bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of the JAK2 V617F Mutation on Survival in Patients with Catastrophic Intra-Abdominal Thromboses.
Authors: McMahon Christine , Abu-Elmagd Kareem , Bontempo Franklin A. , Kant Jeffrey A. , Swerdlow Steven H.
Published: October 2019

Catastrophic intra-abdominal thrombosis can result from a variety of hypercoaguable states, including the chronic myeloproliferative disorders (CMPD), even though the patient’s clinical and laboratory findings may not suggest such a diagnosis. Recently a somatic mutation in the tyrosine kinase, JAK2, has been reported in myeloid cells of 65–97% of patients with polycythemia vera and approximately 23–57% of patients with essential thrombocythemia or chronic idiopathic myelofibrosis, but not in normal individuals. To assess the prevalence and clinical implications of this mutation in the setting of intra-abdominal thrombosis, JAK2V617F genotyping was performed in 42 patients who had suffered catastrophic intra-abdominal thromboses that resulted in visceral transplants, as well as in 20 control subjects. The prevalence of V617F was compared to that of other hypercoaguable states for which molecular testing is routinely performed. The V617F mutation was detected in 7 patients (16.7%), who were not distinguishable from the 35 others on the basis of their peripheral blood counts. JAK2 V617F was not detected in any of the control subjects. Six patients were heterozygous (14.3%) for the factor V Leiden mutation and one patient was heterozygous for prothrombin 20210 G>A, all of whom had unmutated JAK2 genes. Evidence for a lupus anticoagulant was found in 7 patients, including one with the V617F mutation. Six of the 7 patients with the V617F mutation died within 36 months post-transplant from the following causes: an aggressive post-transplant lymphoproliferative disorder, sepsis secondary to bone marrow failure, allograft-liver failure, and three cases of sepsis preceded by mental status changes, possibly due to cerebral vascular disease. The median post-transplant survival of V617F-positive patients was 1.5 years compared to 9.8 years for the V617F-negative patients (ratio: 6.6; 95% CI 6.3–7.0). These results highlight the diagnostic utility of JAK2V617F testing in the setting of abdominal thrombosis and underscore the clinical significance of a positive result. The explanations for major thromboses in JAK2 V617F positive patients with clinically silent CMPD and the significantly shorter survival of the JAK2 V617 positive patients in the setting of visceral transplantion are uncertain. The optimal management of these patients also remains to be determined.

POST-TRANSPLANT SURVIVAL POST-TRANSPLANT SURVIVAL Median Peripheral Blood Counts at Presentation Platelets (109/L) Hemoglobin (g/dl) Hematocrit (%) Leukocytes (109/L) V617F 342 12.2 37.2 6.3 WT 239 11.4 33.3 7.2 p-value 0.22 0.59 0.46 0.89
Monocytosis Is an Independent Risk Factor for Survival in Agnogenic Myeloid Metaplasia.
Authors: Elliott Michelle A. , Dingli David , Schwager Susan M. , Li Chin Y. , Tefferi Ayalew
Published: October 2019

Background: Young (age < 60 years) patients with agnogenic myeloid metaplasia (AMM) are potential candidates for curative therapy with allogeneic stem cell transplantation. In such transplant-eligible patients, median overall survival (OS) is reported to be between 78 and 128 months. Because clinical course in an individual patients is markedly heterogeneous (survival ranges of months to decades), objective prognostic variables are essential for transplant decision making. Accordingly, we recently developed a modified Dupriez prognostic scoring system (PSS) that effectively discriminated between high (2 or more risk factors), intermediate (one risk factor) and low (no risk factor) risk patient groups based on the presence or absence of complete blood count (CBC)-derived adverse parameters; hemoglobin < 10 g/dl, leukocyte count < 4 or > 30 × 109/L, and platelet count < 100 × 109/L (Dingli et al. Cancer2006;106:623). In the current study, we identify absolute monocytosis of ≥ 1 × 109/L as an additional independent risk factor for survival in AMM.

Methods: A consecutive cohort of WHO-defined AMM patients diagnosed before the age of 60 years was identified. The impact of various clinical and laboratory parameters on overall survival was evaluated with univariate and multivariable analysis.

Results: The study included 129 patients (median age 52 years, range 18–60; 69 males) with AMM. An overall median survival of 75 months was univariately affected by platelet count < 100 × 109/L, hemoglobin level of < 10 g/dL, leukocyte count of either < 4 or > 30 × 109/L, monocytosis of ≥ 1 × 109/L, and, where cytogenetic studies were available (n=41), presence of unfavorable cytogenetic abnormalities (p < 0.01 in each instance). On multivariable analysis, all but leukocyte count maintained their significance. The independent prognostic value of monocyte count was also validated against the modified Dupriez score (see above) and thus allowed further refinement of the particular CBC-based PSS that included monocytosis of ≥ 1 × 109/L as a fourth risk factor; low-risk identified patients with none of the 4 risk factors, intermediate-risk with one risk factor, and high-risk with 2 or more risk factors (Figure 1). For comparison, Figure 2 illustrates survival curves according to the original Dupriez PSS.

Conclusion: Monocytosis of ≥ 1 × 109/L is an independent risk factor for inferior survival in AMM.

