post-transplantation (wt 46.6% ± 11.9% vs. Blm−/− 53.5% ± 11.9%, n=6, p=0.33 at 9 weeks post-transplantation). However, at 15 weeks post-transplantation, Blm−/− cells showed higher repopulation than wildtype bone marrow cells (wt 30.7 % ± 7.1 % vs. Blm−/− 69.3% ± 7.1%, n=3, p=0.003), implying Blm−/− cells might gradually accumulate a proliferative advantage over wildtype cells. To assess whether drug treatment may cause sensitivity in Blm−/− cells after transplantation, primary recipients of an equal mixture of Blm−/− and wildtype bone marrow were treated with 1 mg/kg cisplatin biweekly i.p. between 9 to 15 weeks post-transplantation and repopulation capacity was monitored. The repopulation advantage of Blm−/− cells in primary transplants was abolished by cisplatin treatment (wt 61.3 % ± 17.3 % vs. Blm−/− 38.7% ± 17.3%, n=3, p=0.19, in the cisplatin-treated cohort compared to wt 30.7 % ± 7.1 % vs. Blm−/− 69.3% ± 7.1%, n=3, p=0.003, in the control cohort). Thus, a proliferative advantage of progenitors is apparent in Blm−/− bone marrow, but lost after cisplatin-mediated DNA damage. This suggests that defective DNA repair of Blm−/− cell may promote deregulated proliferation of hematopoietic progenitors.
Marrow Derived Muscle Colonies Are a Clonal Phenomenon.
Authors: Abedi Mehrdad , Greer Deborah A. , Foster Bethany A. , Demers Delia A. , Colvin Gerald A. , Dooner Mark S. , Quesenberry Peter J.
Published: April 2020

We have previously established a model of plasticity of marrow to skeletal muscle that is based on transplantation of GFP positive marrow and cardiotoxin injury to the tibialis muscle. In these experiments, we have observed colonies of GFP positive muscle fibers, defined as 3 or more myocytes. We noted that individual GFP positive fibers showed marked heterogeneity of fluorescence. In sharp contrast, colonies of GFP+ cells, showed homogeneous fluorescence. This was confirmed by using confocal microscopy and by measuring the mean fluorescent intensity of the individual cells in the colonies. When the intensity of the green fluorescence in individual fibers was graded to dull, moderate or bright, in 95% of the colonies counted, showed homogenous intensity of green fluorescence while for single GFP fibers dispersed throughout the specimen the probability of 3 cells being the same color was only 14.12%. Therefore the probability of 95% of colonies to have a minimum of 3 homogenous fibers by random association is less than 0.0001. To show that colonies are not from clumps of cells injected into the muscle, we compared the number of the colonies in two different experimental setting where in both GFP positive marrow cells were injected intravenously after 900cGy of radiation followed by cardiotoxin injury to the muscle. Animals who received two cycle of G-CSF mobilization were compared with those who had lineage negative marrow cells directly injected into their muscle, one day after injury. The incidence of muscle colonies remained the same between the groups. The incidence increased in experiments with multiple cycles of mobilization and injury and also in mdx mouse that has spontaneous ongoing regeneration of its muscles. Cotransplantation of marrow cells from yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) transgenics into C57BL/6 mice followed by cardiotoxin injury showed colonies that were either from YFP or CFP cells. No mixed colonies were found. Transplantation of YFP marrow cells before injury followed by CFP cells after injury and vice versa showed colonies only from cells that were infused at the time of transplantation. Colonies were seen only in samples that received CD45, C-Kit and/or Sca positive marrow cells and not in those negative populations. These cells resulted in a GFP+ CD45 negative population 4 weeks after transplantation and produced Myf5+ and MyoD+ myoblasts three days after injury. These data suggests that marrow derived muscle colonies are a clonal phenomenon similar to CFU-S cells in recipient spleens after transplantation.
Monitoring of Bone Marrow Cell Homing in the Infarcted Human Myocardium by PET.
Authors: Hertenstein Bernd , Wollert Kai C. , Hofmann Michael , Meyer Gerd P. , Arseniev Lubomir , Gasner Arnold , Knapp Wolfram H. , Drexler Helmut
Published: April 2020