Figure 1. Mayo score Figure 1. Mayo score Figure 2. Dupriez score Figure 2. Dupriez score
Depletion of Regulatory T Cells Dramatically Improves DC-Based Immunization Against Acute Myeloid Leukemia.
Authors: Delluc Stephanie , Gaston Auguste , Marchiol-Fournigault Carmen , Fradelizi Didier , Regnault Armelle , Sang Kim Hanh Le Quan , Tourneur Lea , Chiocchia Gilles , Buzyn Agnes
Published: October 2019

Dendritic cell (DC)-based vaccination is a promising approach to enhance anti-tumor immunity that could be considered for patients with high risk acute myeloid leukemia (AML). We have already shown in human that DC pulsed with eluted peptides (EP) from autologous AML blasts (DC/EP) are able to induce CD4+ and CD8+ anti-leukemic immune response in vitro (Delluc S, Haematologica, 2005). In order to optimize this vaccination strategy for AML patients we developed a pre-clinical murine model. C57/Bl6 mice were vaccinated by DC pulsed or not with peptides eluted from the syngenic C1498 myelomonocytic leukemic cell line in a prophylactic setting. Injection of DC/EP induced an anti-leukemic response as shown by the cytotoxic activity of CD4 T cells, whereas the injection of unpulsed DC induced a NK cell-mediated cytotoxicity against C1498 cells. In vivo depletion of CD4 T cells or NK cells abrogated the protective effect induced by DC/EP (p=0.02) or DC (p=0.06) vaccination, respectively, confirming their critical role in preventing leukemic outgrowth. However, late C1498 re-challenge showed that the anti-leukemic immune response was insufficient to protect DC/EP vaccinated mice from death, suggesting an ineffective or absent long-lived memory response. Since several populations of T cells have regulatory properties potentially inhibiting anti-tumor responses, we hypothesized that CD25+ cell-depletion in vivo could enhance the protection induced by vaccination. Indeed, we observed a dramatic improvement of the survival of mice treated by an anti-CD25 antibody before vaccination compared to mice vaccinated by DC/EP alone (p<0.01). More interestingly, CD25 depletion allowed the generation of long-lived immune responses since mice were protected from a late re-challenge by C1498 cells. Our results strongly suggest that depletion of regulatory CD25 T cells before DC-based vaccination should be considered for immunotherapy of weakly immunogenic tumors such as AML.
Reconstitution of STAT4 Restores Defective Interferon-gamma Production and Allows Normal IFN-gamma-Dependent Responses after Autologous Stem Cell Transplantation.
Authors: Robertson Michael J. , Chang Hua-Chen , Pelloso David , Kaplan Mark H.
Published: October 2019

Production of IFN-gamma is critical for optimal antitumor immune responses in several preclinical animal models. Patients with lymphoma who have undergone autologous hematopoietic stem cell transplantation exhibit profoundly diminished IFN-gamma production in vivo during IL-12 therapy (Clin Cancer Res.2002; 8: 3383). Furthermore, post-transplant patient peripheral blood mononuclear cells (PBMCs) stimulated directly in vitro with IL-12 secrete little if any IFN-gamma. To determine if restoration of IFN-gamma production would be sufficient to reconstitute IFN-gamma-dependent responses after transplantation, the integrity of IFN-gamma signaling was assessed in post-transplant patient PBMCs. Compared to control subject PBMCs, post-transplant patient PBMCs expressed equivalent levels of CD119 (IFN-gamma receptor alpha) and STAT1. Moreover, tyrosine phosphorylation of STAT1 in response to IFN-gamma did not differ in post-transplant patient as compared to control PBMCs. Thus, IFN-gamma signaling is intact after transplantation. Our prior studies have shown that STAT4 protein levels are decreased by ~97% in post-transplant patient PBMCs, whereas levels of Jak2, Tyk2, and STAT3 are similar to control PBMCs (Blood2005; 106: 963). We wished to demonstrate directly that STAT4 deficiency is the mechanism of defective IFN-gamma production after autologous transplantation. Enriched CD4+ T cells were isolated from post-transplant patient and control PBMCs and cultured under conditions that promote Th1 differentiation. STAT4 deficiency persisted in post-transplant patient CD4+ T cells cultured under Th1 conditions, whereas these cells expressed normal levels of T-bet. Compared to control Th1 cells, post-transplant patient CD4+ T cells cultured under Th1 conditions exhibited markedly reduced IFN-gamma production after restimulation with CD3 mAb. These results are consistent with expected impairment of Th1 differentiation due to profound STAT4 deficiency. STAT4 cDNA was transiently transfected into Th1-cultured CD4+ T cells from post-transplant patients. After stimulation with CD3 mAb plus IL-12, the amounts of IFN-gamma secreted by STAT4-transfected patient cells were equivalent to or higher than those secreted by control subject cells. These data indicate that reconstitution of STAT4 expression is sufficient to restore IFN-gamma production by post-transplant patient PBMCs. As IFN-gamma signaling is normal post-transplant, circumventing STAT4 deficiency should therefore restore IFN-gamma-dependent antitumor immune responses after autologous transplantation. Future studies will determine the molecular mechanisms of STAT4 deficiency after transplantation and develop clinically feasible methods to circumvent this deficiency.
A Novel Mechanism in Generation of Self-Antigen Repertoire - An Unconventional Antigen Translated by a Novel Internal Ribosome Entry Site Elicits Anti-Tumor Humoral Immune Responses.
Authors: Yang Xiao-Feng , Xiong Zeyu , Liu Enli , Yan Yan , Silver Richard T. , Verstovsek Srdan , Prchal Josef , Wang Hong
Published: October 2019

Self-tumor antigens that elicit anti-tumor immune/autoimmune responses in responses to interferon-a (IFN-a) stimulation remain poorly defined. We screened a human testis cDNA library with sera from three polycythemia vera (PV) patients who responded to IFN-a and identified a novel antigen, MPD6. MPD6 belongs to the group of cryptic antigens without conventional genomic structure and is encoded by a cryptic open reading frame located in the 3′untranslated region (3′UTR) of myotrophin mRNA. MPD6 elicits IgG antibody responses in a subset of PV patients, as well as patients with chronic myelogenous leukemia and prostate cancer, suggesting that it is broadly immunogenic. The expression of myotrophin-MPD6 transcripts in tumor cells was upregulated in some tumor cells, but only slightly increased in K562 cells in response to IFN-a treatment. By using bicistronic reporter constructs, we showed that the translation of MPD6 was mediated by a novel internal ribosome entry site (IRES) upstream of the MPD6 reading frame. Furthermore, the MPD6-IRES mediated translation, but not myotrophin-MPD6 transcription, was significantly upregulated in response to IFN-a stimulation. These findings demonstrate for the first time that a novel IRES-mediated mechanism is responsible for the translation of unconventional self-antigen MPD6 in responsive to IFN-a stimulation. The eliciting anti-tumor immune response against unconventional antigen MPD6 in patients with myeloproliferative diseases suggests MPD6 as a potential target of novel immunotherapy. Our results also suggest that the enhancement of IRES-mediated translation of unconventional self-antigen(s) by IFN-a is a novel mechanism of IFN-a enhanced anti-tumor immune/autoimmune responses.
Identification and Characterization of T Cell Epitopes Deduced from RGS5, a Novel Broadly Expressed Tumor Antigen.
Authors: Boß Cristina N. , Grünebach Frank , Brauer Katharina M. , Weinschenk Toni , Kanz Lothar , Stevanovic Stefan , Rammensee Hans-Georg , Brossart Peter
Published: October 2019

Identification of tumor-associated antigens (TAA) has resulted in the development of therapeutic vaccines for the treatment of cancer. We applied an integrated functional genomics approach to identify TAA in malignant tissues of patients with renal cell carcinoma (RCC). A comparative DNA chip analysis of tumor and the corresponding non-malignant tissue from patients with RCC followed by sequencing of peptides bound to the HLA-class I molecules by mass spectrometry was applied to identify novel TAA in RCC. To confirm the immunogenicity of identified epitopes cytotoxic T lymphocytes (CTL) were generated using dendritic cells (DC). Utilizing this approach, two peptides derived from RGS5 binding to either HLA-A*02 or 03 were identified. The key function of regulators of G protein-signalling (RGS) is to bind to G protein α subunits and to stimulate their intrinsic GTPase activity. The hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) thereby is accelerated and the inactive heterotrimer more rapidly restored. Thus, RGS proteins inhibit the biological activity of G proteins. Interestingly, it was recently shown that RGS5 is overexpressed in pericytes of newly developing tumor vessels, indicating that RGS5 plays an important role during tumor angiogenesis.

Using RT-PCR analysis we found that RGS5 is expressed on a broad variety of tumor cells including RCC, colorectal, breast and ovarian cancer, malignant melanoma and multiple myeloma as well as in acute and chronic leukemias making this protein an interesting candidate for the development of vaccination strategies to target the tumor cells and the tumor vessels. CTL that were induced using the RGS5 peptides lysed autologous DC pulsed with the cognate peptide or transfected with in vitro transcribed RGS5 RNA as well as HLA-matched tumor cell lines. The specificity and HLA restriction was confirmed using blocking monoclonal antibodies and in cold-target inhibition assays. We next utilized DC transfected with RGS5 RNA to generate specific CTL. Using this approach we confirmed the processing and presentation of the identified peptides by malignant cells. These CTL lysed tumor cells in an antigen specific manner while sparing non-malignant cells. To analyze the induction of RGS5 specific CTL in an autologous setting in patients with malignant diseases, we used blood samples from a patient with acute myeloid leukemia (AML) in complete remission after chemotherapy and were able to generate specific CTL capable of recognizing autologous AML blasts while sparing non-malignant cells.

Our results demonstrate that RGS5 is a novel tumor rejection antigen expressed in a wide range of malignancies that can be applied to target malignant cells and tumor angiogenesis.
Stimulation of Potent Antigen-Specific T-Cell Responses Using Semiallogenic Dendritic Cells Derived from Human Embryonic Stem Cells.
Authors: Su Zhen , Wei Fusheng , Fesperman Susan , Wang Siqing , Dahm Philipp , Vieweg Johannes
Published: October 2019

The major objective of this study is to develop a novel and broadly applicable immunotherapy platform against cancer and infectious diseases. We hypothesized that human embryonic stem cells (hESC) could serve as a source for generating dendritic cells (DC) with potent immunostimulatory function. One advantage of using hESC-derived DC in clinical settings is the ability to generate virtually unlimited amounts of antigen presenting cells for vaccination. Although hESC-derived DC are not genetically identical to the recipient patient, antigen processing and presentation can be facilitated by matching hESC to recipients that share HLA class I alleles. Another advantage of this technology is that hESC express highly polymorphic HLA class II molecules that serve as major rejection antigens, thereby augmenting the antigen-specific T-cell response in the cancer patient.