Intracoronary transfer of autologous bone marrow cells (BMCs) has been shown to promote recovery of left ventricular (LV) systolic function in patients with acute myocardial infarction. (BOOST Trial; Wollert et al. Lancet, 2004, 364 141-8). Although the mechanisms of this effect remain to be established, homing of BMCs to the infarcted LV is probably a crucial early event. We determined BMC tissue distribution after therapeutic application in nine patients with a first ST-elevation myocardial infarction, who had undergone stenting of the infarct-related artery (all male; median age 43 ys; range 36 – 66). The study was approved by the local ethics committee and all patients provided written informed consent. Time from symptom onset to percutaneous coronary intervention (PCI) was 8 h (3–27) and median maximum CK level was 1767 U/l. Cells were harvested from the posterior iliac crest during short anesthesia with etomidate and midazolam and subjected to 4% gelatine polysuccinate sedimentation to obtain a preparation of unfractionated BMCs. 2.5 ± 0,7 x 108 unfractionated BMCs (10% of the harvested cell number) were radiolabeled with 100 MBq 2′-[18F]-fluoro-deoxyglucose (FDG) and infused into the infarct-related coronary artery (i.c., n=3 patients) or injected via an antecubital vein (i.v., n=3 patients). In 3 additional patients, CD34pos cells were immunomagnetically enriched from unfractionated BMCs (Clinimacs, Miltenyi, Germany), labeled with FDG, and infused i.c. Cell transfer was performed 8±2 days after stenting. Following application of FDG labelled cells all patients received 20 ± 6 x 108 non-labeled BMCs i.c. (i.e. the cell dosage that improved cell function in the BOOST trial). More than 98% of the total radioactivity infused was cell-bound. Cell viability after FDG-labeling was 95±2%. Sixty minutes after cell transfer, all patients underwent 3D-positron emission tomography imaging. After i.c. transfer, 3.4±1.4% of FDG-labeled unfractionated BMCs were detected in the infarcted LV; the remaining activity was found primarily in the liver and spleen. After i.v. transfer, only background activity was detectable in the infarcted LV. After i.c. transfer of FDG-labeled CD34-enriched cells, 25±13% of the total activity was detectable in the infarcted LV. Unfractionated BMCs engrafted in the infarct center and border zone, CD34pos cell homing was more pronounced in the border zone. FDG-labeling can be used to monitor myocardial homing and tissue distribution of BMCs after therapeutic application. I.c. transfer is superior to i.v. application in terms of BMC homing in the the infarcted LV. CD34-enriched cells display a 7-fold higher retention in the infarcted LV as compared to unfractionated BMCs.
Transfusion of Red Blood Cells under Shock Conditions in the Rat Microvasculature.
Authors: Chin-Yee Ian , Statchuk Leslie , Milkovich Stephanie , Xenocostas Anargyros , Ellis Christopher
Published: April 2020

Background RBC transfusion is the mainstay of therapy for resuscitation of patients with hemorrhagic shock. Ex-vivo storage of RBCs for transfusions is associated with a storage lesion which decreases erythrocyte deformability and increases their adhesiveness to vascular endothelium. This may impair microcirculatory flow with deleterious effects on oxygen delivery post-transfusion. The objective of this study was to determine whether transfused stored rat blood impairs the microcirculation following resuscitation from hemorrhagic shock

Methods Donor Sprague Dawley rat RBCs were collected and stored in CPDA-1 under standard blood bank conditions. RBCs were labelled with FITC (fluorescein isothiocyanate) 24 hours before the experiment. RBCs stored for less than 24 hours (fresh) RBCs (n=3) were compared to RBCs stored for 7 days (n=3). To induce shock, forty percent of blood volume was removed from male Sprague Dawley rats. Animals were then resuscitated by injecting 1.5mL of labelled packed RBCs and sufficient saline to restore blood volume via arterial line. The number of adherent labelled RBCs in the extensor digitorum longus muscle (EDL) was detected using intravital video microscopy (20X magnification). RBC velocity, capillary diameters, capillary Hct (hematocrit), arterial Hct, and lactate data was collected at three time points (baseline, shock and resuscitation).

Results Lactate levels increased significantly during shock in both the fresh and stored groups. Arterial Hct decreased in shock and was restored to baseline levels with resuscitation. The stored group had an increased number of stopped cells in the microcirculation compared to their fresh counterparts (p=0.004). There was no statistical significant difference in capillary diameters between groups or time points. Both groups showed an increase in RBC velocity from baseline to resuscitation (stored group doubled, fresh group tripled). Capillary Hct did not change in the fresh group but doubled in the old group from baseline to resuscitation.

Conclusion In our shock model, transfusion of stored RBCs resulted in an increased number of stopped cells and lower RBC velocity in capillaries as compared to fresh RBCs. Stored RBCs also had an increased capillary Hct from baseline to resuscitation and a diminished hyperemic response as compared to fresh RBCs. We conclude that stored RBCs following transfusion behave differently than native or fresh transfused cells in the microcirculation. Further studies are needed to determine the impact of these microcirculatory changes on oxygen availability in transfusion.