In the current study, we have established a novel three-step method to differentiate hESC (H9 cell line) into mature DC sequentially through hematopoietic stem cell and myeloid precursor stages. During the first step, 10–15% CD34+ hematopoietic stem cells were generated by co-culturing hESCs with the bone marrow stromal cell line OP9. During the second step, these CD34+ hematopoietic stem cells were differentiated into CD45+CD33+ myeloid precursors and further expanded in the presence of GM-CSF. In the final step, all myeloid precursors were differentiated into mature DC using a cytokine cocktail including GM-CSF, Flt-3L and TNF-α. Using this method, we were able to generate approximately 1 × 108 mature hESC-derived DCs (hESDC) with ≥ 80% purity. These hESDC exhibited a similar phenotype than monocyte-derived DC with high expression of MHC class I, MHC class II, CD11c, CD54, CD40 and the co-stimulatory molecule CD86. Upon activation with proinflammatory cytokines, the hESDC secreted IL-12p70 and migrated in response to MIP-3β. In mixed lymphocyte reaction assays, hESDC exhibited strong allo-stimulatory capacity. Moreover, peptide-loaded mature hESDCs were able to stimulate antigen-specific CD8+ T-cell responses against the EBV peptide BMLF1280-288 and the MART-1 peptide (ELAGIGILTV) in a HLA-A2-restricted manner. Most importantly, hESDC stimulated HLA-A2+ MART-1 peptide-specific CD8+ T cells in vitro that were capable of recognizing and killing the HLA-A2+ melanoma cell line Malena-3M. These data suggest the development of a scalable DC platform that could be applied in clinical immunotherapy protocols.
The Role of Nitric Oxide in Regulation of Red Blood Cell Deformability.
Authors: He Xiaojun , Azarov Ivan , Gordon Beth , Kim-Shapiro Daniel B. , Ballas Samir K.
Published: October 2019

Nitric Oxide (NO) has been suggested to modulate the deformability of red blood cells (RBCs). Bor-Kucukatay (Bor-Kucukatay et al. Am J Physiol Heart Circ Physiol284: H1577, 2003) found that cells incubated with 1 μM of the NO donor sodium nitroprusside lead to a small but significant increase RBC deformability as measured by ektacytometry. However, no significant effect was seen at lower or higher concentrations of sodium nitroprusside or for any concentration of another NO donor, diethylenetriamine NONOate. Kleinbongard (Kleinbongard et al. Blood10; 3992, 2005) found large increases in red cell deformability as a function of added arginine (the substrate for Nitric Oxide Synthase) by measuring the flow rate through filters. On the other hand, using cell aspiration techniques, Bateman (Bateman et al. Am J Physiol Heart Circ Physiol 280; H2848 H2001) found that NO production during sepsis causes a decrease in RBC deformability. Clearly more work is needed to determine the effects of NO on RBC deformability.

The present work was undertaken to further investigate the effect of NO on normal and sickle RBC deformability. ProLi NONOate, arginine, and nitrite (which can be reduced to NO by hemoglobin (Hb), were incubated with blood at various concentrations over a period of 2 hours. Nitrosyl Hb and MetHb formed due to the interaction between NO and RBCs were quantified by electron paramagnetic resonance spectroscopy. The deformability was measured using a flow channel laser diffraction similar to ektacytometry (Huang et al. Am J Hematol67; 151, 2001, Biophys J85; 2374, 2003) with a stress range from 0 to 1,000 Pa. Diffraction patterns produced by deformed cells were analyzed by Matlab®. The deformability coefficients were compared to the control (n=6 per experiment condition). Our results suggested that ProLi NONOate did not significantly effect the deformability of normal RBCs. In a single case, ProLi NONOate improved the deformability of poorly deformable sickle red cells and this result is being studied further. Using our flow channel assay, we did not find any significant affects of arginine on RBC deformability. In addition, our studies involving nitrite, performed under both oxygenated and deoxygenated conditions, suggested that nitrite has no significant effect on RBC deformability. In summary, NO didn’t significantly affect the deformability of normal RBCs, and its potential effects on sickle RBCs needs to be further investigated.
Study of Deferiprone or Deferoxamine Versus Combination Therapy in Iron-Loaded Thalassemia Patients in Taiwan.
Authors: Peng Ching-Tien , Wu Kang-Hsi , Tsai Chang-Hai , Yang Chao-Ping , Wang Lin-Yen , Jang Ren-Chin , Hsiao Chih-Cheng
Published: October 2019

BACKGROUND: Deferiprone (L1) has been suggested to be an effective oral chelation therapy for regular transfusion thalassemia major patients. The purpose of this study was to test the efficacy either of L1 or DFO alone, or combination use, and also monitor the safety of L1 in Taiwan.

MATERIALS AND METHODS: From April 1999 to December 2005, 114 thalassemic patients from 5 treatment centers were enrolled in this program. L1 at the standard dose of 75mg/kg was given to 57 patients. The mean administered dose of L1 was 72.5mg/kg body weight divided into three doses per day. DFO at the standard dose of 30–50mg/kg/day at least 5 days/week was given to 26 patients, and the mean DFO dose was 46.5mg/kg/day 5 or more days/week. Combined therapy of daily L1 with subcutaneous DFO was given 2 to 6 days each week to the other 31 patients. The mean administered dose of DFO was 45.7mg/kg body weight 2 to 6 days per week, and the mean L1 dose was 71.5mg/kg/day divided into 3 doses per day. The therapeutic efficacy and potential side effects on cardiac and/or hepatic systems of these patients were assessed by left ventricular ejection fraction, T2-weighted magnetic resonance imaging (T2-MRI) and biochemical parameters and liver biopsies.

RESULTS: No significant liver function impairment, neutropenia or arthropathy ever occurred. Only one patient was found to have transient leukopenia (he had a coincidental viral infection at that time). Three patients had temporary G-I upset but no one required discontinuation of L1. The serum ferritin level was significantly lower in 9 patients receiving L1 for more than 6 years (p=0.04), 22 patients receiving L1 for 2–3 years (p<0.01) and 31 receiving the combined therapy (p=0.01), yet significantly higher in those receiving DFO only (p<0.01). The combined therapy group witnessed a more significant ferritin decrease than the L1 alone or DFO alone groups and hepatic function improved or stabilized notably. Besides a decrease of iron, no marked pathohistological changes were observed in the liver biopsies. GPT had a decreasing trend in 3 subgroups of L1 & combination therapy group, however there was no statistic significance. Cardiac studies showed a marked recovery of signal intensity in the heart T2-MRI and increased LVEF, indicating a significant reduction of iron load in the heart in L1 use and in over 50 -month combination therapy groups

CONCLUSIONS: We conclude that in this large group of thalassemia major patients, L1 was a safe long-term method of iron chelation. Combining subcutaneous DFO with L1 therapy is an improved method not only for patients inadequately chelated but also by lowering the DFO dosage to less than 25%. This study also shows that the combined therapy is associated with an improvement in heart function.
Splenic Infarction Due to Concomitant Hereditary Spherocytosis and Sickle Cell Trait.
Authors: Yates Robin , Buchanan George R. , Ginsburg Howard , Fefferman Nancy , Greco M. Alba , Borys Dariusz , Blei Francine
Published: October 2019

The occurrence of hereditary spherocytosis (HS) and sickle cell trait (HbAS) in the same patient is rare, with just 21 cases reported in the literature. Although six of these cases had splenic infarction or sequestration, none were reported as having a precipitating event. However, in persons with HbAS, splenic sequestration or infarction has been reported following exposure to high altitudes, including pressurized commercial aircraft. The interaction of these 2 (HS and HbAS) disorders can be explained by the hypoxic and acidic environment in the spleen. This environment causes spherocytes containing HbAS to be both less deformable and more prone to sickling, which promotes sluggish blood flow within the spleen and leads to sequestration and infarction. We have encountered a 10-year-old Caucasian girl with both HS and HbAS who experienced left upper quadrant pain during and following a flight on a commercial aircraft. Past medical history included episodes, although less severe, of left upper quadrant pain after traveling to high altitudes on automobile trips or on commercial aircraft. Family history included the dual diagnoses of HbAS and HS in the patient’s mother, for which she underwent splenectomy as a teenager, and a diagnosis of HS in her 9-year-old brother. At presentation, she had significant left upper quadrant tenderness upon palpation that was associated with voluntary guarding. There was no rebound or involuntary guarding. The spleen was noted to span 3–4cm below the left costal margin. Laboratory values included a hemoglobin of 10.8 (g/dl), reticulocyte count of 7.5%, total bilirubin of 3.0 (mg/dl), and lactate dehydrogenase of 1160 (U/L), all of which were similar to her baseline values. Abdominal ultrasound and cat scan demonstrated infarction in the inferior pole of her spleen. Despite supportive medical treatment, she required immediate splenectomy to relieve her symptoms and prevent recurrence. Her operative and post-operative courses were uneventful. This first report of a patient with both HS and HbAS who suffered splenic infarction after traveling on a commercial aircraft demonstrates how two different inherited red blood cell abnormalities can interact and lead to clinically significant complications.
Hydroxyurea-Mediated Reactivation of Fetal Genes in Primary Erythroid Cell Cultures from Beta-Thalassemia Patients.
Authors: Calzolari Roberta , Maggio Aurelio , Pecoraro Alice , Borruso Vito , Troia Antonio , Acuto Santina , D’Apolito Danilo , Ferro Leda , Di Marzo Rosalba
Published: October 2019

Fetal hemoglobin (HbF) is frequently increased in the hemoglobinopathies such as sickle cell anemia and b-thalassemia. Epidemiological studies have indicated that an increase in HbF ameliorates the clinical symptoms of these diseases (Rodgers and Rachmilewitz - Bri J. Haemat. 91: 263 -1995).

In sickle cell anemia, HbF containing red blood cells have a lower concentration of sickle hemoglobin (HbS), and the HbF itself inhibits HbS polymerization, decreasing cell sickling process (Eaton and Hofrichter - Science 268:1142 -1995). In b-thalassemia patients, HbF partially compensate HbA deficiency and could potentially improve RBC survival resulting in an increase of hemoglobin levels.

Hydroxyurea (HU) is one of the pharmacological agents currently used to stimulate HbF synthesis in patients with sickle cell anemia and more recently has been tested in clinical trials for b-thalassemia patients too (Olivieri et al. Ann. NY Acad Sci850:100-1998; Rigano et al. Hemogl.21(3): 219- 1997; Dixit et al. Ann. Haematol. 84: 441 -2005).

The mechanism involved in the HU-mediated changes is still unclear. It may involve a selection of a minor pre-existing subpopulation of F-cells that has a growth and/or survival advantage (cellular mechanism). This mechanism may be particularly effective for cells derived from patients with hemoglobinopathies, where F-cells may be resistant to “ineffective erythropoiesis”. An alternative mechanism could involve stimulation of HbF in all or the majority of cell-population by direct induction of g genes (molecular mechanism).

Here we report the analysis on thalassemia patients homozygoutes for Lepore genotype that present high levels of fetal hemoglobin.

We combined the use of primary erythroid cell culture from peripheral blood stem cells of these patients, with primary transcript in situ hybridization (RNA-FISH) of the g and b globin genes to investigate the mechanism of action of hydroxyurea in adult erythroid cells.

RNA-FISH on erythroid cell cultures from these patients reveals that the majority of cells express one g allele only (g: 75.2 %, g:g 19.6%). The analysis in hydroxyurea-treated cultures shows the increase of cells transcribing both g-alleles, indicating the reactivation of fetal genes (g: 58.1%, g:g 40%).

This evidence suggests that the molecular mechanism is involved directly on fetal genes reactivation to increase fetal hemoglobin production in HU-treated patients.
AHSP (Alpha Hemoglobin Stabilizing Protein) Gene Expression during Normal and β-Thalassemic Erythroid Differentiation.
Authors: Ronzoni Luisa , Libani Ilaria V. , Giribaldi Giuliana , Turrini Franco , Rusconi Daniela , Refaldi Chiara , Cappellini Maria Domenica
Published: October 2019

β-thalassemia severity is stricktly related to the amount of free a-chains depending at least in part from different b-thalassemic genotype (β0 /β0, β0/β+, β+ /β+). Recently a new erythroid-specific protein, named AHSP (Alpha Hemoglobin Stabilizing Protein), able to form a stable complex with free α-hemoglobin, has been identified. This protein probably acts as a molecular chaperone to prevent the harmful aggregation and precipitation of α-globin that occurs during normal erythropoiesis and to a greater extent in different β-thalassemias. The aim of this study was to evaluate if the AHSP expression in erythroid cells depends on free α-chains amount. In order to verify this hypothesis, we studied the relative expression of AHSP and globin genes during differentiation of erythroid progenitors derived from peripheral blood of both normal subjects and β-thalassemic patients with different genotypes. Erythroid cultures were set up accordingly to the two phases liquid colture method described by Fibach. Total cytoplasmatic mRNA was extracted by the method of Chomczynsky-Sacchi and quantitative real-time PCR was performed using SYBRGreen to evaluate AHSP, α, β and γ globin genes expression. To quantify gene expression a mathematical model using calibration data (2-ΔΔct) was used and quantification of the house-keeping gene S14 was the internal control. In normal subjects AHSP expression was first detected at proerythroblast stage (on day 3 of culture), peaked at orthocromatic stage (on day 9) and declined to low levels by polychromatophilic-reticulocytes stage (day 13) (a decrease of 65% vs day 9 expression). Globin genes expression reached a steady state (a/non-a ratio value of 0.78 very near to a clinical equilibrium) on day 13. In this situation few free α-globin are detectable explaning the lower need of AHSP expression. In β-thalassemic subjects we observed a persistance of globin gene unbalance with an excess of free α-chains also in the advanced differentiation steps: interestingly the worst was the genotype, the highest was the globin ratio and higher was AHSP expression. In β+/β+ thalassemic patients AHSP expression declined on day 13 (a decrease of 15% vs day 9 expression) and globin gene ratio was 2.4, expression of a persistance of free α-chains excess. In β0 /β+ patients globin gene ratio on day 13 was 2.8 and AHSP expression increased of 2.5 times. Finally in β0/β0 patients globin gene ratio was very high with values up to 9–10; AHSP expression profiles instead were different between different subiects and related not only to globin unbalance but also to ineffective erythropoiesis rate and γ-globin chains persistance. All these findings underline an evident relation between AHSP and globin genes expression, suggesting a probable function of this protein during erythropoiesis.
Defective γδ T-Cell Function and Granzyme B Gene Polymorphism in Newly Diagnosed Breast Care Patients. Expansion with Zoledronic Acid Partially Compensates for This Deficiency.
Authors: Gaafar Ameera , Al-Sulaiman Abdulla , Iqniebi Alia , Al-Sayed Adher , Sahovic Entezam , Chaudhri Naeem A. , Al-Mohareb Fahad , Ajarim Dahish , Tbakhi Abdelghani , Al-Hussein Kkaled , Aljurf Mahmoud
Published: October 2019

It has been well established that γδ T-cells play a role in innate anti-tumor immunity. However, the exact mechanism has not yet been fully elucidated. Most of these responses have been ascribed to Vγ9Vδ2 cells, which represent a major subset of the circulating γδ T-cells in humans (1–10%). IFN-γ and granzyme B are important molecules in the anti-tumor immune responses. Upon stimulation, γδ T-cells rapidly produce IFN-γ and cytotoxic molecules. In the present study we analyzed the immune responses by γδ T-cells in 30 newly diagnosed breast cancer patients and 30 normal controls before and after expansion with zoledronic acid. We also scanned the granzyme B gene polymorphism in breast cancer patients and controls. Our result revealed that γδ-T cells in PBMC were reduced in both frequency and function in breast cancer patients compared with the normal controls. Ex-vivo stimulation of γδ T-cells with zoledronic acid and IL-2 partially compensated for this deficiency, as it stimulates production of IFN- γ and release of cytotoxic molecules by these cells. However, the IFN- γ and granzyme B and cytotoxicity of the expanded γδ T-cells from breast cancer patients remained significantly below normal control. Genotypic analysis of granzyme B gene revealed significantly higher frequency of the RAH haplotype in breast cancer patients compared with normal controls. The prevalence of the wild genotype QPY/QPY was significantly higher in normal controls compared with the breast cancer patients. Cytotoxicity by γδ T-cells against various targets was reduced in breast cancer patients compared to normal controls. In conclusion, our analysis shows a defective immune function of γδ T-cells and granzyme B gene polymorphism in breast cancer patients. The γδ T-cell function defect in these patients can be partially corrected by zoledronic acid. Further studies of γδ T-cell function and granzyme B gene polymorphism in other cancers, as well as the therapeutic use of zoldedronic acid is warranted.
Micro-RNA Profiling in CD4+ T Cell Differentiation.
Authors: Banerjee Arnob , Schambach Felix , Hammond Scott , Reiner Steven
Published: October 2019

Micro-RNAs comprise a class of small noncoding RNAs which have been found to be important regulators of cellular differentiation in multiple species. Previous analysis of micro-RNA expression in the murine hematopoietic system has suggested a role in cell differentiation and the maintenance of cell identity. Naïve progenitor CD4+ T cells respond to a combination of appropriate antigen and other specific signals by undergoing proliferation and further differentiation into one of at least two subsets. T helper 1 (TH1) cells produce high levels of the cytokine IFN-γ and T helper 2 (TH2) cells produce high levels of IL-4, optimizing them for control of intracellular and extracellular pathogens, respectively. It is currently not known whether micro-RNA molecules influence CD4+ T cell differentiation. We have used oligonucleotide arrays to analyze micro-RNA expression profiles of freshly isolated murine CD4+ T cells compared to cells differentiating into TH1 and TH2 subsets. Expression profiles were found to differ significantly between naïve and stimulated CD4+ cells, with fewer differences between TH1 and TH2 subsets. Promising candidate micro-RNAs are being further evaluated by northern blot and genetic studies. Micro-RNA-155 is upregulated on stimulation of CD4+ T cells in multiple oligonucleotide array assays. Micro-RNA-155 is encoded by the BIC oncogene and has been implicated in lymphomagenesis as well as in other malignancies. We have verified the induction of micro-RNA-155 in stimulated helper T cells by northern blot and are studying the effects of this micro-RNA on CD4+ T cell differentiation. Our observations support a role for micro-RNAs in helper T cell differentiation during the immune response.
Serotonergic Mechanisms in Haemostasis: A Connection between Major Depression and Thrombotic Risk.
Authors: Lopez-Vilchez Irene , Serra Montserrat , Alonso Antonio , Navalon Fulgencio , Hernandez Rosa , Gasto Cristobal , Escolar Gines , Galan Ana M.
Published: October 2019

Serotonergic mechanisms implied in both neuronal and platelet functions have been pointed out as a possible link between depressive disorders and cardiovascular risk. Recent studies suggest serotonin (5-HT) as a key element in the generation of a subpopulation of platelets with elevated procoagulant properties (coated-platelets). We have investigated the implication of serotonergic mechanisms in platelet and coagulation activation in samples from healthy donors or from patients suffering major depression. Furthermore, we evaluated the possible prothrombotic role of serotonergic mechanisms and the potential antithrombotic effect of selective serotonin reuptake inhibitors (SSRI) in vitro and compared results with those observed in a group of patients with major depression. Different experimental strategies were used to evaluate platelet function and the activation of coagulation system including standard aggregometry, flow cytometry, perfusion studies with circulating blood to evaluate platelet interaction and fibrin deposition on damaged vessels. Levels of tissue factor (TF) in whole blood and prothrombin fragment F1+2 in plasma samples were also determined. Aggregation studies did not reveal marked differences between control and patients with major depression. The presence of SSRI in plasma decreased up to 30% of the aggregation induced by ADP potentiated with 5-HT. Flow cytometry studies revealed that platelets from patients with major depression showed an enhanced expression of platelet antigens: FV, fibrinogen, CD63, GPIV, GPIb, as well as elevated exposure of anionic phospholipids (p<0.05 vs. controls). Treatment of patients with the SSRI normalized the expression of these antigens vs. control samples. Studies performed under flow conditions only showed a significant increase of the fibrin formation on vascular damaged vessels of patients with major depression vs. controls (71.1±9.5 % vs. 45.8±5.3 %, p<0.05) with corresponding elevations in F1+2 levels. In vitro incubation of blood samples with a SSRI was followed by a 30% of reduction in fibrin formation. Similar tendencies were confirmed in studies performed along clinical treatment of patients with SSRI. A decrease of 20% and 80% of covered surface by fibrin was observed after 8 and 24 weeks of treatment respectively. Levels of whole blood TF of patients suffering depression were found elevated vs. control samples (1.3±0.4 ng/ml vs. 1.0± 0.4 ng/ml respectively) and tended to decrease during the treatment (p<0.05). Our results reveal a more thrombogenic state in patients diagnosed of major depression associated to serotonergic mechanisms. Strategies aimed to control serotonergic mechanisms in platelets may offer a potential therapy for the regulation of the prothrombotic state of patients with major depression, but also in patients with cardiovascular risk.
Contribution of Lipopolysaccharide to Prolylcarboxypeptidase Activation in Endothelial Cells.
Authors: Perkins Ryan , Ngo My-Linh D. , Mahdi Fakhri , Shariat-Madar Zia
Published: October 2019

Bradykinin (BK) participates in the etiology of vascular inflammation and the regulation of local blood flow and blood pressure by causing a vascular hyperemia. The bradykinin generating pathway of human plasma consists of prolylcarboxypeptidase (PRCP), heat shock protein 90 (HSP90), coagulation factor XII (FXII, Hageman factor), and the heteromer complex of prekallikrein (PK) and high molecular weight kininogen (HK). In the presence of HK, PRCP activates PK to generate kallikrein on endothelial cells. Formed kallikrein then cleaves HK to liberate BK. PRCP-induced PK activation is independent of the activated FXII. The activity of PRCP leads to vasodilation. In inflamed tissues, tissue injury or hypotensive bacteremia, BK has been shown to accumulate in the blood or plasma of these patients. In this investigation, we have assessed the possible contribution of lipopolysaccharide (LPS) to PRCP activation in primary cultures of human umbilical vein endothelial cells (HUVECs) using multiple approaches. The role of LPS in HK binding to HUVECs was assessed. The effect of LPS on the endothelial permeability injury was evaluated by histological changes and analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The release of von Willebrand factor (VWF), endothelin-1 (ET-1) and intercellular adhesion molecule-1 (ICAM-1) induced by LPS in HUVEC was determined. PK activation on LPS-pretreated endothelial cells was higher than untreated cell. PRCP inhibitor, z-pro-prolinal, inhibited the production of kallikrein from PK compared to the control. These results suggest that LPS contributes to PRCP overactivation which results in the acceleration of the inflammatory process, exerting its effects via PK activation. Inhibiting the activity of PRCP may exert some beneficial therapeutic action in systemic inflammation, at least in part by inhibiting the production of BK and thus preventing vascular dysfunction.
The Use of Recombinant Activated Factor VII in Australia and New Zealand.
Authors: Dunkley Scott , Cameron Peter A. , Phillips Louise E. , Isbister James
Published: October 2019

Introduction:

The role of Recombinant activated factor VII (rFVIIa, NovoSeven) for off-label indications, including trauma, cardiac surgery and other critical bleeding episodes, remains controversial. Through the Haemostasis Registry, we report on the Australian and New Zealand experience of using rFVIIa.

Methods:

Monash University has established the Haemostasis Registry (with an educational grant from NovoNordisk Pharmaceuticals) to monitor the use of rFVIIa throughout Australia and New Zealand. More than 50 hospitals are contributing data to the Registry.

Results:

Over 600 cases have been reported to the Register. In those where complete data has been received, major areas of use are cardiac surgery (~42%), other surgery (~19%) and trauma (~13%). The reported efficacy of rFVIIa was high (where reported, in >70% cases bleeding decreased or stopped after first dose of rFVIIa) while the reported Adverse Events “possibly” or “probably” linked to rFVIIa use were relatively low (~6%). There were no directly linked adverse events. Despite suffering from conditions where mortality is high, the survival rate in rFVIIa recipients was 69% at 28 days post treatment. These results will be compared with international experience and published trials.

Conclusions:

Although randomized controlled trials are important in establishing the safety and efficacy of new treatments, they do not replace the need for registries, especially for treatments where clinicians believe that withholding treatment may be unethical because of potential life threatening consequences. This problem is made more difficult where there are a wide range of applications. As more data becomes available, the Haemostasis Registry data will help to elucidate the safety and efficacy of rFVIIa and provide important feedback to doctors and hospitals.
Results of a One-Year Registry of Bleeding Complications of Vitamin K Antagonist Therapy outside of a Controlled Population.
Authors: Halbritter Kai M. , Beyer Jan , Oettler Wolfram , Kleinstück Babett , Schellong Sebastian M.
Published: October 2019

Background: The registry was designed to collect data on bleeding complications in patients under Vitamin-K-antagonist therapy (VKA) during a one year period in a predefined area outside a controlled population and to describe clinical management of these complications.

Patients and methods: Between 1.1.05 and 31.12.05, bleeding complications requiring hospital stay in 23 hospitals in eastern saxonia, Germany, were documented. Indication for VKA therapy, type of bleeding, INR at hospital entry, application of packed erythrocytes and fresh-frozen plasma, clinical management and outcome at the end of hospital stay and after 3 months were recorded.

Results: 311 episodes of VKA-associated bleeding were documented. Patients were male in 49%, mean age was 73±10 years. Indication for VKA therapy was atrial fibrillation in 64%, mechanical heart valves in 10%, impaired LV-function in 1%, secondary prophylaxis of VTE in 18% and others in 6%. Bleeding affected the gastrointestinum in 23%, 22% were superficial hematomas, 14% were intracranial, 10% epistaxis, 10% hematuria, 7% intraabdominal/retroperitoneal, 5% were overdoses without clinical signs, 10% involved other sites.

INR on admission was 3.76±2.45, 40% of INR values were above, and 30% were in the targeted INR range. Vitamin K was administered orally in 26% of episodes, FFP in 9%, factor concentrate in 6%. Per episode, 3±2 units of packed erythrocytes were necessary. 8% of patients died during hospital stay. Surgical therapy was chosen in 25% of episodes, 20% were controlled by endoscopy. 3% of episodes were followed by ongoing detoriation of health status. Mean hospital stay was 13±9 days. Mortality was highest in patients with intracranial bleeding (24%). Retroperitoneal/intraabdominal bleedings required the largest amount of blood products and stabilization of coagulation. Three months after the index episode, mortality was 14%.

Conclusions: Complications of VKA-therapy outside of a controlled population occur most often at a supratherapeutic INR level. Gastrointestinal bleeding constitutes the major part of the complications, intra-hospital mortality of 8% is mainly caused by intracranial bleeding.